KR20030097604A - A composition for delivering a lipidized immunoglobulin to an intracellular compartment of a cell, a lipidized protein, and a lipidized antibody - Google Patents

Abstract

The present invention provides methods for targeting proteins such as antibodies to intracellular compartments in eukaryotic cells, enhancing organ uptake of proteins, pharmaceutical compositions of modified proteins for use in human therapy, and methods for preparing modified proteins. It is about. Modified proteins of the invention include bound lipid proteins, wherein one or more acyl groups are bound to the protein through various covalent binding chemistry provided with the carbohydrate side chain. Lipidized antibodies of the invention can be used for diagnostic and therapeutic purposes.

Description

A COMPOSITION FOR DELIVERING A LIPIDIZED IMMUNOGLOBULIN TO AN INTRACELLULAR COMPARTMENT OF A CELL, A LIPIDIZED PROTEIN, AND A LIPIDIZED ANTIBODY }

The present invention provides methods for targeting proteins, such as antibodies, to intracellular compartments in eukaryotic cells, enhancing organ uptake of proteins, pharmaceutical compositions of modified proteins for use in human therapy, and preparing modified proteins. It is about a method. Modified proteins of the invention include a linked lipid moiety wherein one or more acyl groups are bound to the protein via a carbohydrate side chain and provide various covalent chemistries.

Many naturally occurring or modified proteins have been proposed as diagnostics and / or therapeutics for use in human and domestic animals. However, proteins are generally only transported in small quantities along the endothelium of blood vessels and usually cannot cross the cell membrane to access intracellular compartments. Thus, antibodies can be produced against purified intracellular proteins such as, for example, transcription factors, intracellular enzymes, and structural proteins for cell construction, but these antibodies generally cannot enter native cells and disrupt cell membranes. Unless it is able to bind to the intracellular antigen target.

The advent of the monoclonal antibody technique in the mid-1970s opened a new era of medicine. First, researchers and clinicians have approached an almost infinite amount of homogeneous antibodies that can bind to certain antigenic determinants and have various immunological effector functions. Such proteins, known as "monoclonal antibodies," were considered to have great potential, for example, for removing in vivo harmful cells, pathogenic microorganisms and viruses. A method that allows the development of specific monoclonal antibodies with binding specificities corresponding to almost any desired antigenic epitope, including antigens located in intracellular compartments of native cells, is known as "magic bullets." We could provide abundant medicine.

Unfortunately, the development of appropriate therapeutic products based on monoclonal antibodies as well as polyclonal antisera has been significantly hampered by many disadvantages derived from the chemical nature of naturally occurring antibodies. First, as immunoglobulins cannot penetrate the plasma membrane of cells, antibodies generally do not have effective access to intracellular sites and are only internalized primarily as a result of ineffective intracellular entry mechanisms. Second, the antibodies generally do not pass through the vascular membrane (eg, the subcutaneous basement membrane), preventing the antibody from being effectively absorbed into organs and interstitial spaces. Thus, if there is an effective way to introduce specific biologically active immunoglobulin molecules through capillary barriers into intracellular locations, therapies for many important diseases can be developed. For example, the life cycle of retroviruses, such as HIV, involves an intracellular replication step, where specific monoclonal antibodies that are responsive to virus-coding proteins are readily located at the intracellular locations where retrovirus replication occurs. If accessible, the various virus-encoding polypeptides necessary for producing infectious virions in infected cells can be strongly inhibited or blocked.

Immune liposomes have been prepared as potential targeted delivery systems for delivering various molecules contained in liposomes to targeted cells. Immunoliposomes use immunoglobulins as targeting agents, wherein the acylated immunoglobulins are adhered to the lipid bilayer of liposomes and liposomes to specific cell types that have an external antigen to which the acylated immunoglobulin (s) of the immunoliposomes bind. Targets [Connor and Huang (1985) J. Cell Biol. 101 : 582; Huang, L. (1985) Biochemistry 24:29 ; Babbitt et al. (1984) Biochemistry 23 : 3920; Connor et al. (1984) Proc. Natl. Acad. Sci. (USA) 81 : 1715; Huang et al. (1983) J. Biol. Chem. 258 : 14034; Shen et al. (1982) Biochim. Biphys. Acta 689 : 31; Huang et al. (1982) Biochim. Biophys. Acta 716 : 140; Huang et al. (1981) J. Immunol. Methods 46 : 141; and Huang et al. (1980) J. Biol. Chem. 255 : 8015]. Immunoliposomes generally comprise immunoglobulins that are attached to the liposome's lipid bilayer by binding to an acyl substituent of the liposome bilayer via a crosslinking agent such as N-hydroxysuccimid. Thus, crosslinked immunoglobulins bind liposomes and target liposomes to specific cell types with a given foreign antigen by binding to foreign cell antigens. While this method can target liposomes for specific cell types, immunoliposomes have several important drawbacks, particularly limiting applications such as drug delivery media for delivering proteins to intracellular locations.

Attempts have been made to modify proteins to facilitate the transport of proteins into cells through capillary barriers (EP 0 329 185), but no fully satisfactory method has yet been reported in the art. Chemical modification of proteins such as antibodies by nonspecific “cationization” has been reported to increase transvascular and intracellular delivery of proteins (U.S.S.N. 07 / 693,872). However, this method for preparing cationized immunoglobulins significantly reduces the binding affinity of cationized immunoglobulins relative to their corresponding noncationic immunoglobulins in binding to a given epitope (about 90%). In general, cationization includes carbodiimide linkages of diamines such as putrescine or hexanediamine to carboxylates of aspartate and glutamate residues in immunoglobulin polypeptide sequences. Chemical modification of these primary amino acids disrupts its secondary and tertiary structures sufficiently to lose the binding affinity of the immunoglobulin. In addition, the method slightly cationicizes glutamate and aspartate residues located within the variable domains of the immunoglobulin chains, thereby significantly losing binding affinity and / or specificity.

Chemical modification of small molecules has also been proposed as a method of enhancing the transport of small bioactive compounds. Pelner (WO 91/17242) discloses the formation of a lipid complex consisting of a lipid vesicle and a bioactive material contained therein. Pelner et al. (WO 91/16024) disclose cationic lipid compounds that are believed to be useful for enhancing the transfer of small bioactive molecules in plants and animals. Liposomes and polycationic nucleic acids have been proposed as a method of delivering polynucleotides into cells. Liposomes often exhibit a narrow range of cell specificities, and when the DNA is applied externally to liposomes, the DNA often responds to cellular nucleases. Newer polycationic lipospermine compounds exhibit a broad cellular range (Behr et al., (1989) Proc. Natl. Acad. Sci. USA 86 : 6982), and DNA is covered by these compounds. Also, a combination of neutral and cationic lipids has been pointed out as a method of transfecting animal cells (Rose et al., (1991) BioTechniques 10 : 520).

In particular, another attempt to promote drug delivery through the blood-brain barrier is to convert hydrophilic drugs to fat-soluble drugs or to use pharmacological procedures that involve drug latency. Most of the attempts involving a latent process include blocking hydroxyl, carboxyl and primary amine groups on the drug to make the drug more soluble so that it is more easily transported through the blood-brain barrier. Padridge and Shimmel (U.S. Patent No. 4,902,505) disclose chimeric peptides that enhance transport by receptor-mediated trans cytosis.

Thus, methods for facilitating the transport of specific proteins, such as antibodies, through the capillary barrier into cells, targeting agents, such as monoclonal antibodies, and the above-mentioned immunity for treating diseases of humans and animals that are susceptible to treatment with intracellular proteins There is a need in the art for a pharmaceutical composition of globulin.

Summary of the Invention

Conventional methods of enhancing antibody transport into cells by binding cationic substituents to the primary polypeptide sequences of immunoglobulins by relatively nonspecific chain chemistry have been found to have deleterious changes in the secondary, tertiary and / or quaternary structure of the protein. It was observed to produce. This structural change is a clear cause of the loss of binding affinity observed in cationized antibodies. To overcome this, the present invention provides a method for binding lipid substituents to proteins such as immunoglobulins, mainly by covalent binding to the carbohydrate side chains of the protein such that the lipid substituents rarely destroy the protein's biological activity (eg, antigen binding). to provide.

The present invention generally provides a method for preparing a lipidated protein by lipidating carbohydrate moieties on glycoproteins or glycopeptides. In general, the methods of the present invention are used to attach lipids, such as lipoamine, to the polypeptide by covalently binding the carbohydrate moiety on the protein, wherein the carbohydrate moiety is generally chemically oxidized to react with lipoamine to lipidize. Form a protein. The resulting lipidated protein generally has advantageous pharmacodynamic properties, such as increased ability to access the parenchymal cells of various organs through the vascular barrier and increased ability to access intracellular compartments. In one aspect of the invention, the lipidation of a protein, such as an antibody, directed against a transcription factor (e.g. Fos, Jun, AP-1, OCT-1, NF-AT) is in the nucleus of the lipidated protein (s). Improve localization

The invention also provides a method for producing lipidated antibodies that are effectively transported through the capillary barrier in vivo and are internalized in mammalian cells. The method of the invention relates to a method of chemically binding one or more lipid substituents (e.g., lipoamines) to carbohydrate substituents on immunoglobulins to produce carbohydrate-linked lipidated immunoglobulins, wherein the lipidated immunoglobulins Can be localized intracellularly. In another embodiment of the invention, one or more lipid substituents (eg lipoamines) are covalently attached to the non-carbohydrate moiety on the protein or polypeptide (eg, an amide with a side chain carboxyl substituent of an Asp or Glu residue). Chain formation, or by thioester chain formation with Cys residues). In addition, fatty acids may be linked to Arg or Lys residues by side chain amine substituents.

Similarly, lipid substituents can be covalently linked to peptide mimetic compounds. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to template peptides. Non-peptide compounds of this kind are referred to as "peptide pseudo" or "peptide pseudo" (Fauchere, J. (1986) Adv. Drug Res. 15 : 29; Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem 30 : 1229, incorporated herein by reference), usually developed with the aid of computerized molecular modeling. Peptide constructs that are structurally similar to therapeutically useful peptides can be used to provide equivalent therapeutic or prophylactic effects. Generally, the peptidomimide is structurally similar to the polypeptide of the legend (ie, a polypeptide having biological or pharmacological activity), but one or more peptide chains are -CH ₂ NH-, -CH ₂ S-, -CH _2- CH ₂ -, -CH = CH- (cis and _{trans), -COCH 2 -, -CH (} OH) CH 2 -, and the following are known in the art as a chain selected from the group consisting of -CH ₂ SO- literature Optionally substituted by the method described further in: Spatola, AF in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, AF, Vega Date (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley, JS, Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., Int J Pept Prot Res (1979) 14 : 177-185 (-CH ₂ NH-, -CH ₂ CH _2- ); Spatola, AF et al., Life Sci (1986) 38 : 1243-1249 (-CH ₂ -S); Hann, MM, J Chem Soc Perkin Trans I (1982) 307-314 (-CH-CH, cis and trans); Almquist, RG et al., J Med Chem (1980) 23 : 1392-1398 (-COCH _2- ); Jennings-White, C. et al., Tetrahedron Lett (1982) 23 : 2533 (-COCH _2- ); Szelke, M. et al., European Appln. EP 45665 (1982) CA: 97 : 39405 (1982) (-CH (OH) CH _2- ); Holladay, MW et al., Tetrahedron Lett (1983) 24 : 4401-4404 (-C (OH) CH _2- ); and Hruby, VJ, Life Sci (1982) 31 : 189-199 (-CH ₂ -S-); These documents are incorporated herein by reference. Particularly preferred non-peptide chains are -CH ₂ NH-. Such peptide constructs may, for example, produce more economically, greater chemical stability, enhanced pharmacological properties (half-life, uptake, potential, efficacy, etc.), modified specificity (eg, broad spectrum of biological activity), reduction May have significant advantages over polypeptide embodiments, including antigenicity and the like. Lipidation of peptidomine generally involves directing one or more acyl chains or spacers (e.g., amides) to non-interfering position (s) on the peptidobody, as expected by quantitative structural activity data and / or molecular modeling. Covalently linked). These non-interfering positions are generally positions that do not form direct bonds with the macromolecular (s) (eg, receptors) to which the peptidomide binds to produce a therapeutic effect. Lipidation of the peptidomide should hardly interfere with the desired biological or pharmacological activity of the peptide pseudosome.

In addition, the present invention provides intracellular immunotherapeutic targets, such as lipidized antibodies that can enter the intracellular compartments through the vascular membrane, particularly viral encoded gene products (eg, HIV-1 Tat protein) that are essential components of the viral life cycle. , Biologically active intracellular antigens (eg, tumor gene proteins such as c-fos, c-src, c-myc, c-lck (p56), c-fyn (p59), and c-abl), and / Or treatment of lipidated proteins such as lipidated antibodies that bind to dura mater or extracellular antigens (eg, polypeptide hormone receptors such as IL-2 receptor, PDGF receptor, EGF receptor, NGF receptor, GH receptor, or TNF receptor) And diagnostic compositions. Other proteins that may be targeted by lipidated antibodies include c-ras p21, c-her-2 protein, c-raf, any of a variety of G proteins and / or G protein activating proteins (GAPs), NF-AT and Such transcription factors, calcineurin, and cis-trans prolyl isomerase and the like, but are not limited thereto. Lipidized antibodies can be used to detect diagnostic agents, such as radiocontrast agents or magnetic resonance imaging components, in particular organs, tissues, body compartments, cell types, tumors or other anatomical structures (eg, pathological lesions). Can be used to localize at a specific location. Lipidized antibodies can also be used to localize bound therapeutic agents, such as chemotherapy drugs, radiosensitizers, radionuclides, antibiotics, and other agents, at specific locations in the body. In addition, the lipidated antibodies of the invention may be HIV-1 Tat proteins, dura mater or membrane bound antigen targets (e.g., γ-glutamyltranspeptidase, c-ras ^H p21, rasGAP) or extracellular antigen targets (i.e. Intracellular target antigens, such as β-amyloid protein deposits in Alzheimer's disease brain, can be used therapeutically by neutralizing (ie binding to and inactivating the antigen). Lipidized antibodies cross the blood-brain barrier and can react with extracellular antigen targets that are generally inaccessible to immunoglobulins circulating in the blood or lymphatic system. In addition, the lipidated antibody reacts with intracellular portions on the dense protein, such as the cytoplasmic tail of the viral envelope glycoprotein or the protein kinase domain of the proto-oncogene proteins (c-src, c-abl), resulting in a virus with an infectious envelope. Production and kinase activity can be inhibited, respectively.

1 is a structural formula representing various lipoamines that may be used in the present invention. The right column represents branched lipoamine and the left column represents straight lipoamine.

Figure 2 is a schematic diagram of a glycosylated antibody comprising (1) an immunoglobulin tetramer (two light chains bound to two heavy chains), and (2) a schematic diagram of the carbohydrate-bound lipidated immunoglobulins of the present invention. For example, branched chain lipoamine substituents are shown to be bound to the partially oxidized carbohydrate side chain of an immunoglobulin tetramer, but are not limited thereto. Such carbohydrate side chains may be located in the C _H , V _H , C _L and / or V _L regions.

3 shows that the effects of native (ie, non-lipidized) anti-Tat immunoglobulins are deficient in in vitro survival of cells infected with HIV-1, while lipidized anti-Tat immunoglobulins have a beneficial effect. It is shown.

4 shows that native (non-lipidated) anti-Tat antibodies, lipidated anti-gp120 antibodies, or rsCD4 have little effect on inhibiting CAT activity in HLCD4-CAT cells, while lipidized anti-Tat antibodies Significantly inhibits CAT activity (about 75%).

details

Justice

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All methods and materials similar or equivalent to those set forth herein can be used in the practice or testing steps of the present invention, which present preferred methods and materials. For the purposes of the present invention, the following terms are defined as follows:

The 20 common amino acids and their abbreviations used herein are used according to conventional conventions ( Imunology-A Synthesis , 2nd Edition, ES Golub and DR Gren, Massachusetts, United States, Sunderland Sinauer Associates (1991). ), Incorporated herein by reference).

The term "corresponds to" means that the polynucleotide sequence is homologous (i.e., strictly evolutionarily irrelevant but identical) to all or part of the polynucleotide reference sequence, or the polypeptide sequence is a polypeptide reference It means the same as the sequence. In contrast, the term "complementary to" means that the complementary sequence is homologous to all or part of a polynucleotide reference sequence. As an example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

The term "substantial similarity" or "substantial identity" is used to characterize a polypeptide sequence or nucleic acid sequence, wherein the polypeptide sequence has a sequence identity of at least 50% compared to the reference sequence and the nucleic acid sequence. Is at least 70% sequence identity compared to the reference sequence. The percentage of sequence identity is calculated by excluding small amounts of deletions or additions that are up to 25% of the total of the reference sequence. The reference sequence may be a subset of longer sequences, such as a constant domain in an immunoglobulin constant region gene; However, the reference sequence consists of at least 18 nucleotides in the case of polynucleotides and is at least 6 amino residues in length for polypeptides.

The term "natural-generating" as used for an object means that the object can be found in its natural state. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) and that can be isolated from a natural source and that has not been intentionally modified by a human in a laboratory is naturally occurring. Lipoproteins (eg, naturally-occurring isoprenylated or myristylated proteins) that are found in nature and have not been manipulated by humans and that can be separated from an organism are naturally-occurring lipoproteins.

"Glycosylation site" means an amino acid residue that is recognized by eukaryotic cells as a binding site for sugar residues. Amino acids to which carbohydrates are bound, such as fructose, are generally asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. Certain binding sites are usually signaled by amino acid sequences referred to herein as "glycosylation site sequences". The glycosylation site sequence for N-linked glycosylation is -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid other than proline. The major glycosylation site sequence for O-linked glycosylation is-(Thr or Ser) -XX-Pro-, where X is any conventional amino acid. The recognition sequence of glycosaminoglycans (specific forms of sulfated sugars) is -Ser-Gly-X-Gly-, where X is any conventional amino acid. The terms "N-bond" and "O-bond" refer to chemical groups that act as binding sites between sugar molecules and amino acid residues. N-linked sugars are bound via amino groups, and O-linked sugars are bound via hydroxyl groups. However, not all glycosylation site sequences in proteins are necessarily glycosylated; Some proteins are secreted in glycosylated and aglycosylated forms, while others are fully glycosylated in one glycosylation site sequence, but also include other glycosylation site sequences that are not glycosylated. Thus, not all glycosylation site sequences present in polypeptides are essential glycosylation sites for which the sugar residues actually bind. The first N-glycosylation reaction in the biosynthesis process is to insert "core carbohydrates" or "core fructose" ( Proteins, Structures and Molecular Principles , (1984) Creighton (ed.), WH Freeman and Company, New York) , Incorporated herein by reference).

As used herein, “glycosylated cell” means one or more mannose residues in a cell capable of glycosylating a protein, in particular one or more polypeptides expressed in the cell, in particular one or more glycosylation site sequences in a secreted protein By eukaryotic cell capable of adding an N-linked “core fructose” containing and / or adding an O-linked sugar. Thus, glycosylated cells contain one or more enzymatic activities that can catalyze the binding reaction of sugar residues to glycosylation site sequences in the protein or polypeptide, which cells in fact glycosylate one or more expressed polypeptides. do. For example, mammalian cells are generally glycosyl cells, but are not limited to these. Other eukaryotic cells, such as insect cells and yeast, may also be glycosyl cells.

As used herein, the term "antibody" refers to a protein composed of one or more polypeptides substantially encoded by immunoglobulin superfamily genes (eg, The Immunoglobulin Gene Superfamily , AF Williams and AN). Barclay, in Immunoglobulin Genes, T. Honjo, FW Alt, and TH Rabbitts, eds., (1989) Academic Press: San Diego, Calif., Pp. 361-387, incorporated herein by reference). For example, although not particularly limited, an antibody may comprise part or all of the heavy chain and part or all of the light chain, or may comprise only part or all of the heavy chain. Known immunoglobulin genes include kappa, lambda, alpha, gamma (IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ ), delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are variable region genes at the NH ₂ -terminal (about 110 amino acids) and kappa or lambda constant region genes at the COOH-terminus. Is encrypted by Full-length immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids) may be used for variable region genes (about 116 amino acids) and one of the other constant region genes described above, such as gamma (about 330 amino acids). Password is similarly encrypted). Although not particularly limited, antibodies include the following: immunoglobulin fragments (eg, Fab, F (ab) ₂ , Fv), single-stranded immunoglobulins, chimeric immunoglobulins, humanized antibodies, primates Antibodies, and various light chain-heavy chain combinations. Antibodies are produced in glycosyl cells (eg, human lymphocytes, hybridoma cells, yeasts, etc.) or non-glycosyl cells (eg, E. coli ) or synthesized by chemical methods, or polynucleotide templates It can be prepared by an ex vivo detoxification system that directly deciphers using.

As used herein, “lipidated antibody” refers to a lipid derivatization reaction of one or more carbohydrate moieties bound to an immunoglobulin at a glycosylation site (eg, glycyldioctadecylamide, dilauroylphosphatidylethanolamine, or di Octadecylamido refers to an antibody modified by covalent reaction of lipoamine such as glycylspermidine). In general, lipid substituents, such as lipoamine, are covalently linked via a naturally-occurring carbohydrate moiety at a naturally-occurring glycosylation site. However, modified glycosylation site sequences (typically by site-directed mutation of polynucleotides encoding immunoglobulin chains), and / or modified glycosylation patterns (eg, within glycosylated cells other than lymphocytes or of other species). Immunoglobulins) can be produced by expressing immunoglobulin-coding polynucleotides in lymphocytes. Lipid substituents may bind to one or more naturally occurring carbohydrate moieties or non-naturally occurring carbohydrate moieties on an immunoglobulin chain. When antibodies are produced either by direct polypeptide synthesis or by biosynthesis in non-glycosyl cells (eg, phage expression libraries), carbohydrate substituents are usually carried out by chemical or enzymatic modifications for subsequent lipidation reactions. (Alternatively, this carbohydrate may be lipidated before binding to immunoglobulins).

As used herein, “lipidized protein” refers to a modified protein (multimeric proteins, glycoproteins and polypeptides of varying sizes) by binding lipids (eg, lipoamines) via binding of lipids, generally carbohydrate moieties. Inclusive). Lipidated proteins are produced by derivatizing proteins, and the resulting lipidated proteins are distinguished from naturally-occurring lipid-bound proteins and lipoproteins. For biologically active proteins (eg, enzymes, receptors, transcription factors), the lipidation reaction should rarely destroy the biological activity (eg, at least about 15% of the natural biological activity has been lipidated). Must be preserved in protein). Lipidated peptidomides should maintain at least about 25-95% of the pharmacological activity of the corresponding non-lipidized peptidomide.

"Alkyl" means a fully saturated aliphatic group which may be a cyclic or branched or linear chain. Examples of the alkyl group include methyl, ethyl, cyclopropyl, cyclopropylmethyl, sec-butyl, heptyl, dodecyl and the like. All of these groups are unsubstituted or one or more non-interfering substituents such as halogen; C ₁ -C ₄ alkoxy; C ₁ -C ₄ acyloxy; Formyl; Alkylenedioxy; Benzyloxy; Phenyl or benzyl, which may optionally be substituted with one to three substituents respectively selected from halogen, C ₁ -C ₄ alkoxy or C ₁ -C ₄ acyloxy. The term "non-interfering" refers to substituents that do not adversely affect any reactions carried out by the process of the invention. If more than one alkyl group is present in a molecule, each may be separately selected from "alkyl" unless otherwise specified.

The term "alkylene" refers to a divalent radical that is fully saturated as a branched or straight chain radical containing only carbon and hydrogen. The term may also be exemplified by radicals such as methylene, ethylene, n-propylene, t-butylene, i-pentylene, n-heptylene and the like. All of the foregoing are one or more non-interfering substituents, for example halogen; C _1-4 alkoxy; C _1-4 acyloxy; Formyl; Alkylenedioxy; Benzyloxy; Phenyl or benzyl, which may optionally be substituted with one to three substituents each selected from halogen, C _1-4 alkoxy or C _1-4 acyloxy. The term "non-interfering" refers to substituents that do not adversely affect any reaction carried out by the process of the invention. If more than one alkylene group is present in a molecule, each may be separately selected from "alkylene" unless otherwise specified.

"Aryl" is represented by Ar and includes monocyclic aromatic groups having 6 to 20 carbon atoms or includes condensed carbon ring aromatic groups. Examples of the aryl group include phenyl, naphthyl and the like. The aryl group is one or more non-interfering substituents, for example lower alkyl; Lower alkenyl; Lower alkynyl; Lower alkoxy; Lower alkylthio; Lower alkylsulfinyl; Lower alkylsulfonyl; Dialkyl amines; Halogen; Hydroxy; Phenyl; Phenyloxy; Benzyl; Benzoyl; And substituents selected from nitro. Each substituent may be optionally substituted with additional non-interfering substituents.

"Amino" means a -NH ₂ group.

"Alkylcarbonyl" means a-(CHR ₁ ) -CO- group wherein R ₁ is specified at the α position. R ₁ may be hydrogen, alkyl or an amino group. R ₁ is preferably an amino group.

Description of Preferred Embodiments

The present invention provides novel methods of chemically modifying proteins such as antibodies to facilitate passage into cells through capillary barriers. In general, this method involves one or more non-interfering lipid substituents (eg, glycyldioctadecylamide, glycyldiheptadecylamide, text) at reactive sites (eg, periodate-oxidized carbohydrate moieties) in the protein molecule. Covalently linking lysyldihexadecylamide, dilauroylphosphatidyl ethanolamine, and glycyldioctadecadienoylamide). Various non-interfering lipid substituents attach to proteins to produce lipidated proteins, such as the lipidated antibodies of the present invention. For example, the lipids exemplified below can be bound to a protein of interest to provide a lipidated protein, but is not limited thereto; Lipoamines, lipopolyamines and fatty acids (eg, stearic acid, oleic acid, etc.). In general, lipids will be covalently bound to carbohydrates (eg, carbohydrate side chains of glycoproteins) bound to proteins. Naturally occurring carbohydrate side chains are useful for binding to lipoamines, and novel glycosylation sites are genetically engineered into polypeptides by genetic engineering of coding polynucleotides and by expressing coding polynucleotides in glycosylated cells. Provides a glycosylated polypeptide.

Glycosylated proteins can be lipidated to improve the transvascular transport, endotracheal uptake and intracellular localization (including nuclear localization) properties of the lipidated protein. In general, glycosylated polypeptides (e.g. antibodies) are chemically oxidized using an oxidizing agent (e.g. periodicate) to generate carboxyl side groups and / or aldehyde side groups, which then react with lipoamine to lipoproteins. To form a covalent bond (amide or imide, respectively) that binds the amine. In general, chemical oxidation methods partially oxidize some molecules and non-oxidize or completely oxidize the other, but the oxidation reaction of the carbohydrate side chain is a partial oxidation reaction that produces one or more reactive carboxyl or aldehyde groups. However, to be lipidized by reaction with lipoamine, the glycoprotein must be oxidized to produce one or more aldehyde side groups that can react with lipoamine, but also through binding to carboxyl side groups Can be generated. The carboxyl side group or aldehyde side group of the oxidized glycoprotein is a carboxyl group or an aldehyde group having a carbonyl carbon derived from oxidized oligosaccharides, which is either directly or a spacer (e.g., N-linked or O-linked carbohydrate side chains). Covalently bound to the protein via the non-oxidized portion of the Preferably, the carbohydrate chains of the N-linked and O-linked are incompletely oxidized to produce a plurality of reactive aldehyde and carboxyl groups for reacting with lipoamine at each glucosylation site. Most commonly, glycoproteins having one or more complex N-linked persaccharides, for example glycoproteins with branched (mannose) ₃ (β-N-acetylglucose amino) cores, are suitable oxidants, generally Partially oxidized by limited reaction with acid salts. N-acetylglucosamine (NAG), mannose, galactose, fucose (6-deoxygalactose), N-acetylneuraminic acid (sialic acid), glucose, N-acetylmuramic acid, N-acetylgalactose Bound fructose, including amines, xylose, or combinations of such monosaccharide units, is oxidized to react with lipoamine to yield lipidated proteins, more specifically carbohydrate bound lipidated proteins. In addition to non-naturally occurring monosaccharides, persaccharide bound glycoproteins having monosaccharide units other than those specifically exemplified above may also be oxidized to covalently bind lipoamine to form lipidated proteins.

Lipoamines are molecules having at least one acyl group and at least one free amine (ie, primary or secondary amine). The present invention may also be practiced with lipoamines having tertiary amines, which include one or more substituents that can be replaced upon reaction with oxidized carbohydrates. Examples of lipoamines with primary amines are shown in FIG. 1. For example, the present invention can produce lipidated proteins by reacting glycoproteins with straight lipoamines of the formula:

NH ₂ -R- (CH ₂ ) _n -CH ₃

Where

R is di-substituted alkyl (alkylene), preferably methylene (-CH _2- ); 1,4-disubstituted cyclohexyl; Disubstituted aryl (arylene); Preferably 1,4-disubstituted phenyl (phenylene); Amido groups of the formula-(CHR ₁ ) -CO-NH-, wherein R ₁ is hydrogen or an amino group; Alkylcarbonyl, preferably α-amino substituted alkylcarbonyl; Or phosphate diesters, preferably of the formula -CH ₂ -O-PO ₂ -O-,

n is usually an integer from 1 to 50, preferably an integer from about 5 to 30, more preferably an integer from about 10 to 25, most preferably an integer from about 15 to 20. In general, n is selected by the experimenter according to the following criteria: when the molecule to be lipidated is large (ie, a protein of about 10 KD or more), n is about to increase the hydrophobicity of the resulting lipidated protein. Preferably 8 to 12 or more; If the molecule to be lipidated is small (eg oligopeptide), n usually ranges from 2 to 18, but if the hydrophobicity of the lipidated molecule is additionally desired, n can be larger.

The invention may also be practiced with branched chain lipoamines comprising lipoamines of the formula:

Where

R 'is trisubstituted alkyl (preferably CH ₂ -CH <or 1,2,4-trisubstituted cyclohexyl); Trisubstituted aryl (preferably 1,2,4-trisubstituted phenyl); An amido group of formula-(CHR ₁ ) -CO-N <wherein R ₁ is hydrogen or an amino group; Imino groups of the formula -CHR ₂ -NH-CH <(wherein R ₂ is hydrogen or an amino group) or imino groups of the formula CH ₂ -N <; Or phosphate diesters, preferably of the formula -CH ₂ -CH ₂ -O-PO ₂ -O-CH ₂ -CH (CO _2- ) ₂ ,

m and n are each independently selected integers, which are generally 1 to 50, preferably about 5 to 30, more preferably about 10 to 25, and most preferably about 15 to 20. In general, n is selected by one of ordinary skill in the art according to the following criteria: when the molecule to be lipidated is large (ie, at least about 10 kD protein), n is about 8 to increase the hydrophobicity of the final lipidated protein. Preferably 12 to 12 or more; If the molecule to be lipidated is small (eg oligopeptide), n usually ranges from 2 to 18, but if the hydrophobicity of the lipidated molecule is additionally desired, n may be larger.

Essentially all glycoproteins can be lipidized according to the method of the present invention by reacting the oxidized carbohydrate side chains with lipoamine. In Fig. 2, the glycosylated antibody and the carbohydrate-binding lipidated antibody of the present invention are shown schematically. Non-glycosylated proteins can be bound to lipids by binding via a suitable crosslinker (eg, by carbodiimide binding chemistry).

The present invention provides novel lipidated antibodies that can specifically bind to certain intracellular epitopes with strong affinity. Such antibodies readily enter the intracellular compartment and have a binding affinity of about 1 × 10 ⁶ M ⁻¹ or greater, preferably 1 × 10 ⁷ M ⁻¹ to 1 × 10 ⁸ M ⁻¹ , more preferably about 1 × 10 ⁹ M ^-1 or more. Lipidized antibodies usually contain lipid substituents attached to naturally occurring carbohydrate side chains on the feed immunoglobulin chain and constitute antibodies that specifically react with intracellular epitopes, transmembrane epitopes or extracellular epitopes. Because carbohydrates are located at the F _C region of immunoglobulins, chemical modification of carbohydrate residues by lipidation is believed to result in little loss of the affinity of the antibody for antigen (Rodwell et al., (1986) Proc. Natl Acad.Sci. (USA) 83: 2632). In general, a lipidated antibody retains substantial affinity for its antigen, and its affinity can be readily determined by any of several antibody-antigen binding assays known in the art. The antibodies can be produced economically in large quantities, and can be used, for example, for treating various human diseases by various methods.

One form of immunoglobulin consists of the basic structural units of the antibody. This form is a tetramer, consisting of two identical pairs of immunoglobulin chains, each pair containing one light chain and one heavy chain. In each pair, the variable portions of the light and heavy chains together serve to bind antigens, and the constant portions play a role in antibody agonist function. In addition to antibodies, immunoglobulins can be a variety of other forms, such as Fv, Fab, and (Fab ') ₂ as well as bifunctional hybrid antibodies, fusion proteins (eg, bacteriophage expression libraries), and other forms (eg, Lanzavecchia et al. Many, Eur. J. Immunol. 17 , 105 (1987)), single-stranded (eg, Houston et al . , Proc. Natl. Acad. Sci. USA, 85 , 5879-5883 (1988) and Bird et al. , Science , 242 , 423-426 (1988)) (see, Hood et al., "Immunology", Benjamin, New York, 2nd Edition (1984) and Hungkafiler and Hood, Nature , 323 , 15-16 ( 1986)).

Antibodies are produced in glycosyl cells (eg, human lymphocytes, hybridoma cells, yeasts, etc.), non-glycosyl cells (eg, E. coli ), synthesized by chemical methods, or tested using polynucleotide templates. It can be produced by detoxification directly with an endothelial detoxification system. One source of hybridoma cell lines and immunoglobulin-coding polynucleotides is the United States Model Fungi Culture Collection (ATCC, Rockville, Maryland). Methods for expressing heterologous proteins in recombinant hosts, methods for chemical synthesis of polypeptides and methods for in vitro translation are known in the art and are also described in the following references: Maniatis et al., Molecular Cloning : A Laboratory Manual (1989), 2nd Edition, Cold Spring Harbor, New York; Berger and Kimmel, Methods in Enzymology , Vol 152 , Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, CA; Maryfield, J. (1969) J. Am. Chem. Soc. 91 : 501; Kaiken Ai. M. (1981) CRC Crit. Rev. Biochem. 11 : 255; Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986) Science 232 : 342; Kent, S. ratio. H. (1988) Ann. Rev. Biochem. 57 : 957; And offered, al. this. (1980) Semisynthetic Proteins , Willy Publishing; These documents are incorporated herein by reference. Antibodies produced in non-glycosylated cells can bind to lipids by using a bifunctional crosslinking agent, or preferably post-translationally glycosylated in glycosylation systems such as purified dog pancreatic microsomes. (Mucler and Lodish (1986) Cell 44 : 629 and Walter, P. (1983) Meth. Enzymol. 96 : 84, incorporated herein by reference). Alternatively, the polynucleotide encoding the antibody can be isolated from the expression library of selected prokaryotic cells (eg, the combined antibody fragment expression library) and then expressed in glycosylated cells to generate glycosylated antibodies. According to this method, glycosylated antibodies having naturally occurring and / or non-naturally occurring glycosylation patterns can be obtained. Such glycosylated antibodies can be lipidated according to the methods of the present invention.

Glycosylation of immunoglobulins has been shown to have a significant impact on agonist function, structural stability, and rate of secretion from antibody-producing cells (Leather Barrow et al. , Mol. Immunol. 22 : 407 (1985)). Carbohydrate groups that play a role in these features are generally bound to the constant region (C) of an antibody. For example, glycosylation of IgG in asparagine 297 present in the C _H 2 domain allows IgG to function fully to activate the classical pathway of complement-dependent cell lysis (Tao and Morrison, J. Immnol. 143 : 2595 ( 1989). Glycosylation of IgM at asparagine 402 present in the C _H 3 domain is essential for proper combination of antibodies and cytolytic activity (Muraoka and Schulman, J. Immunol. 142 : 695 (1989)). By removing glycosylation sites such as the 162 and 419 positions present in the C _H 1 and C _H 3 domains of IgA antibodies, intracellular degradation and secretion inhibition of at least 90% are induced (Taylor and Wall, Mol. Cell. Biol. 8: 4197 (1988).

Glycosylation of immunoglobulins in the variable region (V) was also observed. Sox and Hood ( Proc. Natl. Acad. Sci. USA 66 : 975 (1970)) reported that about 20% of human antibodies were glycosylated in the V region. Glycosylation of the variable region (V) domain is presumed to be due to the accidental presence of the N-linked glycosylation signal Asn-Xaa-Ser / Thr in the variable region sequence and is recognized in the art to play an important role in immunoglobulin function. It never happened.

Thus, in general, lipidation is preferably performed in antibodies having naturally occurring glycosylation patterns. When the glycosylation site is genetically introduced into the antibody, it is desirable that the new glycosylation site enters the variable or constant region framework region, which does not seem to adversely affect the antigen binding activity of the antibody. In general, new glycosylation sites that are genetically introduced into the antibody are most preferably located in the constant region.

In addition, polypeptide fragments can be prepared that contain only a portion of the primary antibody structure and have carbohydrate side chains that can be derivatized with lipid substituents (eg, lipoamines), which fragments can be one or more immunoglobulin activity (eg, antigens). Binding activity). Such polypeptide fragments may be prepared by proteolytic cleavage of the original antibody by methods known in the art, or by site-directed mutation of the desired position in the expression vector comprising the immunoglobulin protein coding sequence. For example, mutations following CH ₁ to generate Fab fragments, or mutations following junctions to generate (Fab ') ₂ fragments. Single-stranded antibodies can be prepared by linking the V _L and V _H regions with a DNA linker (cf. Houston et al., And Bud et al., Cited above). In addition, as with many genes, since immunoglobulin-related genes each contain one or more distinct functional domains, the gene can be fused to the functional domain of other genes with novel characteristics. have. Nucleic acid sequences for producing immunoglobulins of the present invention can fully express a given antibody, and may include a variety of different polynucleotides (genome or cDNA, RNA, synthetic oligonucleotides, etc.) and components (eg, V, J, D and C regions), as well as can be produced by various techniques. Although methods for combining appropriate synthetic and genomic sequences are currently the most common methods of preparation, cDNA sequences can also be used (see, eg, European Patent Publication No. 0239400 and L. Lykmann et al., Nature, 332, 323-327). (1988)).

DNA sequences encoding immunoglobulins and / or immunoglobulin chains can be obtained using hybridoma clones that can be produced according to methods known in the art (Köhler and Milstein (1976) Eur. J. Immunol. 6 : 511, which is incorporated herein by reference, is also available from several sources (“ATCC List of Cell Lines and Hybridomas”, ATCC, Rockville, MD; incorporated herein by reference). DNA sequences encoding immunoglobulin chains can be obtained by conventional cloning methods known in the art described in various literature (cf. Maniatis et al., Molecular Cloning : A Laboratory Manual, 2nd Edition (1989), Cold Spring) Haber, New York, and Berger and Kimmel, Methods in Enzymology, Vol 152 , Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif., Incorporated herein by reference.

As noted above, the DNA sequence is operably linked to the expression control sequence (ie, placed in a position that allows for the action of the expression control sequence) and then transduced in the host, usually in glycosyl cells. will be. Such expression vectors can generally be replicated as an integral part of episomal or host chromosomal DNA in the host organism. In general, the expression vectors can detect cells transformed with the desired DNA sequence by including markers of selection for tetracycline-resistant or G418-resistant (see, eg, US Pat. No. 4,704,362).

this. E. coli is a prokaryotic host that is particularly useful for cloning the DNA sequences of the present invention. With an appropriate other microbial host to use a Bacillus [e.g., Bacillus subtilis (Bacillus subtilis), and other enterobacteria EXAMPLE, Salmonella (Salmonella), Serratia (Serratia marcescens), and various Pseudomonas (Pseudomonas) species; Include. For such prokaryotic hosts, one may also construct a transgenic expression vector that generally contains a transgenic regulatory sequence suitable for the host cell (eg, origin of replication). In addition, various known promoters may be present in any number, examples include lactose promoter systems, tryptophan (trp) promoter systems, β-galactosidase promoter systems, or phage lambda promoter systems. Such promoters, optionally in combination with operator sequences, generally control expression and may contain ribosomal binding site sequences and the like to initiate and complete transcription and translation. Proteins (eg, antibodies) that are expressed in non-glycosylated cells can be glycosylated after translation in a glycosylation system (muecklers and rodishes, cited above, and incorporated herein by reference).

Other microorganisms such as yeast can also be used for expression. Saccharomyces is a preferred host glycosyl cell, and if necessary, promoters including 3-phosphoglycerate kinase or other enzymes of corresponding function, and expression control sequences such as replication origin, and sequencing, etc. It has a suitable vector to contain.

In addition to microorganisms, tissue cell cultures of mammals can also be used to express and produce the polypeptides of the invention (see "From Genes to Clones" in Wiener, VCH Publishing, New York, New York (1987)). Indeed, many suitable host cell lines have been developed in the art capable of secreting intact immunoglobulins, such as CHO cell lines, various COS cell lines, HeLa cells, preferably myeloma cell lines, etc., and transformed B- Cells or hybridomas. Expression vectors for these cells are expressed in expression control sequences such as replication origins, promoters, enhancers [Queen et al. , Immunol. Rev. , 89 , 49-68 (1986), and essential processing information sites such as ribosomal binding sites, RNA splice sites, polyadenylation sites, and transcription termination factor sequences. Preferred expression control sequences are promoters derived from the immunoglobulin gene, SV40, adenovirus, cytomegalovirus, bovine tumorigenic virus, and the like.

Vectors containing the DNA segment of interest (eg, heavy and light chain coding sequences and expression control sequences) can be delivered into a host cell by known methods, which methods vary depending on the type of host cell. For example, calcium chloride transfection is widely used for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other host cells (typically, many of Molecular Cloning: A, et al. See Laboratory Manual, Cold Spring Harbor Press, (1982).

Once transduced, the complete antibodies, their dimers, their respective light and heavy chains, or the present invention, according to standard methods in the art, including ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, and the like. It can be purified to other immunoglobulin types (see R. Scorps's "Protein Purification", Springer-Berlag Publications, New York (1982)). Almost pure immunoglobulins with a homogeneity of about 90-95% or more are preferred for pharmaceutical use, with homogeneity of 98-99% or more being most preferred. Once purified to a partially or desired homogeneity, the polypeptide can be used therapeutically (including externally) or in the development and performance of assays, immunofluorescent staining, etc. (typically Immunological Methods , Vols. I and II, Lev Kovitz and Furnace compilation, Academy Press, New York, NY (1979, 1981).

In the method of the present invention, it is preferable to use binding fragments such as intact immunoglobulins or Fabs. Typically, the lipidated antibody may be an IgM or IgG isotype of the human body, but constant regions of other mammals may be used if necessary. Lipidized antibodies of the class IgA, IgG, IgM, IgE, IgD can also be generated. The lipidated antibody of the present invention is preferably an antibody of human, rat, bovine, equine, swine, or non-human primate, more preferably an antibody of human or rat. The present invention can be used to produce a variety of lipidated antibodies, including the chimeric antibodies, humanized antibodies, primateized antibodies, Fv fragments, toxin-antibody conjugates, isotope-antibody conjugates, and imaging Include, but are not limited to, topical-antibody conjugates. In vivo imaging, lipidated antibodies are appropriately labeled with a diagnostic label, administered to the patient, and then their positions are detected at various time points. Various methods of labeling antibodies with diagnostic reporters (eg, Tc ⁹⁹ , other radioligands, radiocontrasts or radio-opaque dyes) are known in the art.

Proteins and oligopeptides other than immunoglobulins (ie, polypeptides comprising from 2 to about 50 amino acid residues bound by peptidyl bonds) can be lipidized according to the methods of the invention. Preferred substrates for lipidation through carbohydrate binding are naturally-occurring glycoproteins (e.g., γ-glutamyltranspeptidase, thrombomodulin, glucose transporter proteins), but any polypeptide is a crosslinker (e.g. , N-hydroxysuccinimide) can also be lipidated by covalent linking to the appropriate amino acid side chains. In an alternative embodiment of the invention, one or more lipid substituents (eg lipoamines) are covalently attached to the non-carbohydrate moiety on the protein or polypeptide (eg, amide bonds with Asp or Glu residue side chain carboxyl substituents or Cys Through the formation of thioester bonds with residues). Fatty acids may also be linked to Arg or Lys residues via side chain amine substituents. Examples of non-glycosylated proteins that can be lipidated to enhance transvascular and intracellular transport include, but are not limited to, c-fos, c-myc, c-src, NF-AT, and HMG CoA reductase. It doesn't happen. Naturally occurring lipoproteins, such as physiological farnesylated, geranylgeranylated, and palmitylated natural proteins, are natural products and are not included in the "lipidated proteins" herein.

Lipidized antibodies and pharmaceutical compositions thereof are particularly useful for parenteral administration, ie subcutaneous, intramuscular or intravenous administration. Compositions for parenteral administration will usually comprise an immunoglobulin solution, or a cocktail thereof, dissolved in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and are usually free of particulates. These compositions can be sterilized through conventional known sterilization techniques. The composition contains, if necessary, a pharmaceutically acceptable auxiliary substance necessary for control similar to physiological conditions such as pH adjusting agents and buffers, toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, human albumin, etc. can do. Antibody concentrations in these formulations can vary from about 0.5% by weight or less, usually from about 1% or more to 15% or 20% by weight, and are based primarily on the amount of solution, viscosity, etc., depending on the respective mode of administration selected. Will be chosen.

Thus, a pharmaceutical composition conventional for injection can be prepared containing 1 ml of sterile buffered water and 1-10 mg of lipidated immunoglobulin. Conventional compositions for intravenous injection may contain 250 ml of sterile ring gel solution and 150 mg of antibody. Actual methods of preparing compositions for parenteral administration are known to those skilled in the art and are described, for example, in more detail in Remington's Pharmaceutical Science (15th edition, Mac Press, Eastern, Pennsylvania (1980)), which is incorporated herein by reference. .

The lipidated proteins and antibodies of the invention can be frozen or lyophilized for storage and restored to a suitable carrier prior to use. This method has been found to be effective against conventional immunoglobulins, and lyophilization and restoration techniques known in the art can be used. Lyophilization and reconstitution may show varying degrees of activity (e.g., in the case of conventional immunoglobulins, IgM antibodies tend to have a greater loss of activity than IgG antibodies) and may require adjustment to supplement Those skilled in the art will understand.

Compositions containing the present lipidated proteins (eg antibodies) or cocktails thereof can be administered for prophylactic and / or therapeutic treatments. In therapeutic use, the composition is administered to a patient in an amount sufficient to treat or at least partially inhibit the disease and its complications. A suitable amount to accomplish this is defined as a "therapeutically effective amount." The amount effective for this use depends on the severity of the infection and the general state of the patient's own immune system, but is typically within the range of about 1 to about 200 mg of antibody per dose, and a dosage of 5 to 25 mg is more commonly used. It should be noted that the materials of the present invention can typically be used in severe disease states, i.e. life threatening or potential life-threatening situations.

For prophylactic use, the composition containing the present immunoglobulin or a cocktail thereof is administered to a patient who is not yet in a disease state to enhance patient tolerance. Such amounts are defined as "prophylactically effective amounts". In this use, the exact amount also depends on the patient's state of health and the overall degree of immunity, but typically ranges from 0.1 to 25 mg per dose.

The composition can be administered in a single or multiple dose, and the dosage and pattern can be selected according to the treatment. In any case, the pharmaceutical composition must provide an amount of the lipidated protein and / or lipidated antibody (s) of the invention sufficient to effectively treat the patient.

For diagnostic purposes, the lipidated antibody can be labeled or unlabeled. Unlabeled antibodies can be used with other labeled antibodies (second antibodies) that are reactive with lipidated antibodies, such as antibodies specific for human immunoglobulin constant regions. Alternatively, the lipidated antibody can be labeled directly. Various labels may be used, such as radionuclides, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (specifically hapten), radiocontrasts, metal chelates and the like. Various kinds of diagnostic imaging applications are useful and are known to those skilled in the art. For example, an antibody that binds to a tumor antigen (eg, anti-CEA antibody) may be lipidated to bind radioactive agents or self-imaging materials and, after administration to a patient, to localize the location of the tumor or metastatic lesion. Can be detected, but is not limited to such.

The lipidated immunoglobulins of the present invention can be used for diagnosis and treatment. They can be used to treat cancer, autoimmune diseases, or viral infections, but this is for illustrative purposes and not intended to be limiting. In the case of cancer treatment, antibodies will typically bind antigens that are preferentially expressed in certain cancer cells, such as the c-myc gene product and others known to those skilled in the art. Lipid antibody is mutated proteins, such as pathogens, such as the substituents of 12, 13, 59 or 61 position of the protein (e.g., Ser 12 is replaced with p21 ^ras position) (e.g., neoplastic) sequence with a c- will bind to the ras oncogene product. For the treatment of autoimmune diseases, antibodies will typically bind to key regulatory proteins mainly expressed in activated T-cells, such as NF-AT, and to many other intracellular proteins known to those of skill in the art (eg, see text here). See Cited Fundamental Immunology, 2nd Edition, edited by WE Paul, Raven Publishers (New York, NY). In the case of viral infection treatment, antibodies will typically bind to a variety of virally encoded polymerases and proteins expressed in cells infected by a particular virus, such as HIV-1 Tat, and many other viral proteins known to those of skill in the art ( See, eg, Virology , 2nd edition, BN Field et al., (1990), Raven Publishing (New York, New York).

In addition, kits may be provided that allow the use of the lipidated antibodies of the invention for protection against cell activity or for its detection or for diagnosis of disease or for the presence of intracellular proteins of a selected cell. Thus, the main composition of the present invention may be provided alone in lyophilized form in a container, or may be provided with additional antibodies specific for the desired cell type. Lipidated antibodies which may bind to the label or toxin, or are unbound, are bound to a set of instructions for use with buffers, stabilizers, sterilizers, inert proteins such as serum albumin, etc., such as Tris, phosphate, carbonate, etc. Included together. Typically, these materials will be present in up to about 5% by weight based on the amount of active antibody, and typically the total amount is at least about 0.001% by weight based on the antibody concentration. It will often be desirable to add inert extenders or excipients to dilute the active ingredient, and excipients may be provided in about 1 to 99% by weight of the total composition. If a second antibody capable of binding a lipidated antibody is used in the assay, it will usually be provided in a separate vial. The second antibody is typically bound to a label and then formulated in a manner similar to the antibody preparations described above, and also typically lipidizing the second antibody itself.

The lipidated antibodies of the invention are also suitable for use in improved diagnostic methods and protein purification methods. For example, many intracellular proteins are unstable (ie, have short half-lives and are prone to proteolytic activity) and are prone to aggregation (eg, β-amyloid protein), making purification and / or diagnostic detection difficult. Lipidized antibodies penetrate live cells and bind to specific intracellular target antigens; Such antibody-antigen binding can stabilize the target antigen and block enzymes involved in the degradation of the target antigen (eg, proteases, ubiquitin-binding enzymes, glycosidase), thereby facilitating detection and / or purification of the target antigen. .

In one variation of the invention, a lipidated antibody that specifically binds to an intracellular target antigen is contacted with live cells comprising the intracellular target antigen under physiological conditions (e.g., cell culture state, somatic state) and then suitable. Incubate for the duration of binding (eg, about 10 minutes to several hours). The lipidated antibody specifically binds to the target antigen, forming an antigen-antibody conjugate that is itself less sensitive to denaturation and / or aggregation, which is the target antigen. Typically, these cells are subsequently immobilized to immobilize antigen-antibody conjugates comprising a target antigen bound to a lipidated antibody, usually detected by a labeled second antibody that specifically binds to the lipidated antibody. Examples of preferred labels bound to the second antibody are FITC, rhodamine, horseradish peroxidase conjugate, alkaline phosphatase conjugate, β-galactosidase conjugate, biotinyl moiety, radioisotope and the like. In some embodiments, the second antibody can be lipidized to replace the step of immobilization and / or permeabilization and to wash the cell sample sufficiently to remove nonspecific staining. It is also possible to use the lipidated, labeled first antibody directly and omit the second antibody. Labeled Protein A may be used in place of the second antibody for detection of the first (lipidized) antibody.

Lipidized antibodies can also be used for intracellular therapies, such as binding to certain intracellular target antigens to denature the biochemical properties of the target antigen. For example, multi-subunit proteins, such as heteromultimeric proteins (eg, transcription factors, G-proteins) or homodimeric proteins (eg, polymerized tubulin) may be biochemically active (eg, GTPase activity), or It may have other activities that require intermolecular interaction (s) that can be masked by lipidated antibodies that specifically bind to one or more subunits to prevent functional interaction of the subunits. For example, a lipidated anti-Fos antibody that binds to Jun and binds to a portion of Fos required to form the transcriptionally active AP-1 transcription factor (Fos / Jun heterodimer) blocks the formation of functional AP-1. Can inhibit AP-1 mediated gene transcription. In addition, for example, lipidated anti-ras antibodies bind to epitopes of ras (eg, GTP / GDP-binding sites, portions of ras that bind to subproteins such as GAP) that are required for proper signal transduction function. It is possible to denature the activity of intracellular ras present in living cells.

The following examples are provided for illustration, and the invention is not limited thereto.

EXAMPLE

Example 1

IgG Preparation of Lipidated Bovine

In Bear et al. (1989) Proc. Natl. Acad. Sci. (USA) 86 : 6982] combined glycine residues with dioctadecylamine to obtain glycyldioctadecylamide. One equivalent of benzyloxycarbonyl-glysil-p-nitrophenol and 1.1 equivalent of triethylamine were reacted in CH ₂ Cl ₂ for 5 hours, followed by addition of 10% Pd / C in H ₂ , CH ₂ Cl ₂ / EtOH. The reaction was carried out for 1 hour.

2 mg of bovine IgG (Sigma) was dissolved in 400 μl of 300 mM NaHCO ₃ in 1.5 ml of Eppendorf vial. To this was added 50 μl of freshly prepared NaIO ₄ solution (42 mg / ml in H ₂ O), the vial was sealed with aluminum foil and stirred slowly at room temperature for 90 minutes. The reaction solution was then loaded onto a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na ₂ CO ₃ (fraction 1), and the column was eluted in 500 μl fractions. Fraction number 7 (3 ml to 3.5 ml) contained about 1.6 mg of bovine IgG as measured using the Bradford protein assay.

A solution of glycyldioctadecylamide in DMSO was prepared (5 mg of lipid was added to 1 ml of DMSO and vigorously shaken for several minutes). Under these conditions, the lipid did not completely dissolve. 50 μl of this solution were carefully taken (not containing any undissolved lipids) and added to 350 μl of fraction 7 (obtained as described above) prepared in an Eppendorf vial. The vial was sealed with aluminum foil and the mixture was shaken slowly at room temperature for 20 hours.

Then 100 μl of NaBH ₄ solution (10 mg in 1 ml of H ₂ O) was added. After 1 hour 40 μl of ethanolamine solution (15 μl in 1 ml H ₂ O) was added. After another hour, the reaction mixture was loaded onto a PD-10 column previously equilibrated with 100 mM HEPES buffer (pH 8.5). Fractions containing lipidated IgG (3 to 3.5 ml) were collected and stored on ice.

¹⁴ Labeling with C-acetic anhydride

¹⁴ C-acetic anhydride (500 μCi, amersam) in benzene (10 × 10 ⁶ cpm / μl) was used. A 5 μl aliquot was added twice at 10 minute intervals to a 500 μl fraction containing lipidated IgG contained in an Eppendorf vial. The reaction was left on ice. 500 μl of native IgG solution (800 μg in 100 mM HEPES, pH 8.5) was also treated in the same manner.

After 30 minutes, the vial was warmed to 20-25 ° C. and ¹⁴ C-labeled IgG was isolated from free ¹⁴ C-acetate on a PD-10 column equilibrated with PBS. Incorporated radioactivity was approximately 10 × 10 ⁶ cpm per 500 μg.

Organ Absorption Rate Study

Swiss Albinos male mice (20 g) were used. 100 μl of ¹⁴ C-labeled lipidated IgG or ¹⁴ C-labeled control IgG (approximately 400,000 dpm each) in PBS was administered intravenously by tail vein injection. Mice were killed after 30 minutes or 3 hours, their blood collected in EDTA-containing tubes, and the brain (except cerebellum and brainstem), spleen, one kidney and one mesenchyme dissected. Each organ was homogenized in 1 ml of 10 mM Tris buffer at pH 7.4 and 500 μl aliquots were counted on a Beckman scintillation counter. Protein concentration in the homogenate was determined by Bradford assay (Kumasi Blue). Blood was centrifuged to count 20 μl fractions of plasma. Table 1 shows the absorption rates of ¹⁴ C of the brain, liver, spleen and kidneys, which is expressed as the ratio of 1 μl of organ protein divided by 1 μl of plasma (unit of data is μl / μg protein).

Agency 30 minutes 3 hours Control Lipidated Control Lipidated brain .20 ± .30 .32 ± .08 .94 ± .05 1.75 ± .09 kidney 1.46 ± .31 9.50 ± 2.19 1.17 ± .04 3.54 ± .22 liver 1.26 ± .26 4.95 ± .82 .66 ± .04 7.59 ± .27 spleen 1.08 ± .27 4.54 ± .76 1.13 ± .03 12.0 ± 2.15

The control group used 4 and 6 mice for 30 minutes and 3 hours respectively. The group to which the lipidated IgG was administered used 6 mice at both time points.

Data are shown as mean ± s.e.m.

Example 2

Inhibition of HIV-1 Cytotoxicity with Lipidated Anti-Tat Antibodies

Monoclonal antibodies that specifically bind to the Tat protein of HIV-1 covalently bind glycyldiooctadecylamide after the periodate oxidation of carbohydrates on the antibody to form carbohydrate-lipidated antibodies, which are the final PD Lipidation was carried out according to the method described in Example 1 above, including eluting with -10 column from PBS.

Sup T1 cells were maintained in 24-well plates (100,000 cells per ml in 2 ml of modified RPMI 1640 culture medium). During the first 5 days of the experiment, cells were either (1) added without treatment (two controls), (2) natural anti-Tat antibody (15 μg / ml) added, or (3) lipidated anti-Tat antibody (11.7). μg / ml) was preserved in the added culture. At the end of day 1, HIV-1 IIIB was added to cultures containing 1 well of control cells and native anti-Tat antibody-treated cells or lipidated anti-Tat antibody-treated cells. Viable cells were counted daily. Untreated HIV-infected cells grew to approximately 500,000 cell densities per ml and their number began to decline after about 8 days due to the cytotoxic effects of the virus. Uninfected cells grew to a density of approximately 1,000,000 cells per ml. Treatment of cells infected with native anti-Tat antibodies did not protect the cells from the cytotoxic effects of the virus. In contrast, lipidated anti-Tat antibodies almost completely protected cells from the cytopathic effects of HIV-1 virus. This protection continued for at least about 5 days after stopping treatment with the lipidated antibody. The results are shown in Table 2.

As another experiment, Sup T1 cells were maintained in culture as described in the examples above, then maintained in culture without any treatment and any infection, or infected with HIV-1 IIIB without any treatment. Cultures treated with natural anti-Tat antibody (1 μg / ml) and maintained in infected cultures or treated with lipidized anti-Tat antibody (1 μg / ml) and infected Kept in water. In the last three conditions, the virus was added to the culture at the end of day 1 of culture. Under the last two conditions, natural or lipidated antibodies were present from day 1 to day 7.

Through the data presented in Table 2, native anti-Tat antibodies have very little effect on viable cell number and reverse transcriptase activity, whereas lipidated antibodies significantly protect cells in culture from the cytopathic effects of viruses. It can be seen that the reverse transcriptase activity is also significantly reduced. These results show that lipidated antibodies can inhibit intracellular HIV-1 replication.

Effect of Lipidized Anti-Tat Antibody on Live Cell Number and Reverse Transcriptase Activity in HIV-1 Infected Sup T1 Cells Live cell (× 10 ⁶ ) days of culture Condition One 2 3 5 6 7 8 Untreated, uninfected 1.15 1.28 1.52 1.63 1.69 1.76 1.72 Untreated, infected 1.12 1.2 1.18 1.12 0.92 0.64 0.51 Treatment with natural anti-Tat antibodies, infection 1.12 1.2 1.21 1.15 0.96 0.67 0.51 Treatment with lipidized anti-Tat antibody, infection 1.15 1.26 1.3 1.36 1.17 0.99 0.75 Reverse transcription enzyme activity (cmp / 0 ⁹ cells) days of culture Condition One 2 3 5 6 7 8 Untreated, uninfected 2 2 One 2 2 One One Untreated, infected 3 2 4 175 264 337 367 Treatment with natural anti-Tat antibodies, infection 2 2 3 151 237 259 331 Treatment with lipidized anti-Tat antibody, infection 3 2 2 57 135 179 184

HIV-1-infected Sup T1 cells were treated with anti-Tat antibody or rsCD4 (either at 1 μg / ml) in natural or lipidated form from 1 day prior to addition of HIV-1 virus-containing supernatant to 10 days after infection. Used daily). Cell number and reverse transcriptase activity (RT) in culture were measured daily from 2 days post infection. By 10 days, native anti-Tat had no significant effect on both cell number or RT activity, whereas lipidated anti-Tat antibodies increased cell viability by approximately 70% compared to untreated and infected cells, and RT activity It was suppressed to about the same degree. Incubation was continued for 3 days without additional antibody addition. The effect of the lipidated anti-Tat lasted for 3 days, indicating that the lipidated anti-Tat antibody accumulated in the cells in an amount high enough to be continuously protected against viral infection / replicate. The extent of the effect of lipidated anti-Tat antibody effect on cell viability and RT activity was very similar to the results observed with the same dose of rsCD4. Increasing the lipidated anti-Tat antibody concentration to 10 μg / ml no longer inhibited RT activity.

Example 3

Lipidated Anti-Tat Activity Inhibits Tat Transcriptional Activity Against HIV-1 LTR

HeLa cell lines report HIV-1 long terminal repeats (LTDs) that mediate HIV-1 infection and also operatively bind to and induce its transcription to a bound reporter gene (chloramphenicol acetyltransferase (CAT)) Constant transfection was performed with CD4 expressing polynucleotides, which are membrane receptors containing structures. These cells (HLCD4-CAT) are susceptible to HIV-1 producing a functional Tat protein, so that when the newly synthesized Tat binds to HIV-1 LTR, the bound CAT gene is transcribed. Thus, the extent of CAT expression is roughly proportional to the extent of HIV-1 infection and the activity of Tat protein in cells.

Cultured HeLa cells (3 × 10 ⁵ cells in 1 ml of DMEM containing 10% fetal bovine serum) were subjected to the same concentration (1 μg / ml or 10 μg / ml) of various antibodies (natural or lipidated) or recombinant HIV-containing cell culture supernatant (100 μl) was added after exposure to soluble CD4 (rsCD4) for 1 hour and thoroughly washed. After 24 hours cells were harvested and CAT expression was measured according to the method of Ho et al. (1984). The experiment was repeated four times each and at four different times. 4 shows that lipidized anti-Tat antibodies significantly inhibit CAT activity (up to approximately 75%), whereas native (non-lipidized) anti-Tat antibodies, lipidated anti-gp120 antibodies, or rsCD4 are CAT activities It is not much more effective at suppressing this. These data indicate that lipidated anti-Tat can specifically bind their intracellular target, ie Tat, and inhibit target activity as a transcriptional activator of the LTR / reporter gene construct.

The data also shows that the lipidated anti-Tat antibody migrates into HeLa cells, indicating that no endosomal formation is required for the transport mechanism, since HeLa cells have been reported to have very little phagocytosis.

Example 4

Preparation of Lipidized Immunoglobulins Reactive with Intracellular Proteins

Bear et al . (1989) Proc. Natl. Acad. Sci. (USA) 86 : 6982], glycine dioctadecylamide was obtained by combining glycine residues with dioctadecylamine. One equivalent of benzyloxycarbonyl-glysil-p-nitrophenol and 1.1 equivalent of triethylamine were reacted in CH ₂ Cl ₂ for 5 hours, followed by addition of 10% Pd / C in H ₂ , CH ₂ Cl ₂ / EtOH. And reacted for 1 hour.

Anti-human c-Myc Ig

Glycosylated murine immunoglobulins that specifically bind to human c-myc proteins are subject to certain conditions (Evan et al. (1985) Mol. Cell. Biol. 5 : 3610), which is incorporated herein by reference. ) Hybridoma cell lines MYC CT9-B7.3 (ATCC CRL 1725), MYC CT 14-G4.3 (ATCC CRL1727), and MYC 1-9E 10.2 (ATCC) at RPMI 1640 containing 10% fetal bovine serum CRL 1729) was prepared by incubating each, and the secreted monoclonal antibodies were collected and purified by conventional methods known in the art.

About 2 mg of each purified monoclonal antibody was dissolved in 400 μl of 300 mM NaHCO ₃ in 1.5 ml of Eppendorf vial. 50 μl of freshly prepared NaIO ₄ solution (42 mg / ml in H ₂ O) was added and the vial was sealed with aluminum foil and stirred slowly at room temperature for 90 minutes. The reaction was then loaded onto a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na ₂ CO ₃ (fraction 1), and the column was eluted by 500 μl fractions. Fraction (s) containing at least about 500 μg of IgG were collected as measured using the Bradford protein assay.

A solution of glycyldioctadecylamide in DMSO was prepared (5 mg of lipid was added to 1 ml of DMSO and vigorously shaken for several minutes). Under this condition, the lipid was not completely dissolved. 50 μl of this solution was carefully taken and added to 350 μl of the purified IgG fraction obtained as described above in an Eppendorf vial. The vial was sealed with aluminum foil and the mixture was slowly stirred at rt for 20 h.

Then 100 μl of NaBH ₄ solution (10 mg per 1 ml of H ₂ O) was added. After 1 hour 40 μl of ethanolamine solution (15 μl in 1 ml of H ₂ O) was added. After 1 hour of addition, the reaction mixture was loaded onto a PD-10 column previously equilibrated with PBS. Fractions containing lipidated murine anti-human-myc IgG (3 to 3.5 ml) were collected and stored on ice.

Anti-HMG CoA Reductase Ig

Glycosylated murine immunoglobulins that specifically bind to the intracellular enzyme HMG CoA reductase are described in Goldstein et al. (1983) J. Biol. Chem. 258 : 8450, prepared by culturing hybridoma cell line A9 (ATCC CRL 1811) in DMEM containing 4.5 g / l glucose, 5% equine serum and 2.5% bovine fetal serum, respectively, as described in Ronald antibodies were harvested and purified according to conventional methods known in the art.

Approximately 2 mg of each purified monoclonal antibody was dissolved in 400 μl of 300 mM NaHCO _{3 in} 1.5 ml of Eppendorf vial. 50 μl of freshly prepared NaIO ₄ solution (42 mg per ml of H ₂ O) was added and the vial was sealed with aluminum foil and stirred slowly at room temperature for 90 minutes. The reaction was then loaded onto a PD-10 column (Parciacia) previously equilibrated with 10 mM Na ₂ CO ₃ (fraction 1) and the column eluted at 500 μl fractions. Fraction (s) containing at least about 500 μg of IgG were collected as measured using the Bradford protein assay.

A solution of glycyldioctadecylamide in DMSO was prepared (5 mg of lipid was added to 1 ml of DMSO and vigorously shaken for several minutes). Under this condition, the lipid was not completely dissolved. 50 μl of this solution was carefully taken and added to 350 μl of the purified IgG fraction obtained as described above in the Eppendorf vial. The vial was sealed with aluminum foil and the mixture was slowly stirred at rt for 20 h.

Then 100 μl of NaBH ₄ solution (10 mg per ml of H ₂ O) was added. After 1 hour 40 μl of ethanolamine solution (15 μl in 1 ml of H ₂ O) was added. After an additional hour, the reaction mixture was loaded onto a PD-10 column previously equilibrated with PBS. Fractions containing lipidated anti-HMG CoA reductase IgG (3 to 3.5 ml) were collected and stored on ice.

Example 5

Preparation of Lipidized Immunoglobulins Reacting with Dural Protein

Proc. Natl. Acad. Glycylic dioctadecylamide was prepared by combining glycine residues with dioctadecylamine according to the method described in Sci. (USA) 86: 6982 (Behr et al. (1989)). . Benzyl in CH ₂ Cl ₂ butyloxycarbonyl-glycyl was added to 1 equivalent -p- nitrophenol and triethylamine 1.1 eq., And then allowed to react for 5 _{hours, CH 2 Cl 2 / EtOH 10} % Pd / C, H ₂ of The reaction was carried out for 1 hour.

Anti-Ras Ig

Contains 4.5 g of glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids, 1xBME vitamin, 0.1 mM hypoxanthine, thymidine 0.032 mM, gentamicin 0.05 mg / ml, and 10% serum of fetal bovine Hybridoma cell lines 142-24E5 (ATCC HB 8679; US Pat. Nos. 5,015,571 and 5,030,565, incorporated herein by reference) in DMEM include 1% L-glutamine and HT and 10% fetal bovine serum Hybridoma cells MX (ATCC HB 9158) in DMEM of isolated Iscove, each cultured under specific conditions (US Pat. No. 4,820,631, incorporated herein by reference) to glycosylation specifically binding to ras tumor gene protein Immunoglobulins of rats were prepared and monoclonal antibodies secreted from the hybridoma strain were collected and purified according to conventional methods known in the art.

Approximately 2 mg of each purified monoclonal antibody was dissolved in 400 μl of 300 mM NaHCO _{3 in} 1.5 ml Eppendorf vial. 50 μl of freshly prepared NaIO ₄ solution (42 mg / ml in H ₂ O) was added and the vial was sealed with aluminum foil and stirred slowly at room temperature for 90 minutes. The reaction solution was loaded on a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na ₂ CO ₃ (fraction 1), and the column was eluted with 500 μl fractions. Fraction (s) containing at least about 500 μg IgG were collected as measured using the Bradford protein assay.

A solution of glycyldioctadecylamide in DMSO was prepared (5 mg of lipid in 1 ml of DMSO, vigorously shaken for several minutes). Under this condition, the lipid does not dissolve completely. 50 μl of this solution was carefully taken and added to 350 μl of purified IgG fraction contained in the Eppendorf vial obtained as described above. The vial was sealed with aluminum foil and the mixture was shaken slowly at room temperature for 20 hours.

Then 100 μl of NaBH ₄ solution (10 mg / ml in H ₂ O) was added. After 1 hour, 40 μl of ethanolamine solution (15 μl in 1 ml of H ₂ O) was added. After an additional hour, the reaction mixture was loaded onto a PD-10 column previously equilibrated with PBS. Fractions containing lipidated rat anti-ras IgG (3 to 3.5 ml) were collected and stored on ice.

The hybridoma cell lines described in the above examples can be obtained from the American Type Culture Collection, Rockville, MD; ATCC Cell Lines and Hybridomas (1992) 7th Ed, incorporated herein by reference.

Example 6

Lipidation of Dural Enzymes

The enzyme gamma-glutamyltranspeptidase (GGT: EC 2.3.2.2) is a widely distributed catalyst that catalyzes the degradation of glutathione and other γ-glutamyl compounds by hydrolyzing the γ-glutamyl moiety or transferring it to a suitable receptor. Is an enzyme. GGT is a heterodimeric glycoprotein synthesized as a precursor protein that is glycosylated and then cleaved into two subunits of the aging enzyme. GGT adheres to the cell membrane through the N-terminus of its heavy chain subunit. The active site of this enzyme is on the extracellular portion of the molecule, which is highly glycosylated.

Known methods in the art (Barouki et al. (1984) J. Biol. Chem. 259 : 7970; Curthoys and Hughey (1979) Enzyme 24 : 383; Matsuda et al. (1983) J. Biochem. 93 : 1427; Taniguch et al. (1985) J. Natl. Cancer Inst. 75 : 841; Tate and Meister (1985) Methods Enzymol. 113 : 400; and Toya et al. (1983) Ann. NY Acad. Sci. 417 : 86, incorporated herein by reference). GGT is purified from rat kidney and human liver cancer cell lines, respectively.

Approximately 1 mg of rat and human GGT tablet preparations were dissolved in 400 μl of 300 mM NaHCO ₃ in 1.5 ml Eppendorf vial, respectively. 50 μl of freshly prepared NaIO ₄ solution (42 mg / ml in H ₂ O) was added and the vial was sealed with aluminum foil and stirred slowly at room temperature for 60 minutes. The reaction solution was loaded on a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na ₂ CO ₃ (fraction 1), and the column was eluted with 500 μl fractions. Fraction (s) containing at least about 100 μg of GGT were collected when measured using the Bradford protein assay.

A solution of glycyldioctadecylamide in DMSO was prepared (5 mg of lipid in 1 ml of DMSO, vigorously shaken for several minutes). Under this condition, the lipid does not dissolve completely. 50 μl of this solution was carefully taken and added to 350 μl of purified GGT fraction in the Eppendorf vial obtained as described above. The vial was sealed with aluminum foil and the mixture was slowly stirred at room temperature for 20 hours.

Then 100 μl of NaBH ₄ solution (10 mg / ml in H ₂ O) was added. After 1 hour, 40 μl of ethanolamine solution (15 μl in 1 ml of H ₂ O) was added. After an additional hour, the reaction mixture was loaded onto a PD-10 column previously equilibrated with PBS. Fractions containing lipidized human and rat GGT (3 to 3.5 ml) were collected and stored on ice.

Γ-glutamyltranspeptidase activity on lipidated GGT fractions of the human and rats was assayed by conventional assay procedures (Tate and Meister (1983), supra, incorporated herein by reference), and The specific activity of GGT and GGT in lipidated mice was measured.

The lipidated human GGT and the lipidated rat GGT were radiolabeled by iodine at ¹²⁵ I using chloramine T and approximately 50 μg of this radiolabeled lipidated GGT was injected intraperitoneally. After 24 hours, the mice were killed and tissue samples were taken for radiographic imaging to determine the localization pattern of lipidated GGT present in the various organs.

Example 7

Lipidation and Intracellular Immunostaining of Anti-Actin Antibodies

To demonstrate that lipidated antibodies can localize intracellularly in live cells and bind to intracellular targets, the anti-actin antibodies are lipidated to penetrate the cultured Swiss 3T3 fibroblasts and cytoskeletal protein. The ability to bind actin was measured. Natural anti-actin antibody (unlipidized) was used as a control.

Protein A-purified rabbit anti-actin polyclonal antibodies were lipidated according to the following method. The lipoamine glycyldioctadecylamide was covalently bound to the carbohydrate portion of the anti-actin antibody by periodate oxidation and sodium borohydride reduction reaction. Antibodies at a concentration of about 0.2-1.0 mg / ml were dissolved in 0.8 ml of 300 mM NaHCO ₃ . 50 μl of freshly prepared aqueous NaIO ₄ solution (42 mg / ml) was added and the culture vial was sealed with aluminum foil and stirred slowly at room temperature for 90 minutes. After loading the reaction mixture onto 10mM Na ₂ CO ₃ was pre-equilibrated PD-10 column (Pharmacia, Piscataway, NJ) as a purified and eluted in 10mM Na ₂ CO _3. 50 μl of a 10 mg / ml solution of glycyldioctadecylamide in benzene is added to the fraction containing the antibody (e.g., measured by A ₂₈₀ , Bradford assay) and the reaction is incubated for 20 hours at room temperature with gentle agitation. I was. 100 μl of freshly prepared NaBH ₄ aqueous solution (10 mg / ml) was added and incubated at room temperature for 1 hour, followed by 50 μl of ethanolamine solution (15 μl of ethanolamine dissolved in 1 ml of H ₂ O). After an additional hour at room temperature, the lipidated antibody obtained was purified by chromatography on a PD-10 column equilibrated with phosphate-buffered saline.

ELISA analysis

ELISA assays determined the binding affinity of the lipidated anti-actin and lipidated anti-Tat (see above) to specific antigens as compared to native (unlipidated) anti-actin or anti-Tat antibodies. Lipidation of anti-actin antibodies or anti-Tat antibodies hardly reduced the affinity of the antibody for each antigen as compared to their native (non-lipidated) antibodies.

Intracellular immunostaining

To demonstrate that lipidated anti-actin antibodies can bind to intracellular actin present in living cells, lipidized anti-actin antibodies or native anti-actin antibodies are combined with Swiss 3T3 cell culture for 1 hour. Contact, followed by washing to remove residual anti-actin antibody. The cells were then immobilized and permeabilized to secrete anti-actin antibodies using a fluorescently labeled second antibody. No specific staining was detected in the cells pre-cultured with the native (non-lipidated) anti-actin antibody, whereas specific actin staining (ie stained actin) in the lipidated anti-actin antibody and pre-cultured cells. Cable) is clearly observed. The staining pattern observed when lipidated anti-actin antibody was added prior to immobilization was similar to the staining pattern observed when the native (non-lipidized) anti-actin antibody was incubated with cells after immobilization and permeability. This data demonstrates that lipidated anti-actin antibodies can reach and bind intracellular actin and can be recognized and bound by a labeled second antibody, indicating that the antibody is functional and nearly complete. Shows.

Although the foregoing has been described in some detail for the purpose of clarifying the understanding of the invention, it is obvious that certain changes and modifications can be made within the scope of the claims.

The present invention relates to conventional methods of enhancing antibody transport into cells by binding cationic substituents to the primary polypeptide sequence of immunoglobulins by relatively nonspecific chain chemistry to the secondary, tertiary and / or quaternary structures of the protein. It has been observed to produce deleterious changes. This structural change is a clear cause of the loss of binding affinity observed in cationized antibodies. To overcome this, the present invention provides a method for binding lipid substituents to proteins such as immunoglobulins, mainly by covalent binding to the carbohydrate side chains of the protein such that the lipid substituents rarely destroy the protein's biological activity (eg, antigen binding). to provide.

Claims (3)

A composition for delivering lipidized immunoglobulins to intracellular compartments of cells, comprising lipidized immunoglobulins and excipients, which are lipidized immunoglobulins that have been lipidated by covalently binding one or more lipoamine residues to the carbohydrate side chain of the immunoglobulin. Composition. A lipidated protein, which is a polypeptide lipidated by covalently linking one or more lipoamine residues to a carbohydrate side chain of the protein. A lipidated antibody, wherein the antibody is lipidated by covalently binding at least one lipoamine residue to the carbohydrate side chain of the antibody.