CA2118586A1

CA2118586A1 - Transvascular and intracellular delivery of lipidized proteins

Info

Publication number: CA2118586A1
Application number: CA002118586A
Authority: CA
Inventors: Bernard Malfroy-Camine
Original assignee: Individual
Current assignee: Eukarion Inc
Priority date: 1992-07-13
Filing date: 1993-07-13
Publication date: 1994-01-20
Also published as: NZ255043A; FI941169A; FI941169A0; KR20030097604A; OA09893A; EP0607408A4; JPH07502753A; KR100372119B1; EP0607408A1; RU2157239C2; WO1994001131A1; AU4772793A; RU94024566A

Abstract

The invention provides methods for targeting a protein, such as an antibody, to intracellular compartments in a eukaryotic cell, methods for enhancing organ uptake of proteins, pharmaceutical compositions of modified proteins for use in human therapy, and methods for manufacturing modified proteins. The modified proteins of the invention comprise an attached lipid protein, wherein one or more acyl groups are linked to the protein through a carbohydrate side-chain and various covalent linkage chemistries which are provided. Lipidized antibodies of the invention can be used for diagnostic and therapeutic uses.

Description

2 1 1 8 .~
TRAMSVASCULAR AND INTR~CELLULAR DELIVERY OF LIPIDIZED PROTEINS

FIELD OF THE INVENTION
ThP invention provides methods for targeting a protein, such as an antibody, to intracellular compar~ments in a eukaryotic cell, me~hods ~or enhancing organ up~ake of proteins, pharmaceutical composi~ions of modified proteins for use in human ~herapy, and methods for manufacturing modified protPins. The modified proteins of the invention comprise an attached lipid portion, wherein one or more acyl groups are linked to the protein through a carbohydrate side-chain and 15 ~various covalent linkage chemistries which are provided.

BACKGROUND OF THE INVENTION
Many naturally-occurring or modified proteins have been proposed a~ diagnostic and/or therapeutic agPnts for use in humans and domestic animals~ However, proteins are generally only poorly transported across vascular endothelial membranes, if at all/ and usually cannot ~raverse cellular membranes to gain access to intracellular compartments. Thus, for exampIe, antibodies can be raised against purified intracellular proteins, such as transcription factors, intracellular enzymes, and cy~oarchitec~ural ~tructural proteins, but such antibodies generally are not able to enter intact cells and bind to the intracellular antigen targets unless the cell membrane is disrupted.
The advent of monoclonal antibody technology in the mid 1970's heralded a new age of medicine. For the first time, researchers and clinicians had access to essentially unlimited quantities of uniform antibodies capable of binding to a predetermined antigenic site and having various immunological effector functions. These proteins, known as ~'monoclonal antibodies'l were thought to hold great promise in, e.g., the removal of harmful ce~ls, microbial pathogens, and viruses in vivo. Methods allowing the development of specific monoclonal antibodies having binding specificities directed WO94/01131 PCT/US~3/~6599 2 1 i 8 r~ 8 ~ 2 against almost any desired antigenic epitope, including antigens which are located in intracellular compartments in intact cells, promised a cornucopia of medicinal "magic bullets".
Unfortunately, ~he development of appropriate therapeutic products based on monoclonal antibodies, as well as polyclonal antisera, has been severely hampered by a number o~ drawbacks inherent in the chemical nature of naturally-occurring antibodies. First, antibodi~s are generally not able to efficien~ly gain access to intracellular locations, as immunoglobu~ins are not able to travers~ the plasma membrane of cells, and are typically only internalized, if at all, as a conse~uence of inefficient endocytotic mecha~isms. Second, ~-~antibodies do not generally cross vascular membranes (e.g., subendothelial basement membrane), hampering the efficient ~; uptake of antibodies into organs and interstitial spaces.
Therefore, therapies for many important diseases could be developed if there were an efficient method to get specific, biologically active immunoglobulin molecules across capillary barriers and into i~ltracellular locations. For example, the life cycle of a retrovirus such as HIV involves intracellular replication wherein se~eral viral-encoded polypeptides assential ~or production of infectious virions from an infected cell could ~potentially be inhibited or blocked if specific monoclonal an~ibodies reactive with the viral encoded proteins could read~ily~gain access to the intracellular locations where retroviral~replication occurs.
Immunoliposomes have been produced as a potential targeted delivery system ~for delivering various molecules contained in the liposome to a targated cell. Immunoliposomes e~ploy immunoglobulins as targeting agents, wherein an a y?ated immunoglobulln is anchored in the lipid bilayer of the liposome to target the liposome to particular cell types that have external antigens that are bound by tha acylated immunoglo~ulin(s) of the immunoliposomes ~Connor and Huang ; (1985) J. Cell Biol. lOl: 582; Huang, L. (1985) BiochemistrY
~ 24: 29; Babbitt et al. (1984~ Biochemistr~ 23: 3920; Connor et W094J01131 - PCT/US93/06~99 8 ~

al. (1984) Proc. Natl. Acad. Sci. (U.S.A.) 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.
Bio~hys. Acta 716: 140; Huang et al. (1981) J. Immunol.
Methods 46: 141; and Huang et al. (1980) J. Biol. Chem. 255:
8015). Immunoliposomes generally contain immunoglobulins which are attached to acyl substituents of a liposome bilayer through a crosslinkiny agen~ such as N-hydroxysuccimide and which thus become anchored in the liposome lipid bilayer.
Hence, the crosslinked immunoglobulin is linked to the liposome and serves to ~arget the liposomes to specific cell types bearing a predetermined external antigen by binding to the external cellular antigen. While such methods may serve ~o target liposomes to particular cell types, immunoliposomes suffer from several important drawbacks that have limited their application as drug-deli~ery vehicles, particularly for de~ivering proteins to intracellular locations.
Attempts have been made at modifying proteins so as to facilitate their transport across capillary barriers and into cells (EP 0 329 185), however, no completely satisfactory method has yet been ~eported in the art. Chemical modification of protQins, such as antibodies, by non-specific "cationization" to enhance transvascular and intracellular delivery of proteins, has been reported (U.S.S.N. 07/693,872).
However, present methods for making cationized immunoglobulins lead to a significant~loss of binding affinity (approximately about ~0 percent) of~a cationized immunoglo~ulin for binding to its predetexmined epitope as compared to the comparable non-cationized immunoglobulin. Generally, cationization involves carbodiimide linkage of a diamine, such as putrescine or hexanediamine, to the carboxylates of aspartate and glutamate residues in the immunoglobulin polypeptide sequence.
These chemical modifications of primary amino acids likely disrupt the secondary and tertiary structure of the immunoglobulin sufficiently to account for the loss in binding affinity. Also, present method~ produce some degree of cationization in glutamate and aspartate residues located in 2118~ 6 4 the variable domain of an immunoglobulin chain, which results in significant loss of binding affinity and/or specificity.
Chemical modification of small molecules has also been proposed as a method to augment transport of small bioactiv~ compounds. Felgner (W09l/17242~ discloses forming lipid complexes consisting of lipid vesicles and bioactive substances contained therein~ Felgner et al. (W09l/16024) discloses cationic lipid compounds that are allegedly useful ~or enhancing trans~er of small bioactive molecules in plants and animals. Liposomes and polycationic nucleic acids have been suggested as methods to deliver polynucleotides into cells. Liposomes often show a narrow spectrum of cell ~pecificities, and when ~NA is coated externally on to t~em, ~-~he DNA is often sensitive to cellular nucleases~ Newer polycationic lipospermines compound~ exhibit broad cell ranges ~Behr et aI., (1989) Proc. Natl Acad. Sci. USA 86:6982) and ; DNA is coated by these compounds. In addition, a combination of neutral and cationic lipid has been indicated as a method or transfection of animal cells (Rose et al., (l99l) BioTechni~ues lO:520).
Other approaches to enhancing delivery of drugs, particularly across the;blood-brain barrier, utilize pha~macologic-based procedures involving drug latentiation or the conversion of hydrophilic drugs into lipid-soluble drugs.
2~ The majority of the latentiation approaches involve blocking the hydroxyl, carboxyl and primary amine groups on the drug to make it more lipid~soluble and therefore more easily transported across the~blood-brain barrier. Pardridge and 5chimmel, U~S. Pa~ent 4,902,505, disclose chimeric peptides for enhancing transpor~ by receptor-mediated transcytosis.
Thus, there exists a need in the art for methods of ~facili~ating transport of specific ~roteins, such as antibodies, across capillary barriers and into cells, and for pharmaceutical Gompositions of such iD unoglobulins ~or t~eating human and veterinary diseases which are amenable to treatmen~ with intracellular pro~eins and targeting agents like monoclonal antibodies.

2118 .-~8 ~.i SUMMARY OF THE INVENTION
The prior art me~hod of increasing antibody transport into cells by attaching a cationic substituent to the primary polypeptide sequence of an immunoglo~ulin by a relatively nonspecific linkage chemistry have been observed to produce detrimental alterations in the secondary, tertiary, : andJor quaternary structure of the protein. These structural alterations apparently cause the loss of bindlng affinity observed in cationized an~ibodies. To o~ercome this, the present invention provides methods wherein ~ipid substituents are linked to a protein, such as an immunoglobulin, typically by covalent linkage to.a carbohydrate side chain of the protein such that the lipid substituent does not substantially ~-~estroy the biological acti~i~y of the protein (e.g., antigen binding).
: The invention provides methods for producing lipidized pro~eins, generally by lipidization of a :: carbohydrate moiety on a glycoprotein or glycopeptide. In ..
general, the methods of the invention are used for attaching a 20~ lipid, such as a lipoamine, to a polypeptide, typically by ~:
co~alent linkage of th~ lipid to a carbohydrate moiety on a protein, wherein the carbohydrate moiety generally is chemically oxidized: and reacted with a lipoa~ine to form a lipidized protein. The resultant lipidized protein generally : 25 has advan~ageous pharmacokinetic characteristics, such as an increase~ capacit~ to:cross vascular barriers and acces~
pàrenchymal cells o~ various organs and an increased ability o access intracellular compar~ments. In one aspect of the ~ : in~ention,: lipidization~of proteins, such as antibodies : 30 directed against transcription ~actors (e.g., Fos, Jun, AP-l, : OC~-1, NF-AT~, enhances intranuclear localization of the lipidized protein(:s).~
The invention:also provides methods for producing lipidized antibodies that are efficiently transported across capillary barriers and internalized into mammalian cells in : vivo~ The methods of the invention relate to meth~ds ~or chemicall~ a~taching at least~one lipid substituent ~e.g., .

~ ' 6 lipoamine) to a carbohydrate substituent on an immunoglobulin to produce a carbohydrate-linked lipidized immunoglobulin, wherein the lipidized immunoglobulin is capable of intracellular localization. In alternate embodiments of the invention~ at least one lipid substituent (e~g., lipoamine) is covalently attached to a non-carbohydrate moiety on a protein or polypeptide (e.g., by formation of an amide linkage with a Asp or Glu residue side-chain carboxyl substituent or a thioester linkage with a Cys residue). Also, a fatty acid can be linked to an Arg or Lys residue by the side-chain amine substituents .
Similarly, lipid substitutents can be covalently attached to peptidomimetic compounds. Peptide analogs are ~-~commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed : "peptide mimetics" or ~'peptidomimetics" ~Fauchere, J~ (1986) Adv. Druq Res. l5: 29; Veber and Freidinger (1985) TINS p.392;
and Evans e~ al. (1987) J. Med. Chem 30: 1229, which are ~20 incorporated herein:by reference) and are usually developed with the aid of com~uteriæed molecular modeling. Peptide ;~
mimetics that are structurally similar to therapeutically useful peptides~may be used~to produce an equivalent therapeutic or prophylactic effect. Generally, : ~ 25 peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or ~harmacological activity), but have one or more peptide linkages optionally:rep~aced by a linkage selected from the group consisting of: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=~H- (cis and ~rans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods known in the art and further described in the following references:
5patola, A.F. in "Chemistry and Biochemistry of Amino Acids, ~: Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, : New York, p. 267 (1983); Spatola, A~F., Vega Data (March I983), Vol. l, Issue 3, "Peptide Backbone Modifications"
(general review3; Morley, J.S., Trends Pharm Sci (l980) pp.
: 463-468 (general review); Hudson, D. et al., Int J PePt Prot WO9~/01131 P~T/US93/06599 2118~} $

(1979) 14:177-185 ( CH2NH-, CH2CH2-); Spatola, A.F. et al., L~fe Sci (1986) 38:1243-1249 (-CH2-S); Hann, M.M., J Chem Soc Perkin Trans I (1982) 307-314 (-CH-CH-, cis and trans);
Almquist, R.G. et al., J Med Chem (1980) 23:1392-1398 (-COCH2-S ); Jennings-White, C. et al., Tetrahedron Lett ~1982) 23:2533 ~-COCH2-); Szelke, M. et al., European ~ppln~ EP 45665 (1982) CA~ 39405 (1982) (-CH(OH)CH2-3; Holladay, M.W. et al., Tetrahedron Lett (1983) 24:4401-4404 (-C(OH~CH2-); and Hruby, V.J., ~ife Sci (1982) 31:189-199 (-CH2-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -CH2NH-. Such p~ptide mimetics may have significant advantages over polypepti~ 2mbodiments, including, for example: more economic~ production, greater ~hemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), alteredspecificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and~others. Lipidization of peptidomimetics usually involves covalent attachment of one or more acyl chains, directly or through a spacer (e.g., an amide group), to non-interfering~ position(s) on the peptidomimetic that are predicted ~y~quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do~ not form direct contacts with the macromolecules(s)~(e.g., receptors) to which the 2~5 peptidomimetic binds~to;produce~the therapeutic effect.
Lipidization of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic. ~
The invention~also relates to therapeutic and diagnostic compositions of lipidized proteins, such as lipidized anti~odies,~that can;cross vascular membranes and enter the intracellular compartment, particularly lipidized antibodies that bind to in~rac~ellular immunotherapeutic targets, such as viral-encoded gene products that are ~35 essential components of a~viral life cycle (e.g., HIV-1 Tat protein), to intracellular antigens that are biologically active (e.g., an oncogene protein such as c-fos, c-src, c-myc, .
: : :

W094/01131 PC~/US93/06599 2il8~6 c-lck (p56), c-fyn ~p59~, and c-abl), and/or to transmembrane or extracellular antigens (e.g., polypeptide hormone receptors such as an IL-2 receptor, PDGF receptor, ~GF receptor, NGF
receptor, GH recep~or, or TNF receptor). Other proteins which can be targeted by lipidized antibodies include, but are not limited to, the following: c-ras p21, c-her-2 protein, c-raf, any of the various G proteins and/or G-protein acti~ating proteins (GAPs~, transcription factors such as N~-AT, calcineurin, and cis-trans prolyl isomerases. The lipidized antibodies can be used to loc~lize a diagnostic reagent, such as a radiocontrast agent or magnetic resonance imaging component, to a speci~ic ~ocation in the body, such as a specific organ, tissue, body compartmen~, cell type, neoplasm, ~-~or other anatomical structure (eOg., a pathological lesion).
The lipidiæed antibodies can also be used ~o localize linked therapeutic agents, such as chemotherapy drugs, radiosensitizing agents, radionuclides, antibiotics, and other agents, to specific locations in ~he body~ Alternatively, the lipidized antibodies of the invention can be used therapeutically for neutralizing ~i.e, binding to and thereby : i~activating) an in~raceIlular target antigen, such as HIV-l Tat protein, a tran~membrane or membrane-associated antigen target (e.g., ~-glutamyltra~nspeptidase, c-rasH p21, rasGAP) or an extracellular antigen ~arget (i.e., ~-amyloid pro~ein deposits in the brain of an Alzheimer's disease pa~ient).
:~ Lipidi~.ed antibodies can traverse the bloo~-brain barrier and react with extracellular antigen ~arge s that are generally inaccessible to immunoglobulins which cir~ulate in the blood or lymphatic system~ Lipidized antibodies can also react with intracellular portions on transmembrane proteins, such as cytoplasmic tails of viral envelope glycoproteins or protein kinase domains of protooncogene pro~eins (c-src, c-abl~, and thus inhibit production of infectious enveloped virus or kinase activity, re~pectively.
: BRIEF DESCRIPTION OF THE ~RAWINGS
Figure l shows structural formulae representing WO94/01131 PCT/US93/06~99 21 18 .~}~ ~

various lipoamines that can be used in the invention. The righthand column exemplifies branched-chain lipoamines and the lefthand column exemplifies straight-chain lipoamines.
Fiyure 2 is a schematic representation o~ (l) a 5 glycosylated antibody comprising an immunoglobulin tetramer (two light chains associated with two heavy chains), and (2) a schematic representation of carbohydrate-linked lipidized immunoglobulins of the inven~ion. For example but not limitation, branched-chain lipoamide substituent~ are shown attached to partially oxidized carbohydrate sidechains of an immunoglobulin tetramer. Such carbohydrate sidechains may be located in the C~, VH~ CL~ and/or VL regions~ ;;
Figure 3 shows the beneficial effect o~ a lipidized ~-~anti~Tat immunoglobulin on the ln vitro survival of cells infected with HIV-l as compared to the lack of effect of the native ~i.e., non-lipi~ized) anti-Tat immunoglobulin.
Fig. 4 sh~ws that the lipidized anti-Tat antibody significantly inhibited CAT activity (by approximately 75%), whereas native (unlipidized) anti-Tat antibody, lipidized ~anti-gpl20 antibody, or rsCD4 were far less effective in inhibiting CAT acti~vity in HLCD4-CAT cells.

Definitions ~
~ Unless defined otherwise, all techniîcal and scientifîc terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. ;Ali~hough any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are deifined below.
As used herein, the twenty conventional amino acids and their a~breviations follow con~entional usage (Immunoloq~
- A Synthesis, 2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer ~ssociates, Sunderland, Massachusetts ~l99l), which is incorporatedi herein by reference).
~::

WO94/01131 PCT/US93/06~9g 2~ 8~ lo The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide se~uence is identical to a reference polypeptide sequenc~ In contradistinction, the term "complementary to"
is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. ~or illustration, the nucleotide sequence "TATAC"
corresponds to a re~rence sequence "TATAC" and is complementary to a reference sequence "GTATA'I.
: The terms "substantial similarity" or "substantial identity" as used herein denotes a char~cteristic of a ~ polypeptide sequence or nucleic acid sequence r wherein the polypeptide sequence has at least 50 percent sequence identity compared to a reference sequence, and the nucleic acid sequen~e has at leas~ 70 percent seguence identity compared to :: a reference sequence. The percentage of sequence identity is :~ calculated excluding small deletions or additions which total : 20 less than 25 percen~ of the reference ~equence. The reference ~ seguence may be a subset of a lsrger sequence, such as a :~ : constant region domain:of a constant region immunoglobulin : gene; however, the reference sequence is at least 18 nucleotides long in the case of polynucleotides, and at least : : 25 6 amino residues }ong in the case of a polypeptide.
The term "naturally-occurring" as used herein as ~; applied to an object refers to~the fact that an object can be : found ~n nature. For example, a polypeptide or polynucleotide s~quence that is present in an organism (including v~ruses) 30 that can be isola~ed from a source in nature and which has not been inte~ionally modif ied by man in the laboratory is nakurally-occurring. A lipoprotein (e.g., a naturally occuring isoprenylated or myristylated protein) that can be isolated from àn organism that is found in nature and has not been engineered by man is a naturally-occurring lipoprotein.
"Glycosylation sites" refer to amino acid residues : which are recognized by a eukaryotic cell as locat ons for the W094/01131 - PCT/US93/0659g .

attachment of sugar residues. The amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-linkage), serine (0-linkage), and threonine ~0-linkage) residues. The specific site of attachment is typically signaled by a sequence of amino acids, referred to herein as a "glycosylation site sequence". The glycosylation site sequence for N-linked glycosylation is:
-Asn-X-Ser- or -Asn-X-Thr-, where X may be any of the conventional amino acids, other than proline. The predominant glycosylation site sequence for 0-linked glycosylation is:
-(Thr or Ser)-X-X-Pro- t where X is any conventional amino acid. The recoqnition sequènce for glycosaminoglycans (a specific type of sulfated sugar1 is -Ser.-Gly-X-Gly-, where X
~-~s any conventional amino acid. The terms "N-linked" and "0-linked" refer to the chemical group that serves as the ; attachment site between the sugar molecule and the amino acid residue. N-linked sugars are attached through an amino group;
0-linked sugars are attached through a hydroxyl group.
However, not all glycosylation site sequences in a protein are necessarily glycosylated;~some proteins are secreted in both g~lycosylated and nonglycosylated forms, while others are fully glycosylated at one~glycosylation site sequence but contain an~ther glycosylation~site sequence that is not glycosylated.
Therefore, not all~glycosylation site se~uences that are 25 ~present in a polypeptide~are necessarily glycosylation sites wh~re~sugar residues~are~actual~ly attached. The initial N-glycosy`lation during biosynthesis inserts the "core carbohydrate" or;"core oligosaccharide" (Proteins, Structures and Molecular PrinciPles,~ 984) Creighton (ed.), W.H. Freeman and Company, New York,~which is incorporated herein by reference).
As used herein,~"glycosylating cell" is a cell ; capable of glycosylating~proteins, particularly eukaryotic cells capable of adding an~N-linked "core oligosaccharide"
.
containing at least one~mannose residue and/or capable of addi~g an 0-linked sugar, to at least one glycosylation site sequence in at least one polypeptide expressed in said cell, 21 lg.~ ~ 12 particularly a secre~ed protein. Thus, a glycosylating cell contains at least one enzymatic activity that catalyzes the attachment of a sugar residue to a glycosylating site sequence in a protein or polypeptide, and the cell actually glycosylates at least one expressed polypeptide. For example but not for limitation, mammalian cells are typically glycosylating cells. Other eukaryotic cells, such as insect cells and yeast, may be glycosylating cells.
As used herein, the term "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immuno~lobulin Gene SuPerfamilY, A.F. Williams and A.N.
Barclay, in Immunoqlobulin ~enes, T. Honjo, F.W. Alt, and T.H.
~-~abbitts, eds., (l989) Academic Press: San Diego, CA, pp.361-387, which is incorporated herein by reference). For example, ~; ~ but not for limitation, an antibody may comprise part or all o~ a heavy chain and part or all of a light chain, or may comprise only part or all;of a heavy chain. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma ~20 (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant regiongenes, as~well as the myriad immunoglobulin variable region genes. Full-length i D unoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus~(about 110 amino acids) and a kappa or lambda constant region gene~at the COO~ - terminus. Full-length immunoglobulin "heaYy chains" (about 50 Kd or 446 amino acids), are simi~larly~encoded by a variable region gene (about 1I6 amino acids)~ and~one of the other aforementioned constant region genes, e.q., gamma ~(encoding about 330 amino acids).
Antibodies include, bu~ are not limited tol the fo~lowing:
immunoglobulin fragments~e.~g., Fab, F(ab)2), Fv), single hain immunoglobulins, chimeric immunoglobulins, humanized antibodies, primati;zed antibodies, and various light chain-heavy chain combinations). Antibodies can be produced in glycosylating cells~e.g., human lymphocytes, hybridoma cells, yeast, etc.), in non-glycosylating cells (e.g., in E. coli), or synthesized by ~chemical methods or produced by in vitro : ~ :

W094/01131 PCT/US93/0~599 2118~ ~

translation systems using a polynucleotide template to direct translation As used herein, a ~'lipidized antibody9' is an antibody which has been modified by lipid derivatization (e.g~, by covalent attachment of a lipoamine, such as glycyldioctadecylamide, dilauroylphosphatidylethanolamine~ or dio~tadecylamidoglycylspermidine) of one or more carbohydrate moi~ties ~ttached to an immunoglobulin at a ~lycosylation site. Generally, the lipid substituent, such as a lipoamine, is covalently attached through a naturally-occurring ca~bohydrate moiety a~ a naturally-occurring glycosylation site. However 9 it is possible to produce immunoglobulins that have altered glycosylatlon site sequences ~typically by site-~ irected mutagenesis of polynucleotides encoding immunoglobulin chains) and/or altered glycosylation patterns (e.g., by expression of immunoglobulin-encoding polynucleotides in glycosylating cells other than lymphocytes or in lymphocytes of other species). Lipid substituents can be atta~hed to one or more naturally-occurring or non-naturall~-occurring carbohydrate moiety on an immunoglobulin chain. When an anti~ody is produced by dire~t polypeptide synthesis or by biosynthesis in a non-glycosylating cell (e.g., a phage display library), it will generally be necessary to attach a~carbohydrate substituent by chemical or enzymatic modification for subsequent lipidization : (alternatively, the carbohydrate may be lipidized prior to attachment to the immunoglobulin).
As used herein, a "lipidized protein" refers to a prokein (including multimeric proteins, glycoproteins, and polypepti~es of various sizes) that has been modified by attachment of lipid ~e.g., lipoamine), generally through a carbohydrate moiety~ A lipidized protein is generated by derivatizing a protein such that the resultant lipidized protein is distinct from naturally-occurring lipid-linked proteins and lipoproteins. For proteins that are biologically active (e.g., enzymes, receptors, transcription factors), lipidization should not substantially destroy the biological actiYity (e~g., at least about 15 percent of a native biological activity should be preserved in the lipidized pro~ein). ~ipidized peptidomimetics should retain at least about 25 to 95 percent of the pharmacologic activity of a corresponding non-lipidized peptidomimetic.
"Alkyl" refers to a fully saturated aliphatic group which may be cyclic, branched or straightchain. ~lkyl groups include those exemplified by methyl, ethyl, cyclopropyl, cyclopropylmethyl, sec-butyl, heptyl, and dodecyl~ All of the above can either be unsu~stituted or substituted with one or more nsn-interfering substitutents, e.g., halogen; Cl-C4 alkoxy; C~ C4 acyloxy; formyl; alkylenedioxy; benzyloxy; phenyl or benzyl, each optionally substituted with from l to 3 ~-substituen~s selecked from halogen, Cl-C~ alkoxy or Cl-C4 lS acyloxy. The term "non-interfering" characterizes the substituents as not a~versely affecting any reactions to be performed in accordance with the process of this invention.
If more than one alkyl group is present in a gi~en molecule, each may be îndependently selected from "alkyl" unless otherwise stated.
~ "Alkylenel~ refers to a fully saturated divalent - radical containing only carbon and hydrogen, and which may be a branched or straight chain radical. This term is further exemplified by radicals such as methylene, ethylene, n-propylene, t butylene, i-pentylene, n-heptylene, and the like. All of the above can either be unsubstituted or su~stituted with one or more non-interfering sustituents, e.g., halogen; Cl-C4 alkoxy; Cl-C4 acyloxy; formyl;
alkylenedioxy; benzyloxy; phenyl or benzyl, each optionally substituted with from l to 3 substituents selected from halogen, Cl-C4 alkoxy or Cl-C4 acyloxy. The texm "non-interfering" characterizes the substituents as not adversely af*ecting any reactions to be performed in accordance with the process of this invention. If more ~han one alkylene group is present in a given molecule, each may be independently selected fxom "alkylene" unless othexwise stated.
"~ryl", denoted by Ar, includes monocyclic or W094/01131 PC~/US93/06599 211~S~

condensed carbocyclic aromatic groups having from 6 to 20 carbon atoms. Aryl groups include those exemplified by phenyl and naphthyl. These groups may be substituted with one or more non-interfering subs~ituents, e.g., those selected from lower alkyl; lower alkenyl; lowe~ alkynyl; lower alkoxy; lower alkylthio; lower alkylsulfinyl; lower alkylsulfonyl, dialkylamine; halogen; hydroxy; phenyl; phenyloxy; benzyl;
benzoyl, and nitro. Each substituent may be optionally substituted with additional non-interfering substituents.
"~mino" refers to the group -NH2.
'IAlkylcarbonyl'' refers to the group -(CHRl)-C0-wherein Rl is further designated the a-position. Rl may be hydrogen, alkyl, or an amino group. Pre~erably Rl is an amino ~group .
Description of the Preferred Embodiments In ac~ordance with the present invention, novel methods for chemically modifying proteins, such as antibodies, to facilita~e passage across capillary barriers and into cells are provided. In general, the methods include the covalent attachment of at least one non-interfering lipid substituent (e.g., glycyldioctadlecylamide, glycyldiheptadecylamide, glycyldihexadecylamide, dilauroylphosphatidylethanolamine, and glycyldioctadecadienoylamide) to a reactive site in the ~25 protein molecule (e~g., a periodate-oxidized carbohydrate moiety). Various non-interfering lipid substituents may be : attached to proteins:to~produce lipidized proteins, such as lipidized antibodies of the invention. For example but not for limitation, the following examples of lipids may be conjugat~d to a protein of interest to yield a lipidized protein: lipoamines, lipopolyamines, and fatty acids (e.g., stearic acid, oleic acid, and others). Generally, the lipid will be attached by a covalent linkage to a carbohydrate : linked to the protein (e.g., a carbohydrate side chain of a glycoprotein). Naturally-occurring carbohydrate side~chains are preferably used for linkage to a lipoamine, although noYel glycosylation sites may be engineered into a polypeptide by WO~4/01131 PCT/US93/06599 2î~-3~

genetic manipulation of an encoding polynucleotide, and expression of the encoding polynucleotide in a glycosylating cell to produce a glycosylated pQlypeptide.
Glycosylated proteins can be lipidized to enhance transvascular transport, organ uptake, and intracellular localization of the lipidized proteinl including intranuclear localization. Generally, a glycosylated polypeptide, such as an antibody, i5 chemically oxidized with an oxidizing agen~
(e.g., periodate) to yield pendant carboxyl and/or aldehyde groups, and reac~ed with a lipoamine to form a covalent (amide or imide, respecti~ely) bond linking the lipoamine to the protein. Typically, the oxidation of the carbohy~rate side-chain is a partial oxidation producing at least one reactive ~arboxyl or aldehyde group, although ~enerally chemical oxidation methods will produce some molecules that are partially oxidized and others that are either unoxidized or completely oxidized. However, in order to be lipidized by reaction with a lipoamine, the glycoprotein must be oxidized to produce at least one pendant aldehyde group that can react with a lipoamine, alt:hough it may be possible to produce : lipidized proteins tllrough linkage to pendant carboxyl groups as well. A pendant carboxyl or aldéhyde group of an vxidized glycoprotein is a carboxyl or aldehyde group having a carbonyl carbon derived: from an oxidized oligosaccharide and which is : 25. cova~ently attached to ~he protein, either directly or through : a spacer (e.g., an unoxidized portion of a N- or 0-linked carbohydrate side-chain). Preferahly, N-linked and 0-linked carbohydrate chains are incompletely oxidized to generate a multiplicity of reactive aldehyde and carboxyl groups at each glycosylation position for subsequent reaction with lipoamines. Most usually, glycoproteins having one or more complPx N-linked oligosaccharides, such as those having a branched (mannose) 3 ~-N-acetylglucosamino) core, are partially oxidized by limited reaction with a suitable oxidant, generally periodate. Linked oligosaccharides containing N-acetylglucosamine (NAG), mannose, galactose, fucose (6-deoxygalactose), N-acetylneuraminic acid (sialic acid), WOg~/01131 - PCT/US93/06599 17 2 ~ 8 v glucose, N-acetylmuramic acid, N-acetylgalactosamine, xylose, or combinations of these monosaccharide units can be oxidized and reacted with lipoamines to produce lipidized proteins, more specifically carbohydrate-linked lipidized proteins.
Glycoproteins containing linked oligosaccharides with monosaccharide units other than those specifically lis~ed above for exemplification, including non-naturally occurring monosarcharides, can also be oxidiz~d and covalently linked to a lipoamine to form a lipidized protein.
Lipoamines are molecules having at least one acyl group and at least one free amine (i.e., a primary or second-ary amine). It is believed that the invention can al50 be practiced with lipoamines that have tertiary amines which ~-~omprise at least one substituent that can be displaced by reaction with an oxidize~ carbohydrate. Examples of lipoamines having a primary amine are shown in ~ig. 1. For example, the invention can produce lipidized proteins by reacting a glycoprotein with a s~raight-chain lipoamine of the formula:

NH2-R- ( CH2 ) n~CH3 wher~ R i~: a disubstituted alkyl (alkyl~ne), preferably methylene (-CH2-); a 1,4-disubstituted cyclohexyl;
a disubstituted aryl (aryIene); preferably a 1,4-disubstituted phenyl ~phenylene); an amido group of the formula -(CHRl)-C0 NH- wherein Rl is hydrogen or an amino group; alkylcarbonyl, preferably ~-amino substituted alkylcarbonyl; or a phosphate diester, preferably of the formula -CH2-O-P02-0-. n is an integer which is ~ypically 1 to 50, prefera~ly about 5 to 30, more preferably about 10 to 25, and most usually about 15 to 20. In general~ n is selected at the discretion of the practitioner according to the following guideline: when the molecule to be lipidized is large (i.e., a protein of more than about 10 kD) it is preferred than n is at ~east about 8 to 12 or more to increase the hydrophobicity of the resulting lipidized protein; when the molecule to be lipidized is small 21~)S~J

(e.g., an oligopeptide) n can typically be in the range 2 to 18, but may be larger if additional hydrophobicity of the lipidized molecule is desired.
The in~ention can also be practiced with branched-chain lipoamines, whih, for example, can include lipoaminesof the formula:
NH2-R j - ( CH2 ) n~CH3 (~H2)m where R' is: a trisubstituted alkyl, preferably -CH2-CH< or l,2,4-trisubstituted cyclohex~l; a trisubstituted ~ryl, preferably l,~,4-trisubstituted phenyl; an amido group of the formula -(CHRl)-CO-N< wherein Rl is hydrogen or an amino group; an imino group of the formula CHR2-NH-CH< wherein R2 is hydrogen or an amino group or an imino group of the formula ~
~ : 20 CH2-N<; or a ph~sphate diester, preferably of the formula -CH2-: CH2 0-P0~-0-CH2-CH(C0~-)2. m and n are selected independently : and are integers which are typically l to 50, pre~erably about : 5 to 30, more pre~erably about~lO to 25, and most usually ; about 15 to 20. In general, n is selected at the discretion of the practitioner according to the following guidel'ne: when the mo1ecule to be:lipidized is large (i.e., a protein of more : than about lO kD) it is preferred than m andlor n is at least :; about 8 to 12 or:more~to increase the hydrophobicity of the resulting lipidized proteini~when the molecule to be lipidized is small (e~g., an~o~ligopeptide) n can typically be in the : range 2 to l~, but may:;~e larger if additional hydrophobicity of the lipidized molecule is desired.
Essentially any glycoprotein can be lipidized accvrding to the methods of the invention by reacting a : 35 lipoamine with an oxidiz~ed carbohydrate side-chain. Fig. 2 :: shows schem~tically a~glycosylated antibody and a carbohydra~e-linked lipidized an~ibody of the invention, ~:
respectively. Non-glycosylated protei.ns may be conjugated to a lipid by linkage through a suita~le crosslinking agent ~-(~.g., by carbodiimide linkage chemistry).

WOg4/0l131 PCT/US93/06~99 21185~S

In accordance with the present invention, novel lipidiæed antibodies capable of specifically binding to predetermined intracellular epitopes with strong affinity are provided. These antibodies readily enter the intracellular compartment and have binding affinitiPs of at least about 1 x 106 M-l, preferably 1 x 107 M-1 to ~ x 1o8 M-1, more preferably at least about 1 x 109 M-l or stronger. The lipidized antibodies typically have a lipid substituen~ attached to a naturally-occ~rring carbohydrate side chain on a donor immunoglobulin chain, which composes an antibody specifically reactive with an intracellular, transmembrane, or e~tracellular epitope. Since car~ohydrates are located on the Fc portion of immunoglobulins, chemical modification of the ~arbohydrate residues by lipidization would be unlikely t~
produce a substantial loss of affinity of the antibodies for their antigens (Rodwell et al. (1~86) Proc. Natl. Acad. Sci~
(U.S.A.L 83: 2632). The lipidized antibodies generally retain substantial affinity for their antigen, and the a~idity can be readily measured by ,any of several antibody-antigen binding ~ 20 assays known in the art. The antibodies can be produced : ~ ecQnomically in large quantities and ~ind use, for example, in the treatment of va~ious human disorders by a variety of techniques~
~ One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each ~ pair having one light and one heavy chain. In each pair, the :: light and heavy chain variable regions are together .
respo~sible for binding to an antig~-nl and the constant regîons are responsible ~or the antibo~y effector functions.
In addition to antibodies, immunoglobulins may exist in a variety of o~her forms including, for example, Fv, Fab, and (~ab')2, as we~l as bifunctional hybrid antibodies, fusion proteins (e-.g., bacteriophage display libraries), and other form~ (e.~, Lanzavecchia et al., Eur. ~._Immunol. 17, 105 (1987)) and in single chains ~e.q., Huston et al., Proc Natl~
Acad. Sci. U~S.A.; 85, 5879-5883 (19~8) and Bird e~ al., W~94/0113~ - PCT/US93/06~99 2 ~ 6 2Q

Science, 242, 423-426 (19$8)). (See, generally, Hood et al., i'Immunology", Benjamin, N Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986)).
Antibodies can be produced in glycosylating cells (e.g., human lymphocytes, hybridoma cells, yeast, etc.), in non glycosylating cells ~e.g., in E. coli), or synthesized by chemical methods or produced by in vitro translation systems using a polynucleotide template to direct translation. One ~ource of hybridoma cell lines and immunoglobulin-encoding polynucleotides is American Type Culture Collection, Rockville, MD. Methods for expression of heterologous proteins in recombinant hosts, chemical synthe~is of polypeptides, and ln vitro transla~ion are well known in the ~art and are described further in Maniatis et al., Molecular Cloninq: A_Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor~ N.Y.; Berger and Kimmel, Methods in Enzymoloq~. Volume :~ 52,_Guide to Molecular Clqninq Techni~ues (1987), Academic Press, Inc., San Diego, CA; Merrifield, J. ~1969) J. Am. Chem.
Soc. 91: 501; Chaik~n I.~. (1981) CRC Crit. Rev. Biochem. ll:
255; Kaiser et al.(~989) Science 243: 187; Merrifield, B.
(1986) Science 232: 342; Kent, S.B.H. ~1988) Ann. Rev.
: Biochem. S7: 957; and Offord, R.E. (1980) Semisvnthe~ic Proteins, Wiley Publishing, which are incorporated herein by reference). Antibodies that are produced in non-glycosylating cells can be conjugated to a lipid by use of a bifunctional crosslinking agen~ or pre~erably post-translationally glycosylated in a glycosylation system such as purified canine pancreatic microsomes (MueckIer and Lodish (1g86) Cell 44: 629 and Walter, P. (1983) Meth. Enzvmol. 96: 84, which are incorpora~ed herein by reference). Alternatively, polynucleotides that encode antibodies may be isolated from screened prokaryotic expression libraries, such as combinatorial antibody fragment display libraries, and s~bsequently expressed in glycosylating cells to produce glycosylated antibodies. According to these methods, glyccsylated antibodies may be obtained, having naturally-occurring and/or non-naturally-occurring glycosylation .

WO94/01131 PCT/~S93/065~9 2118~f~

patterns. Such glycosylated antibodies can be lipidized according to the methods of the invention.
Glycosylation of immunoglobulins has been shown to have signi~icant ef~ects on their effector functions, structural stability, and rate of secretion from antibody-producing cells (Leatherbarrow et al., Mol. Immunol. 22: 407 ~1985)). The carbohydrate groups responsible for these properties are generally attached to the constant (C) regions of the antibodies D For example, glycosylation of IgG at asparagine 297 in the CH2 domain is re~uired for full capacity of IgG to activate the classical pathway of complement-dependent cytolysis (Tao and Morrison, J. Immunol. 143: 2595 ~l989)). Glyco~ylation of IgM at asparagine 402 in the CH3 ~omain is necessary for proper assembly and cytolytic activity of the antibody (Muraoka and Shulman, J. Immunol. 142: 695 (1989)). ~emoval of glycosylation sites as positions 162 and 4l9 in the CH1 and C~3 domains of an IgA antibody lead to intracellular degradation and at least 90% inhibition of secretion ~Taylor and Wall, Mol. Cell._Biol. 8: 4197 (1988)).
: Glycosylation of immunoglobulins in the variable (V) ~ region has also been observed. Sox and Hood, Proc. Natl.
:~ ~ A~ad. Sci. USA 66: ~75 (1970), reported that about 20% of : human:antibodies are glycosylated in the V region.
Glycosylation of the V domain is believed to arise from fortuitous occurrences of the N-linked glycosylation signal Asn-Xaa~Ser/Thr in the V region sequence and has not been recogniged in the art as playing an important role in immunoglobulin function.
Therefore, it is generally preferred that ~0 lipidization is p rformed on antibodies having naturally-occurring g1ycosylation patterns. If glycosylation sites are engineered into an antibody, it is preferred that novel glycosylation site be introduced in a constant region or varia~le region framework region, which are less likely to a~versely affect the:antigen binding activity of the antibody.
It is generally most preferred that novel glycosylation sites which are engineered into an antibody are placed in a constant WO94/01131 PCT/US93/06~99 2 1 ~ g ~ 22 region.
Alternatively, polypeptide fragments comprising only a portion of a primary antibody structure and having a carb~hydrate side chain that may be derivatized with a lipid substituent ~e.g., lipoamine) can be produced, which fragments possess one or more immunoglobulin activities (e.g., antigen binding acti~ity~. These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the artf or by site-directed mutagenesis at the desired locations in expression vectors containing sequences encoding immunoglobulin proteins, such as after CHl to produce Fa~ ~ragments or a~ter the hinge region to produce (Fab')2 fragments. Single chain antibodies may be produced by ~oining VL and VH with a DNA linker (see, Huston et al., op.
cit., and Bird et al., op. cit.). Also because like many genes, the immunoglobulin-related genes contain separate functional regions, each having one or more dist}nct biological activities, he genes may be fused to functional regions from other genes having novel properties. Nucleic acid sequences for producing immunoglobulins for the present invention are capable of ultimately expressing the desired an~ibodies and can be formed from a variety of different polynucleotides (genomic or c~NA, RNA, synthetic oligonucleotides, etc.) and components (e.g., V, J, D, and C
; 25 re~ions), as well as by a variety of different techniques.
; ; Joining appropriate synthetic and genomic sequences is presently the most common method of production, but cDNA
sequences may also be utilized (see, European Patent ; Publication No. 023~9400 and L. Reichmann et al., Nature, 332, 323-327 (1988)).;
Immunoglobulins and/or DNA sequences encoding ir~noglobulin chains may be obtained, for exampl~, by hybridoma clones wh1ch can be produced according to methods known i~ the ar~ (Xohler and ~ilstein (1976) Eur. J. Immunol.
6: 5~1, incorporated herein by reference) or can be ob~ained from se~eral sources ("ATCC Catalog of Cell Lines and Hybridomasl~, Amerlcan Type Culture Collection, Rockville, MD, :
.

2118~

which is incorporated herein by reference). DNA sequences encoding immunoglobulin chains can be obtained by conventional cloning methods known in the art and described in various publications, for example, Maniatis et al., Molecular Cloninq:
~ .3~ LI~Lnual, 2nd Ed~, (1989), Cold Spring Harbor, N.Y.
and ~erger and Kimmel, Methods in EnzymoloqY Volume 152, Guide to Molecular Cloninq Techniques (1987), Academic Press, Inc., San Diego, CA, which are incorporated herein by reference.
As stated previously, the DNA sequences will be expressed in hosts, typically glycosylating cells, after the sequences have been opera~ly linked to (i.e., positioned to ensure the functioning of) an expression control sequence.
~-~hese e~pression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will ~contain selection markers, e.g., tetracycline-resistance or G418-resistance, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S.
Patent 4,704,362). ~
E. coli is one prokaryotic host useful particularly for cloning the DNA sequences of the present invention. Other ~ microbial hosts suitabl~e for use include bacilli, such as ;~ ~ Bacillus subtilis, and other En~erobacteriaceae, such as : 25 Salmonella, Serratia, .and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with~the host cell (e.g., an origin of ; replication). In add~ition,~any number of a variety of well-known promoters will be present, such as the lactose p~omoter system, a tryptophan (trp) promoter system, a ~-galactosidase promoter system, or a promoter system from phage lambda. The promoters wi~ll typically~control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Proteins, such as antibodies, that are expressed ~;~ in non-glycosylating cells can be post-translationally 2 ~ 1 8 ~ 24 glycosylated in a glycosylation system (Mueckler ~nd Lodish, op.cit., which is incorporated herein by reference.
Other microbes, such as yeast, may also be used for expression. Saccharomyces is a preferred host glycosylating cell, with suitable vec~ors having expression control ~equences, such as promoters, including 3-phosphoglycerate kinase or other glyco~ytic enzymes, and an origin of replication, termination sequences and the like as desired.
In addition to microorganisms, mammalian tissue cell culture may al50 be used to express and produce the polypeptides of the presen~ invention ~see, Winnacker, "From Genes to Clones," VCH Publishers, N.Y., N.Y. ~1987)).
Eukaryotic cells are actually preferred, because a number of ~uitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include : the C~O cell lines, various COS cell lines, HeLa cells, preferably myeloma cell lines, etc, and trans~ormed ~-cells or hybridomas. Expression vectors for these cells can include expression con~rol se~uences, such as an origin of X0 replication, a promoter, an enhancer (Queen et al., Immunol.
.
B~Y ~ 89, 49-68 (l986)), and necessary processing information : ~ : sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator . sequences. Preferred expression control sequences are ::
~: 25 promoters derived from immunoglobulin genes, SV40, Adeno~irus, cytomegalovirus, Bovine:Papilloma Virus, and the like.
The vectors con~aining the DNA segments of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell by well-known me~hods, which vary depending on the type of ~ellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate ~reatment or electroporation may be used for other cellular hosts. (~See,:generally, Maniatis et al , Molecular Cloning: ~ Laboratory ~anual, Cold Spring Harbor Press, (19B2)~) Once expressed, the whole antibodies, their dimers, .

WO94/01131 21~ ~ PCT/US93/06599 individual light and heavy chains, or other immunoglobulin forms of the present inven~ion, can be purified according to ~tandard procedures of the art, including ammonium sulfate precipitation, affinity co~umnsl column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982)).
Substantially pure immunoglobulins of at least about 90 to 95%
homogeneity are pxeferred, and 98 to 9~% or more homogeneity most preferred, for pharmaceutical uses. Once purified, lQ partially or to homogeneity as desired, the polypeptides may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings, and the like. (See, generally, ~-rmmunoloqical_Methods, Vols. I and II, Lefkovits and Pernis, eds., Academic Press, New York, N.Y~.(1979 and 1981)).
In the methods of the in~ention, intact immunoglobulins or their binding fragments, such as Fab, are pre~erably used. Typically, lipidized antibodies will be of the human IgM or IgG isotypes, but other mammalian constant regions may be utilized as desired. ~ipidized antibodies o~
the IgA, IgG, IgM, IgE, IgD classes may be produced.
: Preferably, the lipidized anti~odi~s of the invention are human/ murine, bovine, equine J porcine, or non-human primate antibodies, more preferably human or murine antibodies. The invention c~n be used to produce lipidized antibodies of various types, including but not limited to: chimeric antibodies, humanized antibodies, primatized antibodies, Fv fra~ments, toxin-antibody conjuga~es, isotope~antibody conjugates, and imaging agent-antibody conjugates~ For in ivo imaging, lipidized antibodies are suitably labeled with a diagnostic label, administered to the patient, and their : location determined at various times following administration~
Various methods of labeling antibodies with diagnostic reporters (e.g., with Tc99, other radioligands, radiocontrast agents or radio opaque dye) are known in the art.
Proteins and oligopeptides (i.e., polypeptides comprising ~rom 2 to about 50 amino acid residues attached in WO94/01131 PCT/U~93/06599 211~e3~& 26 peptidyl linkage) other than immunoglobulins can be lipidized according to the methods the invention. Naturally-occurring glycoproteins (e.g., ~-glutamyltranspeptidase, thrombomodulin, glucose transporter proteins) are preferred substrates for lipidization through carbohydrate linkage, although substantially any polypeptide can be lipidized by covalent attachment through a crosslinking agent (e.g., N-hydroxysuccimide) to a suitable amino acid side chain. In alternate embodiments of the invention, at least one lipid substituent (e.g., lipoamine) is covalently attached to a non-ca~bohydrate moiety on a protein or polypeptide (e.g., by formation of an amide linkage with a Asp or Glu residue side-chain carboxyl substituent or a thioester linkage with a Cys ~-~residue). Also, a fatty acid can be linked to an Arg or Lys residue by the side-chain amine substituents. Examples of non-glycosylated proteins which may be lipidized for enhancing transvascular and intracellular transport include, but are not limited to, the following proteins: c-fos, c-myc, c-src, NF-AT, and HMG CoA reductase. Naturally-occurring lipoproteins, ; 20 such as native proteins which undergo physiological farnesylation, géranylgeranylation, and palmitylation are natural products and~are not defined herein as "lipidized proteins".
The lipidized~antibodies and pharmaceutical 25~; compositions thereof~are particularly useful for parenteral a~ministration, i.e.~,~subcu~aneous~ly, intramuscularly or in-travenously. The compositions for parenteral administration will common~ly comprise a~solution of the immunoglobulin or a ~ cocktail thereof dissolved in an acceptable carrier, 30 ~preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g.~, water, buffered water, 0.4% saline, 0.3%
glycine and the like.~ These solutions are sterile and generally free of;particulate~matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceu~ically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering

3~ 2 1 1 8 7 8 ~ P~/US93/06~99 ~gents, toxicity adjusting agents and the like, f~r example ,odium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, human albumin, etc. The concentration of antibody in these formulations can vary widely, i.e., from less than a~out 0~5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
Thus, a typical pharmaceutical composition for injection could be made up to contain l ml sterile buffered water, and l-lO mgs of lipidized immunoglobulirl~ A typical composition for intravenous infusion could b~ made up to ~ontain 250 ml of sterile Ringer's solution, and 150 mg of antibody. Actual me~hods for preparing parenterally administrable compositions will be known or apparent to those skilled in the ar~ and are described in more detail in, ~or example, Reminqton's_Pharmaceutical_Sclence, 15th ed., Mack Publishing Com~any, Eas~on, Pennsyl~ania (1980), which is incorporated herein by reference.
: The lipidized proteins and antibodies of this invention can be fro~en or lyophilized for storage and reconsti~u~ed in a suitable carrier prior to use. This technique has been shown to be effective with conYentional 25 :immune globulins and art-known lyophilization and reconsti-tution ~echniques can:be employed. I~ will be appreciated by those skilled in the art that lyophilization and recon-stitution can lead to varyin~ degrees of activity loss (e.g., with conventional i~mune globulins, IgM antibodies tend to have greater ac~ivity loss than IgG antibodies) and that use levels may have:to be adjusted to compensate.
~ he compositions;containing the present lipidized pro~eins (e.g., antibodies) or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
In therap~ukic application, compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate 8Ç~ 28 to accomplish this is defined as a ~therapeutically effective dose." Amounts effective for this use will depend upon the severity of the infection and the general state of the patient's own im~une system, but generally range from about l to about 200 mg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used. It must be kept in mind that the materials of this invention may generally be employed in serious disease states, that is life threatening or potentially life-threatening situations~
In prophylactic applications, compositions containiny the present immunoglobulins or a cocktail thereof are administered to a patient not already in a disease state to enhance the patien~'s resistance. Such an amount is ~efined to be a "prophylactically effective dose." In this 15 use, the precise amounts again depend upon the patient's state of health and general:level of immunity, but generally range from O.l to 25 mg per dose.
Single or multiple administrations of the compositions can be carried out with dose levels and pattern ;2:0 being selected,by t~le treating physician~ In any event, the ~ pharmaceutical formulations should pro~ide a guantity of the :~ lipidized proteins and/or lipidized antibody~ies) of this in-: ~ ~ vention sufficient to effectively treat the patient.
For diagnostic purposes, the lipidized antibodies ~ ~ ~ 25 may either be labeled or unlabeled. Unlabeled ,~ntibodies can : ~ be used in combination with other labeled antibodies (second ~ : antibodies)~ t~at are react:ive with:the l~pidized antibody, ;~ : such as antibodies~specific ~or human immunoglobulin constant regions. Alternatively., the lipidized antibodies can be 3Q directly labeled. A wide variety of labels may be employed, n such as~radionuclides,~enzymes, enzyme su~s~rates, en~yme cofactors, enæyme:inhibitors, ligands (particularly haptens), radiocontras~ agents~, metal chelates/ etc. Numerous types of diagnostic imaging applications are available and are well 35 known to those~skilled in the art. For example, but not for limitation, an antibody that binds to a tumor antigen (e.g. t anti-CEA antibodies) may be lipidized and conjugated to a ' WO94/01131 21 1 ~ PCT/US93/06599 radiocontrast agent or magnetic imaging material, injected into a human patient, and detected so as to localize the position of a tumor or metastatic lesion.
The lipidized immunoglobulins of the present invention can ~e used for diagnosis and therapy. By way of illustration and not limitation, they can be used to treat cancer, autoimmune diseases, or viral infections. For treatment of cancer, the antibodies will typically bind to an antigen expressed preferentially in certain cancer cells, ~uch as c-myc gene product and others well known to those skilled in the art~ Preferably, ~he lipidized antibody will bind to a mutant protein, such as a c-ras oncogene product having a pathogenic (e.g., neoplastic~ sequence, such as a substitution ~t position 12, 13, 59, or 61 of the protein (e.g., a Ser at position 12 of p21rag). For treatment of autoimmune disease, the antibodies will typically bind to an critical regulatory protein expressed~primarily in activated T-cells, such as NF-AT, and many other~intracellular proteins well known to those skilled in the art (~e.~g., see Fundamental Immunolo~, 2nd ed., W.~E. Paul, ed., Raven Press: New York, NY, which is incorporated herein ~y reference). For treatment of ~iral infections, the antibodies will typically bind to a protein , expressed in cells infected by a particular ~irus such as the various~viral encoded~polymerases and HIV-l Tat, and many other viral proteins~well known to those skilled in the art (e.g., see Viro~=o~y,~2nd~ed., B.N. Fields et al., eds., (19903, Raven Préss:~New York, NY, which is incorporated herein by reference)~
Kits can also be~supplied for use with the subject lipidized antibodies in~the protection against or detection of a cellular activity~or ~for~the presence of a selected cell intracellular prate;in~or the diagnosis of disease. Thus, the subject composition~of the~present invention may be provided, usually in a lyophilized~form in a container, either alone or in conjunction with~additional antibodies specific for the desired cell~type. The~lipidized antibodies, which may be conjugated to a label or toxin, or unconjugated, are included :: `:

:

WO 94/01131 ~/US93/0~99 2 1~ 30 in the kits with buff ers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert prot~ins, e.g., serum albumin, or the like, and a set of instructions for use.
Generally, these materials will be present in less than about 5% wt. based on the amount of ac~ive antibody, and usually present in total amount of at least a~out 0.001% wt. based again on the antibody concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about l to 99~ wt. of the total composition. Where a second antibody capable of binding to the lipidized antibody is employed in an assay, this will usually be present in a separate vial. The second antibody is typically conjuga~ed to ~-~ la~el and formulated in an analogous manner with the antibody formulations described above, as well as typically also being lipidized itself.
The lipidized antibodies of the present invention are also suited for use in improved diagnostic methods and protein purification methods. For example, many intracellular proteins are unstable (e.g., short half-life, susceptible to proteolysis) or prone to aggregation (e.g., ~-amyloid protein) ~making purification andjor diagnostic detection difficult.
Lipidized antibodies are able to p~netrate living cells and bind to specific intracellular target antigens; such antibody-antigen binding may stabilize the target ankige~ and block ~ enzymes involved in degradation~of the target antigen (e.g., : proteases, ubiquitin-conjugating enzymes, glycosidases) facilitating detection and/or purification of the target antigen.
. In one variation of the invention, a lipidized antibody which specifically binds to an intracellular target antigen is contacted with live cells comprising the intracellular target antlgen under physiological conditions (e.g., cell culture:conditions, soma~ic conditions) and incubated for a suitable binding period (e.g., from about lO
minutes to several hours). The lipidized antibody specifically ~inds to the target antigen forming an antigen-W094/01131 ~ PCT/US~3/06~99 antibody complex which is less susceptible to degradationand/or aggregation that is the target antigen itself.
Typically, the cells are then fixed and permeabilized and the antigen-antibody complex, comprising the target antigen bound to the lipidized antibody, is detected, usually with a labeled secondary antibody that specifically binds the the lipidized antibody. Examples of preferred labels attached to the secondary antibody are: FITC, rhodamine, horseradish peroxidase conjugates, alkaline phosphatase c~njugates, ~-galctosidase conjugates, biotinyl moieties, radioisotopes, andthe like. In some embodiments, the secondary antibody may be lipidized and the fixat.ion and/or permeabilization steps may be omitted and replaced with substantial washing of the cell ~ample to remove non-specific staining. It may also be possible to use a lipidized, labeled primary antibody directly and omit the second antibody. Labelled protein A may also be ~;~ substituted for a secondary antibody for the detection of the primary (lipidized) antibody.
Lipidized antibodies may also be used for 20 ~i~ntracellular therapy, such as for binding to a predetermined intracellular target an~igen and modifying a biochemical property of the target antigen. For example, multi-subunit proteins, such as heteromultimeric proteins (e.g., transcription factors,~G-proteins) or homodimeric proteins (e~.g., polymerized tubulin) may possess a biochemical activity (e~g.~, GTPas~ acti~ity) or other activity that requires intermolecular interaction(s) that may be blocked by a lipidized antibody~that~specifically binds to one or more subunits and prevents~functional interaction of the subunits.
For example, a lipidized~anti-Fos antibody which binds to a portion of Fos (e.g., leucine zipper) required for binding to ; Jun to form a transcript~ionally acti~e AP-l transcription factor (Fos/Jun~heterodimer) may block formation of functional AP-l and inhibit~AP-l-mediated gene transcription. Also for example, a lipidized anti-ras antibody may bind to an epitope of ras which is~required~for its proper signal transduction ~unction (e.g., a~GTP/GDP-binding site, a portion of ras that 21~ & 32 binds an accessory protein such as GAP, or the like3, therebymodifying the activity of intracellular ras in living cells.
The following examples are offered by way of :
illustration, not by way of limitation.

XPERIMENTAL EXAMP~ES

Preparation of a Lipidized_Bovine IqG .. r Glycyldioctadecylamide was obtained by linking a glycine residue to dioctadecylami~e according to the method described by Be~r et al. (1989) Proc. Natl. Acad. Sci.
U.S.A.~ ~6: 6982, which is incorporated herein by refPrence.
~enzyloxycarbonyl-ylycyl-p-nîtropheno~ at 1 equivalent and triethylamine at l.l equivalents in CH2Cl2 are reacted for 5 hour~, follo~ed by addition of H2, 10% Pd/C in CH2Cl21EtOH and reaction for l hour.
Two mg of bovine IgG (Sigma) were dissolved in 400 ~1 of 300 mM NaHC03 in a ~ . 5 ml Eppendorf vial . Fifty ~Ll of a freshly prepared NaIO4 solution (42 mg/ml in H2O~ were added : ~ :and the ~ial was wrap~ed in aluminum foil and gently shaken for 90 min. at room ~emperature. The reaction medium was then loaded on a PD-l0 column (Pharmacia) previously equilibrated w~th l0 mM Na2CO3 (fraction l), and the column was eluted with ~00 ~l fractions.~ Fraction number 7 (be~ween 3 ml and 3.S ml) contaîned approximately l.6 mg of bovine IgG as measured using the Bradford protein assay.
~ solu ion of glycyldioctadecylamide in DMSO was prepared (5 mg of the lipid Into l ml of DMSO, ~igorously vortexed for several minutes). Under those conditions the lipid was not fully dissolved. Fifty ~l of this solution were taken carefully (and did not:contain any undi~solved lipid) and were added to 350 ~l of fraction 7 obtained as described above, in an Eppendorf vial. The vial was wrapped in aluminum foil, and the mixture was gently shaken for 20 h at room temperature.
One hundred ~l of a solution of NaBH4 (l0 mg/ml in WO94/01131 PCT/US93/065~9 211 ~ ~(!6 H2O) were then added. A~ter one hour, 40 ~l of a solution of ethanolamine (15 ~l in l ml H20) were added. After an additional l h, ~he reaction mixture was loaded on a PD-l0 column previously equilibrated in l00 mM HEPES buffer, pH 8.5.
The fraction containing the lipidized IgG (between 3 and 3.5 ml) was collected and stored on i.ce.

Labe~linq with l4C-acetic anhydride 14C-acetic anhydride (500 ~Ci, hmersham) in benzene (lOxl06 cpm/~l) was used. Two 5 ~l aliquots wers added to the 500 ~l fraction containing the lipidized IgG at l0 min interval in an Eppendor~ vial. The reaction was left on ice.
A 500 ~l solution of native IgG (800 ~g in l00 mM HEPES, pH
~8.5) was treated the same way.
~fter 30 min~ the vials were warmed to 20 to 25~C, th l4C~labelled Ig~ were separated from free l4C-acetat~ on a PD-l0 column equilibrated with PBS. Radioa~tivity incorporated was of approximately l0xl06 cpm for 500 ~g.
.
Orqan uptake studies .
Male swiss, albinos mice (20g) were used. One hundred ~l of l4C-labeled lipidized IgG or l4C-labeled control ~ : IgG in PBS (approximately ~00,000 dpm each) were administered ::~ intravenously by tail vein injection. Mice were killed after ~25 30 min or 3 h, their blood collected in ~DTA-containing tubes, and their brain (minus cerebellum and brainstem), spleen, one kidney :and one liver lobe were dissected. Organs were homog~nized in 1 ml 10 mM Tris~buffer, pH 7.4, and 500 ~l aliquots were counted in a Beckman scintillation counter.
Protein concent~ation in these homogenates was determined by the Bradford assay (Coomassie ~lue). The blood was cent~-..uge~ and 20 ~l fractions of the plasma were counted.
Table I shows:the uptake of~l4C in the brain,:liver, spleen and kidney, xpressed as:the ration of radioactivity in l ~g protain of the organ~divided by the radioactivity in l ~l plasma ~data expressed as ~ g protein).

.;

WO 94/01131 PCI /US9~/06599 2 ~
TABLE I
Orqan 3 0 minutes 3 hours Control LiPidized Control Lipidized Brain .20 + .03.32 + .08.94 + .05 1.75 + .09 Kidney1.46 + .319.50 + 2.191.17 + .04 3.54 + .22 Liver1. ~6 ~ . 264 . 95 + . 82. 66 + . 04 7 . 59 + . 27 Spleen1.08 + .274.5~ + .761.13 + .03 12.0 + 2.15 . .
10 Control groups of 4 and 6 mice at 3 0 min and 3 hr, respectively. Groups receiving the lipidized IgG were of 6 mice at both time points . Data are means + s ~ e O m .

~ EXAMPLE 2 nhibition of HIV-l Cy~otoxicity_with Lipidized Anti-Tat Antibod~
A monoclonal antibody which specifically binds the ~: Tat protein of HIV-1-was lipidized according to the method ~ ~:
described in ~xample;~l, ~gy~, involving periodate oxidation 2~ of carbohydrate.on t~e antibody, ~ollowed by covalent attachmen~ of glycyldioctadecylamide to yield a carbohydrate- :
~: : lipidized antibody,:which~was eluted from the final PD-10 ;coIumn with P~S.
: Sup T1 cells were maintained in 24-well plates 25~ ~IO~O,OOO ~ells per ml, ~in 2 ml of modified RPMI 1640 culture medium). Cells were kept in culture with either: (1) no additional treatment~(two controls), (2) in the presence of iadde~ native anti-Tat antibody (15 ~y/ml), or (3) in the ~ presence of the l:ipidized anti-Tat antibody (11~7 ~Ig/ml) ;~
: 30 during the first five:days of the experiment. At the end of the first day, HIV-l IIIB was a~ded to one well of control ~; ~ ; cells and to the cultures containing native anti-Tat antibody-~treated cells:or lipidized anti-Tat: antibody-trea~ed cells.
: ~ :; Yiable cells were counted daily. ~The untreated, HIV-infected ~:~
: 35 cells grew up to a density of approximately 500,000 cells per ml, and their number began to decrease after approximately ~; ~ eight days due to the cyto~oxic effect of the virus.

,:
.

WO94/01131 PCT/US93/0~99 2 ~

Uninfected cells grew up to a density of approximatel~
l,000,000 cells per ml. Treatment of infected cells with the native anti-Tat antibody did not protect the cells from the cytotoxic effect of ~he virus. In contrast the lipidized anti-Tat antibody le~ to an almost complete protection of the cells from the cytopathic effects of the HIV-l virus. This protection continued for at least about 5 days after the treatment with the lipidized antibody was interrupted. The results are presented in Fig. 3.
~0 In another experiment, Sup Tl cells were maintained in culture as described in the previous example and kept in culture without any trea~ment and without any infection, infected with HIV-l IIIB with no treatment, treated with the native anti-Tat antibody (l ~g/ml) and infected, or treated with the lipidized anti-Ta~ antibody (l ~g/ml) and infected.
In the last three conditions, the virus was added at the end of the first day in culture. In the last two conditi~ns, the native or lipidized antibody was present from day l until day 7.
The data presented in Table II show that, while the native anti-Tat antibody has little if any effect on viable cell number and reverse transcriptase activity, the lipidized antibody induced a significant protection of the cells in culture from the cytopathic effect of the virus and a significant decrease in reverse transcriptase activity. The latter suggests that the lipidized antibody could inhibit intracellular ~IV-l replication.

WO94/01131PCT/US93/06~99 Y, - 8 ~b TABLE II
Effect of a Lipidized Anti-Tat ~ntibody on Viable Cell Number and Reverse Transcriptase Activity in Sup T1 Cells Infected with HIV-1 ~iable Cells (x106) Days in Culture Conditions Untreated, Uninfected 1.15 1.28 1.52 ~.63 1.69 1.76 1.72 Untreated, Infected 1.12 1.2 1.1~ 1.12 o.g~ 0.64 0.51 Treated with native 1.12 1.2 1.21 1.15 0~96 0~67 0.51 anti-Tat Ab Infected Treated with lipidized 1.15 1.26 1.3 1.36 1~17 O.gg 0.75 anti-Tat Ab Infected ~5 Re~erse Transcriptase Activity (cpm/109 cells~
Days in Culture Conditions Untreated, Uninfected 2 ~ 1 2 2 Untreated, Infected 3 2 4 175 264 337 367 Treated with native anti-Tat Ab Infected ~ 2 3 1~1 237 259 331 Treated with lîpidized 3 2 2 57 135 179 18~ :
anti-Tab Ab Infected ~IV-1-infec~ed SupT1 cells were treated daily with anti-Tat antibody in native or lipidiæed form or wit~. rsCD4 ~:
(all proteins used at 1 ~g/ml) starting from Day 1 before addition of HIV-l virus containing supernatants until 10 days post infection. Cell numbers and reverse transcriptase activity (RT) in the culture medium were determined every day starting from Day 2 post-infection. By Day 10, the native an~i-Tat still had no significant effect on either cell counts WO94/0113l 2118 ~ o ~ PCT/US93/06599 or RT activity, whereas the lipidized anti-Tat antibody increased cell viability as compared to untreated, infected cells by approximately 70% and decreased RT activity by approximately the same extent. Cultures were continued for 3 days without further addition of antibodies. Effects of the lipidized anti-Tat persisted for the 3 days, indicating that the lipidized anti-Tat antibodies had accumulated in the cells in amounts high enough to pro~ide sustained protection against viral infection/replication. The magnitude of the effects of the lipidized anti-Tat antibody on cell viability and ~T
activity were very similar to those observe~ with rsCD4 at the same dose. Increasing the concentration of the lipidized anti-Tat antibodies to lO ~g/ml did not induce further decrease in RT activity.
Example 3 Ability of Lipidized Anti-Tat to Inhibit the Transcri~tional Activity of Tat on the HIV-l LTR
~ A HeLa cell line stably transfec*ed with a ~polynucleotide expr~sing CD4, the membrane receptor mediating HIV-l infection, and a1so containing a reporter construct ~comprising an HIV-l long terminal repeat (LTR) in operable linkage to and driving transcription of a linked reporter gene (chloramphenicc1 acetyltransferaæe, C~T). These cells (HLCP4-~25 CAT) are susceptible to HIV-1 infection which produces functional Tat protein; the binding of newly synth~ized Tat to the HIV-l LTR leads to transcription of the linked CAT gene.
Thus, the magnitude of CAT expression is approximately pro*ortional to the extent~of HIV-l infection and the activity of Tat protein in the cells~.
Cultured HeLa cells (3 x 105 cells/ml in DMEM with 10~ fetal bo~ine serum)~were exposed to the same concentrations (l or lQ ~g/ml) of ~arious antibodies (in native or lipidized ~ form) or recombinant~soluble CD4 (rsCD4) for 1 hour and were extensively washed prior to~addition of HIV-containing cell culture supernatants (lOO ~l). Twenty-four hours later the cells were harvested and CAT expression was measured by the method of Ho et al. (~984). Each experiment was run in quadruplicate and conducted four different time. Fig. 4 shows ~hat the lipidized anti-Tat antibody significantly inhibited W094tO1131 PCT/US93/V6599 211~8~ 38 CAT activity (by approximately 75%), whereas native (u~lipidized) anti-Tat antibody, lipidized anti-gpl20 antibody, or rsCD4 were far less effective in inhibiting CAT activity.
These data indicate ~hat lipidized anti-Tat was able to specifically bind its intracellular target, Tat, and inhibit the target's activity as a transcriptional activator of the LTR/reporter gene construct.
More~ver, the data showing passage of the lipidized anti-Tat antibody into ~eLa cells indicates that the transport mechanism does not likely require endosome formation, since HeLa cells are reported to undergo lit~le if any phagocytosis.

Example 4 Preparation of Lipidized Immunoalobulins Reactive with an Intracellular Protein Glycyldioctadecylamide is obtained by linking a glycine residue ko dioctadecylamine according to the method ~escribed by Behr et al. (1~89) Proc. Natl. Acad. Sci. ~U.S.A.
~6: 6982, which is incorporated herein by reference.
Benzyloxycarbonyl-glycyl-p-nitrophenol a~ 1 equivalent and triethylamine a~ l.l equivalents in CH2Cl2 are reacted for 5 hours, *ollowed by ~dd:ition of H2, lO~ Pd/C in CH2Cl2/EtOH and reaction for l hour.

Anti-Human c-Mvc Iq Glycosylated murine immunoglobulins that bind pecific~lly to human a-myc protein are prepared by separately culturing the hybridoma cell lines MYC CT9 B7.3 (ATCC CRL
1725), MYC CT 14-G4.3 (ATCC CRL 1727j, and MYC l-9El0.2 (ATCC
CRL 1729) in RPMI 1~40 with lO percent fetal bovine serum under specified conditions (Evan et al. (1985) Mol. Cell. Biol. 5:
36lO, incorporated herein by reference) and the monoclonal antibodie secreted are collected and purified by conventional methods k~o~n in the art.
A~out 2 mg of each purified monoclonal antibody are dissolved in 4CO ~l of 300 mM NaHC03 in a l.5 ml Eppendorf vial. Fifty ~l of a freshly prepared NaIO4 solution (42 mg/ml in H20) is added and the vial is wrapped in aluminum foil and WO94/01131 2 1 ~ 8 ~ 8 ~ PCT/US93/06599 39' gently shaken for ~0 min. at room temperature. The reaction medium is then loaded on a PD-lO column (Pharmacia) previously e~uilibrated with lO mM Na2C03 (fraction l), and the column is eluted with 500 ~l fractions. The fraction(s) containing at least approximately 500 ~g of IgG as measured using the Bradford protein assay are collected.
A solution of glycyldioctadecylamide in DMS0 is prepared (5 mg of the lipid into l ml of DMS0, vigorously vortexed for several minutes). Under those conditions the lipid is not fully dissolved. Fifty ~l of this solution is taken carefully and added to 350 ~l of the puri~ied IgG
fractions obtained as described above, in an Eppendorf vial.
The vial is wrapped in aluminum foil, and the mixture is gently shaken for 2~ h at room temperature.
One hundred ~l of a solution of NaBH4 (lO mg/ml in H2O) is then added. After one hour, 40 ~l of a solution of ethanolamine (15 ~l in 1~ml H20? is added. After an additional l h,~ the reaction mixture is~ loaded on a PD-lO column previously equilibrated in PBS. The fraction containing the 20~ lipidiz~ed murine anti-human-nyc IgG (between 3 and 3.5 ml) is collected and stored~on~ice~.

An~i-HMG CoA Reduc_ se_Iq Glycosylated nurine;i~ unoglobulins that bind 25~'~specifically to the~intracellular~ enzyme HMG CoA reductase are prepared~by separately culturing the hybridoma cell line A9 TCC~CRL~18ll)~in~DMEM~with~4.5 g/l glucose, 5~ hor~e serum and 2.5%~$eta1 bovine~'serum~;'as~described (Goldstein et al.
(1983) 2.~Biol. ~Chem~.~;25~ 8450,~incorporated herein by reference~ and the monoclonal antibodies secreted are collected ; and purified by conventional methods~known in the art.
About 2~mg of~each~purified monoclonal antibody are dissolved~in 400 ~1 of 300~mM~NaHC03~in a l.5 ml ~ppendorf vial.~Fifty ~l of a fres~ly prepared~NaIO4 solution (42 mg/ml in H20);is added and~;the~vial is wrapped in aluminum foil and qent'ly~shaken for 90 min. at room temperature. The reaction ~ ~medium is then loaded~on a PD-lO column (Pharmacia) previously ; ~ equi1lbrated with 10 mM~Na2C03 ~fraotlon ~ and the column is ., . . . ... ~ .. ,. ., .. ~ . ..

~ B 40 eluted with 500 ~l fractions. The fraction(s) con~aining at least approximately 500 ,~lg of IgG as measured using the Bradford protein assay are collected.
A solution of glycyldioctadecylamide in DMSO is pxepared (5 mg o~ the lipid into l ml of DMSO, vigorously vortexed for several minutes). Under those conditions the lipid is not fully dissolved. Fifty ,~l of this solution is taken carefully and added to 350 ,~l o~ the purified IgG
fractions obtained as described above, in an Eppendorf vial.
~0 The ~ial is wrapped in aluminum foil, and the mixture is yently shaken for 20 h at room temperature.
One hundred ,~l of a solution of NaBH4 (l0 mg/ml in H2O) is then added. After one hour, 40 ,ll of a solution of ethanolamine (15 ,~ll in l ml H20) is added. After an additional l h, the reaction mixture is loaded on a PD-l0 rolumn previously equilibrated in PBS. The fraction containing the lipidized anti-HMG CoA re~uctase IgG (between 3 and 3.5 ml) is : collected and stored on ice.
':

Example 5 Pre~aration_of_Lipidized Immunoalobulins Reactive with a : Transmembrane Pr~tein Glycyldioctadecylamide is obtained by linking a glycine residue to di~ctadecylamine according to the methQ~
`2~ described by Behr et al. (l9~) Proc. Natl. Acad. Sci. (U.S.A.) 86: 6982, which is incorporated herein by rePerence.
: B~nzyloxycarbony1-glycyl-p-nitrophenol at 1 equivalent and triethylamine at l.l e~uivalents in CH2Cl2 are reacted for 5 : hours, followed by addition of H2, 10% Pd/C in CH2Cl2/EtOH and 30 reaction for l hour. :~

~ Anti-Ras Iq Glycosylated murine immunoglobulins that bind : specifically to ras onco0ene protein are prepared by separately culturing the hybridoma cell line 142-24E5 (ATCC HB 8679; U.S.
Pats. 5,015,571 and 5,030,565, incorporated herein by reference) in DMEM with 4.5 g/l glucose, 2mM ~-glutamine, lmM
sodium pyruvate, non-essential amino acids, lxBME vitamins, 0.l WO 94~U1131 2 1 1 ~ ~ 8 6 PCT/US93/06~99 ~1 mM hypoxanthine, 0.032mM thymidine, 0.05 mg/ml gentamicin, and 10% fetal bovine serum, and the hybridoma cell MX tATCC HB
9158) in Isco~e's DME~ with 1% L-glutamine and HT and 10 percent fetal bovine serum under specified conditions (U.S.
Patent 4,820, ~31, incorporated herein by reference) and the monoclonal antibodies secreted from the hybridoma lines are collected and purified by conventional methods known în the art.
About 2 mg of each purified monoclonal antibody are dissolved in 400 ~1 of 300 m~ NaHC03 in a 1.5 ml Eppendorf vial. Fifty ~l of a freshly prepared NaI04 solutlon (42 mg/ml in H20) is added and the vial is wrapped in aluminum foil and gently shaken for 90 min. at room temperature. The reaction ~:
medium is then loaded on a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na2C03 (fraction 1), and the column is eluted with 500 ~l fractions. The fraction~s) containing at least approximately 500 ~g of IgG as measured using the : ~ Bradford protein assay are collected.
A solution of glycyldioctadecylamide in DMSO is prepared (5 mg of the:lipid into 1 ml of DMSO, vigorously ~o~texed for ~evera~ minutes). Under those conditions the : lipid is not ~ully di solved. Fifty ~I of this solution is taken carefully and added t~ 350 ~l of the purified IgG
: fractions obtained as described above, in an Eppendorf via-~.
25~: Th2~vlaI is~ wrapped in aluminum foil, and ~he mixture is gently :: shaken for 20 h at room temperature.
One hundred ~l of a solution of NaBH4 (10 mg/ml in H20) is t~en added. After one:hour, 40 ~l of a solution of ethanolamine (15 ~l in l~ml H20) is added. After an additional 1 h, the reaction mixture is loaded on a PD-10 column previously equilibrated in PBS. The fraction containing the : lipidized murine anti-ras IgG :(between 3 and 3.5 ml) i5 collected and stored on ice.
Hybridoma cell lines re~erred to in the above .

examples may be obtained from American Type Culture Collection, Rockville, MD (ATCC Cell Lines and Hybridomas (1992) 7th Ed, which is incorporated herein by reference).

.

21~5~ Exam~le LiPidization of a Transmembrane Enzyme . :~
The enzyme gamma-glutamyltranspeptidase (~GT: EC
2.3~2.2) is a widely distributed enzyme that catalyzes the degradation of glutathione and other ~-glutamyl compounds by hydrolysis of the ~-glutamyl moiety or by its transfer to a suitable acceptor, GGT is a heterodimeric glycoprotein, which is synthesized as a precursor protein that is glycosylated and cleaved into the two subunits of the mature enzyme. GGT is anchored to the cell membrane through the ~-terminal portion ~f its heavy subunit. The active site of the enzyme lies on the extracellular portion;of the molecule, which is heavily glycosylated.
GGT is separately purified from rat kidney and a cultured human hepatoma cell line according to procedures described previously in the art (Barouki et al. (lg84) J. Biol. :-~
Chem. 259: ~70; Curthoys and Hughey (1979) Enzyme 24: 383;
:~ Matsuda et al. (1983) J. Biochem. 93: 1427; Taniguchi et al.
(l985) J. Natl. Cancer Inst. 75: 84li Tate and Meister (1985) Methods Enzvmol. ll3: 400; and Toya et aI. (1983) ~nn. N.Y.
: ~ Acad. Sci. 4l7: 86, which are incorporated herein by ref~ere~c~
About l mg of each of the purified rat and human GGT
preparations are dissolved in 400 ~l of 300 mM NaHC03 in a;~.5 ~25 ml Epp~ndorf vial~ Fifty ~l of a ~reshly prepared NaI04 solu~ion ~42 mg/ml in~H20~: is added and the vial is wrapped in al~minum foil and gent~ly shaken for 60 min. at room temperature. The ~eaction medium is then loaded on a PD-lO
~: ~ column (Pharmacia) previously:equilibrated with lO mM Na2C03 ~fraction l), and the column is eluted with 500 ~l fractiQns.
The fraction(s) containing at least approximately lO0 ~g of GGT
: ~ as measured using the Bradford:protein assay are collected.
A solution of glycyldioctadecylamide in DMS0 is prepared t5 mg of the lipid into l ml of DMS0, vigorously vortexed for several minutes). Under those conditions the lipid is not ~ully diss~olved.~ Fifty ~l o~ this solution is taken carefully and added:to 350 ~l of the purified GGT
fractions obtained as:~described above, in an Eppendor~ vial.

W094/01131 2 1 1 ~ 5 ~ & PCT/US93/06599 The vial is wrapped in aluminum foil, and the mixture is gently shaken for 20 h at room temperature.
One hundred ~l of a solution of NaBH4 (lO mg/ml in H~O) is then added. Af~er one hour, 40 ~l of a solution of S ethanolamine (15 ~l in l ml HZO) is added. After an additional l h, the reaction mixture is loaded on a PD-lO column previously equilibrated in PBS. The fraction containing the lipidized human and rat GGT (between 3 and 3.5 ml) is collected and stored on ice.
The lipidized human and rat GGT fractions are assayed for ~-glutamyltranspeptidase activity by conventional assay procedures (Tate and Meister (1983) op.cit., incorporated herein by reference) and the specific activity of the lipidixed human GGT and lipidized rat GGT is determined.
lS The lipidized human and rat GGT is radiolabeled by iodination with l25I u~ing chloramine T and approximately 50 ~g of the radioiodina~ed lipidized GGT is administered to rats by intraperitoneal injec~ion. After 24 hours, the rats are sacrificed and tissue samples removed for autoradiography to ao : :determine the pattern of localization of the lipidized GGT in : the various organs.

: Example 7 : : Lipidization of an Anti-Actin Antibod~ and Intracellular ,-~
:
~Immunostaininq ~ ~
; In order to demonstrate that lipidized antibodies can ::; localize intracellularly in 1iving cells and bind intracellular targe~s, an anti-actin an~ibody was lipidized and evaluated for its ability to penetrate cultured Swiss 3T3 fibroblasts and bind to the cytoskel tal protein actin. Native anti-actin antibody (unlipidized) was used as a control.
:~ Protein A-purified rabbit anti-actin poly~lonal an~ibodies were lipidized~accordin~ tG the following procedure.
A lipoamine, glycyldioctadecylamide, was co~alently linked to 3S~ the carbohydrate moieties of the anti-actin antibodies by periodate oxidation-sodium borohydride reduction. Antibodies were dissolved in O.8 ml of 300 mM NaHC03 at a concentration of : ~approximately 0.2 to l.O mg/mlO Fifty ~l of a freshly prepared W094/01131 PCTtUS93tO6599 211~ 44 aqueous solution of NaIO4 (42 mg/ml) were added and the incubation vials were wrapped in aluminum foil and gently shaken for 90 minutes at room temperature. The reaction mixture was then purified on a PD-lO column (Pharmacia, Piscataway, NJ) equilibrated in and eluted with lO mM Na2CO3.
Fifty ~l of a lO mg/ml solution of glycyldioctadecylamide in benzene are added to the fraction containing the antibodies te.g., as determined by A280 monitoring, Brad~ord assay) and the resulting reaction was incubated for 20 hours at room temperature with gentle shaking. One hundred ~l of a freshly prepared aqueous solution of NaBH4 (lO mg/ml) was then added and inubated at room temperature for one hour, followed by addition of 50 ~l of ethanolamine solution (15 ~l ethanolamine dissolved in l ml of H20)o After an additional hour at room temperature, the resultant lipidized antibodies were purified by chroma~ography on a PD-lO column equilibrated with phosphate-buffered saline.
ELISA AssaYs Lipidized anti-actin and lipidized anti-Tat (supra) were evaluated for their binding affinity for pecific antigen relative to native (u~lipidized~ anti-actin or anti-Tat antib~dy by ELISA assay. ~ipidization of either the anti-actin antibody or the anti-Tat anti~ody did not produce a measurable ~ loss of a~f~inity of the antibodies ~or their respective ~antigens as compared to their native (unlipidized) antibody.
Int~r-a~ellular Immunostainin~
To demonstrate that lipidized anti-actin antibodies ~are able to bind intracellular actin in live cells, lipidized anti-actin antibody or native anti-ac~in antibody were contacted with cultured Swiss 3T3 cells for l hour, followed by extensive washing to remove residual anti-actin antibodies.
The cells were subsequently fixed and permeabilized and the anti-actin antibodies were detected with a fluorescent-labeled secondary antibody. While no;speci~ic staining could be detected in cel~s preincubated with the native (unlipidized) anti-actin antibody, specific actin staining (e.g., stained actin cables~ was clearly evident in cells preincubated with the lipidized anti-actin antibodies. The staining pattern WO94/~1131 2 1 1 ~ ~ 8 ~ PCT/US93/06~99 observed with the lipidized anti-actin anti~ody applied prior to fixation was similar to that seen using native (unlipidized) anti~actin antibody incubated with the cells following fixation and permeabilization. These data demonstrate that the lipidized anti-actin antibodies were able to reach and bind intracellular actin, and that they could still be recognized and bound by the labeled secondary antibodies, indicating that they were functional and substantially intact.

Although the present invention has been described in ~ome detail by way of illus~ration for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be Fracticed within the scope of the claims.

'`

; ~
.

Claims

1. A method for modifying the pharmacokinetic characteristics of a protein, comprising the steps of:
attaching a lipid substituent to the protein by a covalent linkage to produce a lipidized protein; and recovering the lipidized protein.

2. A method according to Claim 1, wherein the lipid substituent is a lipoamine.

3. A method according to Claim 2, wherein the step of attaching further comprises the steps of oxidizing a carbohydrate on a glycosylated polypeptide to produce an oxidized glycoprotein; and reacting the oxidized glycoprotein with a lipoamine under suitable reaction conditions to form a lipidized protein.

4. A method according to Claim 2, wherein the lipoamine is a straight-chain lipoamine according to the formula:

NH2-R-(CH2)n-CH3 where R is selected from the group consisting of disubstituted alkyl (alkylene); 1,4-disubstituted cyclohexyl;
disubstituted aryl (arylene); amido group of the formula -(CHR1)-CO-NH- wherein R1 is hydrogen or an amino group;
alkylcarbonyl; and phosphate diester; n is 1-50.

5. A method according of Claim 2, wherein the lipoamine is a branched-chain lipoamine according to the formula:
where R' is: a trisubstituted alkyl; a trisubstituted aryl; an amido group of the formula -(CHR1)-CO-N< wherein R1 is hydrogen or an amino group; an imino group of the formula -CHR2-NH-CH< wherein R2 is hydrogen or an amino group or an imino group of the formula -CH2-N<; or a phosphate diester; m is 1-50; n is 1-50; and m and n are selected independently.

6. A method according to claim 5, wherein the branched-chain lipoamine is glycyldioctadecylamide.

7. A method according to Claim 1, wherein the protein is a naturally-occurring glycoprotein.

8. A method according to Claim 1, wherein the protein is encoded by an immunoglobulin superfamily gene.

9. A method according o Claim 8, wherein the immunoglobulin superfamily gene encodes a µ or .gamma. heavy chain.

10. A method according to Claim 7, wherein the naturally-occurring glycoprotein is an antibody.

11. A method according to Claim 10, wherein the antibody is a monoclonal antibody.

12. A method according to Claim 1, wherein the lipidized protein comprises at least one lipoamine residue linked to a carbohydrate side chain.

13. A method for targeting an intracellular protein for binding with an antibody in a cell, comprising contacting the cell with a lipidized antibody which binds specifically with the intracellular protein.

14. A method according to Claim 13, wherein the lipidized antibody comprises at least one lipoamine residue linked to a carbohydrate side chain of an immunoglobulin.

15. A method according to Claim 14, wherein the lipoamine is glycyldioctadecylamide.

16. A method according to Claim 13, wherein the lipidized antibody is administered to a nonhuman animal in vivo.

17. A method according to Claim 16, wherein the lipidized antibody is taken up into an organ to a greater extent than is a comparable naturally-occurring antibody having the same amino acid sequence(s) and the same glycosylation pattern.

18. A composition for therapy or prophylaxis of a disease, comprising a therapeutically effective dosage of a lipidized protein.

19. A composition according to Claim 18, wherein the lipidized protein is an antibody.

20. A composition according to Claim 19, wherein the antibody binds to an intracellular protein.

21. A composition according to Claim 20, wherein the intracellular protein is a viral-encoded protein.

22. A composition according to Claim 21, wherein the viral-encoded protein is a Tat protein encoded by HIV-1.

23. A composition for prophylaxis, comprising a prophylactically effective dosage of a lipidized antibody.

24. A composition comprising a lipidized antibody linked to a diagnostic reporter.

25. A composition according to Claim 18, containing a carbohydrate-linked lipidized protein.

26. A composition according to Claim 25, wherein the carbohydrate-linked lipidized protein is a carbohydrate-linked lipidized immunoglobulin.

27. A pharmaceutically acceptable composition comprising a carbohydrate-linked lipidized immunoglobulin, and an excipient.

28. A method for diagnosing a pathological condition, comprising the steps of:
administering a lipidized antibody comprising a diagnostic reporter to a patient; and detecting a location at which the diagnostic reporter is preferentially localized.