WO2001040277A2 - Carbohydrate-aminated glycoproteins - Google Patents
Carbohydrate-aminated glycoproteins Download PDFInfo
- Publication number
- WO2001040277A2 WO2001040277A2 PCT/US2000/033075 US0033075W WO0140277A2 WO 2001040277 A2 WO2001040277 A2 WO 2001040277A2 US 0033075 W US0033075 W US 0033075W WO 0140277 A2 WO0140277 A2 WO 0140277A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- carbohydrate
- aminated
- glycoprotein
- antibody
- antigen
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1072—Regulatory proteins, e.g. tat, rev, vpt
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- glycoproteins are a diverse group of molecules that have therapeutic and diagnostic potential.
- glycoproteins e.g., antibodies
- the full therapeutic and diagnostic potential of these proteins has not been realized.
- One factor which limits the therapeutic and diagnostic use of glycoproteins is that most glycoproteins do not readily traverse vascular barriers (e.g., the blood brain barrier) or cell membranes.
- vascular barriers e.g., the blood brain barrier
- cell membranes e.g., the blood brain barrier
- intravenously administered glycoproteins frequently do not accumulate in target organs or the interior of cells (e.g., cytoplasm, nucleus, organelles) at therapeutically or diagnostically effective levels.
- Cationized proteins are prepared by substituting acidic carboxylate groups on the side chains of amino acids with polyamines, thereby altering the isoelectric point of the protein (see, for example, Triguero, D., et al, Proc. Natl Acad. Sci. U.S.A., 55:4761-4765 (1989); Triguero, D., et al., J. Pharmacol. Exp. Ther., 255:186-192 (1991); Poduslo, J. F., et al, J.
- cationized proteins are reported to cross vascular barriers, these types of proteins have several disadvantages in regard to therapeutic or diagnostic applications. For example, amino acids with acidic side chains (i.e., aspartate, glutamate) can occur in the active site (catalytic site, binding site) of a protein, and modification of such residues can decrease or destroy the biological activity of the proteins.
- cationized proteins can be profoundly more immunogenic than native proteins (Muckerheide A., et al, J. Immunol, 138:833-837 (1987)). Consequently, even autologous proteins which are cationized may not be suitable for administration to patients.
- Lipidated glycoproteins are modified glycoproteins in which lipid moieties are covalently bonded to the carbohydrate moieties (see, for example, Cruikshank, W. W., et al, Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, 14 ⁇ 93-203 (1997)).
- lipidated proteins are difficult to prepare and have been shown to be more immunogenic than native proteins (Phillips, N.C., et al, J. Immunol, 152:3168-3174 (1994)).
- the invention relates to carbohydrate-aminated glycoproteins which comprise one or more diamine moieties that are bonded to carbon atoms of a carbohydrate moiety of the glycoprotem.
- the diamine moiety can be bonded to the carbohydrate moiety through a double or, preferably, a single covalent bond that is formed between an activated carbon atom of an oxidized carbohydrate moiety and a nitrogen atom of the diamine moiety.
- the diamine moiety can be represented by Structural Formula I or Structural Formula H
- R 1 is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group.
- R 2 and R 3 are each independently -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R 2 and R 3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring.
- X is a C] to about C 12 aliphatic group or aromatic group.
- X is an alkylene group represented by -(CH 2 ) n -, wherein n is an integer from one to about twelve; preferably one to about six.
- the diamine moiety can be represented by Structural Formula I wherein X is an alkylene group represented by -(CH 2 ) .
- the carbohydrate-aminated glycoprotein of the invention can be, for example, a growth factor (e.g., nerve growth factor), or an antibody (immunoglobulin) or antigen-binding fragment thereof.
- Antibodies which have binding affinity for a desired antigen can be carbohydrate-aminated.
- the carbohydrate-aminated antibodies of the invention can be directed against tumor antigens, intracellular proteins or viral proteins, for example.
- the carbohydrate-aminated antibody of the invention is directed against the Tat protein of HIV.
- the carbohydrate-aminated antibody of the invention is directed against Ras, p53, papilloma virus E6, papilloma virus E7, the X protein (pX) of hepatitis B virus, interleukins, cytokines, or cytoskeletal proteins such as actin and cytokeratin.
- the carbohydrate-aminated antibody is a chimeric, human or humanized antibody.
- the carbohydrate-aminated glycoprotein of the invention can further comprise one or more additional moieties, such as a detectable labeling group, a drug or a toxin, provided that the additional moiety is not bonded to the diamine moiety.
- the invention also relates to carbohydrate-aminated glycoproteins (e.g., antibodies, antigen-binding fragments, growth factors) as described herein for use in therapy (including prophylaxis) or diagnosis, and to the use of such carbohydrate- aminated glycoproteins for the manufacture of a medicament for treatment of a particular disease or condition as described herein (e.g., viral diseases, such as HIV infection and AIDS, papilloma virus infection, hepatitis B virus infection, hepatitis C virus infection, infections with intracellular parasites, cancer).
- a particular disease or condition e.g., viral diseases, such as HIV infection and AIDS, papilloma virus infection, hepatitis B virus infection, hepatitis C virus infection, infections with intracellular parasites, cancer.
- the invention also relates to a method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell.
- the method comprises contacting a cell with a carbohydrate-aminated glycoprotem, thereby allowing said carbohydrate- aminated glycoprotein to cross the plasma membrane and enter the cytoplasm and/or organelles of the cell.
- the carbohydrate-aminated glycoprotein to be intracellularly delivered to a cell can be, for example, an antibody or antigen-binding fragment thereof.
- the carbohydrate-aminated antibody to be intracellularly delivered is directed against atumor antigen, an intracellular protein or a viral protein.
- the carbohydrate-aminated antibody is directed against the Tat protein of HIV.
- the carbohydrate-aminated antibody of the invention is directed against Ras, p53, papilloma virus E6, papilloma virus E7 or pX of hepatitis B virus, h certain embodiments of the invention, the carbohydrate-aminated antibody is a chimeric, human or humanized antibody.
- the carbohydrate-aminated glycoprotein to be intracellularly delivered can further comprise one or more additional moieties, such as a detectable labeling group, drug or toxin, provided that the label, drug or toxin is not bonded to the diamine moiety.
- the invention also relates to a method of transvascularly delivering a carbohydrate-aminated glycoprotein to an organ or tissue in a subject.
- the method comprises intravascularly (e.g., by intravenous injection, intraarterial infusion) administering a carbohydrate-aminated glycoprotein to a subject, thereby allowing said carbohydrate-aminated glycoprotein to cross from the lumen of the blood vessel to said organ or tissue.
- the carbohydrate-aminated glycoprotein to be delivered can be, for example, a growth factor (e.g., nerve growth factor), or an antibody or antigen-binding fragment thereof.
- the carbohydrate- aminated glycoprotein is transvascularly delivered to an organ or tissue selected from the group consisting of brain, spinal cord, peripheral nerve, intestine, liver, spleen, kidney, lung, muscle, pancreas, ovary, uterus, prostate, testis, breast, heart, stomach and gallbladder.
- the carbohydrate-aminated antibody to be intracellularly delivered is directed against atumor antigen, an intracellular protein or a viral protein.
- the carbohydrate-aminated antibody to be delivered is directed against to the Tat protein of HIV.
- the carbohydrate-aminated antibody to be delivered is directed against Ras, p53, papilloma virus E6, papilloma virus E7 or pX of hepatitis B virus.
- the carbohydrate-aminated antibody to be delivered is a chimeric, human or humanized antibody.
- the carbohydrate- aminated glycoprotein to be delivered can further comprise one or more additional moieties, such as a detectable labeling group, a drug or a toxin, provided that the label, drug or toxin is not bonded to the diamine moiety.
- the invention also relates to a composition
- a composition comprising a carbohydrate- aminated glycoprotein of the invention and a physiologically acceptable carrier.
- the invention also relates to a method of inhibiting the replication of a virus in a subject, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or antigen-binding fragment thereof which is directed against to a protein involved in replication of the virus.
- the virus is HIV and the carbohydrate-aminated antibody is directed against the Tat protein encoded by HIV.
- the invention also relates to a method of treating a subject infected with an intracellular parasite, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or antigen-binding fragment thereof which is directed against a protein which functions in the proliferation or spread of said parasite.
- the invention further relates to a method of treating a subject having a tumor, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or antigen-binding fragment thereof which is directed against a tumor antigen.
- the tumor antigen is a protein which functions in the proliferation and/or metastasis of a tumor.
- the carbohydrate-aminated antibody to be administered to the subject is directed against Ras, p53, papilloma virus E6, papilloma virus E7 or pX of hepatitis B virus.
- Figure 1 is a graphic representation of an SDS/polyacrylamide gel containing separated heavy and light chains of carbohydrate-aminated, carbohydrate pseudo- aminated and native murine anti-Tat monoclonal antibody.
- Figure 2 is a graph showing that binding of the carbohydrate-aminated and carbohydrate pseudo-aminated murine anti-Tat monoclonal antibodies to the Tat protein of HIV is comparable to the binding of the native anti-Tat monoclonal antibody.
- Figure 3A is a graph showing the binding of 14 C-labeled antibodies.
- Figure 3B is a graph showing the binding of 3 H-labeled antibodies.
- Figure 4 is a graph showing that the ability of 3 H-anti-TAT antibody T5 to traverse Caco cell layers is directly related to levels of amine incorporation attained during the modification process.
- Figure 5 A is a graph showing plasma levels of carbohydrate-aminated bovine IgG and of native IgG over time following intravenous administration.
- Figures 5B-5F are graphs showing that intravenously- administered carbohydrate-aminated bovine IgG is taken up by intestine (B), liver (C), brain (D), kidney (E) and spleen (F) more rapidly and to a greater degree than is the native IgG.
- the invention relates to carbohydrate-aminated glycoproteins which comprise one or more diamine moieties that are bonded to carbon atoms of a carbohydrate moiety of the glycoprotein.
- the diamine moiety can be bonded to the carbohydrate moiety through a double or, preferably, a single covalent bond that is formed between an activated carbon atom of an oxidized carbohydrate moiety and a nitrogen atom of the diamine moiety, as described herein.
- activated carbon atom refers to the aldehydic carbonyl carbon atom which is formed when a carbohydrate moiety containing vicinal hydroxyl groups is oxidized as described herein.
- diamine moiety refers to a chemical moiety which comprises at least two nitrogen atoms (e.g., substituted imino, primary amino, secondary amino, tertiary amino, quaternary ammonium), wherein one of the nitrogen atoms is bonded to the activated carbon atom of the oxidized carbohydrate moiety. It is understood that more than two nitrogen atoms can be present in the diamine moiety.
- a diamine moiety can be provided, for example, by reacting an activated carbon atom of an oxidized carbohydrate moiety with one of a variety of diamine compounds as described herein.
- Preferred carbohydrate-aminated glycoproteins of the invention comprise the presence of one or more diamine moieties represented by Structural Formula I and Structural Formula II.
- R ! is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group.
- R 2 and R 3 are each independently -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R 2 and R 3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring (i.e., heteroaromatic ring (e.g., pyrrole, pyridine) or nonaromatic heterocyclic ring (e.g., pyrrolidine, piperidine, morpholine)).
- heteroaromatic ring e.g., pyrrole, pyridine
- nonaromatic heterocyclic ring e.g., pyrrolidine, piperidine, morpholine
- Suitable substituents for lower alkyl, aromatic, heterocyclic and benzyl groups include, for example, halogen atoms (e.g., fluorine, chlorine, bromine, iodine); lower alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl, neopentyl, cyclohexyl); benzyl; hydroxy; primary, secondary and tertiary amino; quaternary ammonium; and lower alkoxy groups.
- the substituents which are bonded to the nitrogen atom of the secondary and tertiary amino group and the quaternary ammonium nitrogen are each individually a lower alkyl group, a benzyl group or an aromatic group.
- X is a suitable linker group. Suitable linkers include, for example, C, to about C ]2 aliphatic or aromatic groups. When X is an aliphatic group it can be a linear, branched or cyclic aliphatic group which is saturated or which contains one or more units of unsaturation. For example, X can be a linear or branched C 2 -C 12 alkane, alkene or alkyne. Preferably, X is an alkylene group represented by -(CH 2 ) n -, wherein n is an integer from zero to about twelve.
- n is an integer from one to about six (i.e.,methylene, ethylene, propylene, butylene, pentamethylene, hexamethylene).
- X is an ethylene group.
- the moieties represented by the Structural Formulae can be referred to as an aminoalkylamine and an iminoalkylamine, respectively.
- Glycoproteins which can be modified in accordance with the invention include proteins, peptides and active fragments of the foregoing with at least one appended carbohydrate moiety (e.g., N-linked carbohydrate, O-linked carbohydrate).
- Active fragments of a glycoprotein have at least one carbohydrate moiety and retain at least one function of the full length protein, such as a catalytic function or a binding function (e.g., antigen binding).
- the glycoprotein can be a native, recombinant or synthetic protein or peptide which can be produced using any suitable method.
- a protein or peptide which does not contain a carbohydrate moiety can be engineered or modified to produce a glycoprotein.
- an expression vector encoding a protein of interest can be mutated, using conventional methods of recombinant DNA technology, so that the encoded protein includes a consensus amino acid sequence for glycosylation (e.g., AsnXaaSer, AsnXaaThr).
- the expression vector can be further modified so that the encoded protein includes a leader peptide.
- the mutated expression vector can then be introduced into a suitable host cell, which can be maintained under conditions suitable for production of the glycoprotein.
- Host cells which are suitable for production of recombinant glycoproteins include mammalian cells (e.g., CHO cells), insect cells(e.g., SF9 cells), and plant cells (e.g., Zea maize).
- mammalian cells e.g., CHO cells
- insect cells e.g., SF9 cells
- plant cells e.g., Zea maize
- carbohydrate moieties can be bonded directly to functional groups of proteins and peptides using conventional methods of synthetic organic chemistry, and glycopeptides can be produced by solid phase synthesis (see, for example, Meldal, M., et al, Curr. Opin. Chem.
- Preferred carbohydrate-aminated glycoproteins include growth factors, antibodies and antigen-binding fragments thereof.
- growth factor refers to soluble proteins or peptides which have specific effects on particular cells, including growth regulation, immunomodulation and trophic effects.
- Growth factors include the groups of proteins which are commonly referred to as neurotrophic factors (e.g., nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BNDF), IL-6 and the like), hematopoietic growth factors, such as erythropoietin, macrophage colony stimulating factor, stem cell factor and the like, and cytokines, including interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 and the like), interferons (e.g., IFN ⁇ , IFN ⁇ , IFN ⁇ ) and chemokines (e.g., MDM ⁇ , members of the super Ig family, including Major Histocompatibility Complex proteins and complement factors, RANTES, eotaxin, SDF-1, and the like).
- neurotrophic factors e.g., nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain derived neurotrophic
- growth factors such as IL-6 can mediate neurotrophic and/or immunoregulatory effects.
- Preferred growth factors which can be carbohydrate-aminated are those which can be isolated as glycoproteins from the organisms or cells in which they are naturally produced.
- a particularly preferred growth factor which can be carbohydrate-aminated is NGF.
- Antibodies which can be carbohydrate-aminated can be polyclonal or monoclonal, and the terni "antibody” is intended to encompass both polyclonal and monoclonal antibodies.
- the tenns polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
- Multivalent antibodies single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody".
- the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g.. Cabilly et al, U.S. Patent No. 4,816,567; Cabilly et al, European Patent No.
- Humanized antibodies can be produced using synthetic or recombinant DNA technology with standard methods or with other suitable techniques.
- Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al, Nucl Acids Res., 17: 5404 (1989)); Sato, K., et al, Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al, Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P.
- variants can also be readily produced, hi one embodiment, cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993).
- a phage library see e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993.
- Antibodies which are specific for an antigen can be raised against an appropriate immunogen, such as an isolated and/or recombinant protein or portions thereof (including synthetic molecules, such as synthetic peptides). Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express an antigen. In addition, cells expressing a recombinant antigen, such as transfected cells, can be used as immunogens or in screens for antibodies which bind to the antigen (e.g., antibodies which bind to receptors) (See e.g., Chuntharapai et al, J.
- a hybridoma is generally produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0, P3X63Ag8.653 or a heteromyeloma) with antibody producing cells.
- a suitable immortal cell line e.g., a myeloma cell line such as SP2/0, P3X63Ag8.653 or a heteromyeloma
- Antibody producing cells can be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals which have been immunized with the antigen of interest.
- the fused cells can be isolated using selective culture conditions, and cloned by limiting dilution.
- Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
- Suitable methods of producing or isolating antibodies of the desired specificity can be used, including, for example, methods which select a recombinant antibody from a library (e.g., a phage display library), or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see e.g., Jakobovits et al, Proc. Natl Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al, Nature, 362: 255-258 (1993); Lonberg et al, U.S. Patent No. 5,545,806; Surani et al, U.S. Patent No. 5,545,807; Lonberg et al, WO97/13852).
- a library e.g., a phage display library
- transgenic animals e.g., mice
- Active fragments of antibodies including fragments of chimeric, humanized, primatized, veneered or single chain antibodies can also be carbohydrate-aminated. Active fragments are multivalent or monovalent antigen -binding fragments, including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments that have at least one carbohydrate moiety. Active fragments of antibodies can be produced by enzymatic cleavage of antibodies which have at least one carbohydrate moiety that is attached to the protein in, e.g., to the CH,, CL, or hinge region. Such antibodies can be produced by introducing an appropriate consensus amino acid sequence into an immunoglobulin chain.
- a sequence encoding a glycosylation site can be incorporated into a nucleic acid encoding an immunoglobulin chain (e.g., using recombinant techniques, such as site directed mutagenesis), and the resulting nucleic acid can be expressed in a suitable host cell.
- the carbohydrate moiety which is attached to an introduced glycosylation site does not interfere with antigen binding.
- Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site.
- a gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and a hinge region which includes a glycosylation site.
- the carbohydrate-aminated antibody can be of any isotype (e.g., IgM, IgG (IgGl , IgG2, IgG3, IgG4), IgA, IgD, IgE) and can have a kappa or lambda light chain, hi one embodiment, the carbohydrate-aminated antibody is a human antibody, a humanized antibody or a chimeric antibody which includes a human constant region. In another embodiment, the carbohydrate-aminated antibody includes about four to about eighty-five diamine moieties.
- Antibodies which are directed against any desired antigen can be carbohydrate-aminated.
- the carbohydrate-aminated antibodies of the invention retain the binding specificity of the corresponding native antibodies.
- directed against means that the antibody or antigen-binding fragment has binding affinity for a specified antigen.
- An antibody or antigen-binding fragment directed against an antigen can specifically or selectively bind said antigen.
- Antibodies directed against an antigen can be produced using any suitable method. For example, an antibody directed against an antigen can be raised in an animal immunized with said antigen or selected from a library (e.g., by phage display). In one embodiment, the carbohydrate-aminated antibody can specifically bind an intracellular protein.
- intracellular protein refers to proteins and peptides which can be found in the cytoplasm and/or organelles of a cell (e.g., nucleus, mitochondria, Golgi apparatus, lysosome, endoplasmic reticula (rough endoplasmic reticulum, smooth endoplasmic reticulum), peroxisome) and encompasses proteins encoded by intracellular parasites (e.g., Listeria monocytogenes, Cryptosporidium parvum, Toxoplasma gondii, Leischmania sp., Plasmodium sp.), viruses (e.g., human immunodeficiency virus (HIV-1, HIV-2), hepatitis C virus (HCV)), and the cytoplasmic domains of transmembrane proteins (e.g., protein tyrosine kinase receptors).
- intracellular parasites e.g., Listeria monocytogenes, Cryptosporidium parvum, Toxoplasma gond
- the carbohydrate- aminated protein can bind and inhibit the function of a parasite or virus encoded protein which participates in the replication, proliferation and/or spread of the parasite or virus.
- Proteins which participate in the replication, proliferation and/or spread a vims include, for example, viral polymerases (e.g., HIV or HCV reverse transcriptase), viral transactivator proteins (e.g., HIV Tat), viral proteases (e.g., HIV or HCV protease) and the like.
- the carbohydrate- aminated antibody can bind to the Tat protein of HIV (e.g., HIV-1 IIIB).
- the carbohydrate-aminated antibody can bind an antigen expressed by a specific cell type (e.g., lymphocyte, neuron, hepatocyte), tissue or organ (e.g., brain, spinal cord, peripheral nerve, intestine, liver, spleen, kidney, lung, muscle, pancreas, ovary, uterus, prostate, testis, breast, heart, stomach and gallbladder), hi one embodiment, the carbohydrate-aminated antibody can bind to a tumor antigen.
- tumor antigen refers to antigens which are expressed uniquely by tumor cells or at higher levels by tumor cells than by non- tumor cells.
- Tumor antigens include cell surface molecules (e.g., such as Lewis Y, HER-2/neu, disialoganglioside G3, carcinoembrionic antigen, CD30) and molecules (e.g., intracellular molecules) which function in the proliferation and/or metastasis of tumors, such as the protein products of oncogenes (e.g., ras) and tumor suppressor genes (e.g., p53) which contain activating or inactivating mutations.
- the carbohydrate-aminated antibody can bind to an antigen expressed in an organ or tissue selected from brain, spinal cord, peripheral nerve, intestine, liver, spleen and kidney.
- the carbohydrate-aminated glycoprotein of the invention can further have one or more additional moieties, such as a detectable labeling moiety, drug or toxin, which are directly or indirectly bonded to the glycoprotein, provided that any such additional moiety is not bonded, either directly or indirectly, to a diamine moiety.
- additional moieties such as a detectable labeling moiety, drug or toxin, which are directly or indirectly bonded to the glycoprotein, provided that any such additional moiety is not bonded, either directly or indirectly, to a diamine moiety.
- labeling moieties which can be conjugated to glycoproteins include, for example, enzymes (e.g., phosphatases, peroxidases), fluorescent compounds (e.g., fluorescene isothiocyanate), chelating compounds (e.g., ethylenediaminetetraacetic acid (EDTA), di ethyl enetriaminepentaacetic acid (DPT A)), radionuclides ( ,25 I, l u In, technetium-99m) boron adducts and affinity labels (e.g., epitopes, biotin, avidin).
- enzymes e.g., phosphatases, peroxidases
- fluorescent compounds e.g., fluorescene isothiocyanate
- chelating compounds e.g., ethylenediaminetetraacetic acid (EDTA), di ethyl enetriaminepentaacetic acid (DPT A)
- radionuclides ,
- Drugs and toxins which can be conjugated to glycoproteins include, for example, chemotherapeutic agents (e.g., mitomycin C, methotrexate, 5-fluorouracil, cyclohexamine), anti-viral agents (e.g., viral protease inhibitors, for example, ritinovir, squinavir, nelfinavir mesylate), and toxins such as ricin, gelonin and the like.
- chemotherapeutic agents e.g., mitomycin C, methotrexate, 5-fluorouracil, cyclohexamine
- anti-viral agents e.g., viral protease inhibitors, for example, ritinovir, squinavir, nelfinavir mesylate
- toxins such as ricin, gelonin and the like.
- the carbohydrate-aminated glycoproteins of the invention can have physical and biological characteristics (e.g., molecular weight, isoelectric point, binding activity, catalytic activity, immunogenicity) which are substantially the same as those of the corresponding native glycoproteins.
- physical and biological characteristics e.g., molecular weight, isoelectric point, binding activity, catalytic activity, immunogenicity
- the molecular weight and isoelectric point of a glycoprotein as determined by SDS-PAGE and isoelectric focusing, generally do not change when the protein is carbohydrate-aminated, and carbohydrate-aminated glycoproteins are no more immunogenic than the corresponding native glycoproteins.
- the biological activity (e.g., binding activity) of carbohydrate-aminated glycoproteins often remains substantially the same as the activity of native glycoproteins.
- the carbohydrate- aminated glycoprotein often retains at least about 80% or 85% or 90% or 95% or 99% of the biological activity of the native glycoprotein.
- the biological activity is expressed as affinity when it is a binding activity, and as the catalytic rate constant when it is an enzymatic activity.
- the carbohydrate-aminated glycoproteins of the invention have improved phannacokinetic properties in comparison to native glycoproteins. For example, as described herein, organ uptake of carbohydrate-aminated antibody after intravenous administration is significantly enhanced compared with native antibody (Example 2). Additionally, carbohydrate-aminated glycoproteins can be delivered into the cytoplasm and/or organelles of living cells. As described herein, the ability of a carbohydrate-aminated antibody which binds HIV Tat to inhibit HIV replication in cultured human T cells has been studied.
- the Tat protein of HIV is a regulatory protein that can activate the transcription of the viral genome, and is required for viral replication (Derse, D., et al, Virology:, 194:530-536 (1993)).
- the Tat protein is produced in the cytoplasm of HlV-infected cells.
- carbohydrate-aminated anti-Tat antibody inhibited viral replication in cultured human T cells, while unmodified or "carbohydrate pseudo- aminated" anti-Tat did not (Example 1, Table 3).
- Carbohydrate pseudo-aminated glycoproteins e.g., anti-Tat antibody
- have a monoamine moiety e.g., ethylamine
- Such pseudo-aminated glycoproteins can be prepared as described herein (see, Example 1).
- the results of the study also demonstrate that carbohydrate- aminated antibodies can enter the cytoplasm and/or organelles of living cell, and inhibit the activity of intracellular proteins by, for example, binding to the protein.
- the invention is a method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell, comprising contacting a cell with an effective amount of a carbohydrate-aminated glycoprotein, thereby allowing the carbohydrate-aminated glycoprotein to cross the plasma membrane and enter the cytoplasm and/or organelles of said cell.
- the method comprises contacting a carbohydrate-aminated antibody or a carbohydrate-aminated antigen- binding fragment thereof with a cell.
- the carbohydrate- aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be intracellularly delivered is directed against an intracellular protein.
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen- binding fragment thereof to be intracellularly delivered is directed against a tumor antigen or a viral protein.
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be intracellularly delivered is directed against the Tat protein of HIV.
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be intracellularly delivered is directed against an antigen selected from Ras, p53, E6 of papilloma vims, E7 of papilloma vims and pX of hepatitis B vims.
- the carbohydrate-aminated antibody is a chimeric, humanized or human antibody or an active fragment of any of the foregoing.
- the carbohydrate-aminated glycoprotein to be to be intracellularly deliveredfurther has an appended detectable label, dmg or toxin, which is not bonded to the diamine moiety.
- the method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell can be used in vitro and in vivo.
- an effective amount of a carbohydrate-aminated glycoprotein is administered to a subject in need, such as a human having a viral infection.
- the invention in another aspect, relates to a method of inhibiting (reducing or preventing) viral replication in a subject.
- the method comprises, administering an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, which is directed against a protein involved in viral replication and/or infection, to a subject in need thereof.
- the method can be employed to inhibit the replication of any vims, such as a herpes vims (e.g., herpes simplex vims, cytomegalovims, Epstein-Barr vims), a retrovims (e.g., HIV, HCV), a papilloma vims (e.g., human papilloma vims 16) and hepatitis B vims.
- a herpes vims e.g., herpes simplex vims, cytomegalovims, Epstein-Barr vims
- a retrovims e.g., HIV, HCV
- a papilloma vims e.g., human papilloma vims 16
- hepatitis B vims hepatitis B vims.
- the invention is a method of inhibiting the replication of HIV in a subject, comprising administering an effective amount of a
- the invention is a method of inhibiting the replication of papilloma vims in a subject, comprising administering an effective amount of a carbohydrate- aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is directed against the E6 or E7 protein encoded by the vims, to a subject in need thereof.
- the invention is a method of inhibiting the replication of hepatitus B vims in a subject, comprising administering an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is directed against the X protein (pX) encoded by the vims, to a subject in need thereof.
- the invention in another aspect, relates to a method of treating a subject having a tumor, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof, which is directed against a tumor antigen.
- a carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, which is administered to the subject is directed against a protein which functions in the proliferation and/or metastasis of a tumor cell.
- Such proteins include, the protein products of cellular protooncogenes (e.g., ras) or tumor suppressor genes (e.g., p53) which contain activating or inactivating mutations, and protein products of viral oncogenes (e.g.. E6 or E7 of human papilloma virus 16 (HPV16)).
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is administered to the subject is directed against an oncoprotein.
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment th ereof, that is administered to the subject is directed against a viral oncoprotein (e.g., E6 of HPV16, E7 of HPV16).
- a viral oncoprotein e.g., E6 of HPV16, E7 of HPV16.
- the carbohydrate- aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is administered to the subject is directed against Ras.
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is administered to the subject is directed against p53.
- the invention relates to a method of treating a subject infected with an intracellular parasite (e.g., Listeria sp., Cryptosporidium sp,, Toxoplasma sp., Leischmania sp., Plasmodium sp.), comprising administering to the subject an effective amount of a carbohydrate-aminated antibody or carbohydrate- aminated antigen binding fragment thereof which is directed against a protein involved in the proliferation or spread of the parasite, thereby inhibiting the proliferation or spread of the parasite.
- an intracellular parasite e.g., Listeria sp., Cryptosporidium sp,, Toxoplasma sp., Leischmania sp., Plasmodium sp.
- the invention in another aspect, relates to a method of transvascularly delivering a carbohydrate-aminated glycoprotein to an organ or tissue in a subject.
- the method comprises mtravascularly administering (e.g., by intravenous injection, intraarterial injection) a carbohydrate-aminated glycoprotein to a subject, thereby allowing said carbohydrate-aminated glycoprotein to cross from the lumen of the blood vessel to said organ or tissue, hi one embodiment, the tissue or organ to which the carbohydrate-aminated glycoprotein is delivered is selected from the group consisting of brain, spinal cord, peripheral nerve, intestine, liver, spleen, kidney, lung, muscle, pancreas, ovary, uterus, prostate, testis, breast, heart, stomach and gallbladder.
- the tissue or organ to which the carbohydrate-aminated glycoprotein is delivered is selected from the group consisting of brain, intestine, liver, spleen and kidney.
- the invention is a method of transvascularly delivering a carbohydrate- aminated glycoprotein to the brain of a subject, comprising mtravascularly administering (e.g., by intravenous injection, intraarterial injection) a carbohydrate- aminated glycoprotein to the subject.
- the method comprises mtravascularly administering a carbohydrate-aminated antibody or a carbohydrate- aminated antigen-binding fragment thereof to a subject.
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be transvascularly delivered is directed against an antigen expressed in an particular cell type, organ or tissue.
- the carbohydrate- animated antibody or carbohydrate-aminated antigen-binding fragment thereof to be transvascularly delivered is directed against an intracellular protein, a tumor antigen or a viral protein.
- the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be transvascularly delivered is directed against an antigen selected from ras, p53, E6 of papilloma vims, E7 of papilloma vims and pX of hepatitis B vims.
- the carbohydrate-aminated antibody or carbohydrate- aminated antigen-binding fragment thereof transvascularly delivered is directed against the Tat protein of HIV-1.
- the carbohydrate- aminated antibody to be transvascularly delivered is a chimeric, humanized or human antibody or an active fragment of any of the foregoing.
- the carbohydrate-aminated glycoprotein to be transvascularly delivered is a carbohydrate-aminated growth factor.
- the growth factor is nerve growth factor.
- the carbohydrate- aminated glycoprotein to be administered further has an appended detectable label, dmg or toxin, which is not bonded to the diamine moiety.
- a "subject” is preferably a human, but can also be an animal, preferably a mammal, in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, fowl, pigs, horses, fish and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
- domestic animals e.g., dogs, cats, and the like
- farm animals e.g., cows, sheep, fowl, pigs, horses, fish and the like
- laboratory animals e.g., rats, mice, guinea pigs, and the like.
- An effective amount of a carbohydrate-aminated glycoprotein can be administered to a subject to treat (reduce or prevent) disease or for diagnostic purposes.
- an effective amount of a carbohydrate-aminated antibody which is directed against a protein that functions in viral replication can be administered to the subject to treat a viral infection.
- an "effective amount” is an amount sufficient to achieve a desired diagnostic, therapeutic and/or prophylactic effect.
- an effective amount of a carbohydrate-aminated antibody is an amount sufficient to bind to a protein for diagnostic purposes,an amount sufficient to bind to and inhibit the function of a protein, an amount sufficient to bind to and inhibit the function of an intracellular protein and or an amount sufficient to result in accumulation of the carbohydrate- aminated antibody in the cytoplasm and/or organelles of a cell, or in a particular tissue or organ (e.g., brain), thereby having a therapeutic and/or prophylactic effect.
- carbohydrate-aminated glycoprotein administered to the subject will depend on the particular carbohydrate-aminated glycoprotein to be administered, the characteristics of the subject, such as general health, age, sex, body weight and tolerance to d gs as well as the degree, severity and type of disorder the subject has. The skilled artisan will be able to detennine appropriate dosages depending on these and other factors. Typically, an effective amount can range from about 0.001 mg/kg per da> to about 10 mg/kg per day for an adult.
- the carbohydrate-aminated glycoprotein can be administered by any suitable route, including, for example, orally in capsules, suspensions or tablets or by parenteral administration.
- Parenteral administration includes intramuscular, intravenous, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration.
- the carbohydrate-aminated glycoprotein can also be administered orally (e.g., dietary), transdermally, topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) or rectally. Administration can be local or systemic as indicated.
- the preferred mode of administration varies depending upon the particular carbohydrate-aminated glycoprotein chosen. However, parenteral administration is generally preferred.
- the carbohydrate-aminated protein can be administered as a neutral compound or as a salt.
- Salts of compounds containing an amine or other basic group can be obtained, for example, by reacting with a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like.
- Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base; for example, a hydroxide base. Salts of acidic functional groups contain a countercation such as sodium, potassium and the like.
- the carbohydrate-aminated glycoprotein can be administered to the individual as part of a pharmaceutical composition; for example, a therapeutic or diagnostic composition having a carbohydrate-aminated glycoprotein and a physiologically acceptable carrier.
- Compositions for co-therapy can have a carbohydrate-aminated glycoprotein and one or more additional therapeutic agents.
- a carbohydrate-aminated glycoprotein and an additional therapeutic agent can be components of separate compositions which are mixed together prior to administration or administered separately.
- Formulations will vary according to the route of administration selected (e.g., solution, emulsion, capsule).
- Suitable physiological carriers preferably contain inert ingredients which do not interact with the carbohydrate-aminated glycoprotein and/or additional therapeutic agent.
- Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
- Suitable carriers for parenteral administration include sterile water, physiological saline, bacteriostatic saline, phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like.
- Methods for encapsulating compositions are known in the art (Baker, et al, "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).
- the invention relates to a composition comprising a carbohydrate-aminated glycoprotein.
- the composition can be a pharmaceutical composition (e.g., therapeutic or diagnostic composition) comprising a carbohydrate-aminated glycoprotein and a physiologically acceptable carrier.
- the composition comprises a carbohydrate-aminated antibody or a carbohydrate-aminated antigen-binding fragment thereof.
- the composition comprises a carbohydrate-aminated antibody or carbohydrate- animated antigen-binding fragment thereof which is directed against an intracellular protein, a tumor antigen or a viral protein.
- the composition comprises a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against the Tat protein of HIV.
- the composition comprises a carbohydrate- aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against an antigen selected from Ras, p53, E6 of papilloma vims, E7 of papilloma vims and pX of hepatitis B vims.
- the composition comprises a carbohydrate-aminated antibody which is a chimeric, humanized or human antibody or an active fragment of any of the foregoing.
- the composition comprises a carbohydrate-aminated growth factor.
- the carbohydrate-aminated growth factor is nerve growth factor.
- the composition comprises a carbohydrate-aminated glycoprotein which further has an appended detectable label, drug or toxin, which is not bonded to the diamine moiety.
- the carbohydrate-aminated glycoproteins of the invention can be prepared by reacting a glycoprotein with a suitable oxidizing agent, thereby cleaving the carbohydrate ring structure between vicinal hydroxyl groups and converting the hydroxyl groups into aldehyde groups.
- suitable oxidizing agents can be used, with sodium periodate being preferred.
- an aqueous solution containing an oxidizing agent is added to an aqueous solution containing a glycoprotein.
- concentration of glycoprotein is usually between about 1 mg/ml and about 20 mg/ml.
- the amount of oxidizing agent used is generally sufficient to provide about a 1000-fold excess relative to the glycoprotein.
- the amount of oxidizing agent can be adjusted to oxidize the carbohydrate moiety of the glycoprotein to the desired degree.
- the reaction can be carried out from 4°C to room temperature for a period of from 2 hours to about 24 hours. Preferably, the reaction solution is protected from light.
- Suitable diamine compounds comprise at least one, and preferably, two primary amino groups.
- Preferred diamine compounds are ⁇ , ⁇ -diaminoalkanes (e.g., 1, 2- diaminoethane (ethylenediamine), 1, 3-diaminopropane, 1, 4-diaminobutane, 1, 5- diaminopentane, 1, 6-diaminohexane, 1, 7-diaminoheptane, 1, 8-diaminooctane, 1, 9-diaminononane, 1, 10-diaminodecane).
- a particularly preferred diamine compound is 1, 2-diaminoethane.
- the oxidized glycoprotein can be reacted with about a 1000-fold molar excess of diamine compound in an aqueous reaction solvent from 4°C to room temperature for a period of 2 hours to about 24 hours. If desired, the amount of diamine compound used can be adjusted to produce a carbohydrate- aminated glycoprotein having a desired quantity of diamine moieties.
- a diamine compound having a primary amino group is chosen, the resulting carbohydrate-aminated glycoprotein can have a diamine moiety that is bonded to an activated carbon atom of an oxidized carbohydrate moiety through a double bond (i.e., a Schiff s base).
- the Schiff s base can be stabilized by reduction with a suitable reducing agent (e.g., sodium cyanoborohydride, sodium borohydride) to fomi a secondary amine.
- a suitable reducing agent e.g., sodium cyanoborohydride, sodium borohydride
- a suitable reducing agent e.g., sodium cyanoborohydride, sodium borohydride
- about a 50, 000-fold molar excess of reducing agent over glycoprotein is used.
- the carbohydrate-aminated glycoprotein can be purified using conventional methods (e.g., column chromatography (e.g., gel filtration, ion exchange, hydrophobic interaction, affinity), preparative electrophoresis, precipitation).
- column chromatography e.g., gel filtration, ion exchange, hydrophobic interaction, affinity
- preparative electrophoresis precipitation
- one or more additional moieties which are not bonded to the diamine moiety e.g., a labeling moiety, drug or toxin
- suitable methods of protein chemistry e.g., by coupling through thiol, hydroxyl or carboxylate groups.
- the coupling chemistry is chosen so that any additional moieties which are conjugated to the carbohydrate-aminated glycoprotein are not bonded to the diamine moiety.
- the invention relates to a method of preparing a carbohydrate-aminated glycoprotein, comprising reacting a glycoprotein or a carbohydrate containing fragment thereof with an oxidizing agent to generate an activated carbon atom (i.e., an aldehydic carbonyl carbon atom) in the carbohydrate moiety; and reacting said aldehyde group with a diamine compound. If desired, the resulting product can be reacted with a reducing agent.
- an activated carbon atom i.e., an aldehydic carbonyl carbon atom
- EXAMPLE 1 A CARBOHYDRATE-AMINATED ANTIBODY THAT IS DIRECTED AGAINST THE TAT PROTEIN OF HIV PREPARATION OF CARBOHYDRATE-AMINATED ANTI-TAT MONOCLONAL ANTIBODY
- the tube was wrapped in foil to protect it from light, and incubated at room temperature overnight ( ⁇ 18 hours) on a rotator. Following the overnight incubation, the reaction buffer was exchanged for 10 mM sodium carbonate pH 10.6 (Sigma) using an Econo-Pac® 10DG column (BioRad, Hercules, CA).
- a 28 mM solution of ethylenediamine (Sigma) was freshly prepared in water.
- the ethylenediamine solution was added to the solution of oxidized IgG at a ratio of 200 ⁇ L of ethylenediamine solution per milligram of oxidized IgG.
- the tube was wrapped in foil and incubated on a rotator at room temperature for about two hours.
- a freshly-made solution of sodium cyanoborohydride 20 mg/mL in H 2 O) (Sigma) was added to the solution at a ratio of 100 ⁇ L per milligram of oxidized IgG.
- the solution was incubated on a rotator at room temperature for about thirty minutes (tubes were protected from light).
- a carbohydrate pseudo-aminated antibody was produced by the method described above, except ethylamine was substituted for ethylenediamine.
- the quantity of diamine moieties which are bonded to the carbohydrate- aminated IgG was assessed by preparing a carbohydrate-aminated murine anti-Tat HIV-1 IHB monoclonal antibody that was trace labeled with 14 C-ethylenediamine (Sigma).
- the trace labeled carbohydrate-aminated antibody was prepared as described above except that a 29.016 mM solution of ethylenediamine was used in which 10% of the ethylenediamine molecules were l4 C labeled.
- the amount of 14 C incorporated was determined by scintillation counting and the protein concentration of the carbohydrate-aminated antibody was determined using a BCA assay (Pierce, Rockford, IL).
- the molecular weights of the heavy and light chains of the carbohydrate- aminated IgG and the pseudo-aminated IgG were compared to those of the heavy and light chains of the native antibody using SDS-PAGE under reducing conditions. 7-15 ⁇ g of the proteins were mn on a 4-15% Tris gel (BioRad), and the separated heavy and light chains were visualized by staining with Commassie stain (Sigma). (See Figure 1.) The figure shows that the electrophoretic mobilities of the heavy and light chains of the modified antibodies were the same as the mobilities of the native heavy and light chains.
- the immunoreactivity of the carbohydrate-aminated murine anti-Tat antibody was assessed by ELISA.
- Recombinant Tat protein from HIV-l strain IIIB (5 ⁇ g/mL in PBS) (ImmunoDiagnostics, Inc.) was coated onto 96-well microtiter plates by incubation at 4°C overnight. The excess Tat solution was decanted and blocking buffer (Pierce) was added to the wells of the plates. The plates were stored at 4°C until use. The Tat-coated, blocked plates were warmed to room temperature and the blocking buffer was decanted. The plates were then washed three times with wash buffer (PBS/0.2% Tween 20 (polyoxyethylenesorbitan monolaurate); Sigma).
- Duplicate samples of various concentrations of carbohydrate-aminated anti-Tat antibody, pseudo-aminated anti-Tat antibody, native anti-Tat antibody, and non-specific mouse IgGl (a negative control)(Rockland Immunochemicals) were added to the plates, and the plates were incubated overnight at 4°C. After the overnight incubation, the antibody solutions were decanted and the plates were washed three times with wash buffer. Goat anti-mouse IgG conjugated with horseradish peroxidase was added to each well of the plates, and the plates were incubated at room temperature for one hour. After the one hour incubation, the conjugated goat anti-mouse antibody was decanted and the plates were washed three times with wash buffer.
- Tetramethylbenzidine (Pierce) was added to the wells and the plates were incubated for 30 minutes at room temperature. Sulfuric acid (1M) was added to the wells to stop the reaction. The amount of anti-Tat antibody bound to each well was quantified by measuring the optical density of each well at 450 nm. From the results, antibody dilution curves were plotted versus optical density reading.
- the tube was wrapped in foil to protect it from light and incubated at 4°C for 2 hours on a rotator.
- the reaction buffer was exchanged for 10 mM sodium carbonate, pHl 0.6, using an Econo-Pac* 1 ODG column (BioRad). After the buffer exchange, the IgG solution was divided into 3 equal parts.
- One of the solutions containing both radioactive-labeled and unlabeled ethylenediamine was added to each one of the aliquots of IgG at a ratio of 200 ⁇ L of ethylenediamine solution to each milligram of oxidized IgG.
- the resulting three tubes were wrapped in foil and incubated on a rotator at room temperature for about 2.5 hours.
- Immunoreactivity testing was performed by ELISA on each of the three preparations as described in the "Immunoreactivity Testing" example.
- the amount of anti-Tat antibody bound to each well was quantified by measuring the optical density of each well at 450 nm. From the results, antibody dilution curves were plotted versus optical density reading. The results are presented graphically in Figure 3 A.
- the graph shows that the binding of the carbohydrate-aminated I4 C- labeled anti-Tat (T5) antibody is comparable to the binding of the native anti-Tat (T5) antibody regardless of the amount of amine incorporation achieved in the carbohydrate-amination process.
- EXAMPLE 2B 3 H-LABELED ANTI-TAT MAB WITH VARIABLE ETHYLENEDIAMINE CONCENTRATIONS
- the ethylenediamine solutions were added to the solution of IgG at a ratio of 200 ⁇ L of ethylenediamine solution to each milligram of oxidized IgG.
- the tubes were wrapped in foil and incubated on a rotator at room temperature for about 2.5 hours. Then, a freshly-made solution of sodium cyanoborohydride solution
- Transwell ® cell culture plates with 6.5 mm diameter surfaces and 3mm pore size (Costar, Cambridge, MA) were seeded with 120,000 cells in 0.2 ml of complete media above the membrane in the Transwell ® and with 1.2 ml of complete media below the membrane. Resistance was measured with a Millicell ® -Electrical Resistance System (Millipore) every two days until a consistant level of approximately 500 ⁇ was reached. This level is the indicator of a confluent cell layer. At this point, a 50:50 mixture of complete media with radio-labeled antibodies to be tested was filter-sterilized and used to replace the media above the cells growing on the membrane.
- Millicell ® -Electrical Resistance System Millipore
- the data are expressed as a ratio of the radioactivity measured in the media below the cell layer and membrane to the amount of radioactivity added.
- Immunoreactivity testing was perfonned on the three anti-Tat antibody preparations.
- the amount of anti-Tat antibody bound to each well was quantified by measuring the optical density of each well at 450 nm. From the results, antibody dilution curves were plotted versus optical density reading. The results of this study, a comparison of three levels of amine incorporation, are presented graphically in Figure 3B.
- the graph shows that the binding of the carbohydrate-aminated 14 C- labeled anti-Tat (T5) antibody is comparable to the binding of the native anti-Tat (T5) antibody regardless of the amount of amine incorporation achieved in the carbohydrate-amination process.
- EXAMPLE 3 BIODISTRIBUTION OF CARBOHYDRATE-AMINATED ANTI-TAT ANTIBODIES.
- mice Male Balb/c and Swiss mice were injected intraveneously with 50 ⁇ l of ⁇ 0.6 ⁇ g/ml !4 C- carbohydrate-aminated anti-Tat antibody or native anti-Tat antibody.
- Antibodies had been labeled with succinimidyl propionate (NEN LifeSciences, Boston, MA) according to manufacturer's instmctions.
- succinimidyl propionate NNN LifeSciences, Boston, MA
- 0.5 mCi 3 H-Succinimidyl propionate was dried down under a gentle stream of nitrogen gas.
- 0.5 mg of carbohydrate-aminated or native antibody was added immediately and incubated for 36 minutes at 4°C.
- mice were euthanized and blood, brain, testes, kidney, liver and spleen tissues were harvested. Tissues were homogenized, digested with tissue solubilizer ( Beckman) and counted in a scintillation counter (Beckman LS6500). The ratio of dpm in one gram of tissue to dpm in one microliter of plasma was calculated after 1 hour, and is shown in Table 3.
- EXAMPLE 4 THE ENHANCEMENT OF TISSUE AND ORGAN UPTAKE OF CARBOHYDRATE-AMINATED ANTIBODIES AFTER INTRAVENOUS ADMINISTRATION
- mice Male Swiss albino mice ( ⁇ 20 g) were used for pharmacokinetic studies. H- labeled carbohydrate-aminated bovine IgG and control 3 H-labeled bovine IgG were diluted to approximately 500,000 cpm/100 ⁇ L. One hundred ⁇ L aliquots were injected intravenously into the tail vein of the mice. At 1, 4 and 16 hours after the injection, mice were euthanized by cervical dislocation, blood was collected into heparinized tubes and plasma was prepared. The brain, one kidney, the spleen, a segment of intestine and the liver were dissected and homogenized in water.
- the amount of radioactivity in 20 ⁇ L aliquots of the plasma and of the organ/tissue homogenates was determined by scintillation counting. Proteins in the organ/tissue homogenates were quantified by the Bradford assay. Data were expressed as ⁇ L/mg protein (organ uptakes), by dividing the radioactivity recovered in each organ per mg of protein by the amount of radioactivity recovered in 1 ⁇ L of plasma.
- Human T cells were isolated from healthy volunteers and cultured at 2 x 10 6 cells/mL in RPMI 1640 (Biowhittaker) supplemented with 10% fetal bovine semm (Biowhittaker) and penicillin/streptomycin (Biowhittaker). The cells were cultured in 12 well dishes with a lmL capacity. At time zero, 5 multiplicity of infection (MOI) HIV-1 IHB (100 ⁇ L), and 10, 1 or 0.1 mg of anti-Tat antibody preparations (carbohydrate-aminated, carbohydrate pseudo-aminated or native) were added to the culture media. After 5 days, the amount of HIV p24 in the cultures was determined by ELISA (hnmunotech). The amount of p24 detected correlates with the amount of HIV replication occurring in the cultures. The results of two independent tests are shown in Table 4.
- the ability of the modified proteins to elicit an immune response was assessed using Balb/c mice.
- the mice were divided into three groups, with each group having four mice.
- Group I was subcutaneously injected with the carbohydrate-aminated murine anti-Tat antibody.
- Group II was subcutaneously injected with the carbohydrate pseudo-aminated murine anti-Tat antibody, and
- Group III was subcutaneously injected with the native murine anti-Tat antibody.
- the amount of IgG preparation injected was the same for each group. Blood was collected into heparinized tubes, plasma was prepared, and immunological reactivity to the particular IgG preparation which was injected
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Abstract
The invention relates to carbohydrate-aminated glycoproteins (e.g., antibodies, growth factors) which comprise a diamine moiety which is covalently bonded to a carbohydrate moiety through a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein, and to pharmaceutical compositions comprising the carbohydrate-aminated glycoproteins. The carbohydrate-aminated glycoproteins of the invention can traverse the plasma membrane of living cells and are taken up by organs and tissues more rapidly and to a greater extent than native glycoproteins after intravascular administration. The invention also relates to a method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell, and to a method of transvascularly delivering a carbohydrate-aminated glycoprotein to an organ or tissue. The invention also relates to a method of inhibiting the replication of a virus in a subject, comprising administering to the subject a carbohydrate-aminated antibody which inhibits replication of the virus by, for example, binding to a protein necessary for replication. The invention also relates to a method of treating a subject infected with an intracellular parasite, comprising administering to the subject a carbohydrate-aminated antibody which inhibits proliferation and/or spread of the parasite by, for example, binding to a protein which functions in proliferation or spread of the parasite. The invention further relates to a method of treating a subject having a tumor, comprising administering to the subject a carbohydrate-aminated antibody which is directed against a tumor antigen.
Description
CARBOHYDRATE-AMINATED GLYCOPROTEINS
RELATED APPLICATION(S)
This application is a continuation-in-part of Application No. 09/455,598, filed December 6, 1999. The entire teachings of the above application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Glycoprotems are a diverse group of molecules that have therapeutic and diagnostic potential. Several glycoproteins (e.g., antibodies) are currently used clinically. However, the full therapeutic and diagnostic potential of these proteins has not been realized. One factor which limits the therapeutic and diagnostic use of glycoproteins is that most glycoproteins do not readily traverse vascular barriers (e.g., the blood brain barrier) or cell membranes. Thus, intravenously administered glycoproteins frequently do not accumulate in target organs or the interior of cells (e.g., cytoplasm, nucleus, organelles) at therapeutically or diagnostically effective levels.
Specific attempts have been made to prepare chemically modified glycoproteins which retain the biological activity of the native protein, for enhanced organ and cellular uptake. The chemical modifications are cationization and lipidation of the glycoproteins. Cationized proteins are prepared by substituting acidic carboxylate groups on the side chains of amino acids with polyamines, thereby altering the isoelectric point of the protein (see, for example, Triguero, D., et al, Proc. Natl Acad. Sci. U.S.A., 55:4761-4765 (1989); Triguero, D., et al., J. Pharmacol. Exp. Ther., 255:186-192 (1991); Poduslo, J. F., et al, J. Neurochem., 66:1599-1609 (1996)). Although cationized proteins are reported to cross vascular barriers, these types of proteins have several disadvantages in regard to therapeutic or diagnostic applications. For example, amino acids with acidic side chains (i.e., aspartate, glutamate) can occur in
the active site (catalytic site, binding site) of a protein, and modification of such residues can decrease or destroy the biological activity of the proteins. In addition, cationized proteins can be profoundly more immunogenic than native proteins (Muckerheide A., et al, J. Immunol, 138:833-837 (1987)). Consequently, even autologous proteins which are cationized may not be suitable for administration to patients.
Lipidated glycoproteins are modified glycoproteins in which lipid moieties are covalently bonded to the carbohydrate moieties (see, for example, Cruikshank, W. W., et al, Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, 14Λ 93-203 (1997)). However, lipidated proteins are difficult to prepare and have been shown to be more immunogenic than native proteins (Phillips, N.C., et al, J. Immunol, 152:3168-3174 (1994)).
Therefore, a need exists for modified glycoproteins which can traverse vascular barriers and/or cell membranes as well as reduce or eliminate the above referenced problems.
SUMMARY OF THE INVENTION
The invention relates to carbohydrate-aminated glycoproteins which comprise one or more diamine moieties that are bonded to carbon atoms of a carbohydrate moiety of the glycoprotem. The diamine moiety can be bonded to the carbohydrate moiety through a double or, preferably, a single covalent bond that is formed between an activated carbon atom of an oxidized carbohydrate moiety and a nitrogen atom of the diamine moiety. The diamine moiety can be represented by Structural Formula I or Structural Formula H
-NR'-X-NR2R3 =N-X-NR2R3 (I) (II)
In Structural Formulae I and II, R1 is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group. R2 and R3 are each independently -H, a substituted or unsubstituted
lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R2 and R3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring. X is a C] to about C12 aliphatic group or aromatic group. In one embodiment, X is an alkylene group represented by -(CH2)n-, wherein n is an integer from one to about twelve; preferably one to about six. In a particular embodiment, the diamine moiety can be represented by Structural Formula I wherein X is an alkylene group represented by -(CH2) . The carbohydrate-aminated glycoprotein of the invention can be, for example, a growth factor (e.g., nerve growth factor), or an antibody (immunoglobulin) or antigen-binding fragment thereof. Antibodies which have binding affinity for a desired antigen can be carbohydrate-aminated. Thus, the carbohydrate-aminated antibodies of the invention can be directed against tumor antigens, intracellular proteins or viral proteins, for example. In a particular embodiment, the carbohydrate-aminated antibody of the invention is directed against the Tat protein of HIV. hi other particular embodiments, the carbohydrate-aminated antibody of the invention is directed against Ras, p53, papilloma virus E6, papilloma virus E7, the X protein (pX) of hepatitis B virus, interleukins, cytokines, or cytoskeletal proteins such as actin and cytokeratin. In certain embodiments of the invention, the carbohydrate-aminated antibody is a chimeric, human or humanized antibody. The carbohydrate-aminated glycoprotein of the invention can further comprise one or more additional moieties, such as a detectable labeling group, a drug or a toxin, provided that the additional moiety is not bonded to the diamine moiety.
The invention also relates to carbohydrate-aminated glycoproteins (e.g., antibodies, antigen-binding fragments, growth factors) as described herein for use in therapy (including prophylaxis) or diagnosis, and to the use of such carbohydrate- aminated glycoproteins for the manufacture of a medicament for treatment of a particular disease or condition as described herein (e.g., viral diseases, such as HIV
infection and AIDS, papilloma virus infection, hepatitis B virus infection, hepatitis C virus infection, infections with intracellular parasites, cancer).
The invention also relates to a method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell. The method comprises contacting a cell with a carbohydrate-aminated glycoprotem, thereby allowing said carbohydrate- aminated glycoprotein to cross the plasma membrane and enter the cytoplasm and/or organelles of the cell. The carbohydrate-aminated glycoprotein to be intracellularly delivered to a cell can be, for example, an antibody or antigen-binding fragment thereof. In particular embodiments, the carbohydrate-aminated antibody to be intracellularly delivered is directed against atumor antigen, an intracellular protein or a viral protein. In a more particular embodiment, the carbohydrate-aminated antibody is directed against the Tat protein of HIV. In other particular embodiments, the carbohydrate-aminated antibody of the invention is directed against Ras, p53, papilloma virus E6, papilloma virus E7 or pX of hepatitis B virus, h certain embodiments of the invention, the carbohydrate-aminated antibody is a chimeric, human or humanized antibody. The carbohydrate-aminated glycoprotein to be intracellularly delivered can further comprise one or more additional moieties, such as a detectable labeling group, drug or toxin, provided that the label, drug or toxin is not bonded to the diamine moiety. The invention also relates to a method of transvascularly delivering a carbohydrate-aminated glycoprotein to an organ or tissue in a subject. The method comprises intravascularly (e.g., by intravenous injection, intraarterial infusion) administering a carbohydrate-aminated glycoprotein to a subject, thereby allowing said carbohydrate-aminated glycoprotein to cross from the lumen of the blood vessel to said organ or tissue. The carbohydrate-aminated glycoprotein to be delivered can be, for example, a growth factor (e.g., nerve growth factor), or an antibody or antigen-binding fragment thereof. In particular embodiments, the carbohydrate- aminated glycoprotein is transvascularly delivered to an organ or tissue selected from the group consisting of brain, spinal cord, peripheral nerve, intestine, liver, spleen, kidney, lung, muscle, pancreas, ovary, uterus, prostate, testis, breast, heart, stomach and gallbladder. In particular embodiments, the carbohydrate-aminated
antibody to be intracellularly delivered is directed against atumor antigen, an intracellular protein or a viral protein. In a more particular embodiment, the carbohydrate-aminated antibody to be delivered is directed against to the Tat protein of HIV. In other particular embodiments, the carbohydrate-aminated antibody to be delivered is directed against Ras, p53, papilloma virus E6, papilloma virus E7 or pX of hepatitis B virus. In certain embodiments, the carbohydrate-aminated antibody to be delivered is a chimeric, human or humanized antibody. The carbohydrate- aminated glycoprotein to be delivered can further comprise one or more additional moieties, such as a detectable labeling group, a drug or a toxin, provided that the label, drug or toxin is not bonded to the diamine moiety.
The invention also relates to a composition comprising a carbohydrate- aminated glycoprotein of the invention and a physiologically acceptable carrier.
The invention also relates to a method of inhibiting the replication of a virus in a subject, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or antigen-binding fragment thereof which is directed against to a protein involved in replication of the virus. In one embodiment, the virus is HIV and the carbohydrate-aminated antibody is directed against the Tat protein encoded by HIV.
The invention also relates to a method of treating a subject infected with an intracellular parasite, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or antigen-binding fragment thereof which is directed against a protein which functions in the proliferation or spread of said parasite.
The invention further relates to a method of treating a subject having a tumor, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or antigen-binding fragment thereof which is directed against a tumor antigen. In one embodiment, the tumor antigen is a protein which functions in the proliferation and/or metastasis of a tumor. In particular embodiments, the carbohydrate-aminated antibody to be administered to the subject is directed against Ras, p53, papilloma virus E6, papilloma virus E7 or pX of hepatitis B virus.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphic representation of an SDS/polyacrylamide gel containing separated heavy and light chains of carbohydrate-aminated, carbohydrate pseudo- aminated and native murine anti-Tat monoclonal antibody. Figure 2 is a graph showing that binding of the carbohydrate-aminated and carbohydrate pseudo-aminated murine anti-Tat monoclonal antibodies to the Tat protein of HIV is comparable to the binding of the native anti-Tat monoclonal antibody.
Figure 3A is a graph showing the binding of 14C-labeled antibodies. Figure 3B is a graph showing the binding of 3H-labeled antibodies.
Figure 4 is a graph showing that the ability of 3H-anti-TAT antibody T5 to traverse Caco cell layers is directly related to levels of amine incorporation attained during the modification process.
Figure 5 A is a graph showing plasma levels of carbohydrate-aminated bovine IgG and of native IgG over time following intravenous administration.
Figures 5B-5F are graphs showing that intravenously- administered carbohydrate-aminated bovine IgG is taken up by intestine (B), liver (C), brain (D), kidney (E) and spleen (F) more rapidly and to a greater degree than is the native IgG.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to carbohydrate-aminated glycoproteins which comprise one or more diamine moieties that are bonded to carbon atoms of a carbohydrate moiety of the glycoprotein. The diamine moiety can be bonded to the carbohydrate moiety through a double or, preferably, a single covalent bond that is formed between an activated carbon atom of an oxidized carbohydrate moiety and a nitrogen atom of the diamine moiety, as described herein.
As used herein, "activated carbon atom" refers to the aldehydic carbonyl carbon atom which is formed when a carbohydrate moiety containing vicinal hydroxyl groups is oxidized as described herein. As used herein,"diamine moiety" refers to a chemical moiety which comprises at least two nitrogen atoms (e.g., substituted imino, primary amino, secondary amino, tertiary amino, quaternary
ammonium), wherein one of the nitrogen atoms is bonded to the activated carbon atom of the oxidized carbohydrate moiety. It is understood that more than two nitrogen atoms can be present in the diamine moiety. A diamine moiety can be provided, for example, by reacting an activated carbon atom of an oxidized carbohydrate moiety with one of a variety of diamine compounds as described herein.
Preferred carbohydrate-aminated glycoproteins of the invention comprise the presence of one or more diamine moieties represented by Structural Formula I and Structural Formula II.
-NR'-X-NR2R3 =N-X-NR2R3
(I) (II)
In Structural Fonnulae I and II, R! is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group. R2 and R3 are each independently -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R2 and R3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring (i.e., heteroaromatic ring (e.g., pyrrole, pyridine) or nonaromatic heterocyclic ring (e.g., pyrrolidine, piperidine, morpholine)). Suitable substituents for lower alkyl, aromatic, heterocyclic and benzyl groups include, for example, halogen atoms (e.g., fluorine, chlorine, bromine, iodine); lower alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl, neopentyl, cyclohexyl); benzyl; hydroxy; primary, secondary and tertiary amino; quaternary ammonium; and lower alkoxy groups. Preferably, the substituents which are bonded to the nitrogen atom of the secondary and tertiary amino group and the quaternary ammonium nitrogen are each individually a lower alkyl group, a benzyl group or an aromatic group.
X is a suitable linker group. Suitable linkers include, for example, C, to about C]2 aliphatic or aromatic groups. When X is an aliphatic group it can be a
linear, branched or cyclic aliphatic group which is saturated or which contains one or more units of unsaturation. For example, X can be a linear or branched C2-C12 alkane, alkene or alkyne. Preferably, X is an alkylene group represented by -(CH2)n-, wherein n is an integer from zero to about twelve. More preferably, n is an integer from one to about six (i.e.,methylene, ethylene, propylene, butylene, pentamethylene, hexamethylene). Most preferably, X is an ethylene group. When X in Structural Formulae I and π is an alkylene group, the moieties represented by the Structural Formulae can be referred to as an aminoalkylamine and an iminoalkylamine, respectively. Glycoproteins which can be modified in accordance with the invention include proteins, peptides and active fragments of the foregoing with at least one appended carbohydrate moiety (e.g., N-linked carbohydrate, O-linked carbohydrate). Active fragments of a glycoprotein have at least one carbohydrate moiety and retain at least one function of the full length protein, such as a catalytic function or a binding function (e.g., antigen binding). The glycoprotein can be a native, recombinant or synthetic protein or peptide which can be produced using any suitable method. A protein or peptide which does not contain a carbohydrate moiety can be engineered or modified to produce a glycoprotein. For example, an expression vector encoding a protein of interest can be mutated, using conventional methods of recombinant DNA technology, so that the encoded protein includes a consensus amino acid sequence for glycosylation (e.g., AsnXaaSer, AsnXaaThr). If necessary, the expression vector can be further modified so that the encoded protein includes a leader peptide. The mutated expression vector can then be introduced into a suitable host cell, which can be maintained under conditions suitable for production of the glycoprotein. Host cells which are suitable for production of recombinant glycoproteins include mammalian cells (e.g., CHO cells), insect cells(e.g., SF9 cells), and plant cells (e.g., Zea maize). If desired, carbohydrate moieties can be bonded directly to functional groups of proteins and peptides using conventional methods of synthetic organic chemistry, and glycopeptides can be produced by solid phase synthesis (see, for example, Meldal, M., et al, Curr. Opin. Chem. Biol, 7:552-563 (1997)).
Preferred carbohydrate-aminated glycoproteins include growth factors, antibodies and antigen-binding fragments thereof. As used herein, the term "growth factor" refers to soluble proteins or peptides which have specific effects on particular cells, including growth regulation, immunomodulation and trophic effects. Growth factors include the groups of proteins which are commonly referred to as neurotrophic factors (e.g., nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BNDF), IL-6 and the like), hematopoietic growth factors, such as erythropoietin, macrophage colony stimulating factor, stem cell factor and the like, and cytokines, including interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 and the like), interferons (e.g., IFNα, IFNβ, IFNγ) and chemokines (e.g., MDM α, members of the super Ig family, including Major Histocompatibility Complex proteins and complement factors, RANTES, eotaxin, SDF-1, and the like). It is noted that some growth factors such as IL-6 can mediate neurotrophic and/or immunoregulatory effects. Preferred growth factors which can be carbohydrate-aminated are those which can be isolated as glycoproteins from the organisms or cells in which they are naturally produced. A particularly preferred growth factor which can be carbohydrate-aminated is NGF.
Antibodies (immunoglobulins) which can be carbohydrate-aminated can be polyclonal or monoclonal, and the terni "antibody" is intended to encompass both polyclonal and monoclonal antibodies. The tenns polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
Multivalent antibodies, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody". The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g.. Cabilly et al, U.S. Patent No. 4,816,567; Cabilly et al, European Patent No.
0,125,023 Bl; Boss et al, U.S. Patent No. 4,816,397; Boss et al, European Patent No. 0,120,694 Bl ; Neuberger, M.S. et al, WO 86/01533; Neuberger, M.S. et al, European Patent No. 0,194,276 Bl ; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl ; Queen et al, European Patent No. 0 451 216 Bl ; and Padlan, E.A. et α/., EP 0 519 596 Al . See also, Newman, R. et al,
BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al, U.S. Patent No. 4,946,778 and Bird, R.E. et al, Science, 242: 423-426 (1988)) regarding single chain antibodies.
Humanized antibodies can be produced using synthetic or recombinant DNA technology with standard methods or with other suitable techniques. Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al, Nucl Acids Res., 17: 5404 (1989)); Sato, K., et al, Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al, Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P. and J.S. Crowe, Gene, 101: 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced, hi one embodiment, cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993).
Antibodies which are specific for an antigen (e.g., a viral protein, a protein product of a human oncogene) can be raised against an appropriate immunogen, such as an isolated and/or recombinant protein or portions thereof (including synthetic molecules, such as synthetic peptides). Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express an antigen. In addition, cells expressing a recombinant antigen, such as transfected cells, can be used as immunogens or in screens for antibodies which bind to the antigen (e.g., antibodies which bind to receptors) (See e.g., Chuntharapai et al, J. Immunol, 152: 1783-1789 (1994); Chuntharapai et al, U.S. Patent No. 5,440,021).
Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al, Nature 266: 550-552 (1977); Koprowski et al, U.S. Patent No. 4,172,124; Harlow, Ε. and D. Lane, 1988,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.M. et al, Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). For production of monoclonal antibodies, a hybridoma is generally produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0, P3X63Ag8.653 or a heteromyeloma) with antibody producing cells. Antibody producing cells can be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals which have been immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Other suitable methods of producing or isolating antibodies of the desired specificity (e.g., human antibodies or antigen-binding fragments) can be used, including, for example, methods which select a recombinant antibody from a library (e.g., a phage display library), or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see e.g., Jakobovits et al, Proc. Natl Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al, Nature, 362: 255-258 (1993); Lonberg et al, U.S. Patent No. 5,545,806; Surani et al, U.S. Patent No. 5,545,807; Lonberg et al, WO97/13852).
Active fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies can also be carbohydrate-aminated. Active fragments are multivalent or monovalent antigen -binding fragments, including, but not limited to, Fv, Fab, Fab' and F(ab')2 fragments that have at least one carbohydrate moiety. Active fragments of antibodies can be produced by enzymatic cleavage of antibodies which have at least one carbohydrate moiety that is
attached to the protein in, e.g., to the CH,, CL, or hinge region. Such antibodies can be produced by introducing an appropriate consensus amino acid sequence into an immunoglobulin chain. For example, a sequence encoding a glycosylation site can be incorporated into a nucleic acid encoding an immunoglobulin chain (e.g., using recombinant techniques, such as site directed mutagenesis), and the resulting nucleic acid can be expressed in a suitable host cell. Preferably, the carbohydrate moiety which is attached to an introduced glycosylation site does not interfere with antigen binding. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and a hinge region which includes a glycosylation site.
The carbohydrate-aminated antibody can be of any isotype (e.g., IgM, IgG (IgGl , IgG2, IgG3, IgG4), IgA, IgD, IgE) and can have a kappa or lambda light chain, hi one embodiment, the carbohydrate-aminated antibody is a human antibody, a humanized antibody or a chimeric antibody which includes a human constant region. In another embodiment, the carbohydrate-aminated antibody includes about four to about eighty-five diamine moieties.
Antibodies which are directed against any desired antigen can be carbohydrate-aminated. The carbohydrate-aminated antibodies of the invention retain the binding specificity of the corresponding native antibodies. As used herein, "directed against" means that the antibody or antigen-binding fragment has binding affinity for a specified antigen. An antibody or antigen-binding fragment directed against an antigen can specifically or selectively bind said antigen. Antibodies directed against an antigen can be produced using any suitable method. For example, an antibody directed against an antigen can be raised in an animal immunized with said antigen or selected from a library (e.g., by phage display). In one embodiment, the carbohydrate-aminated antibody can specifically bind an intracellular protein. As used herein, the term "intracellular protein" refers to proteins and peptides which can be found in the cytoplasm and/or organelles of a cell (e.g., nucleus, mitochondria, Golgi apparatus, lysosome, endoplasmic reticula
(rough endoplasmic reticulum, smooth endoplasmic reticulum), peroxisome) and encompasses proteins encoded by intracellular parasites (e.g., Listeria monocytogenes, Cryptosporidium parvum, Toxoplasma gondii, Leischmania sp., Plasmodium sp.), viruses (e.g., human immunodeficiency virus (HIV-1, HIV-2), hepatitis C virus (HCV)), and the cytoplasmic domains of transmembrane proteins (e.g., protein tyrosine kinase receptors). In one embodiment, the carbohydrate- aminated protein can bind and inhibit the function of a parasite or virus encoded protein which participates in the replication, proliferation and/or spread of the parasite or virus. Proteins which participate in the replication, proliferation and/or spread a vims include, for example, viral polymerases (e.g., HIV or HCV reverse transcriptase), viral transactivator proteins (e.g., HIV Tat), viral proteases (e.g., HIV or HCV protease) and the like. In one particular embodiment, the carbohydrate- aminated antibody can bind to the Tat protein of HIV (e.g., HIV-1 IIIB).
In another embodiment, the carbohydrate-aminated antibody can bind an antigen expressed by a specific cell type (e.g., lymphocyte, neuron, hepatocyte), tissue or organ (e.g., brain, spinal cord, peripheral nerve, intestine, liver, spleen, kidney, lung, muscle, pancreas, ovary, uterus, prostate, testis, breast, heart, stomach and gallbladder), hi one embodiment, the carbohydrate-aminated antibody can bind to a tumor antigen. As used herein, "tumor antigen" refers to antigens which are expressed uniquely by tumor cells or at higher levels by tumor cells than by non- tumor cells. Tumor antigens include cell surface molecules (e.g., such as Lewis Y, HER-2/neu, disialoganglioside G3, carcinoembrionic antigen, CD30) and molecules (e.g., intracellular molecules) which function in the proliferation and/or metastasis of tumors, such as the protein products of oncogenes (e.g., ras) and tumor suppressor genes (e.g., p53) which contain activating or inactivating mutations. In another embodiment, the carbohydrate-aminated antibody can bind to an antigen expressed in an organ or tissue selected from brain, spinal cord, peripheral nerve, intestine, liver, spleen and kidney.
The carbohydrate-aminated glycoprotein of the invention can further have one or more additional moieties, such as a detectable labeling moiety, drug or toxin, which are directly or indirectly bonded to the glycoprotein, provided that any such
additional moiety is not bonded, either directly or indirectly, to a diamine moiety. A variety of labeling moieties which can be conjugated to glycoproteins include, for example, enzymes (e.g., phosphatases, peroxidases), fluorescent compounds (e.g., fluorescene isothiocyanate), chelating compounds (e.g., ethylenediaminetetraacetic acid (EDTA), di ethyl enetriaminepentaacetic acid (DPT A)), radionuclides (,25I, l uIn, technetium-99m) boron adducts and affinity labels (e.g., epitopes, biotin, avidin). Drugs and toxins which can be conjugated to glycoproteins include, for example, chemotherapeutic agents (e.g., mitomycin C, methotrexate, 5-fluorouracil, cyclohexamine), anti-viral agents (e.g., viral protease inhibitors, for example, ritinovir, squinavir, nelfinavir mesylate), and toxins such as ricin, gelonin and the like.
The carbohydrate-aminated glycoproteins of the invention can have physical and biological characteristics (e.g., molecular weight, isoelectric point, binding activity, catalytic activity, immunogenicity) which are substantially the same as those of the corresponding native glycoproteins. For example, the molecular weight and isoelectric point of a glycoprotein, as determined by SDS-PAGE and isoelectric focusing, generally do not change when the protein is carbohydrate-aminated, and carbohydrate-aminated glycoproteins are no more immunogenic than the corresponding native glycoproteins. In addition, the biological activity (e.g., binding activity) of carbohydrate-aminated glycoproteins often remains substantially the same as the activity of native glycoproteins. For example, the carbohydrate- aminated glycoprotein often retains at least about 80% or 85% or 90% or 95% or 99% of the biological activity of the native glycoprotein. Preferably the biological activity is expressed as affinity when it is a binding activity, and as the catalytic rate constant when it is an enzymatic activity.
The carbohydrate-aminated glycoproteins of the invention have improved phannacokinetic properties in comparison to native glycoproteins. For example, as described herein, organ uptake of carbohydrate-aminated antibody after intravenous administration is significantly enhanced compared with native antibody (Example 2). Additionally, carbohydrate-aminated glycoproteins can be delivered into the cytoplasm and/or organelles of living cells. As described herein, the ability of a carbohydrate-aminated antibody which binds HIV Tat to inhibit HIV replication in cultured human T cells has been studied. The Tat protein of HIV is a regulatory protein that can activate the transcription of the viral genome, and is required for viral replication (Derse, D., et al, Virology:, 194:530-536 (1993)). The Tat protein is
produced in the cytoplasm of HlV-infected cells. During the course of the study it was discovered that carbohydrate-aminated anti-Tat antibody inhibited viral replication in cultured human T cells, while unmodified or "carbohydrate pseudo- aminated" anti-Tat did not (Example 1, Table 3). Carbohydrate pseudo-aminated glycoproteins (e.g., anti-Tat antibody) have a monoamine moiety (e.g., ethylamine) which is bonded to an activated carbon atom in an oxidized carbohydrate moiety of the glycoprotein. Such pseudo-aminated glycoproteins can be prepared as described herein (see, Example 1). The results of the study also demonstrate that carbohydrate- aminated antibodies can enter the cytoplasm and/or organelles of living cell, and inhibit the activity of intracellular proteins by, for example, binding to the protein.
In another aspect, the invention is a method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell, comprising contacting a cell with an effective amount of a carbohydrate-aminated glycoprotein, thereby allowing the carbohydrate-aminated glycoprotein to cross the plasma membrane and enter the cytoplasm and/or organelles of said cell. In one embodiment, the method comprises contacting a carbohydrate-aminated antibody or a carbohydrate-aminated antigen- binding fragment thereof with a cell. In another embodiment, the carbohydrate- aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be intracellularly delivered is directed against an intracellular protein. In another embodiment, the carbohydrate-aminated antibody or carbohydrate-aminated antigen- binding fragment thereof to be intracellularly delivered is directed against a tumor antigen or a viral protein. In a particular embodiment, the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be intracellularly delivered is directed against the Tat protein of HIV. In additionalparticular embodiments, the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be intracellularly deliveredis directed against an antigen selected from Ras, p53, E6 of papilloma vims, E7 of papilloma vims and pX of hepatitis B vims. In certain embodiments, the carbohydrate-aminated antibody is a chimeric, humanized or human antibody or an active fragment of any of the foregoing. In an additional embodiment, the carbohydrate-aminated glycoprotein to be to be intracellularly deliveredfurther has an appended detectable label, dmg or toxin, which is not bonded to the diamine moiety.
The method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell can be used in vitro and in vivo. For in vivo applications, an effective amount of a carbohydrate-aminated glycoprotein is administered to a
subject in need, such as a human having a viral infection.
In another aspect, the invention relates to a method of inhibiting (reducing or preventing) viral replication in a subject. The method comprises, administering an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, which is directed against a protein involved in viral replication and/or infection, to a subject in need thereof. The method can be employed to inhibit the replication of any vims, such as a herpes vims (e.g., herpes simplex vims, cytomegalovims, Epstein-Barr vims), a retrovims (e.g., HIV, HCV), a papilloma vims (e.g., human papilloma vims 16) and hepatitis B vims. In one embodiment, the invention is a method of inhibiting the replication of HIV in a subject, comprising administering an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is directed against the Tat protein encoded by HIV, to a subject in need thereof. In particular embodiments, the invention is a method of inhibiting the replication of papilloma vims in a subject, comprising administering an effective amount of a carbohydrate- aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is directed against the E6 or E7 protein encoded by the vims, to a subject in need thereof. In another particular embodiment, the invention is a method of inhibiting the replication of hepatitus B vims in a subject, comprising administering an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is directed against the X protein (pX) encoded by the vims, to a subject in need thereof.
In another aspect, the invention relates to a method of treating a subject having a tumor, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof, which is directed against a tumor antigen. In certain embodiments, the carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, which is administered to the subject, is directed against a protein which functions in the proliferation and/or metastasis of a tumor cell. Such proteins include, the protein products of cellular protooncogenes (e.g., ras) or tumor suppressor genes (e.g., p53) which contain activating or inactivating mutations, and protein products of viral oncogenes (e.g.. E6 or E7 of human papilloma virus 16 (HPV16)). In one embodiment, the carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is administered to the subject, is directed against an oncoprotein. In certain embodiments, the carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment
th ereof, that is administered to the subject is directed against a viral oncoprotein (e.g., E6 of HPV16, E7 of HPV16). In a particular embodiment, the carbohydrate- aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is administered to the subject, is directed against Ras. In another particular embodiment, the carbohydrate-aminated antibody or carbohydrate-aminated antigen binding fragment thereof, that is administered to the subject, is directed against p53. hi another aspect, the invention relates to a method of treating a subject infected with an intracellular parasite (e.g., Listeria sp., Cryptosporidium sp,, Toxoplasma sp., Leischmania sp., Plasmodium sp.), comprising administering to the subject an effective amount of a carbohydrate-aminated antibody or carbohydrate- aminated antigen binding fragment thereof which is directed against a protein involved in the proliferation or spread of the parasite, thereby inhibiting the proliferation or spread of the parasite.
In another aspect, the invention relates to a method of transvascularly delivering a carbohydrate-aminated glycoprotein to an organ or tissue in a subject. The method comprises mtravascularly administering (e.g., by intravenous injection, intraarterial injection) a carbohydrate-aminated glycoprotein to a subject, thereby allowing said carbohydrate-aminated glycoprotein to cross from the lumen of the blood vessel to said organ or tissue, hi one embodiment, the tissue or organ to which the carbohydrate-aminated glycoprotein is delivered is selected from the group consisting of brain, spinal cord, peripheral nerve, intestine, liver, spleen, kidney, lung, muscle, pancreas, ovary, uterus, prostate, testis, breast, heart, stomach and gallbladder. In a particular embodiment, the tissue or organ to which the carbohydrate-aminated glycoprotein is delivered is selected from the group consisting of brain, intestine, liver, spleen and kidney. In another particular embodiment, the invention is a method of transvascularly delivering a carbohydrate- aminated glycoprotein to the brain of a subject, comprising mtravascularly administering (e.g., by intravenous injection, intraarterial injection) a carbohydrate- aminated glycoprotein to the subject. In another embodiment, the method comprises mtravascularly administering a carbohydrate-aminated antibody or a carbohydrate- aminated antigen-binding fragment thereof to a subject. In another embodiment, the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be transvascularly delivered is directed against an antigen expressed in an particular cell type, organ or tissue. In another embodiment, the carbohydrate- animated antibody or carbohydrate-aminated antigen-binding fragment thereof to be transvascularly delivered is directed against an intracellular protein, a tumor antigen
or a viral protein. In more particular embodiments, the carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof to be transvascularly delivered is directed against an antigen selected from ras, p53, E6 of papilloma vims, E7 of papilloma vims and pX of hepatitis B vims. In another particular embodiment, the carbohydrate-aminated antibody or carbohydrate- aminated antigen-binding fragment thereof transvascularly delivered is directed against the Tat protein of HIV-1. In another embodiment, the carbohydrate- aminated antibody to be transvascularly delivered is a chimeric, humanized or human antibody or an active fragment of any of the foregoing. In an additional embodiment, the carbohydrate-aminated glycoprotein to be transvascularly delivered is a carbohydrate-aminated growth factor. In a particular embodiment, the growth factor is nerve growth factor. In an additional embodiment, the carbohydrate- aminated glycoprotein to be administered further has an appended detectable label, dmg or toxin, which is not bonded to the diamine moiety. A "subject" is preferably a human, but can also be an animal, preferably a mammal, in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, fowl, pigs, horses, fish and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
An effective amount of a carbohydrate-aminated glycoprotein can be administered to a subject to treat (reduce or prevent) disease or for diagnostic purposes. For example, an effective amount of a carbohydrate-aminated antibody which is directed against a protein that functions in viral replication can be administered to the subject to treat a viral infection.
An "effective amount" is an amount sufficient to achieve a desired diagnostic, therapeutic and/or prophylactic effect. For example, an effective amount of a carbohydrate-aminated antibody is an amount sufficient to bind to a protein for diagnostic purposes,an amount sufficient to bind to and inhibit the function of a protein, an amount sufficient to bind to and inhibit the function of an intracellular protein and or an amount sufficient to result in accumulation of the carbohydrate- aminated antibody in the cytoplasm and/or organelles of a cell, or in a particular tissue or organ (e.g., brain), thereby having a therapeutic and/or prophylactic effect.
The amount of carbohydrate-aminated glycoprotein administered to the subject will depend on the particular carbohydrate-aminated glycoprotein to be administered, the characteristics of the subject, such as general health, age, sex, body weight and tolerance to d gs as well as the degree, severity and type of disorder the subject has. The skilled artisan will be able to detennine appropriate dosages
depending on these and other factors. Typically, an effective amount can range from about 0.001 mg/kg per da> to about 10 mg/kg per day for an adult.
The carbohydrate-aminated glycoprotein can be administered by any suitable route, including, for example, orally in capsules, suspensions or tablets or by parenteral administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration. The carbohydrate-aminated glycoprotein can also be administered orally (e.g., dietary), transdermally, topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) or rectally. Administration can be local or systemic as indicated. The preferred mode of administration varies depending upon the particular carbohydrate-aminated glycoprotein chosen. However, parenteral administration is generally preferred.
The carbohydrate-aminated protein can be administered as a neutral compound or as a salt. Salts of compounds containing an amine or other basic group can be obtained, for example, by reacting with a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base; for example, a hydroxide base. Salts of acidic functional groups contain a countercation such as sodium, potassium and the like.
The carbohydrate-aminated glycoprotein can be administered to the individual as part of a pharmaceutical composition; for example, a therapeutic or diagnostic composition having a carbohydrate-aminated glycoprotein and a physiologically acceptable carrier. Compositions for co-therapy can have a carbohydrate-aminated glycoprotein and one or more additional therapeutic agents. Alternatively, a carbohydrate-aminated glycoprotein and an additional therapeutic agent can be components of separate compositions which are mixed together prior to administration or administered separately. Formulations will vary according to the route of administration selected (e.g., solution, emulsion, capsule). Suitable physiological carriers preferably contain inert ingredients which do not interact with the carbohydrate-aminated glycoprotein and/or additional therapeutic agent. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Suitable carriers for parenteral administration include sterile water, physiological saline, bacteriostatic saline, phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions
(such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al, "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).
In another aspect, the invention relates to a composition comprising a carbohydrate-aminated glycoprotein. The composition can be a pharmaceutical composition (e.g., therapeutic or diagnostic composition) comprising a carbohydrate-aminated glycoprotein and a physiologically acceptable carrier. In one embodiment, the composition comprises a carbohydrate-aminated antibody or a carbohydrate-aminated antigen-binding fragment thereof. In another embodiment, the composition comprises a carbohydrate-aminated antibody or carbohydrate- animated antigen-binding fragment thereof which is directed against an intracellular protein, a tumor antigen or a viral protein. In a particular embodiment, the composition comprises a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against the Tat protein of HIV. In other particular embodiments, the composition comprises a carbohydrate- aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against an antigen selected from Ras, p53, E6 of papilloma vims, E7 of papilloma vims and pX of hepatitis B vims. In certain other embodiments, the composition comprises a carbohydrate-aminated antibody which is a chimeric, humanized or human antibody or an active fragment of any of the foregoing. In another embodiment, the composition comprises a carbohydrate-aminated growth factor. In a particular embodiment, the carbohydrate-aminated growth factor is nerve growth factor. In an additional embodiment, the composition comprises a carbohydrate-aminated glycoprotein which further has an appended detectable label, drug or toxin, which is not bonded to the diamine moiety. The carbohydrate-aminated glycoproteins of the invention can be prepared by reacting a glycoprotein with a suitable oxidizing agent, thereby cleaving the carbohydrate ring structure between vicinal hydroxyl groups and converting the hydroxyl groups into aldehyde groups. A variety of suitable oxidizing agents can be used, with sodium periodate being preferred. Generally, an aqueous solution containing an oxidizing agent is added to an aqueous solution containing a glycoprotein. The concentration of glycoprotein is usually between about 1 mg/ml and about 20 mg/ml. The amount of oxidizing agent used is generally sufficient to provide about a 1000-fold excess relative to the glycoprotein. However, the amount of oxidizing agent can be adjusted to oxidize the carbohydrate moiety of the glycoprotein to the desired degree. The reaction can be carried out from 4°C to
room temperature for a period of from 2 hours to about 24 hours. Preferably, the reaction solution is protected from light.
The resulting glycoprotein, which has an oxidized carbohydrate moiety with an activated carbon atom, can then be reacted with a suitable diamine compound. Suitable diamine compounds comprise at least one, and preferably, two primary amino groups. Preferred diamine compounds are α,ω-diaminoalkanes (e.g., 1, 2- diaminoethane (ethylenediamine), 1, 3-diaminopropane, 1, 4-diaminobutane, 1, 5- diaminopentane, 1, 6-diaminohexane, 1, 7-diaminoheptane, 1, 8-diaminooctane, 1, 9-diaminononane, 1, 10-diaminodecane). A particularly preferred diamine compound is 1, 2-diaminoethane. The oxidized glycoprotein can be reacted with about a 1000-fold molar excess of diamine compound in an aqueous reaction solvent from 4°C to room temperature for a period of 2 hours to about 24 hours. If desired, the amount of diamine compound used can be adjusted to produce a carbohydrate- aminated glycoprotein having a desired quantity of diamine moieties. When a diamine compound having a primary amino group is chosen, the resulting carbohydrate-aminated glycoprotein can have a diamine moiety that is bonded to an activated carbon atom of an oxidized carbohydrate moiety through a double bond (i.e., a Schiff s base). If desired, the Schiff s base can be stabilized by reduction with a suitable reducing agent (e.g., sodium cyanoborohydride, sodium borohydride) to fomi a secondary amine. Generally, about a 50, 000-fold molar excess of reducing agent over glycoprotein is used.
The carbohydrate-aminated glycoprotein can be purified using conventional methods (e.g., column chromatography (e.g., gel filtration, ion exchange, hydrophobic interaction, affinity), preparative electrophoresis, precipitation). If desired, one or more additional moieties which are not bonded to the diamine moiety (e.g., a labeling moiety, drug or toxin) can be directly or indirectly conjugated to the carbohydrate-aminated glycoprotein using suitable methods of protein chemistry (e.g., by coupling through thiol, hydroxyl or carboxylate groups). It is understood that the coupling chemistry is chosen so that any additional moieties which are conjugated to the carbohydrate-aminated glycoprotein are not bonded to the diamine moiety. hi another aspect, the invention relates to a method of preparing a carbohydrate-aminated glycoprotein, comprising reacting a glycoprotein or a carbohydrate containing fragment thereof with an oxidizing agent to generate an activated carbon atom (i.e., an aldehydic carbonyl carbon atom) in the carbohydrate moiety; and reacting said aldehyde group with a diamine compound. If desired, the
resulting product can be reacted with a reducing agent.
EXEMPLIFICATION
EXAMPLE 1 : A CARBOHYDRATE-AMINATED ANTIBODY THAT IS DIRECTED AGAINST THE TAT PROTEIN OF HIV PREPARATION OF CARBOHYDRATE-AMINATED ANTI-TAT MONOCLONAL ANTIBODY
OXIDATION
A solution of murine anti-Tat HIV-1 IIIB (T5) monoclonal antibody (IgGl)(product #1102, ImmunoDiagnostics, Inc., Bedford, MA) in 300 mM sodium bicarbonate pH 8.8 (Sigma, St. Louis, MO) was added to a reaction tube. A freshly- made solution of sodium periodate (80 mg/mL in H2O)(Sigma) was then added to the tube at a ratio of 17 μL of periodate solution per milligram of IgG. The tube was wrapped in foil to protect it from light, and incubated at room temperature overnight (~18 hours) on a rotator. Following the overnight incubation, the reaction buffer was exchanged for 10 mM sodium carbonate pH 10.6 (Sigma) using an Econo-Pac® 10DG column (BioRad, Hercules, CA).
AMI ATION AND REDUCTION
A 28 mM solution of ethylenediamine (Sigma) was freshly prepared in water. The ethylenediamine solution was added to the solution of oxidized IgG at a ratio of 200 μL of ethylenediamine solution per milligram of oxidized IgG. The tube was wrapped in foil and incubated on a rotator at room temperature for about two hours. Then, a freshly-made solution of sodium cyanoborohydride ( 20 mg/mL in H2O) (Sigma) was added to the solution at a ratio of 100 μL per milligram of oxidized IgG. The solution was incubated on a rotator at room temperature for about thirty minutes (tubes were protected from light). After the incubation, additional freshly-made sodium cyanoborohydride solution ( 20 mg/mL in H2O) was added at the same ratio as above. The resulting solution was incubated on a rotator at room temperature for at least 2.5 hours to overnight (tubes were protected from light). Following the incubation, the reaction buffer was exchanged for 10 mM phosphate buffered saline, pH 7.2 (PBS; Sigma) using an Econo-Pac® 10DG column (BioRad).
PREPARATION OF CARBOHYDRATE PSEUDO-AMINATED ANTI-TAT MONOCLONAL ANTIBODY
A carbohydrate pseudo-aminated antibody was produced by the method described above, except ethylamine was substituted for ethylenediamine.
QUANTIFICATION OF CARBOHYDRATE AMINATION OF ANTI-TAT MONOCLONAL ANTIBODY
The quantity of diamine moieties which are bonded to the carbohydrate- aminated IgG was assessed by preparing a carbohydrate-aminated murine anti-Tat HIV-1 IHB monoclonal antibody that was trace labeled with 14C-ethylenediamine (Sigma). The trace labeled carbohydrate-aminated antibody was prepared as described above except that a 29.016 mM solution of ethylenediamine was used in which 10% of the ethylenediamine molecules were l4C labeled. The amount of 14C incorporated was determined by scintillation counting and the protein concentration of the carbohydrate-aminated antibody was determined using a BCA assay (Pierce, Rockford, IL). The specific activity of the labeled carbohydrate-aminated antibody and the number of ethylenediamine moieties incorporated per IgG molecule were calculated. These studies revealed that the number of diamine moieties bonded to the carbohydrate-aminated IgG ranged from about 4 to about 85. The analysis of a typical synthesis is presented in Table 1.
Table 1 : Analysis of Carbohydrate Amination of IgG, Example 1
ELECTROPHORETIC ANALYSIS
The molecular weights of the heavy and light chains of the carbohydrate- aminated IgG and the pseudo-aminated IgG were compared to those of the heavy and light chains of the native antibody using SDS-PAGE under reducing conditions. 7-15 μg of the proteins were mn on a 4-15% Tris gel (BioRad), and the separated heavy and light chains were visualized by staining with Commassie stain (Sigma). (See Figure 1.) The figure shows that the electrophoretic mobilities of the heavy and light chains of the modified antibodies were the same as the mobilities of the native heavy and light chains. It can be concluded that the molecular weights of monomeric heavy and light chains of the antibody are relatively unchanged in the native, carbohydrate-aminated and carbohydrate pseudo-aminated forms. Thus, the
carbohydrate amination of the antibody did not significantly change the molecular weight of the protein.
IMMUNOREACTIVITY TESTING
The immunoreactivity of the carbohydrate-aminated murine anti-Tat antibody was assessed by ELISA. Recombinant Tat protein from HIV-l strain IIIB (5 μg/mL in PBS) (ImmunoDiagnostics, Inc.) was coated onto 96-well microtiter plates by incubation at 4°C overnight. The excess Tat solution was decanted and blocking buffer (Pierce) was added to the wells of the plates. The plates were stored at 4°C until use. The Tat-coated, blocked plates were wanned to room temperature and the blocking buffer was decanted. The plates were then washed three times with wash buffer (PBS/0.2% Tween 20 (polyoxyethylenesorbitan monolaurate); Sigma). Duplicate samples of various concentrations of carbohydrate-aminated anti-Tat antibody, pseudo-aminated anti-Tat antibody, native anti-Tat antibody, and non- specific mouse IgGl (a negative control)(Rockland Immunochemicals) were added to the plates, and the plates were incubated overnight at 4°C. After the overnight incubation, the antibody solutions were decanted and the plates were washed three times with wash buffer. Goat anti-mouse IgG conjugated with horseradish peroxidase was added to each well of the plates, and the plates were incubated at room temperature for one hour. After the one hour incubation, the conjugated goat anti-mouse antibody was decanted and the plates were washed three times with wash buffer. Tetramethylbenzidine (Pierce) was added to the wells and the plates were incubated for 30 minutes at room temperature. Sulfuric acid (1M) was added to the wells to stop the reaction. The amount of anti-Tat antibody bound to each well was quantified by measuring the optical density of each well at 450 nm. From the results, antibody dilution curves were plotted versus optical density reading.
The results of this study, which are presented graphically in Figure 2, demonstrate that binding of the carbohydrate-aminated anti-Tat antibody is comparable to that of the native anti-Tat antibody.
EXAMPLE 2A : ANTI-TAT MAB WITH VARIABLE CONCENTRATIONS OF ,4C-ETHYLENEDIAMINE (ED) LABELING
PREPARATION
A solution of murine anti-Tat HIV-1 III B (T5) monoclonal antibody (IgGl) in 0.2 M potassium phosphate buffer, pH 7.5, was added to a reaction tube. A freshly-made solution of sodium periodate (80 mg/mL) was then added to the tube at a ratio of 35 μl of periodate solution per milligram of IgG. The tube was wrapped in foil to protect it from light and incubated at 4°C for 2 hours on a rotator. Following the incubation, the reaction buffer was exchanged for 10 mM sodium carbonate, pHl 0.6, using an Econo-Pac* 1 ODG column (BioRad). After the buffer exchange, the IgG solution was divided into 3 equal parts.
Three solutions of ethylenediamine, having concentrations of 3 mM, 30 mM and 300 mM, respectively, were freshly prepared in water. 14C-labeled ED (Sigma) was added to each of the three solutions as follows: 3 mM and 2 μL of ,4C-labeled ED;
30 mM and 20 μL of I4C-labeled ED; and
300 mM and 200 μL of ,4C-labeled ED.
One of the solutions containing both radioactive-labeled and unlabeled ethylenediamine was added to each one of the aliquots of IgG at a ratio of 200 μL of ethylenediamine solution to each milligram of oxidized IgG. The resulting three tubes were wrapped in foil and incubated on a rotator at room temperature for about 2.5 hours.
Then, a freshly-made solution of sodium cyanoborohydride (20mg/mL in water)(Sigma) was added to each of the IgG solutions at a ratio of 100 μL of solution to each milligram of oxidized IgG. The solutions were incubated on a rotator in the dark at 4°C for 30 min. After the incubation, additional freshly-made sodium cyanoborohydride (20mg/mL in water) was added in the same ratio as above. Each of the resulting solutions was protected from light and incubated on a rotator at 4°C for an additional 1.5 hours. Following the incubation, the reaction buffer was exchanged for 10 mM phosphate buffered saline, pH 7.4, (Sigma) using an Econo-Pac® 10DG column (BioRad).
QUANTIFICATION
Table 2: Analysis of Amine Incorporation of T5 Antibody, Example 2A
Concentration of ED Ratio of moles ED to moles IgG
3 mM 7
30 mM 14
300 mM 81
Immunoreactivity testing was performed by ELISA on each of the three preparations as described in the "Immunoreactivity Testing" example. The amount of anti-Tat antibody bound to each well was quantified by measuring the optical density of each well at 450 nm. From the results, antibody dilution curves were plotted versus optical density reading. The results are presented graphically in Figure 3 A. The graph shows that the binding of the carbohydrate-aminated I4C- labeled anti-Tat (T5) antibody is comparable to the binding of the native anti-Tat (T5) antibody regardless of the amount of amine incorporation achieved in the carbohydrate-amination process.
EXAMPLE 2B: 3H-LABELED ANTI-TAT MAB WITH VARIABLE ETHYLENEDIAMINE CONCENTRATIONS
PREPARATION
A solution of murine anti-Tat HIV-1 III B (T5) monoclonal antibody (IgGl) in 0.2 M potassium phosphate buffer, pH 7.5, (Sigma) was added to a reaction tube in which 3H-labeled n-succinimidyl propionate (NEN Life Sciences, Boston, MA) had been dried down under nitrogen gas. The solution was mixed overnight at 4°C. Unbound radioactivity was separated from bound radioactivity by using an Econo- Pac® 10 DG column (BioRad). A BCA (BioRad) protein assay was done and incorporation of radioactivity was measured using a Beckman model LS 6500 scintillation counter (Beckman, Fullerton, CA). Specific activity was determined.
A freshly made solution of sodium periodate (80 mg/mL)(Sigma) was then added to the tube at a ratio of 35 μl of periodate solution per milligram of IgG. The
tube was wrapped in foil to protect it from light and incubated at 4°C for 2 hours on a rotator. Following the incubation, the reaction buffer was exchanged for 10 mM sodium carbonate pH 10.6 (Sigma) using an Econo-Pac* 10DG column (BioRad). After the buffer exchange, the IgG solution was divided into 3 equal parts. Three solutions of ethylenediamine were freshly prepared in water: 3 mM,
30 mM and 300 mM. The ethylenediamine solutions were added to the solution of IgG at a ratio of 200 μL of ethylenediamine solution to each milligram of oxidized IgG. The tubes were wrapped in foil and incubated on a rotator at room temperature for about 2.5 hours. Then, a freshly-made solution of sodium cyanoborohydride solution
(20mg/mL in water)(Sigma) was added to each solution at a ratio of 100 μL of solution to each milligram of oxidized IgG. The solutions were incubated on a rotator in the dark at 4°C for 30 min. After the incubation, additional freshly-made sodium cyanoborohydride (20mg/mL in water)(Sigma) was added in the same ratio as above. The resulting solution was protected from light and incubated on a rotator at 4°C for an additional 1.5 hours. Following the incubation, the reaction buffer was exchanged for 10 mM phosphate buffered saline, pH 7.4 (Sigma) using an Econo- Pac® 1 ODG column (BioRad).
CELL CULTURE Human colon cancer cells (Caco-2) (HTB-37)(American Type Tissue
Culture, Manassas, VA) were grown in Eagles Minimum Essential Media (EMEM) (BioWhittaker, Walkersville, MD), supplemented with 20% Fetal Bovine Semm (FBS) (BioWhittaker) and 2 mM 1-glutamine (complete media) (BioWhittaker). The cells were grown to confluence in a humid incubator in an atmosphere of 5%CO2 and temperature of 37°C. For use in the determination of cell permeability,
Transwell® cell culture plates with 6.5 mm diameter surfaces and 3mm pore size (Costar, Cambridge, MA) were seeded with 120,000 cells in 0.2 ml of complete media above the membrane in the Transwell® and with 1.2 ml of complete media below the membrane. Resistance was measured with a Millicell® -Electrical Resistance System (Millipore) every two days until a consistant level of approximately 500 Ω was reached. This level is the indicator of a confluent cell layer. At this point, a 50:50 mixture of complete media with radio-labeled antibodies to be tested was filter-sterilized and used to replace the media above the cells growing on the membrane. After 24 and 48 hours, 100 μl samples of media
were removed from below the cell membrane and measured in a scintillation counter for radioactivity. Resistance was tested at each time point to assure the cell membrane remained intact. Duplicate and triplicate samples were done as controls. An unmodified but radiolabeled anti-Tat antibody was included in the experiments. The 50:50 antibody-media mixture was added to the Transwell® plate with no cell barrier.
QUANTIFICATION
The data are expressed as a ratio of the radioactivity measured in the media below the cell layer and membrane to the amount of radioactivity added. Immunoreactivity testing was perfonned on the three anti-Tat antibody preparations. The amount of anti-Tat antibody bound to each well was quantified by measuring the optical density of each well at 450 nm. From the results, antibody dilution curves were plotted versus optical density reading. The results of this study, a comparison of three levels of amine incorporation, are presented graphically in Figure 3B. The graph shows that the binding of the carbohydrate-aminated 14C- labeled anti-Tat (T5) antibody is comparable to the binding of the native anti-Tat (T5) antibody regardless of the amount of amine incorporation achieved in the carbohydrate-amination process.
The results of experiments using carbohydrate-aminated anti-Tat monoclonal antibodies having different levels of amine incorporation, with measurements taken after 24 hours, are presented graphically in Figure 4. Each result represents an average of three trials. The experiments demonstrate that the ability of 3H-anti-TAT antibody T5, to traverse Caco cell layers appears to be directly related to the level of amine incorporation achieved during the modification process.
EXAMPLE 3 : BIODISTRIBUTION OF CARBOHYDRATE-AMINATED ANTI-TAT ANTIBODIES.
Male Balb/c and Swiss mice were injected intraveneously with 50 μl of ~0.6 μg/ml!4C- carbohydrate-aminated anti-Tat antibody or native anti-Tat antibody. Antibodies had been labeled with succinimidyl propionate (NEN LifeSciences, Boston, MA) according to manufacturer's instmctions. 0.5 mCi3H-Succinimidyl propionate was dried down under a gentle stream of nitrogen gas. 0.5 mg of carbohydrate-aminated or native antibody was added immediately and incubated for
36 minutes at 4°C. An equal volume of 1M Tris buffer (BioRad) was added, and an EconoPac® 10 DG column (BioRad) was used to exchange the buffer into phosphate buffered saline, pH 7.4 (Sigma). One hour after injection, mice were euthanized and blood, brain, testes, kidney, liver and spleen tissues were harvested. Tissues were homogenized, digested with tissue solubilizer ( Beckman) and counted in a scintillation counter (Beckman LS6500). The ratio of dpm in one gram of tissue to dpm in one microliter of plasma was calculated after 1 hour, and is shown in Table 3.
TABLE 3
Organ uptake (u,l plasma/ g tissue)
Organ Native CaAb (fold increase over native)
Brain 20.4 ± 6.9 27.5 ± 18 (x 1.3)
Spleen 114.5 ± 36.6 302.9 ± 277 (x 2.6)
Kidney 157 ± 52.9 409.9 ± 270 (x 2.6)
Liver 97.5 ± 28.8 202.3 ± 163 (x 2.1)
Testis 30.3 ± 5.9 58.7 ± 49 (x 1.9)
EXAMPLE 4 : THE ENHANCEMENT OF TISSUE AND ORGAN UPTAKE OF CARBOHYDRATE-AMINATED ANTIBODIES AFTER INTRAVENOUS ADMINISTRATION
CARBOHYDRATE AMINATION OF 3H-LABELED BOVINE IgG WITH
1 ,4-DIAMINOBUTA_NE
Ten mg of bovine IgG (Sigma) were dissolved into 1 mL of HEPES buffer (950 mM, pH 7.4). Five 10 μL aliquots of 3H-acetic anhydride (5.2 Ci/mmole) in benzene (Sigma) were added to the antibody solution which was kept on ice. After approximately 45 minutes, the radiolabeled antibody was purified on a PD-10 column (Pharmacia) equilibrated in 300 mM NaHCO3. One hundred μL of an 80
mg/mL solution of Na m-peiodate (sodium meta-periodate) in water were added to a 500 μL aliquot of the 3H-labeled antibody (1.14 million cpm/10 mL). The reaction tube was protected from light by aluminum foil, and was incubated on a rotator at room temperature for about 90 minutes. After incubation, the oxidized 3H-labeled antibody was purified on a PD-10 column equilibrated in 10 mM sodium carbonate. To a 500 μL fraction from the PD-10 column containing the oxidized 3H-labeled antibody (approximately 500,000 cpm/10 μL), a 50 μL aliquot of a 32 mg/ml putrescine (1 ,4-diaminobutane) solution in water was added. After overnight incubation at room temperature, 100 μL of a solution of sodium borohydride (10 mg/mL in water) were added. After approximately one hour, 40 μL of a 15 μL/mL solution of ethanolamine in water were added. The 3H-labeled carbohydrate- aminated antibodies were then purified on a PD-10 column equilibrated in PBS.
ORGAN UPTAKE STUDY
Male Swiss albino mice (~20 g) were used for pharmacokinetic studies. H- labeled carbohydrate-aminated bovine IgG and control 3H-labeled bovine IgG were diluted to approximately 500,000 cpm/100 μL. One hundred μL aliquots were injected intravenously into the tail vein of the mice. At 1, 4 and 16 hours after the injection, mice were euthanized by cervical dislocation, blood was collected into heparinized tubes and plasma was prepared. The brain, one kidney, the spleen, a segment of intestine and the liver were dissected and homogenized in water. The amount of radioactivity in 20 μL aliquots of the plasma and of the organ/tissue homogenates was determined by scintillation counting. Proteins in the organ/tissue homogenates were quantified by the Bradford assay. Data were expressed as μL/mg protein (organ uptakes), by dividing the radioactivity recovered in each organ per mg of protein by the amount of radioactivity recovered in 1 μL of plasma.
The amount of carbohydrate-aminated IgG and of native IgG which remained in the plasma of injected mice decreased over time (Figure 5A). However, the results show that the carbohydrate-aminated IgG was taken up more rapidly and to a greater extent than native IgG by all organs examined (i.e., intestine, liver, brain, kidney and spleen) (Figures 5B-5F).
EXAMPLE 5: THE INHIBITION OF REPLICATION OF HIV BY
CARBOHYDRATE-AMINATED ANTI-TAT ANTIBODY
Human T cells were isolated from healthy volunteers and cultured at 2 x 106 cells/mL in RPMI 1640 (Biowhittaker) supplemented with 10% fetal bovine semm (Biowhittaker) and penicillin/streptomycin (Biowhittaker). The cells were cultured in 12 well dishes with a lmL capacity. At time zero, 5 multiplicity of infection (MOI) HIV-1 IHB (100 μL), and 10, 1 or 0.1 mg of anti-Tat antibody preparations (carbohydrate-aminated, carbohydrate pseudo-aminated or native) were added to the culture media. After 5 days, the amount of HIV p24 in the cultures was determined by ELISA (hnmunotech). The amount of p24 detected correlates with the amount of HIV replication occurring in the cultures. The results of two independent tests are shown in Table 4.
Table 4: Amount of HIV p24 in T Cell Cultures (ng/mL)
NT = not tested
The results are the average of four cultures. Cell viability ranged from 75% to 90%) for all cultures. The results presented in Table 4 clearly demonstrate that carbohydrate-aminated anti-Tat antibody, but not carbohydrate pseudo-aminated or native antibody, can inhibit the replication of HIV in cells. Therefore, because Tat is an intracellular product of HIV-1 infection, it can be deduced that intracellular uptake of the anti-Tat antibody can be increased by modification to the carbohydrate-aminated form.
EXAMPLE 6: ASSESSMENT OF p24 PRODUCTION
Normal, cultured human T cells were infected with 5 MOI (multiplicity of infection) HIV-1 III B. At the same time, 10 μg of carbohydrate-aminated anti-Tat antibody were added in media. The cultures were continued for 5 days and the p24 levels were assessed to measure replication of the vims. Cell supernatants were harvested, centrifuged to remove the cells, and the supernatant subjected to quantitation by commercial p24 ELISA kits (available from hnmunotech, Miami, FL) according to manufacturer's specification. The ELISA kits have a sensitivity limit of 50pg/ml. The results of this study are shown in Table 5.
TABLE 5
Sample p24 level Cell I viability
(ng/mL) (%)
+ Control 357 63 11 Amines/IgG 28 51 3 Amines/IgG 112 65 1 amine/IgG 231 71
EXAMPLE 7 : IMMUNOGENICITY TESTING
The ability of the modified proteins to elicit an immune response, hereinafter refered to as the immunogenicity, was assessed using Balb/c mice. The mice were divided into three groups, with each group having four mice. Group I was subcutaneously injected with the carbohydrate-aminated murine anti-Tat antibody. Group II was subcutaneously injected with the carbohydrate pseudo-aminated murine anti-Tat antibody, and Group III was subcutaneously injected with the native murine anti-Tat antibody. The amount of IgG preparation injected was the same for each group. Blood was collected into heparinized tubes, plasma was prepared, and immunological reactivity to the particular IgG preparation which was injected
(carbohydrate-aminated, carbohydrate pseudo-aminated or native) was determined by Ouchterloney assay at predetermined time points. Groups I and II were also evaluated for reactivity to the native IgG. The Ouchterloney assays were run for 48 hours with inspection at 24 hours and at 48 hours. The schedule for injections, blood draws, and the results of the Ouchterloney assays are presented in Table 6.
Assays in which no precipitin bands were detected were scored negative.
The data presented in Table 6 demonstrate that carbohydrate amination of murine IgG did not increase the immunogenicity of the antibody.
Table 6: Results of Immunogenicity Testing
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in fomi and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Claims
1. A carbohydrate-aminated glycoprotein, comprising a diamine moiety which is covalently bonded to a carbohydrate moiety through a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein.
2. The carbohydrate-aminated glycoprotein of Claim 1 wherein said diamine moiety is represented by -NR'-X-NR2R3 or =N-X-NR2R3 ; wherein:
R1 is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group; R2 and R3 are each independently -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R2 and R3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring; and X is a C, to about C,2 aliphatic or aromatic group.
3. The carbohydrate-aminated glycoprotein of Claim 2 wherein X is an alkylene group and said diamine moiety is represented by
-NR'-(CH2)n-NR2R3 or =N-(CH2)n-NR2R3; wherein n is an integer from one to about twelve.
4. The carbohydrate-aminated glycoprotein of Claim 3 wherein n is an integer from one to about six.
5. The carbohydrate-aminated glycoprotein of Claim 3 wherein the diamine moiety is an aminoalkylamine moiety represented by 
wherein n is an integer from one to about six.
6. The carbohydrate-aminated glycoprotein of Claim 5 wherein n is two.
7. The carbohydrate-aminated glycoprotein of Claim 1 wherein said carbohydrate-aminated glycoprotein is a carbohydrate-aminated antibody or a carbohydrate-aminated antigen-binding fragment thereof.
8. The carbohydrate-aminated glycoprotein of Claim 7 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a tumor antigen.
9. The carbohydrate-aminated glycoprotein of Claim 7 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against an intracellular protein.
10. The carbohydrate-aminated glycoprotein of Claim 7 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a protein selected from the group consisting of
Ras, p53, E6 of papilloma vims, E7 of papilloma vims, pX of hepatitis B vims, interleukins, cytokines, and cytoskeletal proteins such as actin and cytokeratin.
11. The carbohydrate-aminated glycoprotein of Claim 7 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a viral protein.
12. The carbohydrate-aminated glycoprotein of Claim 1 1 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against the Tat protein of HIV.
13. The carbohydrate-aminated glycoprotein of Claim 7 wherein said carbohydrate-aminated antibody is a carbohydrate-aminated chimeric, carbohydrate-aminated human or carbohydrate-aminated humanized antibody.
14. The carbohydrate-aminated glycoprotein of Claim 1 wherein said carbohydrate-aminated glycoprotein is a carbohydrate-aminated growth factor.
15. The carbohydrate-aminated glycoprotein of Claim 14 wherein said carbohydrate-aminated growth factor is carbohydrate-aminated nerve growth factor.
16. The carbohydrate-aminated glycoprotein of Claim 1 wherein a detectable label, dmg or toxin is covalently bonded to said carbohydrate-aminated glycoprotein, and wherein said detectable label, drug or toxin is not bonded to the diamine moiety.
17. A method of intracellularly delivering a carbohydrate-aminated glycoprotein to a cell, comprising contacting said cell with said carbohydrate-aminated glycoprotein thereby allowing said carbohydrate-aminated glycoprotein to cross the plasma membrane and enter the cytoplasm and/or organelles of said cell, wherein said carbohydrate-aminated glycoprotein comprises a diamine moiety which is covalently bonded to a carbohydrate moiety through a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein.
18. The method of Claim 17 wherein said diamine moiety is represented by -NR'-X-NR2R3 or =N-X-NR2R3 ; wherein:
R1 is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group;
R2 and R3 are each independently -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R2 and R3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring; and
X is a C, to about C12 aliphatic or aromatic group.
19. The method of Claim 18 wherein X is an alkylene group and said diamine moiety is represented by
-NR'-(CH2)n-NR2R3 or -N-(CH2)n-NR2R3; wherein n is an integer from one to about twelve.
20. The method of Claim 19 wherein n is an integer from one to about six.
21. The method of Claim 19 wherein the diamine moiety is an aminoalkylamine moiety represented by
-NR'-(CH2)n-NR2R3; wherein n is an integer from one to about six.
22. The method of Claim 21 wherein n is two.
23. The method of Claim 17 wherein said carbohydrate-aminated glycoprotein is a carbohydrate-aminated antibody or a carbohydrate-aminated antigen- binding fragment thereof.
24. The method of Claim 23 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a tumor antigen.
25. The method of Clain 23 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against an intracellular protein.
26. The method of Claim 23 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a protein selected from the group consisting of Ras, p53, E6 of papilloma vims, E7 of papilloma vims, pX of hepatitis B vims, interleukins, cytokines, and cytoskeletal proteins such as actin and cytokeratin.
27. The method of Claim 23 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a viral protein.
28. The method of Claim 27 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against the Tat protein of HIV.
29. The method of Claim 23 wherein said carbohydrate-aminated antibody is a carbohydrate-aminated chimeric, carbohydrate-aminated human or carbohydrate-aminated humanized antibody.
30. The method of Claim 23 wherein a detectable label, dmg or toxin is covalently bonded to said carbohydrate-aminated glycoprotein, and wherein said detectable label, drug or toxin is not bonded to the diamine moiety.
31. A method of transvascularly delivering a carbohydrate-aminated glycoprotein to an organ or tissue in a subject, comprising mtravascularly administering said carbohydrate-aminated glycoprotein to said subject thereby allowing said carbohydrate-aminated glycoprotein to cross from the lumen of the blood vessel to said organ or tissue, wherein said carbohydrate-aminated glycoprotein comprises a diamine moiety which is covalently bonded to a carbohydrate moiety tlirough a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein.
32. The method of Claim 31 wherein said organ or tissue is selected from the group consisting of brain, spinal cord, peripheral nerve, intestine, liver, spleen, kidney, lung, muscle, pancreas, ovary, utems, prostate, testis, breast, heart, stomach and gallbladder.
33. The method of Claim 31 wherein said organ or tissue is selected from the group consisting of brain, intestine, liver, spleen and kidney.
34. The method of Claim 31 wherein said diamine moiety is represented by -NR'-X-NR2R3 or =N-X-NR2R3 ; wherein:
R1 is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group; R2 and R3 are each independently -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R2 and R3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring; and X is a C] to about C12 aliphatic or aromatic group.
35. The method of Claim 34 wherein X is an alkylene group and said diamine moiety is represented by
-NR'-(CH2)n-NR2R3 or =N-(CH2)n-NR2R3; wherein n is an integer from one to about twelve.
36. The method of Claim 35 wherein n is an integer from one to about six.
37. The method of Claim 35 wherein the diamine moiety is an aminoalkylamine moiety represented by
-NR'-(CH2)n-NR2R3; wherein n is an integer from one to about six.
38. The method of Claim 37 wherein n is two.
39. The method of Claim 31 wherein said carbohydrate-aminated glycoprotein is a carbohydrate-aminated antibody or a carbohydrate-aminated antigen- binding fragment thereof.
40. The method of Claim 39 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a tumor antigen.
41. The method of Claim 39 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against an intracellular antigen.
42. The method of Claim 39 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a protein selected from the group consisting of Ras, p53, E6 of papilloma virus, E7 of papilloma vims, pX of hepatitis B vims, interleukins, cytokines, and cytoskeletal proteins such as actin and cytokeratin.
43. The method of Claim 39 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a viral protein.
44. The method of Claim 43 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against the Tat protein of HIV.
45. The method of Claim 39 wherein said carbohydrate-aminated antibody is a carbohydrate-aminated chimeric, carbohydrate-aminated human or carbohydrate-aminated humanized antibody.
46. The method of Claim 31 wherein said carbohydrate-aminated glycoprotein is a carbohydrate-aminated growth factor.
47. The method of Claim 46 wherein said carbohydrate-aminated growth factor is carbohydrate-aminated nerve growth factor.
48. The method of Claim 31 wherein a detectable label, dmg or toxin is covalently bonded to said carbohydrate-aminated glycoprotein, and wherein said detectable label, dmg or toxin is not bonded to the diamine moiety.
49. A composition comprising a carbohydrate-aminated glycoprotein, wherein said carbohydrate-aminated glycoprotein comprises a diamine moiety which is covalently bonded to a carbohydrate moiety through a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein and a physiologically acceptable carrier.
50. The composition of Claim 49 wherein said diamine moiety is represented by
-NR'-X-NR2R3 or =N-X-NR2R3 ; wherein: R1 is -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted aromatic group;
R2 and R3 are each independently -H, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted aromatic group, or R2 and R3 taken together with the nitrogen atom to which they are bonded form a substituted or unsubstituted heterocyclic ring; and X is a C, to about C12 aliphatic or aromatic group.
51. The composition of Claim 49 wherein said carbohydrate-aminated glycoprotein is a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof.
52. The composition of Claim 51 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a tumor antigen.
53. The composition of Claim 51 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against an intracellular protein.
54. The composition of Claim 51 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a protein selected from the group consisting of Ras, p53, E6 of papilloma vims, E7 of papilloma vims, pX of hepatitis B vims, interleukins, cytokines, and cytoskeletal proteins such as actin and cytokeratin.
55. The composition of Claim 51 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a viral protein.
56. The composition of Claim 55 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen -binding fragment is directed against the
Tat protein of HIV.
57. The composition of Claim 51 wherein said carbohydrate-aminated antibody is a carbohydrate-aminated chimeric, carbohydrate-aminated human or carbohydrate-aminated humanized antibody.
58. The composition of Claim 49 wherein said carbohydrate-aminated glycoprotein is a carbohydrate-aminated growth factor.
59. The composition of Claim 58 wherein said carbohydrate-aminated growth factor is carbohydrate-aminated nerve growth factor.
60. The composition of Claim 49 wherein a detectable label, dmg or toxin is covalently bonded to said carbohydrate-aminated glycoprotein, and wherein said detectable label, dmg or toxin is not bonded to the diamine moiety.
61. A method of inhibiting replication of a vims in a subject, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against a protein involved in said viral replication thereby inhibiting replication of said vims, wherein said carbohydrate-aminated glycoprotein comprises a diamine moiety which is covalently bonded to a carbohydrate moiety through a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein.
62. The method of Claim 61 wherein said vims is HIV and said carbohydrate- aminated antibody is directed against the Tat protein encoded by HIV.
63. The method of Claim 61 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a protein selected from the group consisting of Ras, p53, E6 of papilloma virus, E7 of papilloma vims, pX of hepatitis B vims, interleukins, cytokines, and cytoskeletal proteins such as actin and cytokeratin.
64. A method of treating a subject infected with an intracellular parasite, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against a protein involved in the proliferation or spread of said parasite, wherein said carbohydrate-aminated glycoprotein comprises a diamine moiety which is covalently bonded to a carbohydrate moiety through a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein.
65. The method of Claim 64 wherein said subject is infected with Listeria sp., Cryptosporidium sp., Toxoplasma sp., Leischmania sp. or Plasmodium sp.
66. A method of treating a subject having a tumor, comprising administering to said subject an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against a tumor antigen, wherein said carbohydrate-aminated glycoprotein comprises a diamine moiety which is covalently bonded to a carbohydrate moiety tlirough a single or double bond that is formed between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein.
67. The method of Claim 66 wherein said tumor antigen is a protein which functions in the proliferation and/or metastasis of said tumor.
68. The method of Claim 67 wherein said carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment is directed against a protein selected from the group consisting of Ras, p53, E6 of papilloma virus, E7 of papilloma vims, pX of hepatitis B vims, interleukins, cytokines, and cytoskeletal proteins such as actin and cytokeratin.
69. Use, for the manufacture of a medicament for treating disease caused by replication of a vims in an individual, of an effective amount of a carbohydrate-aminated antibody or carbohydrate-aminated antigen-binding fragment thereof which is directed against a protein involved in said viral replication and which is suitable for inhibiting replication of said virus, wherein said carbohydrate-aminated glycoprotein comprises a diamine moiety which is covalently bonded to a carbohydrate moiety through a single or double bond that is fomied between a nitrogen atom of said diamine moiety and a carbon atom of an oxidized carbohydrate moiety of said glycoprotein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU20642/01A AU2064201A (en) | 1999-12-06 | 2000-12-06 | Carbohydrate-aminated glycoproteins |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US45559899A | 1999-12-06 | 1999-12-06 | |
| US09/455,598 | 1999-12-06 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2001040277A2 true WO2001040277A2 (en) | 2001-06-07 |
| WO2001040277A3 WO2001040277A3 (en) | 2002-06-27 |
Family
ID=23809488
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2000/033075 WO2001040277A2 (en) | 1999-12-06 | 2000-12-06 | Carbohydrate-aminated glycoproteins |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU2064201A (en) |
| WO (1) | WO2001040277A2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11002646B2 (en) | 2011-06-19 | 2021-05-11 | DNA Genotek, Inc. | Devices, solutions and methods for sample collection |
| US11046949B2 (en) | 2014-04-10 | 2021-06-29 | Dna Genotek Inc. | Method and system for microbial lysis using periodates |
| US11572581B2 (en) | 2002-06-07 | 2023-02-07 | DNA Genotek, Inc. | Compositions and methods for obtaining nucleic acids from sputum |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4237479A1 (en) * | 1992-11-06 | 1994-05-11 | Behringwerke Ag | Process for the production of conjugates from a specific binding partner and a carbohydrate-containing protein |
| ATE241384T1 (en) * | 1995-03-22 | 2003-06-15 | Jackson H M Found Military Med | PREPARATION OF IMMUNOGENIC CONSTRUCTS USING SOLUBLE CARBOHYDRATES ACTIVATED BY ORGANIC CYANYLATION REAGENTS |
-
2000
- 2000-12-06 AU AU20642/01A patent/AU2064201A/en not_active Abandoned
- 2000-12-06 WO PCT/US2000/033075 patent/WO2001040277A2/en active Application Filing
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11572581B2 (en) | 2002-06-07 | 2023-02-07 | DNA Genotek, Inc. | Compositions and methods for obtaining nucleic acids from sputum |
| US11002646B2 (en) | 2011-06-19 | 2021-05-11 | DNA Genotek, Inc. | Devices, solutions and methods for sample collection |
| US11536632B2 (en) | 2011-06-19 | 2022-12-27 | DNA Genotek, Inc. | Biological collection system |
| US11549870B2 (en) | 2011-06-19 | 2023-01-10 | DNA Genotek, Inc. | Cell preserving solution |
| US11592368B2 (en) | 2011-06-19 | 2023-02-28 | DNA Genotek, Inc. | Method for collecting and preserving a biological sample |
| US11046949B2 (en) | 2014-04-10 | 2021-06-29 | Dna Genotek Inc. | Method and system for microbial lysis using periodates |
| EP3129482B1 (en) * | 2014-04-10 | 2021-12-15 | DNA Genotek Inc. | Method and system for microbial lysis using periodates |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2064201A (en) | 2001-06-12 |
| WO2001040277A3 (en) | 2002-06-27 |
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