CA1168150A - Targeting conjugates of albumin and therapeutic agents - Google Patents
Targeting conjugates of albumin and therapeutic agentsInfo
- Publication number
- CA1168150A CA1168150A CA000392658A CA392658A CA1168150A CA 1168150 A CA1168150 A CA 1168150A CA 000392658 A CA000392658 A CA 000392658A CA 392658 A CA392658 A CA 392658A CA 1168150 A CA1168150 A CA 1168150A
- Authority
- CA
- Canada
- Prior art keywords
- albumin
- enzyme
- therapeutic agent
- linking
- agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6815—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
Abstract
"TARGETING CONJUGATES OF ALBUMIN
AND THERAPEUTIC AGENTS"
ABSTRACT OF THE DISCLOSURE
The invention provides conjugates of a therapeutic agent with carrier albumin and a targeting agent and methods for their preparation. The albumin is included in an amount sufficient to mask antigenicity of the therapeutic agent. The targeting agent used has binding specificity for receptor sites on cells towards which it is desirable to direct the therapeutic agent. Insulin, immunoglobulin G, antibody against human pancreatic tumor cell, and antibody against hyaluronic acid are exemplified as targeting agents.
AND THERAPEUTIC AGENTS"
ABSTRACT OF THE DISCLOSURE
The invention provides conjugates of a therapeutic agent with carrier albumin and a targeting agent and methods for their preparation. The albumin is included in an amount sufficient to mask antigenicity of the therapeutic agent. The targeting agent used has binding specificity for receptor sites on cells towards which it is desirable to direct the therapeutic agent. Insulin, immunoglobulin G, antibody against human pancreatic tumor cell, and antibody against hyaluronic acid are exemplified as targeting agents.
Description
~ 3
2 The present ;nvention relates to conjugates of therapeutic
3 agents for the treatment of disease ;n mammals.
The term therapeutic agent includes not only the large number of drugs used in the treatment of disease, but also a large 6 number of enzymes which are used ;n the treatment of genetic, metabolic and malignant diseases. These diseases ;nclude a wide range of Inborn 8 Errors of Metabol~sm in which specific enzymes in the body are either g deficient or deFective. Suitable enzyme replacement might provide an appropriate therapy. In other diseases, cancer for example, certain cells 1l have been shown to be sensitive to a spec;f;c enzyme. For example, 12 in Acute Lymphocytic Leukemia the tumor cells are sensitive to the enzyme L-asparaginase. The tumor cells;have an absolute requirement for exogenous L-asparagine and cannot survive in the presence of L-asparaginase, the enzyme responsible for the removal of the sub-16 strate. Enzyme treatment of this type ;s usually termed enzyme therapy.
There are problems associated with the injection of many 18 of the therapeutic agents, especially enzymes, into the body. Firstly, 19 the therapeutic agent may ~e foreign to the body and can therefore cause the body's immune system-to mount a rejection reaction. The 21 therapeutic agen~ is then rapidly cleared from the body following each 22 subsequent administration. This rejection reaction, in its severest ~3 form called an anaphylactic reaction, may threaten the life of the 24 recipient Secondly, especially in the case of enzymes, the therapeut;c 26 agent is frequently heat labile and sensitive to proteolysis by other 27 circulating enzymes. Rapid bioinactivation of the therapeutic agent 28 can occur. This may necessitate repeated administration of large 29 dsse oF the therapeutic agent. In addition to the expense and bother which this causes, there is al~o the increased risk of complicating the 31 above described immunological reaction, thereby increasing the possibility 32 of anaphylaxis.
l Thirdly, it ;s often necessary or desirable to deliver or 2 target the therapeutic agent to specific body cells or organs requiring3 action. For instance, in many enzyme defiiciency diseases the defect
The term therapeutic agent includes not only the large number of drugs used in the treatment of disease, but also a large 6 number of enzymes which are used ;n the treatment of genetic, metabolic and malignant diseases. These diseases ;nclude a wide range of Inborn 8 Errors of Metabol~sm in which specific enzymes in the body are either g deficient or deFective. Suitable enzyme replacement might provide an appropriate therapy. In other diseases, cancer for example, certain cells 1l have been shown to be sensitive to a spec;f;c enzyme. For example, 12 in Acute Lymphocytic Leukemia the tumor cells are sensitive to the enzyme L-asparaginase. The tumor cells;have an absolute requirement for exogenous L-asparagine and cannot survive in the presence of L-asparaginase, the enzyme responsible for the removal of the sub-16 strate. Enzyme treatment of this type ;s usually termed enzyme therapy.
There are problems associated with the injection of many 18 of the therapeutic agents, especially enzymes, into the body. Firstly, 19 the therapeutic agent may ~e foreign to the body and can therefore cause the body's immune system-to mount a rejection reaction. The 21 therapeutic agen~ is then rapidly cleared from the body following each 22 subsequent administration. This rejection reaction, in its severest ~3 form called an anaphylactic reaction, may threaten the life of the 24 recipient Secondly, especially in the case of enzymes, the therapeut;c 26 agent is frequently heat labile and sensitive to proteolysis by other 27 circulating enzymes. Rapid bioinactivation of the therapeutic agent 28 can occur. This may necessitate repeated administration of large 29 dsse oF the therapeutic agent. In addition to the expense and bother which this causes, there is al~o the increased risk of complicating the 31 above described immunological reaction, thereby increasing the possibility 32 of anaphylaxis.
l Thirdly, it ;s often necessary or desirable to deliver or 2 target the therapeutic agent to specific body cells or organs requiring3 action. For instance, in many enzyme defiiciency diseases the defect
4 is intracellular. Due to the lack of a functioning enzyme, the substrate which accumulates in the body cell is compartementalized in 6 intracellular organelles termed lysosomes. Frequently, the substrate 7 is stored only in specific tissues or organs. Thus to effect enzyme 8 replacement it may be necessary to target the enzymes to these specific9 sites. This has proven to be a major limitation to the treatment of such fatal childhood énzyme deficiency diseases as lipid storage 11 diseases and glycogen storage diseases.
12 Sorne, but not all, of the above problems have been solved13 in the prior art by conjugating the therapeutic agent with a carrier.
14 Carriers are divided into two groups, those that cause highly specific binding to cell-surface receptors and those that do not. The former 16 type of carrier will hereinafter be referred to as a targeting agent.
17 Targeting of drugs is well docurnented, see for example 18 the revlew article by M. J. Poznansky and L. G. Cleland in Drwg 19 Delivery Systems, ed. Julianog Oxford University Press, New York, 1980, pg. 253 - 315. Targeting agents are molecules, frequently bio-21 logical macromolecules, which bind to specific receptor sites on the sur-22 faces of body cells. Known targeting agents include serum hormones, 23 antibodies against cell surface antigens, and lectins.
24 It is also known in the prior art to protect therapeutic agents by conjugation with an appropriate carrier. Conjugates of 26 albumin and therapeutic agents are documented in the above-referenced 27 review article and in an article by M. J. Poznansky and D. Bhardwaj in 28 Canadian Journal oF Physiology and Pharmacology, 58 , 1980, pg. 322-325.
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1 These albumin conjugates have been shown to be both non-immunogenic and 2 non-antigenic, see for instance M. H. Remy and M. J. Poznansky, The Lancet, 3 July 8, 1978, pg. 68 - 70. The albumin ;s believed to mask the antigenic sites on the therapeutic agent such that the recip;ent recogn;zes the conjugate as self and therefore does not mount an ;mmune response. Further, 6 these albumin conjugates have been shown to be more resistant to bioin-activation than was the free therapeutic agent.
8 To my knowledge, no attempt has been made to target these g albumin-protected therapeutic agents. Prior to the present invention it was not known whether a targeting agent would be effective in targeting 11 such a large and complex molecule. Further, it was not known whether12 attachment of a targeting agent would interfereiw;th the biological 13 act;vity of the albumin-therapeutic agent conjugate.
:
I have discovered that conjugates of albumin and therapeutic 16 agent can be made targetable by chem;cally linking the conjugate to a 17 targeting agent having binding specificity for receptor sites on body 18 cells against which it is desirable to direct the therapeutic agent.
19 Known target;ng agents, including serum hormones, cell-surface directed antibodies, and lectins, are suitable for this purpose.
21 While it might have been eY~pected that the albumin carrier22 molecule would mask or interfere with the targetability.~ the-targeting 23 agent once it was l;nked to the therapeutic agent - albumin conjugate, 24 th;s was Found not to be the case. The targeting agent, linked to the conjugate, was found to reta;n its ab;lity to specifically deliver the 26 conjugate to speciF;c cellular surface receptors. Further, the targeting27 agent was Found not to interfere with the ab;lity of the body cell to 28 utilize the therapeut;c agent once the conjugate was delivered to the cell.
29 This was not a predictable property of the conjugates of the present invention.
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1 The albumin carrier ;s included ~n the conjugate in an 2 amount sufficient to mask the antigenici~ty of the therapeutic agent.
3 The term 'to mask the antigenic;ty' ;s meant to infer that the conjugate 4 does not illicit an immune response. The al~umin used is most preferably homologous to the mammal intended as the recipient. Since in most 6 cases the intended recipient is human, human serum albumin is preferred.
7 Other sources of albumin, for example bov;ne serum albumin and horse 8 serum albumin are useful for therapy in cattle and horses respectively.
9 As explained, the targeting agent allows for delivery of the conjugates to specific cellular surface receptors. The cho;ce of 11 targeting a~ent therefore depends on the desired site of delivery. The 12 present invention exemplifies the use of insulin, immunoglobulin G, ant~ibody against human pancreatic tumor cells, and antibody against 14 hepatocytes as targeting agents. Insulin ;s an example of a serum hormone while the rest are examples of cell-surface directed antibodies.
16 The therapeutic agent in the conjugates is chosen from the 17 known drugs and enzymes used in the treatment of disease in mammals.
18 The conjugates of the present invention are illustrated with three 19 examples of therapeutic agents, ~-1,4-glucosidase9 superoxide dismutase, and L-asparaginase. Each of these enzymes is chem;cally linked to 21 carrier albumin and one of the above-listed target;ng agents to 22 demonstrate the targetability and effectiveness of the conjugates.
23 A deficiency of the a~l,4-glucosidase enzyme in humans is 24 known as Pompe's disease or Type II glycogen-storage disease. The deficiency causes death, usually by cardio-respiratory failure, before 26 the age of 2 years. The absence of this enzyme results in the intra-27 cellular accumulation of glycogen in what are believed to be lysosomes in 28 the liver and in both respiratory and cardiac muscle. The conjugates 29 of the present invention with ~-1,4-glucosidase are shown to retain the enzyme activity after cross-linking to the albumin carr;er and the 31 targeting agent. The conjugates are also shown to be non-immunogenic.
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1 Further, the conjugates are shown to greatly enhance the amount of the 2 ~-1,4-glucosidase enzyme delivered to muscle cells.
, 3 As mentioned previously, L-asparaginase has the potential , .
4 of being an important therapeutic agent against Acute Lymphocytic Leukemia. Conjugates of L-asparaginase, albumin and targeting agent 6 are shown to be non-immunogenic, biologically active and targetable to 7 specific cell types~ for example human pancreatic tumor cells.
8 Superoxide dismutase (SOD) may be used as a therapeutic g agent in the treatment of rheumatoid arthritis where it may act to lo reduce superoxide free radicals. The conjugate of SOD, albumin and 1l antibodies against hyaluronic acid may be targeted to joint tissue known 12 to be susceptible to this Form of arthritis.
The process used to chemically link the therapeutic agent to the carrier albumin and in turn to the targeting agent tlepends on the functional groups of the particular therapeutic agent and 16 targeting agent. The linking process should be chosen to preserve water solubility of the final conjugate product, to preserve the 18 activity of the therapeutic agent, and also to preserye the site 19 specific binding capabilîty of the targeting agent. In most cases the l;nking process utilizes a cross-linking agent between the therapeutic 21 agent and the carrier albumin and between the carrier albumin and the 22 targeting agent. Glutaraldehyde, sodium periodate and water soluble 23 carbodiimide are exemplified as suitable cross-linking agents in the 24 specific conjugates illustrated herein.
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1 Broadly stated the invention provides a novel composition 2 oF matter, comprising~ in water soluble, sterile and non-immunogen;c 3 form, a therapeutic agent chemically linked to an albumin carrier, 4 and a targeting agent chemically linked to the albumin, the amount of albumin beîng sufficient to mask the antigenicity of the therapeutic 6 agent, and the targeting agent having binding specificity for receptor 7 sites on cells against which it is desirable to direct the therapeutic 8 agent.
g The present invention also provides a process for producing a water soluble, sterile and non-immunogenic conjugate of 11 a therapeutic agent with an albumin carrier and a targeting agent, 12 comprising the steps of: (a) chemically linking the th0rapeutic 13 agent with an amount of the albumin carrier sufficient to mask the antigenicity of the therapeutic agent; (b) chemically linking the resulting complex of the therapeutic agent and the albumin with a 16 targeting agent having binding speci-Ficity for recptor sites on cells 17 against which it is desirable to direct the therapeutic agent~ said 18 binding specificity being retained after the linking reaction.
1 :i6~5Q
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2 The three component conjugates ~f the present in~ention 3 are useful in delivering therapeu~ic agents to specific body 4 cells, t.issues or organs in mammals for the treatment of disease.
The therapeutic agents which can be delivered in this form are usually those drugs and enzymes for which the characteristics of 7 avoiding immunological reactivity by antigenicity masking and site ~ specific targeting are desired. Exemplary drugs include known 9 alkylating agents, antibiotics, antiparasitic agents and chelating agents. The enzymes useful in the conjugates include th~se enzymes 11 causitive of enzyme deficiency diseases and those enzymes ;ntended 12 for enzyme therapy. A partial list of enzyme deficiency diseases 13 together with the enzyme responsible for the deficiency is included in 14 Table I. Exemplary enzymes for enzyme therapy purposes include L-asparaginase, uricase and superoxide dismutase. Persons skilled 16 in the art will know of other therapeutic agents which would be 17 desirably conjugated in accordance with the present invention.
3 Disease Enzyme .
4 Acatalasia Catalase Albinism Tyrosinase 6 Alcaptonuria Homogentisic Acid Oxidase 7 Cholesteryl Ester Deficiency Lecithin Cholesterol Acyltransferase 8 Cystathioninuria Cystathionase 9 Disaccharide Intolerance III Lactase Fructose Intolerance Fructose-l-Phosphate Aldolase 11 Fructosuria Hepatic Fructokinase 12 Galactosemia Galactose-l-Phosphate Uridyl Transferase 13 Gangliosidosis (GMl) ~-Galactosidase A, B, C, 14 Gaucher's Disease Glucocerebrosidase ~6PD Deficiency Glucose-6-Phosphate Dehydrogenase 16 Glycogen Storage Disease I Glucose-6-Phosphatase 17 Glycogen Storage Disease II a-l ,4-Glucosidase 18 Glycogen Storage Disease III Amylo-1,6-Glucosidase 19 Glycogen Storage Disease V Muscle Phosphorylase Glycogen Storage Disease VI Liver Phosphory1ase 21 Glycogen Storage Disease VII Muscle Phosphofructokinase 22 Glycogen Storage Disease VIII Liver Phosphorylase Kinase 23 Hemolytic Anemia Glucose-6-Phosphate Dehydrogenase 24 Hemolytic Anemia Phosphoglycerate Kinase Hemolytic Anemia Pyruvate Kinase 26 Histidinemia Histidase 27 Homocytinuria I Cystathionine Synthetase 28 Hydroxyprolinemia Hydroxyproline Oxidase 29 Hyperlipoproteinemia II Lipoprotein Lipase Hyperlysinemia Lysine-Ketoglutarate Reductase I ~ 6 ~ ~ 5 ~
1 TABLE I (Continued) 2 _isease_ Enzyme 3 Hypoglycemia (Acidosis) Fructose-1,6-Diphosphatase 4 Immunodeficiency Disease Adenosine Deaminase Intestinal Lactase Deficiency Lactase 6 Krabbe's Disease A ~-Galactosidase 7 Lesch~Nyhan Syndrome Hypoxanthine-Guanine Phosphoribosyl 8 Transferase 9 Mannosidosis ~-Mannosidase Maple Sugar Urine Disease Keto Acid Decarboxylase 11 Metachromatic Leu.kodystrophy ArylsulPatase A
12 Muoopolysaccharidosis I a-L Iduronidase 13 Mucopolysaccharidosis III Heparin Sulphate Sulphatase 14 Mucopolysaccharidosis VI Arylsulfatase B
Mucopolysaccharidosis VII ~-Glucoronidase 16 Niemann Pick Disease Sphingomyelinase 17 Orotic Aciduria II Orotidylic Decarboxylase 18 Pentosuria L-Xylulose Reductase 19 Phenylketonuria Phenylalanine Decarboxylase Pyruvate Carboxylase Def. Pyruvate Carboxylase 21 Richner-Hanhart Syndrome Tyrosine Aminotransferase 22 Sandhoff's Disease Hexosaminidase A, B
23 Tay-Sachs Disease Hexosaminidase A
24 Tyrosinem~a Tyrosine Transaminase Xanth;nuria Xanthine Oxidase l l S8 ~
1 The albumin carrier is included in the conjugate in a molar 2 excess to the therapeutic agent in order to mask the ant;genicity of 3 the therapeutic agent. Homologous albumin is most preferably used in 4 the conjugate so as not to cause the conjugate to triyger an immune response ;n the recipient mammal. Heterologous albumin may be used only 6 if it does not illicit a pronounced immunological response in the 7 recipient mammal.
8 The particular targeting agent included in the conjugate 9 is one which has binding specificity for specific receptor sites on cells against which it is desirable to direct the therapeutic 11 agent. The targeting agent is selected from serum hormones, cell-12 surface directed antibodies and lectins which are known to have receptor 13 sites on specific body cells. Exemplary of suitable targeting agents 14 are insulin, glucagon, epidermal growth factor, low-density lipoprotein, human chorionic gonadrotropin, thyroid stimulating hormone, 16 asialoglycoproteins, mannosyl-term;nal glycoproteins, endorphins, 17 enkephalins, transferrin, melanotropin, cell-surface directed anti-18 bodies (e.g. antibodies against tumor specific antigens , cell surface 19 antigens~, cell surface receptors), human growth hormone, a 2-macroglobulin, melanotropin, plant and human lectins (e.g. peanut lectin, 21 wheat germ lectin, concanavilin A, protein A), and galactose term;nal 22 glycoproteins. The amount of targeting agent included in the conjugate 23 has not been found to be critical; however, a molar excess of the 24 targeting agent to the therapeutic agent has been found to improve the delivery of the conjugate to the desired cells.
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1 To prepare the thearpeutic agent - albumin-targeting agent 2 conjugates, the therapeutic agent is first chemically linked to the 3 molar excess of albumin. The result;ng theYapeut;c agent-album;n 4 conjugate ;s thereafter chemically linked to the preferred molar excess of targeting agent. This order oF linking is preFerably used so that 6 the binding sites of the targeting agent rema~n substantially clear 7 for binding to receptor sites.
8 The functionality of the therapeutic agent and targeting g agent usually require that a cross-linking agent be used for each of the chemical linking steps. The particular cross-linking agent chosen 11 will of course depend on the functional;ty of the specific components 12 being linked. The cross-linking agents will usually utilize carboxyl 13 groups, amino groups, sulfhydral groups or sugar residues on one or 14 both of the components to be linked.
A large number of cross-linking agents are known, see 16 for ex~mple the previously referenced review article by Poznansky and 17 Cleland (1980). A partial list of suitable cross-linking agents includes 18 glutaraldehyde, water-soluble carbodiimides, sodium periodate 19 (periodate oxidation), dithiothreotol (disulfide reduction), diisocyanate, cyanuric chloride, mixed anhydrides, imidoesters, bisdiazobenzidine, 21 cyanogen bromlde, p, p'-difluoro-m, m'-dinitrophenyl sulphone, N-22 succininidyl-4~iodoacetylaminobenzoate, and diazonium salts.
23 The conditions for the cross-link;ng reaction, for 24 example pH, temperature and degree of cross-linking are chosen such that the biological activity of the therapeutic agent, the binding 26 specificity of the targeting agent and the water solubility of the final 27 conjugate are maintained. Each of these characteristics of the final 28 conjugate can be tested for and the cross-linking conditions adjusted 29 accordingly by persons skilled in the art.
I 3 &~3 1 5 ~3 1 The conjugates of the present invention are utilized in a 2 sterile form to treat diseases in warm blooded mammals. To that end 3 the conjugate is injected intramuscularly, subcutaneously, intravenously,4 intraperitoneally, intracranially or intradermally, depending on the desired site of action, into the recipient patient. Alternatively 6 there may be topical applications oF~:the con~ugates. The dosage used 7 will be dependent on such factors as the type and severity of the 8 disease, the size and species of the recipient patient, the toxicity 9 of the therapeutic agent and the degree of targeting attained by the conjugate. The dosage can therefore be worked out by routine experiments 1l with each of the conjugates.
12 The present invention is exemplified by the following 13 specific embodiments which are meant to be merely illustrative and not 14 limitative of the invention.
Example 1 16 L-Aparaginase-Albumin-Insulin Conjugate Cross-Linked with Glutaraldehyde 17 and Carbodiimide 18 L-Asparaginase (5 mg) obtained from E. co~ was chemically 19 linked to homologous albumin (25 mg3 obtained from mouse or human by react~on with 50 ~ 1 of 25% glutaraldehyde in 4 ml of phosphate 21 buffered saline (0.1 M potassium phosphate pH 6.8). The reaction 22 was performed at 4C for 4 hours in the presence of asparagine (5 mg) 23 in order to protect the active site of the enzyme. The reaction was 24 halted with the addition of glycine (50 mg). The product was then separated from the unreacted monomeric components by dialysis, pressure 26 ultrafiltration or molecular sieve chromatography. The isolated product 27 was then cross-linked to bovine or porcine (15 mg) insulin by reacting 28 the same with water soluble 1-ethyl-3-(3-dimethylaminopropyl) I 1 6S ~ 5 ~
1 carbodiimide HCl ~ECDI - 10 mg~ at 4C for 2 hours. The end product 2 was separated and purified by molecular sieve ch w matography ~hich 3 ind~cated that the final product had a molecular ~eight ranging from 4 9 x 105 to 1.4 x 106 The calculated mole ratio-was 1.5:15:90, L-asparaginase:albumin:insulin.
6 The resulting L-asparaginase-album;n-insulin conjugate was 7 assayed for enzyme activity in accordance with the technique of Mashburn 8 and Wriston, Arch. Biochem. Biophys., 1964~ 105 , pg. 450 452. The g product (enzyme:albumin:insulin = l:lO:Ç0, based on starting quantities) was found to retain about 70% of the starting enzyme activity, as 11 reported in Table II. This represen~s a significant amount of enzyme 12 activity since, as will be shown below, the enzyme is now in a 13 protected form.
14 To demonstrate the resistance of the product conjugate to proteolytic inactivation, equal amounts of the enzyme in free and 16 conjugated form were incubated with 5 units of trypsin (Sigma Chemical, 17 St. Louis, Mic.). The enzyme activity monitored as a function of time 18 is reported in Table III. The results show that the enzyme conjugated 19 in accordance with this invention was much more resistant to b;oinactivation than was the free enzyme.
21 To test the binding specificity of the enzyme conjugate, 22 the-end product was labelled covalently with 125Iodine and then incubated 23 with mouse spleen cells. The % b;nd;ng of the enzyme was determined 24 after 20 minutes at 24C in accordance with the procedure of J. R. Gavin, et al., Proc. Natl. Acad. Sci., U.S.A., 71, (1974), 84 - 90. The 26 results, as reported in Table IV, show that the enzyme conjugate binds 27 to the spleen cells, which are known to possess insulin receptors, to 28 a much greater extent than does the free L-asparaginase or the L-29 asparaginase-albumin conjugate. The insulin is therefore shown to be an effective targeting agent. Conjugates having insulin to albumin ~ 5 ~
1 molar ratios ranging from 1:1 to 1:10 (data is given for 1:6) were 2 found to retain the binding qualities of the insulin.
3 The L-asparaginase-albumin conjugates (absent the -targetlng 4 agent~ were shown to be non-immunogPnic in both tissue culture and whole animal experiments in accordance w;th techniques reported by Remy and 6 Poznansky, The Lancet ii, Jul~ 8, 1978, pg. 68 - 70. The addition of 7 insulin to the conjugates does not affect the immunogenicity of the 8 of the resulting conjugate. The immune response to the free L-9 asparaginase enzyme is compared to the immune response from the L-asparaginase-albumin conjugates in Table V~ The L-asparaginase-albumin 11 conjugates tested had ratios of enzyme to albumin ranging from 1:5 12 to 1:20. As indicated in the table a molar excess of 10:1 albumin to 13 enzyme is sufficient to mask any antigenicity of the enzyme.
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~ ~ ~ 8 1 5 3 % Activity 4 Enzyme Preparation Enzyme Activ;ty Retained L-asparag;nase 400 units~mg**
6 L-asparag;nase-albumin 310 units/mg** 77.5 7 L-asparaginase-albumin-IgG 290 units/mg** 72.5 8 L-asparaginase-albumin-(Fab')2 . 301 units/mg** 69.8 - 9 L-asparaginase-albumin-insulin 280 units/mg** 70.0 ~-1,4-glucos;dase 8.5 units/mg***
11 ~-1,4-glucosldase-albumin 6.6 units/mg*** 77.6 12 ~-1,4-glucosidase-albumin-IgG 6.0 units/mg*** 70.5 13 ~-1,4-glucos;dase-albumin-insulin 5.8 un;ts/mg*** 68.2 :
.
14 * % activity retained calculated on a per mg enzyme basis ** E.CoR~ L-asparaginase assayed as per (Mashburn & Wriston, 1964, 16 Arch. Biochem. Biophys. 105, 450 - 452.
17 *** 1, 4-glucosidase assayed as per (de Barsy et al., 1972, Eur. J.
18 Biochem. 31, 156-165) Data is for ~ 4-glucosidase from human 19 placenta.
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3 .. .~ - T 1/2 at 37C ~ *
4 Enzyme Preparat;on 5 Un;ts Trypsin L-asparaginase 10 min.
6 L-asparaginase-albumin-insulin 120 m;n.
7 * Equal amounts of enzyme in free and polymeric form were incubated 8 wi~h 5 units of Trypsin (Sigma Chemical, St. Louis MI~ and the enzyme 9 act;vity was monitored as a functiorl of time as per Tabl~e II.
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B8~50 1 Example 2 2 L-Asparaginase-Albumin-Insulin Conjugate~ Cros~-Linked with Glutaraldehyde 3 Following the procedure oF Example 1, L-asparaginase waslinked to homologous albumin with the glutaraldehyde cross-linking agPnt. The resulting enzyme-albumin complex was then cross-linked to 6 insulin using the same glutaraldehyde cross-linking conditions. A
7 molar ra~io of 1:14:60 of L-asparaginase:albumin:insulin was used. The 8 separated conjugate had a molecular weight of 1.2 x 106 Daltons.
g The physiological properties of this conjugate were very1~ similar to those of the conjugates produced in accordance with Example 1.
: .
~ 11 Example 3 ;
12 ~-1,4-Glucosidase-Albumin-Insulin Conjugate Cross-Linked with Glutaraldehyde 13 or Carbodiimide Human placental ~-1,4-glucosidase (2 mg, 150 units) was cross-linked with human albumin (20 mg) using 5 ~g of glutaraldehyde.
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16 The reaction conditions of Example 1 were maintained except that the enzyme substrate included was p-nitrophenyl glucoside. The resulting 18 enzyme-album;n complex was then cross-linked`to insulin (2 mg) us;ng g either glutaraldehyde or ECDI, again in accordance with the conditions of Example 1. The separated end product had a molecular weight of 21 1 x 106 Daltons when a molar ratio of 1:12:12 of en y me:albumin:insulin 22 was used. Some properties~of these conjugates are shown in Tables II, 28 IV and V.
24 The end produc~ was tested for enzyme activity in accordance with the procedure of de Barsy et al., Eur. J. Biochem.,31, 1972, 26 pg. 156 - 165. As indicated in Table II, the conjugate retained 27 enzyme act~vity against an artificial substrate p-nitrophenyl glucose, 28 against maltose and against its natural substrate human glycogen 29 (from liver).
1 1 ~ 8 1 5 ~
1 The binding specificity of the enzyme conjugate was tested 2 by covalently labell;ng the conjugate ~ith ~25Iod;ne and then incubating the conjugate with mouse spleen cells cr chick em~ryonic muscle cells 4 ;n accordance with the procedure ind;cated in Example 1. The data ;n Table IV ;llustrates the bindin~ of the enzyme-albumin-insulin conjugate ~ to cells known to be high in insulin receptor activity. By subcelluler -~ 7 fractionation of the tissue, ;t was found that the conjuga-te had been 8 internalized by the cell and was associated with a lysosomal fraction.
g The enzyme can be located within the cell in a fraction rich in acid phosphatase act;vity known to be contained within lysosomes. Thus 1l the insulin targeting agent was shown not to interfere with the abllity .
`;~ 12 of the body cell to utilize the ~-1,4-glucosidase therapeutic agent.
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. .
i ~ 6 ~ ~ 5 2 P~EFERENTIAL 3INDING OF INSULIN-CONJUGATED
3 ENZYME-AL3UMrN CONJUGATES TO SPLEEN CELLS
.
Spleen Cells Muscle Cells*
6 Enzyme Preparation % Uptake 7 L-~sparaginase 0.81 8 L-Qsparaginase-Albumin Z.78 `~ g L-Asparaginase-Albumin-Insulin 22.70 " ., ~-1,4-Glucosidase 6.5 8.7 11 ~-1,4-Glucosidase-Albumin 6.9 9.0 12 ~ 4-Glucosidase-Albumin-Insulin 28.1 3U.l 13 * Enzyme preparations were labelled covalently with 125Iodine and 14 then co-incubated with either mouse spleen cells or chick embryonic muscle cells an~ the % binding of the enzyme determined after 20 16 min. at 24C.
~ 5 2 IMMUNOGENICITY OF ENZYME AND ENZrME-ALBUMIN* CONJUGhTES
3 Enzyme Preparation _mmunogenicity**
4 L-Asparaginase*** ~+~
L-Asparaginase-Albumin (1:5) 6 L-Asparaginase-Albumin (1:10) 7 L-Asparaginase-Albumin (1:20) 8 ~-1,4-Glucosidase**** ~+
g ~-1,4-Glucosidase-Albumin (1:10) ~-1,4-Glucosidase-Albumin-Insulin (1:10:60) 11 * Homologous albumin is used in all cases: rabbit albumin if 12 immuno~enicity testing is to be performed in rabbits and mouse 13 albumin if testing is performed in mice. Testing is as described 14 (Remy & Poznansky, The Lancet, ii, (1978), 68 - 70).
** Immunogenicity scored on radioimmunoassay as - no reaction, 16 ~ slight reaction to **~ strong reaction.
17 *** L-asparaginase is from E. co~.
18 **** ~-1,4-Glucosidase is from human placenta 1 ~ 6 ~ ~ 5 1 Example 4 2 L-Asparaginase-Albumin-IgG Conjugate Cross-Linked with Glutaraldehyde 3 and Sodium Periodate 4 L-asparag~nase-albumin conjugates were prepared using glutaraldehyde as a cross-linking agent and the conditions set forth 6 in Example 1 ~2.5 mg L-asparaginase~ 20 mg ~lbumin, 5 ~g glutaraldehyde 7 and 5 mg L-asparagine). The resulting conjugate was then cross-linked 8 to a monoclonal antibody (5.0 mg of anti-H-2k, an antibody against the g mouse histocompatibility antigen H-2k, prepared according to Kennett, R. H. et al. (1980) "Monoclonal Antibody Hybridomas: A New Dimension 11 in Biological Analyses. Plenum Press, New York) using 5 mg of sodium 12 periodate for 2 hours at 4C. The cross-linking procedure utilizes a sugar residue on the Fc fragment oF the antibody and the amino groups on the enzyme-albumin conjugate. The resultant conjugate was separated as described in Example 1 and was found to have a^molecular weight of 16 1.1 x 106 Daltons and a calculated mole ratio of 1:10:2, L-17 asparaginase:albumin:anti-H-2k antibody.
18 Using methods similar to those described in Example 19 the 19 resulting conjuyate was found to retain enzyme activity (see Table II) toward the substrate L-asparagine. Two types of experiments indicate 21 that the conjugate binds preferentially to cells which possess the 22 corresponding H-2k antigen but not to cells which possess a different 23 histocompatibility antigen, the H-2d antigen. When 1 ~g of conjugate 24 (labelled wlth 125I) is incubated w;th 5 x 106 Balb/Ccr spleen cells which contain the H-2d antigen less than 2% of the conjugate binds to 26 the cells. When the same amount of conjugate is incubated with the same 27 number of spleen cells from C3H mice which contain the H-2k antigen 28 45% of the conjugate was found bound to the cells after a 2 hour incubation 29 at 18C. This exper;ment is an in-vitro experiment where the cells were grown in a tissue culture flask. Table VI demonstrates that the ~ ~8~5~
1 preferential binding of the targeted conjugates persists in whole 2 animal experiments. A significant increase in the retent;on of 125I~
3 labelled conjugate is observed ~hen the conjugate (containing the anti-H-4 2k antibody) is injected ;nto Balb/Ccr m;ce (wh;ch possess the H-2d antigen) which have been innoculated with tumor cells (RC3HED cells 6 which possess the H-2k antigen). Th;s suggests strongly that the 7 conjugate is binding preferentially to the tumor cells in the whole 8 animal experiment. The use of the histocompatibility ant;gen (H-2d 9 and H-2k in mice) presents a convenient cell surface antigen commonly found in varying mouse strains. The analagous use of human histo-11 compatibility antigens as cell surface targets (i.e. using the cor-12 responding antibody) might be expected to be useful if this provides 13 a proper target for a given enzyme or drug therapy.
~ 3 ~
1 TABLE VI.
3% 125I-Labelled Enzyme Remaining 4 15 h 24 h 48 h Free L~Asparaginase . 9% 4% 3%
6 L-Asparaginase-Albumin 41% 17% 11%
7 L-Asparaginase-Albumin-Anti-8 H-2k Antibody 74% 51% 37%
9Balb/Ccr mice possessing the H-2d antigen were injected with C63HED
tumor cells which possess the H-2k antigen. 125I-labelled enzyme 11 preparations were injected intravenously ;nto the mice and the %
12 lahel remaining determined after varying time periods.
~' .
':
.
- 2~ ----~ 1 6 ~ ~ ~ 0 l Example S
2 L-Asparag;nase-Albumin-(Fab'~2 Fragment.of IgG Conjugates . . .
3 L-Asparaginase-al~umin c~njugates~ were prepared using the 4 glutaraldehyde cross-linking agent and the conditions set forth in Example 4. The resulting conjugate was then cross-linked to an equal 6 molar quantity of the (Fab')2 fragment of the monoclonal anti-H-2k 7 antibody using either sodium periodate or ECDI as the cross-linking 8 agent. Tbe resulting end conjugate had a molecular weight of about l x 9 106 Daltons. The procedure was exactly~as in Examplé 4 except~that only the (Fab' )2 fragment of the ant,i,body molecule was use~d.
ll In testing procedures similar to those of Examples 1 and 4, 12 the enzyme-albumin-(Fab')2 conjugate was found to retain both L-13 asparaginase activity (see Table II) and binding affinity to cells 14 possessing the H-2k antigen. The bind;ng specificity was shown to exist in both t;ssue culture and whole animal (mouse) exper1ments as 16 dçscribed in Example 4.
17 The IgG targeting agent used in Example 4 included both 18 the Fc fragments and the (Fab')2 fragments. The use of the ~Fab')2 l9 fragments only in the present example is preferred since conjugates of the (Fab')2 fragments retain the ligand properties of the antibody 21 for binding specifically to antigen receptors~ while the possibility 22 of the conjugate binding to the less specific Fc receptors is removed.
23 Fc receptors are found on a wider range of cell ~ypes. Further, use of 24 the (Fab')2 fragment in place of the entire IgG molecule has the added advantage o~ rendering the entire polymeric complex less immunogenic 26 because of the absence of the Fc fragment.
t. ~ V
l Example 6 2 ~-1,4-Glucosidase-Albumin-Immunoglobulin Conjuyates 3 Conjugates of ~-1,4-glucosidase and albumin were produced 4 in accordance with the conditions of Example 3. The conjugates were then chemically linked to a heterologous rabbit immunoglobulin 6 preparation that had been prepared against isolated rat hepatocytes.
7 The mole ratio was 1:10:2~ ~-1,4-glucosidase:albumin:immunoglobulin.
8 Both the ECDI or glutaraldehyde cross-link;ng condi~ions were found to 9 be successful in this linking step. The separated enzyme-albumin-immuno-gl~bul;in conjugate had a ~ ecular weight of 1.2 x 106 Daltons and was 1l composed of an average~of one molec~n e enzyme, twelve molecules albumin 12 and one and a half molecules~of antibody.
13 Testing procedures similar to those used in the previous 14 examples showed that the final conjugate retained enzymatic activity (see Table II). The complex also was shown to be taken up preferentially 16 by rat hepatocytes over other cell types in whole animal experiments 17 (see Table VII). Comparison of the data in Table VII for the ~-1,4-18 glucosidase-albumin-immunoglobulin (control) conjugate and the ~-1,4-19 glucosidase-albumin ilmmunoglobulin (anti-hepatocyte) conjugate shows that preferential uptake by hepatocytes occurs only when the immunoglobulin 21 molecule is directed against hepatocytes.
22 The prepared enzyme-albumin-immunoglobulin conjugates were 23 found to give similar results whether the intact immunoglobulin 24 molecules or only the (Fab')2 fragments were used as the targeting agent.
~ 16~.5~
,~
2 TARGETING OF ENZYME-ALgUMrN-IMMUNOGLOBULIN
. , Enzyme Preparation Hepatocyte!Kupffer Cells ~-1,4-Glucosidase 0.10 6 ~-1,4~Glucosidase-Albumin 0.17 7 ~-1,4-Glucosidase-Albumin-Immunoglobulin (Control)* 0.16 8 Immunoglobulin (Anti-Hepatocyte) 0.91 9 ~-1,4-Glucosidase-Albumin-Immunoglobulin (Anti-Hepatocyte) 1.23 ., 11 125I-labelled enzyme and enzyme conjugate preparations were injected 12 into rats at time zero . After 90% of the label had cleared from the circulation, the liver was excised. The Kup~fer cells and hepatocytes were then separated and the percent label in each fraction was determined.
,:
* Control immunoglobulin was one which was not directed against rat 16 hepatocytes.
, I ~ 3 L 5 (i 1 Example 7 2 L-Aspara~inase-Albumin-Human Pancreatic Tumor Cell Antibody Conjugates 3 L-Asparaginase was cros~s-linked with human serum albumin 4 and antibody in accordance with the pracedure set forth in Example 4 with the exception that the antibody was a monoclonal antibody directed 6 against human pancreatic tumor cells. Human pancreatic tumor cells 7 were grown in suspension culture in accordance with the techniques described by Yunis, A. A. et al., Int. J. Cancer, 19, (1977), pg. 128.
9 Monoclonal antibodies against the cells were produced as described in the Kennett reference cited in Example 4.
11 In accordance with the previous test procedures, the enzyme 12 conjugate was shown to retain enzyme activity and to be non-i~munogenic 13 and resistant to bioinactivation. These test results were similar to 14 those obtained for the L-asparaginase conjugates of Example 4. The enzyme-albumin-antibody conjugates were found to be significantly 16 more cytotoxic to human pancreatic tumor cells grown in tissue culture 17 than L-asparaginase alone, L-asparaginase linked to albumin, 18 L-asparaginase linked to albumin and a non-specific antibody or non-19 specific monoclonal antibody, or the monoclonal antibody against pancreatic tumor cell itself (see Table VIII).
21 This is an important finding s;nce there is no known 22 effect;ve treatment for cancer of the pancreas and yet Yunis and co-workers 23 in Int. J. Can.g 19, ~1977)j pg. 128 - 135, have demonstrated that human 24 pancreatic tumor cells in tissue culture are asparaginase sensitive.
- 136~0 l TABLE ~III
-2 CYTOTOXICITY OF L-ASPARA~INASE-CONJUGATES TO
3 HUMAN PANCREATIC TUMO~ CELLS G~OWN IN
4 T~SSUE CULTURE
~ ' Dose Required to Inhibit 6 Enzyme PreParation Growth for Three Days 7 L-asparaginase 0.08 Units*
8 L-asparaginase-albumin 0.02 Units*
9 L-asparaginase-albumin~antibody (contrcl) 0.03 Units*
L-asparaginase-albumin-antibody (expt.) 0.005 Units*
11 (expt. = monoclonal ant;body against 12 pancrea~ic (human) tumor cells) `' 13 A number (5 x 105) of human pancreatic tumor cells were seeded in a 14 t1ssue culture flask at time zero and the dose required to completely inhibit tumor cell growth over a period of 3 days for the different ^ 16 enzyme prepara~ions was determined. The monoclonal antihody alone was `~ 17 ineffective at inhibiting tumor cell growth at the concentration used 18 in the conjugate.
'''';
19 * Units are defined in the Mashburn paper cited in Example 1.
1 3 ~ ~ ~ S V
1 Example 8 2 Superox~de Dismutase-Album;n-Hyaluronic Acid Ant;body Conjugates 3 In a manner analogous to Example 13 glutaraldehyde was 4 used to link the enzyme superoxide dismutase (from hog liver) to albumin to antibcdies against hyaluronic acid (rabbit antisera). Using 6 a molar ratio of 1:10~1 of enzyme to albumin to antibody a conjugate 7 having a molecular weight of 1.1 x 106 was formed.
8 In test procedures analogous to those of Example 1, the 9 conjugate was found to resist bioinact;vation and to be non-immunogenic.
In addition3 the conjugate showed a high affinity for the substrate 11 hyaluronic acid.
12 The benefit of cross-linking superoxide dismutase to albumin13 has been shown previously, see Wong, Cleland and Poznansky, Agents and 14 Actions, (1980), 10, pg. 231 - 244. The present conjugates with the antibody against hyaluronic acid can be targeted against sites con-16 taining hyaluronic acid to reduce inflammation associated with rheumatoid17 arthrit;s.
18 While the present invention has been disclosed in connection19 with the preferred embodiment thereof, it should be understood that Z0 there may be other embodiments which fall within the spirit and scope 21 of the invention as defined by the following claims.
12 Sorne, but not all, of the above problems have been solved13 in the prior art by conjugating the therapeutic agent with a carrier.
14 Carriers are divided into two groups, those that cause highly specific binding to cell-surface receptors and those that do not. The former 16 type of carrier will hereinafter be referred to as a targeting agent.
17 Targeting of drugs is well docurnented, see for example 18 the revlew article by M. J. Poznansky and L. G. Cleland in Drwg 19 Delivery Systems, ed. Julianog Oxford University Press, New York, 1980, pg. 253 - 315. Targeting agents are molecules, frequently bio-21 logical macromolecules, which bind to specific receptor sites on the sur-22 faces of body cells. Known targeting agents include serum hormones, 23 antibodies against cell surface antigens, and lectins.
24 It is also known in the prior art to protect therapeutic agents by conjugation with an appropriate carrier. Conjugates of 26 albumin and therapeutic agents are documented in the above-referenced 27 review article and in an article by M. J. Poznansky and D. Bhardwaj in 28 Canadian Journal oF Physiology and Pharmacology, 58 , 1980, pg. 322-325.
.
s .;
- ~ 3 6~15(~
1 These albumin conjugates have been shown to be both non-immunogenic and 2 non-antigenic, see for instance M. H. Remy and M. J. Poznansky, The Lancet, 3 July 8, 1978, pg. 68 - 70. The albumin ;s believed to mask the antigenic sites on the therapeutic agent such that the recip;ent recogn;zes the conjugate as self and therefore does not mount an ;mmune response. Further, 6 these albumin conjugates have been shown to be more resistant to bioin-activation than was the free therapeutic agent.
8 To my knowledge, no attempt has been made to target these g albumin-protected therapeutic agents. Prior to the present invention it was not known whether a targeting agent would be effective in targeting 11 such a large and complex molecule. Further, it was not known whether12 attachment of a targeting agent would interfereiw;th the biological 13 act;vity of the albumin-therapeutic agent conjugate.
:
I have discovered that conjugates of albumin and therapeutic 16 agent can be made targetable by chem;cally linking the conjugate to a 17 targeting agent having binding specificity for receptor sites on body 18 cells against which it is desirable to direct the therapeutic agent.
19 Known target;ng agents, including serum hormones, cell-surface directed antibodies, and lectins, are suitable for this purpose.
21 While it might have been eY~pected that the albumin carrier22 molecule would mask or interfere with the targetability.~ the-targeting 23 agent once it was l;nked to the therapeutic agent - albumin conjugate, 24 th;s was Found not to be the case. The targeting agent, linked to the conjugate, was found to reta;n its ab;lity to specifically deliver the 26 conjugate to speciF;c cellular surface receptors. Further, the targeting27 agent was Found not to interfere with the ab;lity of the body cell to 28 utilize the therapeut;c agent once the conjugate was delivered to the cell.
29 This was not a predictable property of the conjugates of the present invention.
~ ~ 6 ~
1 The albumin carrier ;s included ~n the conjugate in an 2 amount sufficient to mask the antigenici~ty of the therapeutic agent.
3 The term 'to mask the antigenic;ty' ;s meant to infer that the conjugate 4 does not illicit an immune response. The al~umin used is most preferably homologous to the mammal intended as the recipient. Since in most 6 cases the intended recipient is human, human serum albumin is preferred.
7 Other sources of albumin, for example bov;ne serum albumin and horse 8 serum albumin are useful for therapy in cattle and horses respectively.
9 As explained, the targeting agent allows for delivery of the conjugates to specific cellular surface receptors. The cho;ce of 11 targeting a~ent therefore depends on the desired site of delivery. The 12 present invention exemplifies the use of insulin, immunoglobulin G, ant~ibody against human pancreatic tumor cells, and antibody against 14 hepatocytes as targeting agents. Insulin ;s an example of a serum hormone while the rest are examples of cell-surface directed antibodies.
16 The therapeutic agent in the conjugates is chosen from the 17 known drugs and enzymes used in the treatment of disease in mammals.
18 The conjugates of the present invention are illustrated with three 19 examples of therapeutic agents, ~-1,4-glucosidase9 superoxide dismutase, and L-asparaginase. Each of these enzymes is chem;cally linked to 21 carrier albumin and one of the above-listed target;ng agents to 22 demonstrate the targetability and effectiveness of the conjugates.
23 A deficiency of the a~l,4-glucosidase enzyme in humans is 24 known as Pompe's disease or Type II glycogen-storage disease. The deficiency causes death, usually by cardio-respiratory failure, before 26 the age of 2 years. The absence of this enzyme results in the intra-27 cellular accumulation of glycogen in what are believed to be lysosomes in 28 the liver and in both respiratory and cardiac muscle. The conjugates 29 of the present invention with ~-1,4-glucosidase are shown to retain the enzyme activity after cross-linking to the albumin carr;er and the 31 targeting agent. The conjugates are also shown to be non-immunogenic.
.:
1 Further, the conjugates are shown to greatly enhance the amount of the 2 ~-1,4-glucosidase enzyme delivered to muscle cells.
, 3 As mentioned previously, L-asparaginase has the potential , .
4 of being an important therapeutic agent against Acute Lymphocytic Leukemia. Conjugates of L-asparaginase, albumin and targeting agent 6 are shown to be non-immunogenic, biologically active and targetable to 7 specific cell types~ for example human pancreatic tumor cells.
8 Superoxide dismutase (SOD) may be used as a therapeutic g agent in the treatment of rheumatoid arthritis where it may act to lo reduce superoxide free radicals. The conjugate of SOD, albumin and 1l antibodies against hyaluronic acid may be targeted to joint tissue known 12 to be susceptible to this Form of arthritis.
The process used to chemically link the therapeutic agent to the carrier albumin and in turn to the targeting agent tlepends on the functional groups of the particular therapeutic agent and 16 targeting agent. The linking process should be chosen to preserve water solubility of the final conjugate product, to preserve the 18 activity of the therapeutic agent, and also to preserye the site 19 specific binding capabilîty of the targeting agent. In most cases the l;nking process utilizes a cross-linking agent between the therapeutic 21 agent and the carrier albumin and between the carrier albumin and the 22 targeting agent. Glutaraldehyde, sodium periodate and water soluble 23 carbodiimide are exemplified as suitable cross-linking agents in the 24 specific conjugates illustrated herein.
.~
1 Broadly stated the invention provides a novel composition 2 oF matter, comprising~ in water soluble, sterile and non-immunogen;c 3 form, a therapeutic agent chemically linked to an albumin carrier, 4 and a targeting agent chemically linked to the albumin, the amount of albumin beîng sufficient to mask the antigenicity of the therapeutic 6 agent, and the targeting agent having binding specificity for receptor 7 sites on cells against which it is desirable to direct the therapeutic 8 agent.
g The present invention also provides a process for producing a water soluble, sterile and non-immunogenic conjugate of 11 a therapeutic agent with an albumin carrier and a targeting agent, 12 comprising the steps of: (a) chemically linking the th0rapeutic 13 agent with an amount of the albumin carrier sufficient to mask the antigenicity of the therapeutic agent; (b) chemically linking the resulting complex of the therapeutic agent and the albumin with a 16 targeting agent having binding speci-Ficity for recptor sites on cells 17 against which it is desirable to direct the therapeutic agent~ said 18 binding specificity being retained after the linking reaction.
1 :i6~5Q
:.
2 The three component conjugates ~f the present in~ention 3 are useful in delivering therapeu~ic agents to specific body 4 cells, t.issues or organs in mammals for the treatment of disease.
The therapeutic agents which can be delivered in this form are usually those drugs and enzymes for which the characteristics of 7 avoiding immunological reactivity by antigenicity masking and site ~ specific targeting are desired. Exemplary drugs include known 9 alkylating agents, antibiotics, antiparasitic agents and chelating agents. The enzymes useful in the conjugates include th~se enzymes 11 causitive of enzyme deficiency diseases and those enzymes ;ntended 12 for enzyme therapy. A partial list of enzyme deficiency diseases 13 together with the enzyme responsible for the deficiency is included in 14 Table I. Exemplary enzymes for enzyme therapy purposes include L-asparaginase, uricase and superoxide dismutase. Persons skilled 16 in the art will know of other therapeutic agents which would be 17 desirably conjugated in accordance with the present invention.
3 Disease Enzyme .
4 Acatalasia Catalase Albinism Tyrosinase 6 Alcaptonuria Homogentisic Acid Oxidase 7 Cholesteryl Ester Deficiency Lecithin Cholesterol Acyltransferase 8 Cystathioninuria Cystathionase 9 Disaccharide Intolerance III Lactase Fructose Intolerance Fructose-l-Phosphate Aldolase 11 Fructosuria Hepatic Fructokinase 12 Galactosemia Galactose-l-Phosphate Uridyl Transferase 13 Gangliosidosis (GMl) ~-Galactosidase A, B, C, 14 Gaucher's Disease Glucocerebrosidase ~6PD Deficiency Glucose-6-Phosphate Dehydrogenase 16 Glycogen Storage Disease I Glucose-6-Phosphatase 17 Glycogen Storage Disease II a-l ,4-Glucosidase 18 Glycogen Storage Disease III Amylo-1,6-Glucosidase 19 Glycogen Storage Disease V Muscle Phosphorylase Glycogen Storage Disease VI Liver Phosphory1ase 21 Glycogen Storage Disease VII Muscle Phosphofructokinase 22 Glycogen Storage Disease VIII Liver Phosphorylase Kinase 23 Hemolytic Anemia Glucose-6-Phosphate Dehydrogenase 24 Hemolytic Anemia Phosphoglycerate Kinase Hemolytic Anemia Pyruvate Kinase 26 Histidinemia Histidase 27 Homocytinuria I Cystathionine Synthetase 28 Hydroxyprolinemia Hydroxyproline Oxidase 29 Hyperlipoproteinemia II Lipoprotein Lipase Hyperlysinemia Lysine-Ketoglutarate Reductase I ~ 6 ~ ~ 5 ~
1 TABLE I (Continued) 2 _isease_ Enzyme 3 Hypoglycemia (Acidosis) Fructose-1,6-Diphosphatase 4 Immunodeficiency Disease Adenosine Deaminase Intestinal Lactase Deficiency Lactase 6 Krabbe's Disease A ~-Galactosidase 7 Lesch~Nyhan Syndrome Hypoxanthine-Guanine Phosphoribosyl 8 Transferase 9 Mannosidosis ~-Mannosidase Maple Sugar Urine Disease Keto Acid Decarboxylase 11 Metachromatic Leu.kodystrophy ArylsulPatase A
12 Muoopolysaccharidosis I a-L Iduronidase 13 Mucopolysaccharidosis III Heparin Sulphate Sulphatase 14 Mucopolysaccharidosis VI Arylsulfatase B
Mucopolysaccharidosis VII ~-Glucoronidase 16 Niemann Pick Disease Sphingomyelinase 17 Orotic Aciduria II Orotidylic Decarboxylase 18 Pentosuria L-Xylulose Reductase 19 Phenylketonuria Phenylalanine Decarboxylase Pyruvate Carboxylase Def. Pyruvate Carboxylase 21 Richner-Hanhart Syndrome Tyrosine Aminotransferase 22 Sandhoff's Disease Hexosaminidase A, B
23 Tay-Sachs Disease Hexosaminidase A
24 Tyrosinem~a Tyrosine Transaminase Xanth;nuria Xanthine Oxidase l l S8 ~
1 The albumin carrier is included in the conjugate in a molar 2 excess to the therapeutic agent in order to mask the ant;genicity of 3 the therapeutic agent. Homologous albumin is most preferably used in 4 the conjugate so as not to cause the conjugate to triyger an immune response ;n the recipient mammal. Heterologous albumin may be used only 6 if it does not illicit a pronounced immunological response in the 7 recipient mammal.
8 The particular targeting agent included in the conjugate 9 is one which has binding specificity for specific receptor sites on cells against which it is desirable to direct the therapeutic 11 agent. The targeting agent is selected from serum hormones, cell-12 surface directed antibodies and lectins which are known to have receptor 13 sites on specific body cells. Exemplary of suitable targeting agents 14 are insulin, glucagon, epidermal growth factor, low-density lipoprotein, human chorionic gonadrotropin, thyroid stimulating hormone, 16 asialoglycoproteins, mannosyl-term;nal glycoproteins, endorphins, 17 enkephalins, transferrin, melanotropin, cell-surface directed anti-18 bodies (e.g. antibodies against tumor specific antigens , cell surface 19 antigens~, cell surface receptors), human growth hormone, a 2-macroglobulin, melanotropin, plant and human lectins (e.g. peanut lectin, 21 wheat germ lectin, concanavilin A, protein A), and galactose term;nal 22 glycoproteins. The amount of targeting agent included in the conjugate 23 has not been found to be critical; however, a molar excess of the 24 targeting agent to the therapeutic agent has been found to improve the delivery of the conjugate to the desired cells.
:
~ ~ ~ 8~
1 To prepare the thearpeutic agent - albumin-targeting agent 2 conjugates, the therapeutic agent is first chemically linked to the 3 molar excess of albumin. The result;ng theYapeut;c agent-album;n 4 conjugate ;s thereafter chemically linked to the preferred molar excess of targeting agent. This order oF linking is preFerably used so that 6 the binding sites of the targeting agent rema~n substantially clear 7 for binding to receptor sites.
8 The functionality of the therapeutic agent and targeting g agent usually require that a cross-linking agent be used for each of the chemical linking steps. The particular cross-linking agent chosen 11 will of course depend on the functional;ty of the specific components 12 being linked. The cross-linking agents will usually utilize carboxyl 13 groups, amino groups, sulfhydral groups or sugar residues on one or 14 both of the components to be linked.
A large number of cross-linking agents are known, see 16 for ex~mple the previously referenced review article by Poznansky and 17 Cleland (1980). A partial list of suitable cross-linking agents includes 18 glutaraldehyde, water-soluble carbodiimides, sodium periodate 19 (periodate oxidation), dithiothreotol (disulfide reduction), diisocyanate, cyanuric chloride, mixed anhydrides, imidoesters, bisdiazobenzidine, 21 cyanogen bromlde, p, p'-difluoro-m, m'-dinitrophenyl sulphone, N-22 succininidyl-4~iodoacetylaminobenzoate, and diazonium salts.
23 The conditions for the cross-link;ng reaction, for 24 example pH, temperature and degree of cross-linking are chosen such that the biological activity of the therapeutic agent, the binding 26 specificity of the targeting agent and the water solubility of the final 27 conjugate are maintained. Each of these characteristics of the final 28 conjugate can be tested for and the cross-linking conditions adjusted 29 accordingly by persons skilled in the art.
I 3 &~3 1 5 ~3 1 The conjugates of the present invention are utilized in a 2 sterile form to treat diseases in warm blooded mammals. To that end 3 the conjugate is injected intramuscularly, subcutaneously, intravenously,4 intraperitoneally, intracranially or intradermally, depending on the desired site of action, into the recipient patient. Alternatively 6 there may be topical applications oF~:the con~ugates. The dosage used 7 will be dependent on such factors as the type and severity of the 8 disease, the size and species of the recipient patient, the toxicity 9 of the therapeutic agent and the degree of targeting attained by the conjugate. The dosage can therefore be worked out by routine experiments 1l with each of the conjugates.
12 The present invention is exemplified by the following 13 specific embodiments which are meant to be merely illustrative and not 14 limitative of the invention.
Example 1 16 L-Aparaginase-Albumin-Insulin Conjugate Cross-Linked with Glutaraldehyde 17 and Carbodiimide 18 L-Asparaginase (5 mg) obtained from E. co~ was chemically 19 linked to homologous albumin (25 mg3 obtained from mouse or human by react~on with 50 ~ 1 of 25% glutaraldehyde in 4 ml of phosphate 21 buffered saline (0.1 M potassium phosphate pH 6.8). The reaction 22 was performed at 4C for 4 hours in the presence of asparagine (5 mg) 23 in order to protect the active site of the enzyme. The reaction was 24 halted with the addition of glycine (50 mg). The product was then separated from the unreacted monomeric components by dialysis, pressure 26 ultrafiltration or molecular sieve chromatography. The isolated product 27 was then cross-linked to bovine or porcine (15 mg) insulin by reacting 28 the same with water soluble 1-ethyl-3-(3-dimethylaminopropyl) I 1 6S ~ 5 ~
1 carbodiimide HCl ~ECDI - 10 mg~ at 4C for 2 hours. The end product 2 was separated and purified by molecular sieve ch w matography ~hich 3 ind~cated that the final product had a molecular ~eight ranging from 4 9 x 105 to 1.4 x 106 The calculated mole ratio-was 1.5:15:90, L-asparaginase:albumin:insulin.
6 The resulting L-asparaginase-album;n-insulin conjugate was 7 assayed for enzyme activity in accordance with the technique of Mashburn 8 and Wriston, Arch. Biochem. Biophys., 1964~ 105 , pg. 450 452. The g product (enzyme:albumin:insulin = l:lO:Ç0, based on starting quantities) was found to retain about 70% of the starting enzyme activity, as 11 reported in Table II. This represen~s a significant amount of enzyme 12 activity since, as will be shown below, the enzyme is now in a 13 protected form.
14 To demonstrate the resistance of the product conjugate to proteolytic inactivation, equal amounts of the enzyme in free and 16 conjugated form were incubated with 5 units of trypsin (Sigma Chemical, 17 St. Louis, Mic.). The enzyme activity monitored as a function of time 18 is reported in Table III. The results show that the enzyme conjugated 19 in accordance with this invention was much more resistant to b;oinactivation than was the free enzyme.
21 To test the binding specificity of the enzyme conjugate, 22 the-end product was labelled covalently with 125Iodine and then incubated 23 with mouse spleen cells. The % b;nd;ng of the enzyme was determined 24 after 20 minutes at 24C in accordance with the procedure of J. R. Gavin, et al., Proc. Natl. Acad. Sci., U.S.A., 71, (1974), 84 - 90. The 26 results, as reported in Table IV, show that the enzyme conjugate binds 27 to the spleen cells, which are known to possess insulin receptors, to 28 a much greater extent than does the free L-asparaginase or the L-29 asparaginase-albumin conjugate. The insulin is therefore shown to be an effective targeting agent. Conjugates having insulin to albumin ~ 5 ~
1 molar ratios ranging from 1:1 to 1:10 (data is given for 1:6) were 2 found to retain the binding qualities of the insulin.
3 The L-asparaginase-albumin conjugates (absent the -targetlng 4 agent~ were shown to be non-immunogPnic in both tissue culture and whole animal experiments in accordance w;th techniques reported by Remy and 6 Poznansky, The Lancet ii, Jul~ 8, 1978, pg. 68 - 70. The addition of 7 insulin to the conjugates does not affect the immunogenicity of the 8 of the resulting conjugate. The immune response to the free L-9 asparaginase enzyme is compared to the immune response from the L-asparaginase-albumin conjugates in Table V~ The L-asparaginase-albumin 11 conjugates tested had ratios of enzyme to albumin ranging from 1:5 12 to 1:20. As indicated in the table a molar excess of 10:1 albumin to 13 enzyme is sufficient to mask any antigenicity of the enzyme.
, "
~ ~ ~ 8 1 5 3 % Activity 4 Enzyme Preparation Enzyme Activ;ty Retained L-asparag;nase 400 units~mg**
6 L-asparag;nase-albumin 310 units/mg** 77.5 7 L-asparaginase-albumin-IgG 290 units/mg** 72.5 8 L-asparaginase-albumin-(Fab')2 . 301 units/mg** 69.8 - 9 L-asparaginase-albumin-insulin 280 units/mg** 70.0 ~-1,4-glucos;dase 8.5 units/mg***
11 ~-1,4-glucosldase-albumin 6.6 units/mg*** 77.6 12 ~-1,4-glucosidase-albumin-IgG 6.0 units/mg*** 70.5 13 ~-1,4-glucos;dase-albumin-insulin 5.8 un;ts/mg*** 68.2 :
.
14 * % activity retained calculated on a per mg enzyme basis ** E.CoR~ L-asparaginase assayed as per (Mashburn & Wriston, 1964, 16 Arch. Biochem. Biophys. 105, 450 - 452.
17 *** 1, 4-glucosidase assayed as per (de Barsy et al., 1972, Eur. J.
18 Biochem. 31, 156-165) Data is for ~ 4-glucosidase from human 19 placenta.
', .~
~ 5 1 TABLE III.
3 .. .~ - T 1/2 at 37C ~ *
4 Enzyme Preparat;on 5 Un;ts Trypsin L-asparaginase 10 min.
6 L-asparaginase-albumin-insulin 120 m;n.
7 * Equal amounts of enzyme in free and polymeric form were incubated 8 wi~h 5 units of Trypsin (Sigma Chemical, St. Louis MI~ and the enzyme 9 act;vity was monitored as a functiorl of time as per Tabl~e II.
., .~
,.,.~
B8~50 1 Example 2 2 L-Asparaginase-Albumin-Insulin Conjugate~ Cros~-Linked with Glutaraldehyde 3 Following the procedure oF Example 1, L-asparaginase waslinked to homologous albumin with the glutaraldehyde cross-linking agPnt. The resulting enzyme-albumin complex was then cross-linked to 6 insulin using the same glutaraldehyde cross-linking conditions. A
7 molar ra~io of 1:14:60 of L-asparaginase:albumin:insulin was used. The 8 separated conjugate had a molecular weight of 1.2 x 106 Daltons.
g The physiological properties of this conjugate were very1~ similar to those of the conjugates produced in accordance with Example 1.
: .
~ 11 Example 3 ;
12 ~-1,4-Glucosidase-Albumin-Insulin Conjugate Cross-Linked with Glutaraldehyde 13 or Carbodiimide Human placental ~-1,4-glucosidase (2 mg, 150 units) was cross-linked with human albumin (20 mg) using 5 ~g of glutaraldehyde.
. ~
16 The reaction conditions of Example 1 were maintained except that the enzyme substrate included was p-nitrophenyl glucoside. The resulting 18 enzyme-album;n complex was then cross-linked`to insulin (2 mg) us;ng g either glutaraldehyde or ECDI, again in accordance with the conditions of Example 1. The separated end product had a molecular weight of 21 1 x 106 Daltons when a molar ratio of 1:12:12 of en y me:albumin:insulin 22 was used. Some properties~of these conjugates are shown in Tables II, 28 IV and V.
24 The end produc~ was tested for enzyme activity in accordance with the procedure of de Barsy et al., Eur. J. Biochem.,31, 1972, 26 pg. 156 - 165. As indicated in Table II, the conjugate retained 27 enzyme act~vity against an artificial substrate p-nitrophenyl glucose, 28 against maltose and against its natural substrate human glycogen 29 (from liver).
1 1 ~ 8 1 5 ~
1 The binding specificity of the enzyme conjugate was tested 2 by covalently labell;ng the conjugate ~ith ~25Iod;ne and then incubating the conjugate with mouse spleen cells cr chick em~ryonic muscle cells 4 ;n accordance with the procedure ind;cated in Example 1. The data ;n Table IV ;llustrates the bindin~ of the enzyme-albumin-insulin conjugate ~ to cells known to be high in insulin receptor activity. By subcelluler -~ 7 fractionation of the tissue, ;t was found that the conjuga-te had been 8 internalized by the cell and was associated with a lysosomal fraction.
g The enzyme can be located within the cell in a fraction rich in acid phosphatase act;vity known to be contained within lysosomes. Thus 1l the insulin targeting agent was shown not to interfere with the abllity .
`;~ 12 of the body cell to utilize the ~-1,4-glucosidase therapeutic agent.
'`
:..
,,~ , .
'~
. .
i ~ 6 ~ ~ 5 2 P~EFERENTIAL 3INDING OF INSULIN-CONJUGATED
3 ENZYME-AL3UMrN CONJUGATES TO SPLEEN CELLS
.
Spleen Cells Muscle Cells*
6 Enzyme Preparation % Uptake 7 L-~sparaginase 0.81 8 L-Qsparaginase-Albumin Z.78 `~ g L-Asparaginase-Albumin-Insulin 22.70 " ., ~-1,4-Glucosidase 6.5 8.7 11 ~-1,4-Glucosidase-Albumin 6.9 9.0 12 ~ 4-Glucosidase-Albumin-Insulin 28.1 3U.l 13 * Enzyme preparations were labelled covalently with 125Iodine and 14 then co-incubated with either mouse spleen cells or chick embryonic muscle cells an~ the % binding of the enzyme determined after 20 16 min. at 24C.
~ 5 2 IMMUNOGENICITY OF ENZYME AND ENZrME-ALBUMIN* CONJUGhTES
3 Enzyme Preparation _mmunogenicity**
4 L-Asparaginase*** ~+~
L-Asparaginase-Albumin (1:5) 6 L-Asparaginase-Albumin (1:10) 7 L-Asparaginase-Albumin (1:20) 8 ~-1,4-Glucosidase**** ~+
g ~-1,4-Glucosidase-Albumin (1:10) ~-1,4-Glucosidase-Albumin-Insulin (1:10:60) 11 * Homologous albumin is used in all cases: rabbit albumin if 12 immuno~enicity testing is to be performed in rabbits and mouse 13 albumin if testing is performed in mice. Testing is as described 14 (Remy & Poznansky, The Lancet, ii, (1978), 68 - 70).
** Immunogenicity scored on radioimmunoassay as - no reaction, 16 ~ slight reaction to **~ strong reaction.
17 *** L-asparaginase is from E. co~.
18 **** ~-1,4-Glucosidase is from human placenta 1 ~ 6 ~ ~ 5 1 Example 4 2 L-Asparaginase-Albumin-IgG Conjugate Cross-Linked with Glutaraldehyde 3 and Sodium Periodate 4 L-asparag~nase-albumin conjugates were prepared using glutaraldehyde as a cross-linking agent and the conditions set forth 6 in Example 1 ~2.5 mg L-asparaginase~ 20 mg ~lbumin, 5 ~g glutaraldehyde 7 and 5 mg L-asparagine). The resulting conjugate was then cross-linked 8 to a monoclonal antibody (5.0 mg of anti-H-2k, an antibody against the g mouse histocompatibility antigen H-2k, prepared according to Kennett, R. H. et al. (1980) "Monoclonal Antibody Hybridomas: A New Dimension 11 in Biological Analyses. Plenum Press, New York) using 5 mg of sodium 12 periodate for 2 hours at 4C. The cross-linking procedure utilizes a sugar residue on the Fc fragment oF the antibody and the amino groups on the enzyme-albumin conjugate. The resultant conjugate was separated as described in Example 1 and was found to have a^molecular weight of 16 1.1 x 106 Daltons and a calculated mole ratio of 1:10:2, L-17 asparaginase:albumin:anti-H-2k antibody.
18 Using methods similar to those described in Example 19 the 19 resulting conjuyate was found to retain enzyme activity (see Table II) toward the substrate L-asparagine. Two types of experiments indicate 21 that the conjugate binds preferentially to cells which possess the 22 corresponding H-2k antigen but not to cells which possess a different 23 histocompatibility antigen, the H-2d antigen. When 1 ~g of conjugate 24 (labelled wlth 125I) is incubated w;th 5 x 106 Balb/Ccr spleen cells which contain the H-2d antigen less than 2% of the conjugate binds to 26 the cells. When the same amount of conjugate is incubated with the same 27 number of spleen cells from C3H mice which contain the H-2k antigen 28 45% of the conjugate was found bound to the cells after a 2 hour incubation 29 at 18C. This exper;ment is an in-vitro experiment where the cells were grown in a tissue culture flask. Table VI demonstrates that the ~ ~8~5~
1 preferential binding of the targeted conjugates persists in whole 2 animal experiments. A significant increase in the retent;on of 125I~
3 labelled conjugate is observed ~hen the conjugate (containing the anti-H-4 2k antibody) is injected ;nto Balb/Ccr m;ce (wh;ch possess the H-2d antigen) which have been innoculated with tumor cells (RC3HED cells 6 which possess the H-2k antigen). Th;s suggests strongly that the 7 conjugate is binding preferentially to the tumor cells in the whole 8 animal experiment. The use of the histocompatibility ant;gen (H-2d 9 and H-2k in mice) presents a convenient cell surface antigen commonly found in varying mouse strains. The analagous use of human histo-11 compatibility antigens as cell surface targets (i.e. using the cor-12 responding antibody) might be expected to be useful if this provides 13 a proper target for a given enzyme or drug therapy.
~ 3 ~
1 TABLE VI.
3% 125I-Labelled Enzyme Remaining 4 15 h 24 h 48 h Free L~Asparaginase . 9% 4% 3%
6 L-Asparaginase-Albumin 41% 17% 11%
7 L-Asparaginase-Albumin-Anti-8 H-2k Antibody 74% 51% 37%
9Balb/Ccr mice possessing the H-2d antigen were injected with C63HED
tumor cells which possess the H-2k antigen. 125I-labelled enzyme 11 preparations were injected intravenously ;nto the mice and the %
12 lahel remaining determined after varying time periods.
~' .
':
.
- 2~ ----~ 1 6 ~ ~ ~ 0 l Example S
2 L-Asparag;nase-Albumin-(Fab'~2 Fragment.of IgG Conjugates . . .
3 L-Asparaginase-al~umin c~njugates~ were prepared using the 4 glutaraldehyde cross-linking agent and the conditions set forth in Example 4. The resulting conjugate was then cross-linked to an equal 6 molar quantity of the (Fab')2 fragment of the monoclonal anti-H-2k 7 antibody using either sodium periodate or ECDI as the cross-linking 8 agent. Tbe resulting end conjugate had a molecular weight of about l x 9 106 Daltons. The procedure was exactly~as in Examplé 4 except~that only the (Fab' )2 fragment of the ant,i,body molecule was use~d.
ll In testing procedures similar to those of Examples 1 and 4, 12 the enzyme-albumin-(Fab')2 conjugate was found to retain both L-13 asparaginase activity (see Table II) and binding affinity to cells 14 possessing the H-2k antigen. The bind;ng specificity was shown to exist in both t;ssue culture and whole animal (mouse) exper1ments as 16 dçscribed in Example 4.
17 The IgG targeting agent used in Example 4 included both 18 the Fc fragments and the (Fab')2 fragments. The use of the ~Fab')2 l9 fragments only in the present example is preferred since conjugates of the (Fab')2 fragments retain the ligand properties of the antibody 21 for binding specifically to antigen receptors~ while the possibility 22 of the conjugate binding to the less specific Fc receptors is removed.
23 Fc receptors are found on a wider range of cell ~ypes. Further, use of 24 the (Fab')2 fragment in place of the entire IgG molecule has the added advantage o~ rendering the entire polymeric complex less immunogenic 26 because of the absence of the Fc fragment.
t. ~ V
l Example 6 2 ~-1,4-Glucosidase-Albumin-Immunoglobulin Conjuyates 3 Conjugates of ~-1,4-glucosidase and albumin were produced 4 in accordance with the conditions of Example 3. The conjugates were then chemically linked to a heterologous rabbit immunoglobulin 6 preparation that had been prepared against isolated rat hepatocytes.
7 The mole ratio was 1:10:2~ ~-1,4-glucosidase:albumin:immunoglobulin.
8 Both the ECDI or glutaraldehyde cross-link;ng condi~ions were found to 9 be successful in this linking step. The separated enzyme-albumin-immuno-gl~bul;in conjugate had a ~ ecular weight of 1.2 x 106 Daltons and was 1l composed of an average~of one molec~n e enzyme, twelve molecules albumin 12 and one and a half molecules~of antibody.
13 Testing procedures similar to those used in the previous 14 examples showed that the final conjugate retained enzymatic activity (see Table II). The complex also was shown to be taken up preferentially 16 by rat hepatocytes over other cell types in whole animal experiments 17 (see Table VII). Comparison of the data in Table VII for the ~-1,4-18 glucosidase-albumin-immunoglobulin (control) conjugate and the ~-1,4-19 glucosidase-albumin ilmmunoglobulin (anti-hepatocyte) conjugate shows that preferential uptake by hepatocytes occurs only when the immunoglobulin 21 molecule is directed against hepatocytes.
22 The prepared enzyme-albumin-immunoglobulin conjugates were 23 found to give similar results whether the intact immunoglobulin 24 molecules or only the (Fab')2 fragments were used as the targeting agent.
~ 16~.5~
,~
2 TARGETING OF ENZYME-ALgUMrN-IMMUNOGLOBULIN
. , Enzyme Preparation Hepatocyte!Kupffer Cells ~-1,4-Glucosidase 0.10 6 ~-1,4~Glucosidase-Albumin 0.17 7 ~-1,4-Glucosidase-Albumin-Immunoglobulin (Control)* 0.16 8 Immunoglobulin (Anti-Hepatocyte) 0.91 9 ~-1,4-Glucosidase-Albumin-Immunoglobulin (Anti-Hepatocyte) 1.23 ., 11 125I-labelled enzyme and enzyme conjugate preparations were injected 12 into rats at time zero . After 90% of the label had cleared from the circulation, the liver was excised. The Kup~fer cells and hepatocytes were then separated and the percent label in each fraction was determined.
,:
* Control immunoglobulin was one which was not directed against rat 16 hepatocytes.
, I ~ 3 L 5 (i 1 Example 7 2 L-Aspara~inase-Albumin-Human Pancreatic Tumor Cell Antibody Conjugates 3 L-Asparaginase was cros~s-linked with human serum albumin 4 and antibody in accordance with the pracedure set forth in Example 4 with the exception that the antibody was a monoclonal antibody directed 6 against human pancreatic tumor cells. Human pancreatic tumor cells 7 were grown in suspension culture in accordance with the techniques described by Yunis, A. A. et al., Int. J. Cancer, 19, (1977), pg. 128.
9 Monoclonal antibodies against the cells were produced as described in the Kennett reference cited in Example 4.
11 In accordance with the previous test procedures, the enzyme 12 conjugate was shown to retain enzyme activity and to be non-i~munogenic 13 and resistant to bioinactivation. These test results were similar to 14 those obtained for the L-asparaginase conjugates of Example 4. The enzyme-albumin-antibody conjugates were found to be significantly 16 more cytotoxic to human pancreatic tumor cells grown in tissue culture 17 than L-asparaginase alone, L-asparaginase linked to albumin, 18 L-asparaginase linked to albumin and a non-specific antibody or non-19 specific monoclonal antibody, or the monoclonal antibody against pancreatic tumor cell itself (see Table VIII).
21 This is an important finding s;nce there is no known 22 effect;ve treatment for cancer of the pancreas and yet Yunis and co-workers 23 in Int. J. Can.g 19, ~1977)j pg. 128 - 135, have demonstrated that human 24 pancreatic tumor cells in tissue culture are asparaginase sensitive.
- 136~0 l TABLE ~III
-2 CYTOTOXICITY OF L-ASPARA~INASE-CONJUGATES TO
3 HUMAN PANCREATIC TUMO~ CELLS G~OWN IN
4 T~SSUE CULTURE
~ ' Dose Required to Inhibit 6 Enzyme PreParation Growth for Three Days 7 L-asparaginase 0.08 Units*
8 L-asparaginase-albumin 0.02 Units*
9 L-asparaginase-albumin~antibody (contrcl) 0.03 Units*
L-asparaginase-albumin-antibody (expt.) 0.005 Units*
11 (expt. = monoclonal ant;body against 12 pancrea~ic (human) tumor cells) `' 13 A number (5 x 105) of human pancreatic tumor cells were seeded in a 14 t1ssue culture flask at time zero and the dose required to completely inhibit tumor cell growth over a period of 3 days for the different ^ 16 enzyme prepara~ions was determined. The monoclonal antihody alone was `~ 17 ineffective at inhibiting tumor cell growth at the concentration used 18 in the conjugate.
'''';
19 * Units are defined in the Mashburn paper cited in Example 1.
1 3 ~ ~ ~ S V
1 Example 8 2 Superox~de Dismutase-Album;n-Hyaluronic Acid Ant;body Conjugates 3 In a manner analogous to Example 13 glutaraldehyde was 4 used to link the enzyme superoxide dismutase (from hog liver) to albumin to antibcdies against hyaluronic acid (rabbit antisera). Using 6 a molar ratio of 1:10~1 of enzyme to albumin to antibody a conjugate 7 having a molecular weight of 1.1 x 106 was formed.
8 In test procedures analogous to those of Example 1, the 9 conjugate was found to resist bioinact;vation and to be non-immunogenic.
In addition3 the conjugate showed a high affinity for the substrate 11 hyaluronic acid.
12 The benefit of cross-linking superoxide dismutase to albumin13 has been shown previously, see Wong, Cleland and Poznansky, Agents and 14 Actions, (1980), 10, pg. 231 - 244. The present conjugates with the antibody against hyaluronic acid can be targeted against sites con-16 taining hyaluronic acid to reduce inflammation associated with rheumatoid17 arthrit;s.
18 While the present invention has been disclosed in connection19 with the preferred embodiment thereof, it should be understood that Z0 there may be other embodiments which fall within the spirit and scope 21 of the invention as defined by the following claims.
Claims (23)
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for producing a water soluble, sterile, non-immunogenic conjugate of a therapeutic agent with an albumin carrier and a targeting agent, comprising the steps of:
(a) chemically linking the therapeutic agent with an amount of albumin carrier sufficient to mask the antigenicity of the therapeutic agent, (b) chemically linking the resulting complex of the therapeutic agent and the albumin with a targeting agent having binding specificity for receptor sites on body cells against which it is desirable to direct the therapeutic agent, said binding specificity being retained after the linking reaction.
(a) chemically linking the therapeutic agent with an amount of albumin carrier sufficient to mask the antigenicity of the therapeutic agent, (b) chemically linking the resulting complex of the therapeutic agent and the albumin with a targeting agent having binding specificity for receptor sites on body cells against which it is desirable to direct the therapeutic agent, said binding specificity being retained after the linking reaction.
2. The process as set forth in claim 1, wherein:
the targeting agent is insulin.
the targeting agent is insulin.
3. The process as set forth in claim l, wherein:
the targeting agent is an antibody directed against human pancreatic tumor cells.
the targeting agent is an antibody directed against human pancreatic tumor cells.
4. The process as set forth in claim 1, wherein:
the targeting agent is an immunoglobulin.
the targeting agent is an immunoglobulin.
5. The process as set forth in claim 1, wherein:
the targeting agent is the (Fab')2 fragment of an immuno-globulin G molecule.
the targeting agent is the (Fab')2 fragment of an immuno-globulin G molecule.
6. The process as set forth in claim 2, 3 or 4, wherein:
the therapeutic agent is an enzyme.
the therapeutic agent is an enzyme.
7. The process as set forth in claim 2, wherein:
the therapeutic agent is an enzyme selected from the group consisting of .alpha.-1,4-glucosidase and L-asparaginase; and the albumin carrier is homologous albumin
the therapeutic agent is an enzyme selected from the group consisting of .alpha.-1,4-glucosidase and L-asparaginase; and the albumin carrier is homologous albumin
8. The process as set forth in claim 4, wherein:
the therapeutic agent is an enzyme selected from the group consisting of .alpha.-1,4-glucosidase and L-asparaginase; and the albumin carrier is homologous albumin.
the therapeutic agent is an enzyme selected from the group consisting of .alpha.-1,4-glucosidase and L-asparaginase; and the albumin carrier is homologous albumin.
9. The process as set forth in claim 7, which further comprises:
linking the enzyme to the albumin with glutaraldehyde;
and linking the insulin to the enzyme-albumin complex with a cross-linking agent selected from the group consisting of glutaraldehyde and carbodiimide.
linking the enzyme to the albumin with glutaraldehyde;
and linking the insulin to the enzyme-albumin complex with a cross-linking agent selected from the group consisting of glutaraldehyde and carbodiimide.
10. The process as set forth in claim 8, which further comprises:
linking the enzyme to the albumin with glutaraldehyde;
and linking the immunoglobulin to the enzyme-albumin complex with a cross-linking agent selected from the group consisting of sodium periodate, glutaraldehyde or carbodiimide.
linking the enzyme to the albumin with glutaraldehyde;
and linking the immunoglobulin to the enzyme-albumin complex with a cross-linking agent selected from the group consisting of sodium periodate, glutaraldehyde or carbodiimide.
11. The process as set forth in claim 1, wherein:
the targeting agent is an antibody against human pancreatic tumor cells 5 the therapeutic agent is L-asparaginase, and the albumin carrier is homologous albumin.
the targeting agent is an antibody against human pancreatic tumor cells 5 the therapeutic agent is L-asparaginase, and the albumin carrier is homologous albumin.
12. The process as set forth in claim 11, which further comprises:
linking the enzyme to the albumin with glutaraldehyde;
and linking the antibody to the enzyme-albumin complex with sodium periodate.
linking the enzyme to the albumin with glutaraldehyde;
and linking the antibody to the enzyme-albumin complex with sodium periodate.
13. The process as set forth in claim 1, wherein:
the targeting agent is an antibody against hyaluronic acid;
the therapeutic agent is superoxide dismutase; and the albumin carrier is homologous albumin.
the targeting agent is an antibody against hyaluronic acid;
the therapeutic agent is superoxide dismutase; and the albumin carrier is homologous albumin.
14. The process as set forth in claim 13, which further comprises:
linking the therapeutic agent to the carrier albumin with glutaraldehyde, and linking the targeting agent to the albumin-therapeutic agent complex with glutaraldehyde.
linking the therapeutic agent to the carrier albumin with glutaraldehyde, and linking the targeting agent to the albumin-therapeutic agent complex with glutaraldehyde.
15. A composition of matter, prepared by the process of claim 1, comprising:
in water soluble, sterile and non-immunogenic form, a therapeutic agent chemically linked to an albumin carrier, and a targeting agent chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the therapeutic agent, and the targeting agent having binding specificity for receptor sites on cells against which it is desirable to direct the therapeutic agent.
in water soluble, sterile and non-immunogenic form, a therapeutic agent chemically linked to an albumin carrier, and a targeting agent chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the therapeutic agent, and the targeting agent having binding specificity for receptor sites on cells against which it is desirable to direct the therapeutic agent.
16. The composition of matter, prepared by the process of claim 2.
17. The composition of matter, prepared by the process of claim 3.
18. The composition of matter, prepared by the process of claim 4.
19. The composition of matter, prepared by the process of claim 5.
20. The composition of matter, prepared in accordance with the process of claims 7 or 9, comprising:
in water soluble, sterile and non-immunogenic form, an enzyme selected from .alpha.-1,4-glucosidase or L-asparaginase, chemically linked to a homologous albumin carrier, and an insulin targeting agent chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the enzyme.
in water soluble, sterile and non-immunogenic form, an enzyme selected from .alpha.-1,4-glucosidase or L-asparaginase, chemically linked to a homologous albumin carrier, and an insulin targeting agent chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the enzyme.
21. The composition of matter, prepared in accordance with the process of claim 8 or 10, comprising:
in water soluble, sterile and non-immunogenic form, an enzyme selected from .alpha.-1,4-glucosidase and L-asparaginase, chemically linked to a homologous albumin carrier, and an immunoglobulin targeting agent chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the enzyme.
in water soluble, sterile and non-immunogenic form, an enzyme selected from .alpha.-1,4-glucosidase and L-asparaginase, chemically linked to a homologous albumin carrier, and an immunoglobulin targeting agent chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the enzyme.
22. The composition of matter , prepared in accordance with the process of claim 11 or 12, comprising:
in water soluble, sterile and non-immunogenic form, L-asparaginase chemically linked to a homologous albumin carrier, and an antibody against human pancreatic tumor cells chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the L-asparaginase.
in water soluble, sterile and non-immunogenic form, L-asparaginase chemically linked to a homologous albumin carrier, and an antibody against human pancreatic tumor cells chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the L-asparaginase.
23 The composition of matter, prepared in accordance with the process of claim 13 or 14, comprising:
in water soluble, sterile and non-immunogenic form, superoxide dismutase chemically linked to a homologous albumin carrier, and an antibody against hyaluronic acid chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the superoxide dismutase.
in water soluble, sterile and non-immunogenic form, superoxide dismutase chemically linked to a homologous albumin carrier, and an antibody against hyaluronic acid chemically linked to the albumin carrier, the amount of albumin being sufficient to mask antigenicity of the superoxide dismutase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000392658A CA1168150A (en) | 1981-12-18 | 1981-12-18 | Targeting conjugates of albumin and therapeutic agents |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000392658A CA1168150A (en) | 1981-12-18 | 1981-12-18 | Targeting conjugates of albumin and therapeutic agents |
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| Publication Number | Publication Date |
|---|---|
| CA1168150A true CA1168150A (en) | 1984-05-29 |
Family
ID=4121650
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000392658A Expired CA1168150A (en) | 1981-12-18 | 1981-12-18 | Targeting conjugates of albumin and therapeutic agents |
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|---|---|
| CA (1) | CA1168150A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987003205A1 (en) * | 1985-11-22 | 1987-06-04 | Coral Sociedade Brasileira De Pesquisas E Desenvol | Enzyme-coupled antibodies |
| FR2603287A1 (en) * | 1986-06-02 | 1988-03-04 | Debiopharm Sa | PROCESS FOR THE PREPARATION OF METALLOPROTEIN COMPLEXES AND NEW DERIVATIVES THEREOF |
| WO1988007365A2 (en) * | 1987-04-01 | 1988-10-06 | David F Ranney | Biodadhesion drug carriers for endothelial and epithelial uptake and lesional localization of therapeutic and diagnostic agents |
| EP0302473A2 (en) * | 1987-08-04 | 1989-02-08 | Bristol-Myers Squibb Company | Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells |
| WO1992000763A1 (en) * | 1990-07-03 | 1992-01-23 | Akzo N.V. | Immunoreactive compound |
| US5108759A (en) * | 1987-04-01 | 1992-04-28 | Ranney David F | Endothelial envelopment drug carriers |
| WO1994013817A1 (en) * | 1992-12-04 | 1994-06-23 | Me Medical Enzymes Ag | Genetically engineered glutaminase and its use in antiviral and anticancer therapy |
| US5672334A (en) * | 1991-01-16 | 1997-09-30 | Access Pharmaceuticals, Inc. | Invivo agents comprising cationic metal chelators with acidic saccharides and glycosaminoglycans |
| US5707604A (en) * | 1986-11-18 | 1998-01-13 | Access Pharmaceuticals, Inc. | Vivo agents comprising metal-ion chelates with acidic saccharides and glycosaminoglycans, giving improved site-selective localization, uptake mechanism, sensitivity and kinetic-spatial profiles |
| US6106866A (en) * | 1995-07-31 | 2000-08-22 | Access Pharmaceuticals, Inc. | In vivo agents comprising cationic drugs, peptides and metal chelators with acidic saccharides and glycosaminoglycans, giving improved site-selective localization, uptake mechanism, sensitivity and kinetic-spatial profiles, including tumor sites |
| WO2002089852A1 (en) * | 2001-05-10 | 2002-11-14 | Bio Vector Solutions Limited | Soluble protein-polymer systems for drug delivery |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987003205A1 (en) * | 1985-11-22 | 1987-06-04 | Coral Sociedade Brasileira De Pesquisas E Desenvol | Enzyme-coupled antibodies |
| FR2603287A1 (en) * | 1986-06-02 | 1988-03-04 | Debiopharm Sa | PROCESS FOR THE PREPARATION OF METALLOPROTEIN COMPLEXES AND NEW DERIVATIVES THEREOF |
| BE1002130A3 (en) * | 1986-06-02 | 1990-07-24 | Debiopharm Sa | PROCESS FOR THE PREPARATION OF METALLOPROTEIN COMPLEXES AND NEW DERIVATIVES THEREOF. |
| US5707604A (en) * | 1986-11-18 | 1998-01-13 | Access Pharmaceuticals, Inc. | Vivo agents comprising metal-ion chelates with acidic saccharides and glycosaminoglycans, giving improved site-selective localization, uptake mechanism, sensitivity and kinetic-spatial profiles |
| US5108759A (en) * | 1987-04-01 | 1992-04-28 | Ranney David F | Endothelial envelopment drug carriers |
| WO1988007365A2 (en) * | 1987-04-01 | 1988-10-06 | David F Ranney | Biodadhesion drug carriers for endothelial and epithelial uptake and lesional localization of therapeutic and diagnostic agents |
| WO1988007365A3 (en) * | 1987-04-01 | 1988-11-17 | David F Ranney | Biodadhesion drug carriers for endothelial and epithelial uptake and lesional localization of therapeutic and diagnostic agents |
| US4925678A (en) * | 1987-04-01 | 1990-05-15 | Ranney David F | Endothelial envelopment drug carriers |
| AU607494B2 (en) * | 1987-04-01 | 1991-03-07 | Access Pharmaceuticals, Inc. | Biodadhesion drug carriers for endothelial and epithelial uptake and lesional localization of therapeutic and diagnostic agents |
| EP0302473A2 (en) * | 1987-08-04 | 1989-02-08 | Bristol-Myers Squibb Company | Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells |
| EP0302473A3 (en) * | 1987-08-04 | 1990-01-17 | Bristol-Myers Company | Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells |
| WO1992000763A1 (en) * | 1990-07-03 | 1992-01-23 | Akzo N.V. | Immunoreactive compound |
| US5672334A (en) * | 1991-01-16 | 1997-09-30 | Access Pharmaceuticals, Inc. | Invivo agents comprising cationic metal chelators with acidic saccharides and glycosaminoglycans |
| US7052689B2 (en) | 1991-12-04 | 2006-05-30 | Me Medical Enzymes Ag | Method for producing therapeutically suitable glutaminase |
| WO1994013817A1 (en) * | 1992-12-04 | 1994-06-23 | Me Medical Enzymes Ag | Genetically engineered glutaminase and its use in antiviral and anticancer therapy |
| US6312939B1 (en) | 1992-12-04 | 2001-11-06 | Me Medical Enzymes Ag | Genetically engineered glutaminase and its use in antiviral and anticancer therapy |
| US6106866A (en) * | 1995-07-31 | 2000-08-22 | Access Pharmaceuticals, Inc. | In vivo agents comprising cationic drugs, peptides and metal chelators with acidic saccharides and glycosaminoglycans, giving improved site-selective localization, uptake mechanism, sensitivity and kinetic-spatial profiles, including tumor sites |
| WO2002089852A1 (en) * | 2001-05-10 | 2002-11-14 | Bio Vector Solutions Limited | Soluble protein-polymer systems for drug delivery |
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