WO1990014844A2

WO1990014844A2 - Sugars as cleavable linkers for the delivery and release of agents in native form

Info

Publication number: WO1990014844A2
Application number: PCT/US1990/003008
Authority: WO
Inventors: Ananthachari Srinivasan; Vivekananda M. Vrudhula
Original assignee: Neorx Corporation
Priority date: 1989-06-06
Filing date: 1990-05-30
Publication date: 1990-12-13
Also published as: WO1990014844A3

Abstract

The present invention provides a cleavable conjugate whose linker contains a labile bond that is cleavable under a variety of mild conditions, including weakly acidic. Since the agent may be bonded directly to the linker, cleavage can result in release of native agent. The targeting molecule employed in the invention may be an intact molecule, a fragment thereof, or a functional equivalent thereof. In a particularly preferred embodiment, the targeting molecule is a monoclonal antibody directed towards a tumor-associated antigen in man. Another aspect of the invention provides a method for isolating a compound. The compound binds covalently to a solid phase which has been derivatized with the linker described above and is released in native form by a variety of mild conditions. An additional aspect of the invention provides a method for introducing into a compound a free sulfhydryl, amino, or hydroxyl group by use of a reagent structurally related to the linker described above.

Description

SUGARS AS CLEAVABLE LINKERS FOR THE DELIVERY AND RELEASE OF AGENTS IN NATIVE FORM

Technical Field

The present invention relates generally to cleavable conjugates that permit release of an agent in native form under mild conditions and to methods for making and using these conjugates.

Background of the Invention

A reoccurring problem in medicine is that, due to the lack of specificity of the agents used for treatment of illnesses, the patient is often the recipient of a new set of maladies from the therapy. This scenario is common especially in the treatment of the various forms of cancer.

An approach taken to circumvent the nonspecificity of the agents used to treat diseases is to couple an agent to a carrier that possesses some degree of specificity. A number of molecules have been utilized as carriers in agent delivery systems, but with limited success. Carrier molecules such as liposomes, proteins, and polyclonal antibodies have been used in conjunction with a broad spectrum of pharmaceutical or cytotoxic agents including radioactive compounds, agents which bind DNA, antimetabolites, agents which act on cell surfaces, and protein synthesis inhibitors.

With the discovery of a method to isolate antibodies with a single specificity, i.e., monoclonal antibodies (MAbs), came the hope that agents could now be delivered to selected cells via "immunoconjugates." "Immunoconjugates" are covalently bonded hybrid molecules composed of a recognition portion, such as an antibody molecule, an antibody fragment, or a functional equivalent thereof, and a biologically active portion, such as a toxin, toxin fragment, a drug, a biological response modifier, or a radioisotope. Immunoconjugates have enormous potential as potent anti-tumor agents, due to the selectivity imparted to the hybrid molecules by the antibody portion of the immunoconjugate. The exquisite selectivity of antibodies or antibody fragments permits delivery of increased doses of cytotoxic, inhibitory or radiolabeled moieties to a defined population of cells.

Although the MAb carrier systems have gone far to solve the cell-specificity problem, other problems remain. In particular, the design of the compound used to link the agent to the MAb is important. First, where the agent is only active, or at least more potent, when free from the MAb carrier, the linker needs to be cleavable in order to release the agent. Second, where the agent is only active, or at least more potent, when none of the linker remains attached following the cleavage, the labile bond must be the one formed between the linker and the agent. Third, the type of labile bond used should be chosen on the basis of the location, i.e., inside or outside a cell, of the release-inducing factor.

A number of different cleavable linker groups have been described previously. The mechanisms for release of an agent from these linker groups include cleavage by reduction of a disulfide bond, by irradiation of a photo- labile bond, by hydrolysis of derivatized amino acid side chains, by serum complement-mediated hydrolysis, and acid- catalyzed hydrolysis. Some of these mechanisms are susceptible to release of the agent prior to having reached the specific cell, tissue or organ. Other of these mechanisms will provide faithful external delivery; however, they are inappropriate where the actual target site of the agent is inside a cell. Where an agent activates or inactivates a cell by binding to an intracellular component, the carrier- agent conjugate must be internalized and then the agent released.

A way to achieve internalization of a carrieragent conjugate is to take advantage of a cell's receptor- mediated endocytosis pathway. Antibodies are one example of a carrier that will bind to cell surface receptors and be internalized. Receptors which are internalized by receptor-mediated endocytosis pass through acidified compartments known as endosomes or receptosomes. Thus, the carrier-agent conjugate will be exposed transiently to an acidic pH.

Blattler et al., in U.S. Patent No. 4,569,789, describe a drug delivery system which is formed by reaction of an active substance with a maleic anhydride moiety. The active substance is released upon cleavage of the amide bond. The patent purports that cleavage occurs under mildly acidic conditions, yet the patent discloses that at pH 5 only about 15% is cleaved after five hours. Even at pH 4 for five hours, less than 50% is cleaved.

Thus, there is a need in the art for a carrier- agent conjugate which releases the agent by cleavage under mild conditions. The present invention fulfills this need and further provides other related advantages.

Summary of the Invention

Briefly stated, the invention in one aspect provides a cleavable conjugate. The conjugate has the formula (I):

where:

T is a targeting molecule;

W is a methylene, methylenoxy, or methylene- carbonyl group or combinations thereof;

n is 0 to 10; Q₁, Q₂ and Q₃ are independently selected from H, OH, O-alkyl, O-acyl, and derivatives thereof, with the proviso that Q₁, Q₂ and Q₃ are not all H;

n' is 1 to 2;

X is an H, alkyl group of C₆ or less, or alkoxy group of C₆ or less; and

Z is an agent.

In another aspect, the present invention provides a method for delivering to the cytoplasm of a target cell an agent free of its targeting molecule carrier. The method comprises the step of administering to a warmblooded animal a diagnostically or therapeutically effective dose of a cleavable conjugate having the formula

(I):

where:

T is a targeting molecule;

n is 0 to 10;

Q₁, Q₂ and Q₃ are independently selected from H, OH, O-alkyl, O-acyl, and derivatives thereof, with the proviso that Q₁, Q₂ and Q₃ are not all H;

n' is 1 to 2;

X is an H, alkyl group of C₆ or less, or alkoxy group of _C6 or less; and

Z is an agent.

Upon delivery of the conjugate to a target cell, the bond joining the agent to the conjugate is cleaved, thereby releasing the agent. The bond is cleavable under a variety of conditions, including mildly acidic conditions, and is accelerated by heat. Since the agent may be bonded directly to the linker, cleavage can result in release of native agent.

A related aspect of the present invention provides a method for isolating a compound. The method comprises the steps of conjugating to a solid phase, via R, a reagent having the formula (II):

where:

R' is a chemically reactive group;

W is a methylene, methylenoxy, or methylene- σarbonyl group or combinations thereof;

n is 0 to 30;

Q₁, _Q2 and Q₃ are independently selected from H,

OH, O-alkyl, O-acyl, and derivatives thereof, with the proviso that Q₁, Q₂ and Q₃ are not all H;

n' is 1 to 2;

X is an H, alkyl group of C₆ or less, or alkoxy group of C₆ or less; and

R is a chemically reactive group, with the provisos that R is attached directly or indirectly to one of the carbons designated α, β or Y, that when R is attached to α then Q₁ is H, that when R is attached to ß then Q₂ is H, that when R is attached to γ then Q₃ is H, and that R and R' are not the same.

Thus, a derivatized solid phase is formed. The derivatized solid phase is contacted with a sample solution in which a compound containing a group capable of reacting with R' is present. The compound binds to the derivatized solid phase, thereby removing the compound from the sample solution. The bound compound is released from the derivatized solid phase by a variety of conditions, including mildly acidic conditions, and heat accelerates the reaction.

In yet another aspect, the present invention provides a method for introducing into a compound a free sulfhydryl, free amino, or free hydroxyl group. The method comprises the steps of reacting a compound with a reagent having the formula (III):

where:

R is a chemically reactive group;

n is 0 to 30;

Y is S, N, or 0;

X is H, alkyl group of C₆ or less, or alkoxy group of C₆ or less;

n' is 1 to 2; and

to form a reagent-linked compound. The reagent-linked compound is cleaved at the bond between Y and the ring. Depending upon whether Y is an S, 0, or N, a free sulfhydryl, free hydroxyl, or free amino group, respectively, will be added to the compound. The cleavage occurs under a variety of conditions, including mildly acidic conditions, and heat accelerates the reaction.

Other aspects of the invention will become evident upon reference to the following detailed description. Detailed Description of the Invention

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter.

Targeting molecule - any molecule, i.e., a protein or a non-protein, that has the capacity to bind to a defined population of cells; and may be an intact molecule, a fragment thereof, or a functional equivalent thereof; and may be genetically engineered.

Protein - as used herein, includes proteins, polypeptides, and peptides; and may be an intact molecule, a fragment thereof, or a functional equivalent thereof; and may be genetically engineered; an example is an antibody.

Antibody - as used herein, includes both polyclonal and monoclonal antibodies; examples of antibody fragments include F(ab')₂, Fab', Fab and Fv.

As noted above, the present invention provides cleavable conjugates. Conjugates in which one of the components is a targeting molecule have enormous potential as potent anti-tumor agents. This is due to the selectivity imparted to the conjugate by the targeting molecule portion. The exquisite selectivity of antibodies, for example, permits delivery of increased doses of agents, such as those that are cytotoxic, inhibitory, radiolabeled, or biological response modifiers.

One advantage of the conjugates described is that cleavage occurs under mildly acidic conditions. For example, subjecting a conjugate to pH 5 results in substantially complete cleavage. The release mechanism may proceed as follows. An equivalent of acid is thought to initially protonate the ring 0. This is followed by formation of a stable carbocation. A second equivalent of acid results in the release of the agent and the formation of an aldehyde or ketone on the compound linking the agent to the targeting molecule. Another advantage of the conjugates of the present invention is that cleavage results in the release of the agent without any of the linker remaining attached. Popular cleavable linkers are those bifunctional reagents with a disulfide bond interposed between two reactive end groups. Cleavage of the disulfide bond by the addition of a reducing compound leaves one end of the bifunctional reagent still attached to the agent. Many agents, however, are inactivated by the permanent addition of a linker, or fragment thereof, to their structure. The conjugates of the present invention are cleaved at the bond formed between the agent and the compound linking the agent to the targeting molecule. Therefore, the present invention provides a way of releasing an agent in native form at the target site.

Yet another advantage of the present invention is the ease of preparation of the linking compound, due in part to the commercial availability of the reagents needed.

The cleavable conjugates of the present invention have the formula (I):

The elements of the conjugate depicted in formula I include the following. W is a group that functions as a "spacer arm" and may be useful to distance the targeting moleculue from the agent. Groups which may be used include methylene (-CH₂-), methyleneoxy (-CH₂-O-), methylenecar bonyl (-CH₂-CO-), amino acids, or combinations thereof. The number, n, of groups such as these would be typically 0 to 30 and preferably 0 to 10. W, or T where n is 0, may be attached to one of the ring positions designated as α, ß and Y. Because the number of methylene ring carbons at the β position is defined by n', which may be greater than one, the β position includes additional- points for attachment of a W or a T to the ring.

Q₁, Q₂ and Q₃ are independently selected from H, OH, O-alkyl, O-acyl, and derivatives thereof. The ring size of the compound may be increased above 5 by an increase in the number, n', of ring methylene groups. Preferred are 5-membered rings and 6-membered rings. X may be a hydrogen (H) or another substituent, preferably an alkyl group of C₆ or less or an alkoxy group of C₆ or less.

Z is an agent. Preferred agents include drugs, toxins, biological response modifiers, radiodiagnostic compounds and radiotherapeutic compounds. Preferred drugs include cancer chemotherapeutic agents. Preferred toxins include bacterial exotoxins and plant toxins. Particularly preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Preferred radionuclides for the radiodiagnostic and radiotherapeutic compounds include Cu-64, Cu-67, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212.

T is a targeting molecule, and, as used herein, includes a chemical group or groups ("linking function"), if any, used to attach the targeting molecule to form a conjugate. That is, a targeting molecule may be bonded to W directly or via a linking function, or when n is 0, it may be bonded to a ring-carbon directly or via a linking function. It will be evident to one skilled in the art that a variety of linking functions may be employed within the present invention and examples are described below.

A targeting molecule has the capacity to bind to a defined population of cells. The targeting molecule may bind through a receptor, substrate, antigenic determinant, or other binding site on the target cell population. Preferred targeting molecules useful within the present invention include antibodies; peptides, such as bombesin, gastrin-releasing peptide, RGD peptide, substance P, neuromedin-B, neuromedin-C, and metenkephalin; and hormones, such as estradiol, neurotensin, melanocyte-stimulating hormone, follice-stimula ting hormone, lutenizing hormone, and human growth hormone. Other suitable targeting molecules include serum proteins, fibrinolytic enzymes, and biological response modifiers, such as interleukin, interferon, erythropoietin and colony- stimulating factor. Analogs of the above-listed targeting molecules that retain the ability to bind to the defined target cell population may also be used within the present invention. In addition, synthetic targeting proteins and peptides may be designed and made to "fit" a particular characterized epitope (binding site). That is, a synthetic targeting protein/peptide would be designed to bind a specific epitope in a "lock and key" fashion. Within the present invention, antibodies, bombesin and gastrin- releasing peptide and their analogs are particularly preferred targeting molecules.

When an antibody is employed in the present invention, it may be an intact molecule, a fragment thereof, or a functional equivalent thereof. Examples of antibody fragments are F(ab')₂ , Fab', Fab, and Fv. While polyclonal antibodies may be employed in the present invention, monoclonal antibodies (MAbs) are preferred, especially those directed toward a tumor-associated antigen in man. Particularly preferred MAbs are anti-TAC, or other interleukin 2 receptor antibodies; 9.2.27 and NR-ML-05 to human melanoma associated proteoglycan; NR-Lu-10 to 37-40 kilodalton pancarcinoma glycoprotein and OVB3 to an as yet unidentified antigen. Genetically engineered antibodies or fragments thereof of these and other MAbs may be employed as well.

Within the present invention, monoclonal antibodies were prepared by immunizing rodents or other animals and/or are developed by harvesting human lympho- cytes from patients bearing malignancies and immortalizing the antibody secretion of the cells by standard hybridoma technology (e.g., Geffer et al., Somatic Cell Genet. 3:231, 1977). Alternatively, polyclonal antiserum is prepared by harvesting serum from animals following immunization with tumor cells or other defined tumor-associated antigens or harvesting from humans who have or have had exposure to tumors or tumor-associated antigens, and subjecting the serum to standard purification techniques. Antibodies were screened for specificity using a standard radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA) against the appropriate targets. Screening was performed with normal human tissues to select antibody with appropriate tumor specificity.

The conjugates of the present invention may be produced by a variety of methods. For example, a targeting molecule and an agent may be conjugated to a compound having the formula (II):

where W, n, Q₁-3, n', and X are defined as described above.

R and R' are chemically reactive groups. R is attached indirectly, or directly when n is 0, to one of the carbons designated α, β or γ. A targeting molecule is conjugated to the compound via R and an agent via R'. Generally, R and R' are not the same.

R is a chemically reactive group. The group may be strongly electrophilic or nucleophilic and thereby be available for reacting directly with a targeting molecule.

Alternatively, the group may be a weaker electrσphile or nucleophile and therefore require activation prior to the conjugation with a targeting molecule. This alternative would be desirable where it is necessary to delay activation of R until an agent is conjugated to the compound in order to prevent the reaction of the agent with R. In either scenario, R is chemically reactive. The scenarios differ by whether following addition of an agent, R is reacted directly with a targeting molecule or is reacted first with one or more chemicals to render R capable of reacting with a targeting molecule. A discussion of reactions illustrative of the activation of R is found below.

The step of conjugating a targeting molecule may be performed by joining the targeting molecule by direct reaction with R. Alternatively, it may be desirable to include before the step of conjugation a preparatory step. For example, a targeting molecule may be itself derivatized in preparation for direct reaction with R. The derivatization of a targeting molecule includes reaction with any of the numerous bifunctional reagents reported in the literature. An example of a derivative form is where an amino group on a targeting agent is modified by reaction with a reagent, such as iminothiolane, to introduce a sulfhydryl group on the targeting molecule.

A direct reaction with R by a derivatized or underivatized targeting molecule is intended to mean that R is capable of reacting with the derivatized or underivatized targeting molecule. For example, R may be a carbonyl-containing group, such as an anhydride or an acid halide, or an alkyl group containing a good leaving group, e.g., a halide. The latter class of compounds may be represented by alkyl X', where X' stands for the leaving group. As another example, R may be a nucleophilic group, such as an amino or sulfhydryl group, which is capable of reacting with a derivatized targeting molecule, e.g., containing a maleimide group.

Yet another way to perform a step in preparation for conjugation of an underivatized or derivatized target ing molecule is to convert R. Examples of conversions of R include where R is a carboxyl group and is then activated. Activation of a carboxyl group includes formation of an "active ester," such as a succinimidyl ester. The term "active ester" is known to refer to esters which are highly reactive in nucleophilic substitution reactions. In the present invention, the targeting molecule would be the nucleophile.

Another example of a conversion is where R is a succinimide derivative containing a protective group, such as phenylsulfonyl. Upon removal of the group, the succinimide is converted to a maleimide which is highly reactive in nucleophilic addition reactions. Alternatively, R may be an amino, sulfhydryl, or hydroxyl group and the conversion comprises reaction with a bifunctional reagent. It will be evident to one skilled in the art that a variety of bifunctional reagents, both homobifunctional and hetero- bifunctional, may be employed within the present invention.

The above discussion regarding R is applicable to the other chemically reactive group, R', on compound (II). As with the targeting molecule, an agent may be reacted in its native form or derivatized in preparation for reaction with R'. The selection of certain chemically reactive groups, e.g., Br or OCH₃, for R' permits the attachment of an agent directly, e.g., via an amino group on the agent, to the ring. Consequently, cleavage of such conjugates results in the release of the agent without any of the compound remaining attached.

Instead of attaching an agent first and a targeting molecule second in the formation of a conjugate, the order may be reversed. Specifically, first a targeting molecule is conjugated to a compound, whose structure (II) is depicted above, via R, and then an agent is reacted via R' on the compound attached to the targeting molecule. The above discussion regarding the compound, agent, targetin molecule, and chemical reactions is relevant to this variation as well. An additional aspect of the present invention provides a method for delivering to the cytoplasm of a target cell an agent free of its targeting molecule carrier. The method comprises the step of administering to a warmblooded animal a diagnostically or therapeutically effective dose of a cleavable immunoconjugate having the formula (I):

where W, n, Q₁-3, n', α, β, Y, X, T, and Z are defined above. A preferred warm-blooded animal is man. Preferred targeting molecules and agents include those described above.

The agent may be diagnostically and/or therapeutically effective. Preferred agents include those described above. A particularly preferred diagnostic agent is a compound containing ^99mTc. A diagnostically effective dose of a cleavable conjugate incorporating such an agent is generally from about 10 to about 30, typically from about 15 to about 25, and preferably from about 18 to about 20 mCi per 75 kg body weight. A particularly preferred therapeutic agent is a drug or a toxin, such as Pseudomonas exotoxin A. A therapeutically effective dose of a cleavable conjugate incorporating such a toxin agent is generally from about 1 to about 100 and preferably from about 1 to about 10 ng per 75 kg body weight. The precise dose for a particular cleavable conjugate is dependent upon the targeting molecule used, e.g., antibodies vary with respect to the number of receptors and their affinity for the receptors, and the agent used, e.g., toxins vary with respect to their potency. It will be evident to one skilled in the art how to determine the optimal effective dose for a particular cleavable conjugate.

The step of administering to a warm-blooded animal a diagnostically or therapeutically effective dose of a cleavable conjugate having formula I sets in motion a sequence of events in vivo that results in the agent portion of the conjugate being delivered free of the targeting molecule portion to the cytoplasm of a target cell. The targeting molecule of the conjugate imparts the selectivity which permits delivery to and binding at the surface of a specific cell. A conjugate of formula I is susceptible to cleavage by pH less than or equal to 6.0, and the acid-catalyzed cleavage is accelerated by heating above room temperature, about 23°C. Since targeting molecules such as antibodies bind to cell surface receptors which are internalized into the cytoplasm via acidified compartments, it is believed that the release to the cytoplasm of an agent from a conjugate of the type described herein is the result of this transient exposure to acidic pH. Further, because warm-blooded animals such as man have normal body temperatures above 23°C, body heat may be a factor in the release. The delivery of an agent free of its targeting molecule carrier to the cytoplasm of a targeted cell increases its potency as compared to the agent when irreversibly linked to its carrier. This method is useful to diagnose, stage, evaluate or treat diseases such as cancer in humans.

A related aspect of the present invention provides a method for isolating compounds containing a group capable of reacting with a chemically reactive group. The isolation of a compound, e.g., from a reaction mixture, is often a difficult and/or tedious process. It was widely believed that the attachment to a solid phase of a reagent with an affinity for a compound was the panacea for the problems with earlier isolation procedures.

In theory, the undesired compounds are removed simply by washing the solid phase. In practice, however, washing conditions sufficient to remove the impurities often result in removal of the compound of interest. This is due to the fact that the compound is only held to the solid phase by noncovalent interactions with the reagent. While covalent attachment of the compound to the solid phase is preferable from a washing standpoint, it can make recovery of the compound off the solid phase impossible. The method of , the present invention provides a way to attach covalently a compound of interest and thereby facilitate removal of undesired compounds, yet nevertheless permit easy recovery of the compound of interest from the solid phase.

This method of isolating compounds containing a group capable of reacting with a chemically reactive group comprises the following steps. To a solid phase is conjugated, via R, a reagent having the formula (II):

to form a derivatized solid phase. The derivatized solid phase is contacted with a sample solution in which a compound containing an available nucleophilic group is present, such that the compound binds to the derivatized solid phase, thereby removing the compound from the sample solution. The compound bound to the derivatized solid phase may be released.

The elements of the reagent depicted above in formula II include the following: R', W, n, Q₁-3, n', α, β, γ, X and R, which are defined above. R is a chemically reactive moiety which may be a nucleophile or an electrophile. When the solid phase contains available nucleophilic groups, such as a free amino group, R is an electrophile such as an activated ester. Conversely, when the solid phase contains electrophilic groups, R is a nucleophile. Examples of suitable solid phases include controlled pore glass and preformed polymers, such as polyvinyls, polyacrylamides, polydextrans, and polystyrenes.

The step of conjugating the reagent to the solid phase attaches the former to the latter via R, thereby forming a derivatized solid phase. The above discussion regarding R and R' is applicable here as well. A sample solution, in which a compound containing a group capable of reacting with R' is present, is contacted with the derivatized solid phase. Reactions of a compound with R' of a reagent include both attachment of the compound to the reagent with R' (or its reaction product) interposed, as well as attachment of the compound directly to a ring-carbon of the reagent with R' having been displaced. The step of contacting results in covalent attachment of the compound to the derivatized solid phase. Following the step of contacting, it may be desirable to wash the solid phase to remove noncovalently bound compounds.

The covalently bound compound may be released from the solid phase by a vrriety of ways. For example, cleavage of the bond formed between a heteroatom and the ring-carbon, which bore R', of the derivatized solid phase may be achieved by mildly acidic conditions and be accelerated by heat. In particular, cleavage occurs by decreasing the pH of a solution contacting the solid phase to 6.0 or lower or by raising the temperature above 23°C in the presence of pH 6.0 or lower.

Alternatively, an undesired compound or compounds, rather than a compound of interest, may contain a group capable of reacting with R'. In this situation, the undesired compound or compounds will bind to a derivatized solid phase and the compound of interest will not bind. Therefore, the method may be used to purify by a single technique one or more compounds from a reaction mixture so long as the compounds of interest or the impurities, but not both, contain groups capable of reacting with R'.

Another way to use this type of reagent to isolate a compound is to bind the compound to the reagent before contacting with a solid phase. For example, a sample solution in which a compound containing a group capable of reacting with R' is present may be reacted with a reagent whose structure is as described above. The step of reacting results in covalent attachment of the reagent to the compound. A reaction mixture is thereby formed wherein a derivatized compound is present. The reaction mixture is then contacted with a solid phase capable of selectively binding, covalently or noncovalently, the derivatized compound, thereby removing the derivatized compound from the reaction mixture. The bound compound may be released in native form by a variety of ways, including those described above.

Yet another aspect of the present invention includes a method for introducing into a compound a free sulfhydryl, free amino, or free hydroxyl group. The two reagents widely used for the introduction of -SH groups in proteins are S-acetyl mercaptoacetic acid succinimidate ester (SATA) and mercaptoacetyl succinic anhydride (SAMSA). In both cases, the antibody reacts to form an amide bond. In the case of SATA, the antibody (Ab) after the reaction is stored as Ab-Lys-NH-COCH₂SCOCH₃ at -20°C from which free sulfhydryl (-SH) groups are generated by treatment with hydroxylamine. In the case of SAMSA, the amine from the protein reacts with the anhydride to form Ab-Lys-NHCO-CH₂- CH(SH)-COOH. Both reagents suffer from several disadvantages. First, the S-acetyl protecting group is base labile, i.e., can be hydrolyzed at pH 7-8. Ideally that is the pH necessary for the conjugation of antibody with electrophiles. Hydrolysis of thioacetyl groups effectively competes with displacement reactions. Second, thioesters are by themselves active esters. Amines will react with thioesters to give N-acetyl compounds . Third , in the case of mercaptoacetyl succinic anhydride, reaction of a Fab fragment of an antibody gives several components including aggregates. This results directly from competing reactionsof the proteins with the reagents. The method of the present invention overcomes the above problems and, furthermore, reagents like hydroxylamine need not be used to generate the free -SH group.

This method for introducing into a compound a free -SH, -NH₂, or -OH group comprises the steps of reacting a compound with a reagent having the formula (III):

to form a reagent-linked compound and cleaving the reagent- linked compound at the bond between Y and the ring, thereby introducing into the compound a free sulfhydryl, free amino, or free hydroxyl group, depending upon whether Y is S, N, or O, respectively.

The elements of the reagent depicted above in formula III include the following: W, n, Q_1-3, n', and X, which are defined above. Y is a heteroatom. Preferred heteroatoms include sulfur (S), oxygen (0), or nitrogen (N). R is a chemically reactive group. The group may be a nucleophile or an electrophile. The selection is generally determined by whether the reactive group on a compound is nucleophilic or electrophilic. For example, where the reactive group on a compound is a nucleophile, such as an amino group, R is an electrophile such as an activated ester or an anhydride. An exception to this method of selecting R is where it is desired to form a disulfide bond between a compound and R. In that situation, a sulfhydryl group on the compound and a sulfhydryl group on the reagent, i.e., R is -SH, may be oxidized to form a disulfide bond. Because disulfide linkages are cleavable, e.g., by reducing agents, it is possible to create a reversibly modified compound. For example, where Y is N and R is -SH and the reagent and a compound are joined by a disulfide bond, the result is the addition to a compound of an amino group that can be removed.

Any compound containing a functional group capable of reacting with the reagent via R may be employed in the present invention. The functional group may be present on a native form of a compound or be added to it. Preferred compounds are drugs and proteins generally, e.g., antibodies.

When the compound is reacted with the reagent, the former is attached to the latter via R, thereby forming a reagent-linked compound. The reagent-linked compound is then cleaved at the bond between Y and a ring carbon, thereby forming compounds with free sulfhydryl, free amino, or free hydroxyl groups, depending upon whether Y is an S, N, or O, respectively. The step of cleaving may be achieved by a variety of ways, including exposing the reagent-linked compound to mildly acidic conditions, heat, or divalent cations. In particular, cleavage occurs by decreasing the pH to 6.0 or lower or by raising the temperature to at least 37°C in the presence of pH 6.0 or lower. The preferred pH range is 5.0 - 6.0.

The step of cleaving results in Y remaining attached to the compound. Therefore, introduction of a desired free heteroatom into a compound is achieved by selection of the appropriate heteroatom for Y. For example, if the addition of a free amino group to a compound is desired, then the heteroatom selected for Y is N.

A preferred use of the method with proteins is to introduce a free sulfhydryl group. For example, an amino group of a protein, e.g., a lysine residue, may be used as the nucleophilic group to react with a form of the reagent where Y is S. When the resulting reagent-linked protein is cleaved, the net effect is to convert a free amino group on the protein to a free sulfhydryl group. Another preferred use is to introduce an amino group.

To summarize the examples which follow, Example I provides the preparation of cleavable conjugates utilizing

MAb 9.2.27 and ethiophos, melphalan or adriamycin. Example

II describes an in vitro assay and the biodistribution and toxicology of the cleavable conjugates utilizing adriamycin.

Example III discloses isolation of an amine utilizing a glucoronide and a derivatized agarose. Example IV describes the introduction of a free amino group into intact MAb 9.2.27.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES EXAMPLE I Preparation of Cleavable Conjugates

A. Preparation of Derivatized Agents

Scheme 1

Preparation of 3: To a stirred solution of the glucoronide 1 (10 mmol) in absolute dioxane (100 mL) is added in portions a solution of bis-cyclohexylammonium salt of ethiophos 2 (10 mmol) in dioxane followed by triethyl- amine (1 eq.). The reaction mixture is refluxed for several hours. The insoluble part is filtered. The filtrate is diluted with water to opalescence, cooled in an ice-water bath, and ammonia gas is bubbled into it until saturation. The reaction mixture is stored at 0°C overnight. Volatiles are evaporated in vacuo. The residue is diluted in methanol, a solution of sodium iodide in acetone is added, and then centrifuged to obtain 3 as a disodium salt. The disodium salt is converted to alkylammonium or pyridinium salt by passing through alkyl ammonium or pyridinium Dow-50 according to the procedure of D. B. Towbridge et al. (J. Amer. Chem. Soc. 94:3816, 1972).

Scheme 2

1

Preparation of 3,5-Dideoxy-5-isothiocyanatomethyl- 1,2-di-O-acetyl-α-D-erythro-pentofuranoside_9:

i) 3-Deoxy-1,2,-O-isopropylidine-α-D-erythro- pentofuranoside 5 is prepared from 4 according to the procedure of Murray and Prokop (D. H. Murray and J. Prokop in "Synthetic Procedures in Nucleic Acid Chemistry," edited by W. Zorbach and R. S. Tipson, Interscience Publishers, New York, 1968, pp. 193-97).

ii) The combined oxidation and nitroaldol condensation sequence (conversion of 5 to 6 ) is carried out according to Vrudhulas's modified procedures of Mock and Moffatt's methodology (V. M. Vrudhula, F. Kappler and A. Hampton, J. Med. Chem. 30:888, 1987, and G. A. Mock and J. G. Moffatt, Nucleic Acids Res. 10:6223, 1982). Conversion of the isopropylidine derivative 6 to the diacetyl derivative is accomplished using acetic anhydride-boron trifluoride in ether according to the procedure of S. Lesage and A. S. Perlin, Can. J. Chem. 56:2889, 1978.

iii) To a suspension of preproduced Adam's catalyst in methanol is added a solution of the nitroalkane 7 (100 mg of catalyst/l g of the compound), and the reduction is carried out in a Paar hydrogenator at 45 psi for 20 hours. The catalyst is filtered through celite and evaporated in vacuo to give the amino sugar 8 , which is converted to the isothiocyanate 9 according to the procedure of H. A. Staab and G. Walther, Liebiqs. Ann. Chem. 657:104, 1962, using thiocarbonyldiimidazole.

Preparation of 10: An equimolar mixture of melphalan and the isothiocyanate are fused at 145°-150°C, and the isolation of the product is carried out according to the method of T. Sato, "Synthetic Procedures in Nucleic Acid Chemistry," edited by W. Zorbach and R. S. Tipson, Interscience Publishers, New York, 1968, pp. 264-68.

Scheme 3

Bn = beznyl

Preparation of 14:

i) The requisite starting material 11 is available according to the procedure of M. L. Wolform and S. Hanessian, J. Org. Chem. 27 : 1800 , 1962, from D-glucose.

ii) The aldehyde 1 1 is reacted with BnOOC-CH=PPh₃ under Wittig reaction conditions to the α, β-unsaturated ester 12.

iii) The benzylester 12 (1 mmol) is hydrogenated in methanol containing Pd-C (10%) (100 mg/mmol) in a Paar apparatus for 24 hours. At the end of the reaction, the catalyst is removed by filtration, and the filtrate containing 13 is again hydrogenated in the presence of 50 mg of Adam's catalyst to the saturated derivative. The catalyst is removed by filtration and the solvent is evaporated to give the product.

iv) The preceding product is stirred with 20 mL of 1% hydrogen chloride-methanol for 6 hours and evaporated to give methoxy sugar derivative 14.

Preparation of the disaccharide 15:

i) To a solution of 1 mmol of adriamycin in 2 mL of pyridine 2.1 mmol of t-butyldimethylsilylchloride is added, and the solution is stirred for 2-3 hours at room temperature. Pyridine is removed in vacuo, and the residue is chromatographed over silica gel to yield the bis-t- butyldimethylsilyl derivative.

ii) The silyl derivative of adriamycin (1 mmol) is fused with 14 and the product 15 is isolated in pure state by liquid chromatography. Preparation of the active ester 16: The above acid is dissolved in 1:1 acetonitrile-water and an equimolar amount of 2,3,5,6-tetrafluorophenol is added, followed by 3-5 equivalents of 1-(3-diethylaminopropyl)-3- ethyl carbodiimide hydrochloride, and the mixture is stirred for 10-15 hours at room temperature. The solvents are removed in vacuo, and the product is isolated by liquid chromatography.

To a solution of the above compound in 5-10 mL of tetrahydrofuran, 2 mL (per mmol of the compound) of tetra- n-butyl ammonium fluoride is added and the solution is stirred for 1 hour. The active ester hydrochloride is isolated from the mixture by liquid chromatography. B. Conjugation of TFP ester 16 with Monoclonal

Antibody 9.2.27

The monoclonal antibody 9.2.27 is directed to a melanoma-associated antigen and is prepared according to Morgan et al., Hybridoma 1:27-35, 1981. An isopropanol solution of 16 (25 μl total volume) is transferred to a vial containing buffered antibody solution (pH 8.5-10) of at least 1 mg antibody per 1 ml. The conjugation mixture is warmed at 37°C for 20 minutes. The modified antibody is purified either using gel permeation chromatography or small pore filtration (e.g., Centricon ultra centrifugation).

EXAMPLE II

In Vitro Assay and Biodistribution With

Cleavable Conjugates

A. In Vitro Assay for Drug Conjugates

1. Day 1: Cell Plating and Plate Setup. a) Growth of human carcinoma cells, raised in DMEM.

b) Cells are usually adherent lines and thus should be lifted with trypsin/EDTA. Cells should be passaged into microtiter plates at least 18-24 hours prior to the start of an assay.

c) Count cells in hemocytometer. Cells should have a viability greater than 80% as measured by trypan blue exclusion.

d) Cells are plated in 100 μl of DMEM at an appropriate concentration such that control wells will have just reached confluency by the end of the assay. For example, for the particular cell lines used, 3000 to 3500 cells per well is sufficient. Fill the outside rows of 96 well plates with sterile water to prevent evaporation.

e) Incubate microtiter plates for a minimum of 18 hours in a 37°C humidified incubator before the addition of drugs and drug conjugates.

2. Day 2: Addition of Drugs and Drug Conjugates a) Exact ID50 values may vary from cell line to cell line. It is well within the skill of an investigator of ordinary skill in this art to determine the appropriate dilution range of the samples to be tested. Usually the

ID50 can be found within the range of 2000 nM to 0.2 nM for doxorubicin and 200 nM to 0.02 nM for doxorubicin conjugates for both the positive and negative cell lines for the antibody used. b) Set up 5 ml snap cap tubes in a test tube rack to make 5 serial 10-fold dilutions for each sample to be tested plus controls.

c) Pipet 900 uL of medium to last 4 tubes of dilution series. With the first tube, add the appropriate medium in order to start the dilution series at 2000 nM for doxorubicin and 200 nM for dox conjugates. Total volume of a tube should be 1000 uL.

d) Add the appropriate amount of sample to the first tube of the dilution series. Mix by repetitive pipeting (approximately 5 times). Discard the pipet in the drug waste. Remove 100 uL of sample/medium from the first tube and place in tube number 2 of the dilution series. Mix by repetitive pipeting, and continue the procedure through the dilution sequence.

e) Once all samples have been diluted, remove cells (that had been set up the previous day) from the incubator. For each dilution of the samples, place 100 uL into the wells of the microtiter plate in triplicate on the positive and negative cell lines.

f) Add 100 uL of medium to the control wells (wells without drug or conjugate sample added).

g) Incubate the cells at 37°C in a CO₂ humidified incubator for 18-24 hours.

h) Once completed, dispense of waste material in drug refuse.

3. Day 3: Removal of Drugs from Plates a) Remove the cells from incubator.

b) Gently remove sample/medium from all wells of microtiter plate using a multichannel pipeter.

c) Rinse the wells with PBS.

d) Add 200 uL of medium per well.

e) Place the plate into the incubator. f) Repeat procedure for other plates. 4 . Day 5 : MTT Assay ( 3- ( 4 , 5-dimethylthiaszol-2-yl ) -2 , 5- diphenyl-2H-tetrazolium bromide )

Assay:

a) A mixture of 50 uL of medium to 10 uL of MTT solution is used per well. Thus, for an entire plate of 60 wells, 3 mis of medium plus 600 uL of MTT solution is used. Mix the two prior to adding to cells.

b) At this point of the assay, sterility is no longer a major precaution.

c) The medium/MTT solution is removed from the wells,

d) Add 60 uL of medium/MTT solution to each well.

e) Incubate at 37°C for cleavage of MTT to occur. Optimal times may vary with cell lines used, but 30 minutes is suitable for most purposes.

f) After the 30 minute incubation, the medium/MTT solution is removed.

g) After the medium/MTT is removed, add 100 uL of DMSO per well.

h) Plates can be read immediately after addition of the DMSO .

i) Tap the sides of the microtiter plate to ensure complete dissolving/mixing of the formazin crystals.

j) Measure the absorbance on an ELISA plate reader. Use a test wavelength of 570 nm and reference wavelength of 630 nm. Also use the T/R setting on the Dynatech ELISA reader. B. Biodistribution and Toxicology of Cleavable Conjugates

C-14-adriamycin (commercially available from Amersham Corporation, Ilinois) is used to prepare compound 15 and compound 16 according to the procedure described in Scheme 3 (Example I). Compound 16 is conjugated to anti-melanoma antibody 9.2.27 according to the procedure in Example I.B. Tumor localization and biodistribution of conjugates are examined in a nude mouse xenograft model of human melanoma, according to the method of K.M. Hwang et al., Cancer Research. 450 : 959 (1986). Animals are sacrificed at 20 hours post-injection and organs are blotted, weighed and counted. A percent dose per gram is calculated for each tissue. In addition, serum half-life is estimated by retroorbital sampling of whole blood.

Mice are administered different doses of drug-linker-anti-TAC and drug-linker-9.2.27 conjugates intraperitoneally to determine an LD₁,,. Following administration of C-14 adriamycin-linker-9.2.27 conjugates, the resultant tumor localization and biodistribution of the conjugates are determined. Nonspecific toxicity of drug-linker-anti-TAC conjugates is also measured in cynomologous monkeys. Monkeys are monitored for liver enzyme levels and are observed for other relevant symptoms including loss of appetite, nausea and increased body temperature.

EXAMPLE III

Isolation of an Amine-Containing Compound A. Derivatization of an Amine in a Biological Fluid

A solution of a biological fluid containing an amine to be isolated is treated with the glucoronide shown in Scheme 1 (Example I) at pH 7-8. Using this procedure all the amines are converted to aminals. The biological fluid is adjusted to about pH = 9-10 and the ester is converted to a sodium salt.

B. Coupling of the Above Acid to Hydroxymethyl Sepharose

Commercially available hydroxymethyl sepharose is washed and swelled according to the procedure described in Affinity Chromatography-Principles and Methods (Pharmacia) and stored in 4°C.

The above acid (from section A) is incubated with the solid phase pH 7.4 with 20-30 molar excess of EDCI (water-soluble carbodiimide). The coupling is done overnight to ensure all the derivatized amine is condensed with the hydroxyl groups (forming ester bonds). This method can be applied to aminohexyl sepharose also. In the latter case the aminal in the biological fluid is attached via the -COOH of the glucoronide as an amide. The conjugate thus prepared is stored at 4°C.

C. Isolation of Amine The conjugate is loaded into a column and is washed several times with water and pH 7.4 buffer to remove other biological material. Finally the desired amine is isolated by passing pH 4-5 buffer through the column. EXAMPLE IV

Introduction of a Free NH₂ Group into an Antibody

or Other Proteins A. Preparation of an Aminal Derivative of Monoclonal

Antibody 9.2.27

1. Synthesis of Protected Aminal.

A solution of tri-O-acetyl α-bromo-D-xylose in dry dioxane is treated with N-trifluoroacetylaminocaproic acid containing a 2 equimolar amount of trietylamine. The reaction mixture is refluxed for several hours. The insoluble part is filtered. The filtrate is evaporated and the residue is dissolved in methylene chloride and washed with water. The organic layer is dried over anhydrous sodium sulfate and evaporated to give the N-trifluoroacetyl aminal. 2. Conjugation of the Above Acid with Monoclonal Antibody 9.2.27.

A solution of the antibody is prepared according to the method described in Example I.B. An isopropanol-water solution (isopropanol not to exceed 30%) of the above acid is transferred to a vial containing a pH 7.4 buffered antibody solution. The antibody concentration is at least 1.0 mg/mL. To this solution 10-15 equivalents of the above acid and 20-30 equivalents of EDCI (water-soluble carbodiimide) are added and the mixture is incubated at 37°C for 2-3 hours. The derivatized antibody is isolated by gel permeation chromatography.

B. Removal of N-trifluoroacetyl Group to Regenerate

the Aminal

The above antibody solution is stirred with a 10% solution of aqueous piperidine (1 mL for approximately 1 mg of protein) for 10-15 hours at 0º-5°C. After treatment with piperidine, the protein is purified by passing over a PD-10 column. The protein obtained has the introduced amine group protected as an aminal.

C. Generation of Free Amines

The buffered solution of the above-protein is deoxygenated using a nitrogen purge. The pH of the solution is then decreased to pH 5.0-5.5 using 0.1 N HCl. The acidified antibody is incubated at 37°C for 1 hour to ensure complete removal of the xylose protecting moiety and complete generation of amino groups.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims

1. A cleavable conjugate having the formula:

where: T is a targeting molecule;

W is a methylene, methylenoxy, or methylenecarbonyl group or combinations thereof;

n is 0 to 10;

Q₁, Q₂ and Q₃ are independently selected from H, OH,

O-alkyl, O-acyl, and derivatives thereof, with the proviso that Q₁, Q₂ and Q₃ are not all H;

n' is 1 to 2;

X is an H, alkyl group of C₆ or less, or alkoxy group of

C₆ or less; and

Z is an agent.

2. A cleavable conjugate having the formula:

where: T is a targeting molecule;

n' is 1 to 2;

X is an H, alkyl group of C₆ or less, or alkoxy group of C₆ or less; and

Z is an agent;

for use as an active therapeutic substance.

3. A cleavable conjugate having the formula:

₃

where: T is a targeting molecule;

n is 0 to 10;

n' is 1 to 2;

X is H, alkyl group of C₆ or less, or alkoxy group of

C₆ or less; and

Z is a diagnostically or therapeutically effective agent;

for use within a method for delivering to the cytoplasm of a target cell an agent free of its targeting molecule carrier.

4. The cleavable conjugate of claims 1, 2 or 3 wherein the targeting molecule is selected from the group consisting of antibodies, peptides, hormones, serum proteins, fibrinolytic enzymes, biological response modifiers, and analogs thereof.

5. The cleavable conjugate of claim 4 wherein the antibody is a monoclonal antibody.

6. The cleavable conjugate of claims 1, 2 or 3 wherein the agent is selected from the group consisting of drugs, toxins, biological response modifiers, radiodiagnostic compounds, radiotherapeutic compounds, and analogs thereof.

7. The cleavable conjugate of claim 6 wherein the drug is a cancer chemotherapeutic agent.

8. The clevable conjugate of claim 6 wherein the radionuclide of the radiodiagnostic compounds and the radiotherapeutic compounds is selected from the group consisting of Cu-64, Cu-67, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, 1-123, 1-125, 1-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212.

A compound having the formula:

where: R' is a chemically reactive group;

n is 0 to 10;

Q₁ , Q₂ and Q₃ are independently selected from H, OH,

n' is 1 to 2;

X is H, alkyl group of C₆ or less, or alkoxy group of

C₆ or less; and R is a chemically reactive group, with the provisos that R is attached directly or indirectly to one of the carbons designated α, ß or γ, that when R is attached to α then Q₁ is H, that when R is attached to ß then Q₂ is H, that when R is attached to Y then Q₃ is H, and that R and R' are not the same.

10. A method for isolating a compound, comprising the steps of:

conjugating to a solid phase, via R, a reagent having the formula:

where: R' is a chemically reactive group;

n is 0 to 30;

Q₁, Q₂ and Q₃ are independently selected from H, OH, O-alkyl, O-acyl and derivatives thereof, with the proviso that Q₁, Q₂ and Q₃ are not all H;

n' is 1 to 2;

X is an H, alkyl group of C₆ or less, or alkoxy group of

C₆ or less; and

R is a chemically reactive group, with the provisos that R is attached directly or indirectly to one of the carbons designated α, β or Y, that when R is attached to α then Q₁ is H, that when R is attached to β then Q₂ is H, that when R is attached to Y then Q₃ is H, and that R and R' are not the same;

to form a derivatized solid phase; and

contacting said derivatized solid phase with a sample solution in which a compound containing a group capable of 6eacting with R' is present, such that said compound binds to said derivatized solid 'phase, thereby removing said compound from said sample solution.

11. The method of claim 10 wherein the solid phase is controlled pore glass, polyvinyl, polyacrylamide, polydextran, or polystyrene.

12. The method of claim 10, additionally including, after the step of contacting, washing the solid phase to remove noncovalently bound compounds.

13. The method of claim 10, additionally including, after the step of contacting, releasing said bound compound from said derivatized solid phase.

14. A method for introducing into a compound a free sulfhydryl, free amino, or free hydroxyl group, comprising the steps of:

reacting a compound with a reagent having the formula:

where: R is a chemically reactive group;

n is 0 to 30;

Y is S, N, or 0;

X is H, alkyl group of C₆ or less, or alkoxy group of C₆ or less;

n' is 1 to 2; and Q₁ , Q₂ and Q₃ are independently selected from H, OH, O-alkyl, O-acyl, and derivatives thereof, with the proviso that Q₁, Q₂ and Q₃ are not all H;

to form a reagent-linked compound; and

cleaving the reagent-linked compound at the bond between Y and the ring, thereby introducing into said compound said free sulfhydryl, free amino, or free hydroxyl group, depending upon whether Y is S, N, or O, respectively.

15. The method of claim 14 wherein the compound is a protein or a drug.

16. A compound having the formula:

where: R is a chemically reactive group;

n is 0 to 30;

Y is S, N, or O;

X is H, alkyl group of C₆ or less, or alkoxy group of C₆ or less;

n' is 1 to 2; and

Q₁, Q₂ and Q₃ are independently selected from H, OH,

O-alkyl, O-acyl, and derivatives thereof, with the proviso that Q₁, Q₂ and Q₃ are not all H.