Prodrug activation using catalytic antibodies

Details Prodrug activation using catalytic antibodies Prodrug activation using catalytic antibodies

1999-05-25

2001-07-31

Peselev, Elli (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C548S204000, C549S375000, C562S463000, C568S448000

Reexamination Certificate

active

06268488

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The field of this invention is prodrug activation. More particularly, the present invention pertains to a compound that includes an active therapeutic agent attached to a blocking group, methods for making such compounds and methods of converting such compounds to active therapeutic agents using antibodies having aldolase activity.
BACKGROUND OF THE INVENTION
Drug therapy can be limited by nonspecific toxicity. To overcome this limitation, several approaches towards a site-selective therapy have been suggested. Selective therapy can be based on the enzymatic activation of a prodrug at a target site. Unless the target displays a specific enzymatic activity that can be used for prodrug activation (Denmeade et al.,
Cancer Res
58, 2537-2540, 1998), the enzymatic activity has to be conjugated to an antibody that binds to a target cell surface antigen selectively expressed at the target site. The antibody-enzyme conjugate is injected first. Once it has accumulated at the site and has been cleared from the periphery, the prodrug is administered. The prodrug is selectively activated by the targeted enzymatic activity. One molecule of enzyme catalyzes the activation of many molecules of prodrug. This inherent amplification feature of the system allows the generation of high drug concentrations at the target site. The concept of antibody-directed enzyme prodrug therapy, termed ADEPT, has been developed by Bagshawe, Senter, and others (Bagshawe et al.,
Br. J. Cancer
58, 700-703, 1988; Senter et al.,
Proc. Natl. Acad. Sci.
USA 85, 4842-4846, 1988; Niculescu-Duvaz, et al.,
Adv. Drug Delivery Rev.
26, 151-172, 1997). A number of antigens that are expressed on the surface of cells have been shown to be effective targets for antibody-mediated therapy. Thus, the antibody component is not the critical parameter for ADEPT. By contrast, the requirements for the enzyme component for ADEPT are difficult to achieve. First of all, selective prodrug activation requires the catalysis of a reaction that must not be accomplished by endogenous enzymes in blood and normal tissue of the patient. Enzymes of non-mammalian origin that meet these needs are, however, likely to be highly immunogenic, a fact that makes repeated administration impossible. There is a need in the art, therefore, for improved ADEPT compounds and methods.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides a compound according to formula I, below
In formula I, X is a heteroatom of a target molecule and Y is absent or a self-immolative linker such as shown below:
Each R is independently hydrogen, C
1
-C
6
alkyl, C
1
-C
6
alkenyl, C
5
-C
6
aryl or a heterocycle containing five or six ring atoms. In one embodiment, the heteroatom is a nitrogen, oxygen or sulfur atom in a functional group of the target molecule. Preferred target molecules are therapeutic agents or fluorescent molecules. Exemplary and preferred therapeutic agents include anti-tumor agents such as a cytotoxic agent, a microtubule stabilizing agent or an antibiotic. A preferred antibiotic is an anthracycline antibiotic such as doxorubicin or a therapeutically active analog thereof. A preferred microtubule stabilizing agent is paclitaxel, epothilone, or a therapeutically active analog thereof.
In another aspect, a compound of the present invention includes a compound having the structure II, below
where X, Y and R are defined in reference to formula I.
An especially preferred compound of this invention has the structure
where R
9
is CH
3
, CH
2
F, CH
2
Cl, CH
2
CH
3
, CH
2
OOCCH
3
or CH═CH
2
and each B is independently H,
where Y and R are as defined above and with the proviso that two Bs are H.
In another aspect, this invention provides a of converting an inactive molecule to an active molecule. The process includes the step of exposing the inactive molecule to an agent that catalyzes a retro-Michael reaction. A preferred inactive molecule is a compound according to formula I or II, above. A preferred agent that catalyzes a retro-Michael reaction is a protein. A preferred is an antibody, the catalytic activity of which is inhibited by a &bgr;-diketone compound. Exemplary and preferred such antibodies are 38C2 or 33F12. The process can occur in vitro, in situ or in vivo. In one embodiment, the antibody is a bifunctional antibody that specifically immunoreacts with a cell surface antigen of a target cell such as a tumor cell or a virus-infected cell. The antibody can be a single chain antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings that form a portion of the specification
FIG. 1
shows prodrug activation via a tandem retro-aldol-retro-Michael reaction. X stands for heteroatoms N, O, or S.
FIG. 2
shows synthesis of generic linkers 3 and 4 which can be used to mask functionally active amine and hydroxy groups of drugs, respectively.
FIG. 3
shows doxorubicin prodrug activation via a tandem retro-aldol-retro-Michael reaction catalyzed by antibody 38C2.
FIG. 4
shows growth inhibition of LIM1215 human colon carcinoma cells in vitro by doxorubicin (▪) and prodoxorubicin (□)(Bars indicate SD; n=4). The data were determined using the assay described in FIG.
5
and are summarized in Table 1. The dashed line indicates 50% decrease in the cell density as compared to the untreated control. Note the reduced capacity of prodoxorubicin for cell growth inhibition.
FIG. 5
shows growth inhibition of LIM1215 human colon carcinoma cells by prodoxorubicin in the presence of antibody 38C2. Cells in quadruplicate wells in a 96-well plate were lysed 120 h after drug addition and the activity of the cytoplasmic enzyme lactate dehydrogenase released from the cells was detected using a colorimetric assay. The intensity of the red color correlates with the number of cells in the well. Controls are shown in black columns. (dox=doxorubicin; prodox=prodoxorubicin; MVK=methyl vinyl ketone; bars indicate SD; n=4).
FIG. 6
shows camptothecin prodrug activation via a tandem retro-aldol-retro-Michael reaction catalyzed by antibody 38C2 followed by a spontaneous lactamization.
FIG. 7
shows growth inhibition of HT29 human colon carcinoma cells by procamptothecin in the presence of increasing concentrations of antibody 38C2. (▴) untreated control; (□) 1 &mgr;M procamptothecin; (▪) 1 &mgr;M camptothecin; (bars indicate SD; n=4).
FIG. 8
shows in vivo activity of antibody 38C2. Mice were injected with 100 &mgr;l of 15 mg/ml 38C2 IgG in PBS on day 0. The concentration of 38C2 IgG in mouse sera was studied as a function of time after the injection. Activity was calculated based on the antibody 38C2-catalyzed conversion of the fluorogenic aldol sensor methodol into fluorescent 6-methoxy-2-naphtaldehyde (11). Typical data derived from one mouse are shown. The initial 38C2 IgG concentration on day 0 can be estimated to be about 6 &mgr;M based on a blood volume of 1.5 ml and 1.5 mg injected antibody. Catalysis was not detectable in sera from mice injected with 100 &mgr;l of 15 mg/ml control antibody in PBS.
FIG. 9
gives the top strand nucleotide (SEQ ID NO:1) and bottom strand (SEQ ID NO 5) and amino acid residue (SEQ ID NO:2) sequence of the catalytic fragment of antibody 38C2.
FIG. 10
gives the top strand nucleotide (SEQ ID NO:3) and bottom strand (SEQ ID NO 6) and amino acid residue (SEQ ID NO:4) sequence of the catalytic fragment of antibody 33F12.


REFERENCES:
patent: 5001115 (1991-03-01), Sloan
Senter, et al., “Anti-tumor Effects on Antibody-Alkaline Phosphatase Conjugates in Combination with Etoposide Phosphate”,Proc. Natl. Acad. Sci. USA 85: 4842-4846 (1988).
Bagshawe, et al., “A Cytotoxic Agent can be Generated Selectively at Cancer Sites”,Br. J. Cancer 58: 700-703 (1988).
Miyashita, et al., “Prodrug Activation via Catalytic Antibodies”,Proc. Natl. Acad. Sci. USA 90: 5337-5340 (1993).
Campbell, et al., “Antibody-Catalyzed Prodrug Activation”,J. Am. Chem. Soc. 116: 2165-2166 (1994).
Wentworth, et al., “Toward Antibody-Directed “Abzyme” Prodrug

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