Method for in vivo delivery of active compounds using reagent conjugate

• US 7,713,528 B1
• Filed: 06/07/1995
• Issued: 05/11/2010
• Est. Priority Date: 02/18/1993
• Status: Active Grant

• Current Assignee: Enzo Therapeutics Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Therapeutics Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Goltz, Sherry P., Rabbani, Elazar
• Primary Examiner(s): Saoud; Christine J
• Assistant Examiner(s): LOCKARD, JON MCCLELLAND
• Application Number:
  US08/486,062
• Time in Patent Office: 5,452 Days
• Field of Search: 424/101,9,1.11,178.1 530/391.3,391.5,388.8,230.4 930/300,350,387.1 435/7.1
• US Class Current: 424/178.1
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 47/6897 Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
• CPC Class Codes: A61K 51/0491 Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
• CPC Class Codes: A61K 51/0495 Pretargeting
• CPC Class Codes: A61K 51/0497 conjugates with a carrier being an organic compounds
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

This invention relates to novel reagent conjugates and a novel multi-step process for delivering active compounds to target analytes of interest in a patient for diagnostic and therapeutic purposes. According to the process, two novel reagents are bound to each other by linkage of the sequence-specific components they contain. The first reagent, which is comprised of a target recognition component and a first sequence-specific component, is introduced into the patient and allowed to achieve maximal localization on the target cells. The second reagent, which is comprised of an active compound component and a second sequence-specific component is then introduced into the patient, thereby forming a complex with the first reagent via the recognition and binding of the sequence-specific components of the two reagents to form the reagent conjugate of the invention. The active compound component is thereby efficiently and specifically delivered to the target analyte.

Claims

1. A method for visualizing specific target analytes in a subject which comprises:

(1) providing a reagent conjugate useful for delivering active compounds to specific target analytes into a subject, said conjugate comprising

(a) a first reagent comprising a specific target recognition component which comprises a Fab′

antibody fragment which is covalently attached to a first sequence-specific component comprising a first oligonucleotide comprised of 2′

-chlorodeoxy-guanosine which is about 20 bases long; and

(b) a second reagent comprising an active compound component linked to a second sequence-specific component which specifically recognizes and binds to said first sequence-specific component, said second reagent-comprised of ⁹⁰y bound to a 1,2-diaminocyclohexane-tetraacetate (DCTA) chelating moiety, said second sequence-specific component comprising a second oligonucleotide, said DCTA chelating moiety being covalently attached through the allylamino group at the 5-position of a cytidyl residue in said second oligonucleotide which comprises the sequence [(2′

-chloro-deoxycytosine)₃(2′

-chloro-deoxycytosine-5-allylamine)]₅, and

wherein said first and second sequence-specific components (a) and (b) have been rendered nuclease resistant by the presence of one or more nucleotide analogs to said oligonucleotides;

(2) introducing into said subject an effective amount of said reagent conjugate; and

(3) visualizing said specific target analytes.

2. The method of claim 1, wherein said introducing step 2) is carried out by an administration means comprising intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or combinations thereof.

3. The method of claim 1, wherein said target analytes comprise low or high molecular weight molecules, molecular complexes or cell surface components, and combinations thereof.

4. The method of claim 1, wherein said target analytes comprise biological systems or parts thereof which comprise abnormal eukaryotic cells, cells with proteins not generally found on cell surfaces, cells with proteins found only at a defined stage on the life cycle of the organism, or pathogenic components of cells, tissues, and organs, and combinations thereof.

5. The method of claim 4, wherein said abnormal eukaryotic cells comprise cancer cells.

6. The method of claim 1, wherein said target analytes comprise biological systems selected from cells, tissues and organs bearing cell surface epitopes, or combinations thereof.

7. The method of claim 6, wherein said cells, tissues or organs bearing cell surface epitopes are located in a lymph node, an inflammation, a tumor, or an abscess in said subject.

8. A method for delivering active compound components to specific target analytes in a subject which comprises:

(1) providing;

(a) a first reagent comprising a specific target recognition component which comprises a Fab′

antibody fragment which is covalently attached to a first sequence-specific component comprising a first oligonucleotide comprised of 2′

-chlorodeoxy-guanosine which is about 20 bases long; and

(b) a second reagent comprising an active compound component linked to a second sequence-specific component which specifically recognizes and binds to said first sequence-specific component, said second reagent comprised of ⁹⁰y bound to a 1,2-diaminocyclohexane-tetraacetate (DCTA) chelating moiety, said second sequence-specific component comprising a second oligonucleotide, said DCTA chelating moiety being covalently attached through the allylamino group at the 5-position of a cytidyl residue in said second oligonucleotide which comprises the sequence [(2′

-chloro-deoxycytosine)₃ (2′

-chloro-deoxycytosine-5-allylamine)]₅;

wherein said first and second sequence-specific components (a) and (b) have been rendered nuclease resistant by the presence of one or more nucleotide analogs to said oligonucleotides;

(2) introducing into said subject an effective amount of said first reagent;

(3) allowing said first reagent to equilibrate to achieve sufficiently maximal localization on said specific targets analytes;

(4) introducing into said subject an effective amount of said second reagent; and

(5) allowing said second reagent to bind to said first reagent, thereby delivering said active compound components to the specific target analytes in said subject.

9. The method of claim 8, wherein said introducing step (2) is carried out by an administration means comprising intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or combinations thereof.

10. The method of claim 8, wherein any of said first reagent that is unbound to said target analyte is cleared from the subject'"'"'s circulatory system by administering to said subject a clearing agent following step (3).

11. The method of claim 8, wherein said target recognition component of the first reagent is introduced and allowed to equilibrate on said target analytes prior to the introduction and binding of said first sequence specific component, thereby forming said first reagent in vivo.

12. The method of claim 8, wherein said second sequence-specific component is introduced and allowed to bind to said first sequence-specific component following step (3) and prior to the introduction and binding of said active compound component, thereby forming said second reagent in vivo.

13. The method of claim 8, wherein said target analytes comprise biological systems or parts thereof which comprise abnormal eukaryotic cells, cells with proteins not generally found on cell surfaces, cells with proteins found only at a defined stage on the life cycle of the organism, or pathogenic components of cells, tissues, and organs, and combinations thereof.

14. The method of claim 13, wherein said abnormal eukaryotic cells comprise cancer cells.