Covalently-linked complexes and methods for enhanced cytotoxicity and imaging

• US 5,169,933 A
• Filed: 08/07/1989
• Issued: 12/08/1992
• Est. Priority Date: 08/15/1988
• Status: Expired due to Fees

• Current Assignee: NeoRx Corporation
• Original Assignee: NeoRx Corporation
• Inventors: Nichols, Everett J., Fritzberg, Alan R., Abrams, Paul G., Morgan, A. Charles Jr., Anderson, David C.
• Primary Examiner(s): Nucker, Christine M.
• Assistant Examiner(s): Kim, Kay K.
• Application Number:
  US07/390,241
• Time in Patent Office: 1,219 Days
• Field of Search: 424/1.1, 424/9, 424/85.91, 530/395, 530/324, 530/325, 530/326, 530/327, 530/328, 530/329, 530/330, 530/377, 530/399, 530/403, 530/408, 530/409, 435/188
• US Class Current: 530/391.3
• CPC Class Codes: A61K 2123/00 Preparations for testing in vivo
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 51/1093 conjugates with carriers being antibodies
• CPC Class Codes: A61K 51/1096 radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/10 containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

Covalently-linked complexes (CLCs) for targeting a defined population of cells, comprising a targeting protein; a cytotoxic agent; and an enhancing moiety, wherein the enhancing moiety is capable of promoting CLC-target cell interaction are disclosed. Methods for using the claimed CLCs to obtain enhanced in vivo cytotoxicity and enhanced in vivo imaging are also described.

Claims

1. A covalently-linked complex (CLC) for targeting a defined population of cells, comprising:

a targeting protein;

a cytotoxic agent; and

an enhancing moiety, wherein the enhancing moiety promotes CLC-target cell interaction and exhibits alpha helical structure at acidic pH and substantially unfolds at physiological pH and exhibits little or no tertiary structure.

2. The covalently-linked complex of claim 1 wherein the targeting protein is covalently linked to the cytotoxic agent and to the enhancing moiety.

3. The covalently-linked complex of claim 1 wherein the cytotoxic agent is covalently linked to the targeting protein and to the enhancing moiety.

4. The covalently-linked complex of claim 1 wherein the enhancing moiety is covalently linked to the targeting protein and to the cytotoxic agent.

5. The covalently-linked complex of claim 1 wherein the targeting protein is selected from the group consisting of an antibody, an antibody fragment, an antigen-binding portion of an antibody, a biologically active peptide, a hormone, a growth factor, a biological response modifier, an enzyme, biotin, avidin, analogs thereof that retain the capacity to bind to the defined population of cells, and synthetic targeting proteins.

6. The covalently-linked complex of claim 1 wherein the cytotoxic agent is a radionuclide;

a toxin or a fragment or analog thereof;

a drug or an analog thereof;

a cytotoxic peptide;

or a combination of the foregoing.

7. The covalently-linked complex of claim 6 wherein the radionuclide is selected from the group consisting of gamma-emitters, positron-emitters, Auger electron-emitters, X-rayemitters, fluorescence-emitters, beta-emitters and alpha-emitters.

8. The covalently-linked complex of claim 6 wherein the radionuclide is selected from the group consisting of ¹⁸⁸ Re, ¹⁸⁶ Re, ²⁰³ Pb, ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ⁷⁷ Br, ²¹¹ At, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁹⁸ Au, ¹⁹⁹ Ag, ¹²³ I, ¹³⁰ I, ¹³³ I, ¹³⁵ I, ⁴⁷ Sc, ⁷² As, ⁷² Se, ⁸⁸ Y, ¹⁰⁰ Pd, ^101m Rh, ¹¹⁹ Sb, ¹²⁸ Ba, ¹⁹⁷ Hg, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁵ Br, ⁷⁶ Br, ^99m Tc, ¹¹ C, ¹³ N, ¹⁵ O and ¹⁸ F.

9. The covalently-linked complex of claim 1 wherein the enhancing moiety is a translocating/internalizing moiety, an anchoring peptide, an accessory moiety, an intracellular retention moiety, a combination peptide, a fusion peptide or a combination thereof.

10. The covalently-linked complex of claim 9 wherein the translocating/internalizing moiety is selected from the group consisting of aa1-aa2-aa3-EAALA(EALA)₄ -EALEALAA-amide, TAT protein 37-62 fragment, CFITKALGISYGRKKRRQRRRPPQGS, growth factor-derived peptides, peptides containing the sequence CMHIESLDSYTC or CMYIEALDKYAC, estrogens, anti-estrogens peptides of apo-lipoprotein A-1 and B, melittin, bombolittin, delta hemolysin, pardaxins, alamethicin, calcitonin, corticotrophin releasing factor, beta endorphin, glucagon, parathyroid hormone, pancreatic polypeptide, signal sequences, hidden hydrophobic domains, anti-clathrin antibody or fragments thereof, pore-forming proteins, and analogs, derivatives and combinations thereof.

11. The covalently-linked complex of claim 10 wherein aa1 is cysteine or lysine.

12. The covalently-linked complex of claim 10 wherein aa2 and aa3 are selected from the group consisting of arginine, lysine, neutral amino acids and peptide spacers having 1-40 amino acids.

13. The covalently-linked complex of claim 9 wherein the enhancing moiety is covalently linked to the targeting protein or the cytotoxic agent through a peptide spacer having 1-40 amino acids.

14. The covalently-linked complex of claim 13 wherein the peptide spacer is CDNDNDDNDDGGG or CRQRQRRQRRGGG.

15. The covalently-linked complex of claim 1 wherein the enhancing moiety is covalently linked to the targeting protein or the cytotoxic agent through a peptide spacer having 1-40 amino acids.