Methods for isolating compounds using cleavable linker bound matrices

• US 5,141,648 A
• Filed: 03/29/1991
• Issued: 08/25/1992
• Est. Priority Date: 12/02/1987
• Status: Expired due to Fees

• Current Assignee: NeoRx Corporation
• Original Assignee: NeoRx Corporation
• Inventors: Hylarides, Mark D., Vrudhula, Vivekananda M., Srinivasan, Ananthachari, Fitzner, Jeffrey N.
• Primary Examiner(s): Nucker, Christine M.
• Assistant Examiner(s): KIM, KAY KWANG AE
• Application Number:
  US07/678,535
• Time in Patent Office: 515 Days
• Field of Search: 210/656, 210/635, 210/198.2, 210/502.1, 530/344, 530/412, 530/413, 530/417, 530/427, 530/811, 530/812, 530/816, 536/112, 536/51, 536/127, 525/279, 525/283, 525/284, 436/527, 436/529, 436/531, 436/541, 436/824, 435/181, 435/815, 549/499, 546/244, 546/245, 546/248
• US Class Current: 210/635
• CPC Class Codes: A61K 2121/00 Preparations for use in therapy
• CPC Class Codes: A61K 2123/00 Preparations for testing in vivo
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: A61K 47/6889 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
• CPC Class Codes: A61K 51/0491 Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
• CPC Class Codes: A61K 51/1093 conjugates with carriers being antibodies
• CPC Class Codes: C07H 15/04 attached to an oxygen atom of the saccharide radical
• CPC Class Codes: C07H 15/12 attached to a nitrogen atom of the saccharide radical
• CPC Class Codes: C07H 15/18 Acyclic radicals, substituted by carbocyclic rings
• CPC Class Codes: C07H 15/252 Naphthacene radicals, e.g. daunomycins, adriamycins
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: Y10S 435/815 by sorption
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 530/811 Peptides or proteins is immobilized on, or in, an inorganic carrier
• CPC Class Codes: Y10S 530/816 Attached to the carrier via a bridging agent

Abstract

The present invention provides cleavable conjugates whose linkers contain 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 invention also provides methods for producing cleavable conjugates. Preferred agents include drugs, toxins, biological response modifiers, radiodiagnostic compounds, radiotherapeutic compounds, and derivatives thereof. 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. The invention further provides methods for delivering to the cytoplasm of a target cell an agent free of its targeting molecule carrier. A diagnostically/therapeutically effective dose of a cleavable conjugate is administered to a warm-blooded animal such as man.

Another aspect of the invention provides methods for isolating a compound. The compound binds covalently to a solid phase which has been derivatized with the linkers described above and is released in native form by a variety of mild conditions.

An additional aspect of the invention provides methods for introducing into a compound a free sulfhydryl, amino, or hydroxyl group by use of reagents structurally related to the linkers described above. Preferred uses of the method are to add a free amino or a free sulfhydryl group to a protein, such as an antibody, or a drug.

Claims

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

conjugating to a solid phase a reagent having the formula;

##STR31## where;

R is a chemically reactive moiety wherein R is attached directly or indirectly to one of the carbons designated α

or β

;

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

n is 0 to 30;

Y is an O, S or NR'"'"', wherein R'"'"' is an alkyl group of C₆ or less;

n'"'"' is 1 to 2; and

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

to form a derivatized solid phase; and

contacting said derivatized solid phase with a sample solution in which a compound containing an available nucleophilic group is present, such that said compound binds to said derivatized solid phase, thereby removing said compound from said sample solution.

2. The method of claim 1 wherein the solid phase is controlled pore glass, polyvinyl, polyacrylamide, polydextran, or polystyrene.

3. The method of claim 1 wherein R is an amino group, sulfhydryl group, hydroxyl group, carbonyl-containing group, or alkyl X'"'"', where X'"'"' is a leaving group.

4. The method of claim 1 wherein Y is an O and n'"'"' is 1.

5. The method of claim 1 wherein the available nucleophilic group of the compound is a sulfhydryl, amino, or hydroxyl group.

6. The method of claim 1, additionally including, after the step of contacting, washing the solid phase to remove noncovalently bound compounds.

7. The method of claim 1, additionally including, after the step of contacting, releasing said bound compound from said derivatized solid phase.

8. The method of claim 7 wherein the step of releasing comprises releasing the bound compound by decreasing the pH to 6.0 or lower.

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

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

##STR32## where;

R'"'"' is a chemically reactive group;

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

n is 0 or 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 γ

, 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 γ

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 reacting with R'"'"' is present, such that said compound binds to said derivatized solid phase, thereby removing said compound from said sample solution.

10. The method of claim 9 wherein the solid phase is controlled pore glass, polyvinyl, polyacrylamide, polydextran, or polystyrene.

11. The method of claim 9, additionally including, after the step of contacting, washing the solid phase to remove noncovalently bound compounds.

12. The method of claim 9, additionally including, after the step of contacting, releasing said bound compound from said derivatized solid phase.

13. The method of claim 12 wherein the step of releasing comprises releasing the bound compound by decreasing the pH to 6.0 or lower.