# US 5,346,687 A · Direct radiolabeling of antibody against stage specific embryonic antigen for diagnostic imaging

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- Number: US 5,346,687 A
- Document ID: US5346687A
- Status: Granted
- Kind: A
- Application number: 07/816,476
- Filed: 1992-01-03
- Granted: 1994-09-13
- Earliest claimed: 1989-08-09
- Assignees: Rhomed Incorporated
- Inventors: Buck A. Rhodes
- Web page: https://patentgrep.com/patent/US5346687A

## CPC

- C07K 14/52: Cytokines; Lymphokines; Interferons
- A61K 51/10: Antibodies or immunoglobulins; Fragments thereof {, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody}
- C07K 14/655: Somatostatins
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2121/00: Preparations for use in therapy
- A61K 2123/00: Preparations for testing in vivo
- Y10S 530/866: involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Antibody against stage specific embryonic antigen-1 is radiolabeled by direct means with a radionuclide for use in detection of occult abscess and inflammation. Radiolabeling is accomplished by partial reduction of the disulfide bonds of the antibody using Sn(II), or using other reducing agents followed by the addition of Sn(II), removal of excess reducing agent and reduction by-products, and addition of a specified amount of radionuclide reducing agent, such as stannous tartrate. The resulting product may be store frozen or lyophilized, with radiolabeling accomplished by the addition of the radionuclide.

## Claims

### Claim 1

A method of radiolabeling antibody against stage specific embryonic antigen-1 with a radionuclide to obtain stable labeling, comprising the steps of:
a) incubating the antibody against stage specific embryonic antigen-1 with a Sn (II) agent, the period of incubation being sufficient to reduce available disulfide bonds to thiolate groups and allow formation of Sn (II)-containing and sulfur-containing complexes, while preventing excessive fragmentation of the antibody;
b) purifying the reduced antibody to remove uncomplexed Sn (II) agents and other impurities yet retaining Sn (II) in a sufficient amount to reduce the radionuclide and not generate significant radio-chemical impurities, the radionuclide to be added in a subsequent step; and
c) radiolabeling the purified antibody with the Sn (II)-containing and sulfur-containing complexes by adding the radionuclide, whereby the Sn (II) agent reduces the radionuclide and the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes.

### Claim 2 (depends on 1)

The method of claim 1 wherein the source of Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0.

### Claim 3 (depends on 1)

The method of claim 1 wherein the source of Sn (II) agent comprises a member selected from the group consisting of stannous tartrate, stannous glucoheptonate, stannous gluconate, stannous phosphonate, stannous chloride, and stannous fluoride.

### Claim 4 (depends on 1)

The method of claim 1 wherein the antibody against stage specific embryonic antigen-1 is a product of the MCA-480 hybridoma.

### Claim 5 (depends on 1)

The method of claim 1 wherein following step b), prior to step c), the purified antibody with Sn (II)-containing and sulfur-containing complexes is frozen in a vial, whereby the frozen purified antibody with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step c) by the addition of the radionuclide to the vial.

### Claim 6 (depends on 1)

The method of claim 1 wherein following step b), and prior to step c), the purified antibody with Sn (II)-containing and sulfur-containing complexes is lyophilized in a vial, whereby the lyophilized purified antibody with Sn (II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling in step c) by the addition of the radionuclide to the vial.

### Claim 7 (depends on 1)

The method of claim 1 wherein 85 percent or more of the radionuclide is strongly bonded to the antibody.

### Claim 8 (depends on 1)

The method of claim 1 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate.

### Claim 9 (depends on 1)

The method of claim 1 wherein the radionuclide comprises a member selected from the group consisting of radioisotopes of indium, gold, silver, mercury, technetium, rhenium and copper.

### Claim 10

A method of producing reduced oxidation resistant intact antibody against stage specific embryonic antigen-1 for subsequent labeling with a radionuclide to obtain stable labeling, comprising the ordered steps of:
a) incubating the intact antibody against stage specific embryonic antigen-1 with a first reducing agent, the period of incubation being sufficient to reduce available disulfide bonds to thiolate groups while preventing excessive fragmentation of the antibody;
b) adding a source of first Sn (II) agent to the reduced intact antibody in a sufficient amount to form Sn (II)-containing and sulfur=containing complexes, resulting in formation of complexes between sulfur and Sn (II) which protect against subsequent reoxidation prior to purification and radiolabeling, the reduced intact antibody to purified in a subsequent step, and the radionuclide to be added in a subsequent step;
c) purifying the rescued intact antibody to substantially remove the first reducing agent and impurities;
d) freezing the purified reduced intact antibody containing Sn (II)-containing and sulfur-containing complexes in a vial, whereby the frozen purified reduced antibody containing Sn(II)-containing and sulfur-containing complexes can be maintained for an indefinite period before radiolabeling by the addition of the radionuclide; and
e) radiolabeling the purified reduced intact antibody by adding the radionuclide, whereby the complexed first Sn(II) agent reduces the radionuclide and the reduced radionuclide and reduced antibody form stable radionuclide-containing and sulfur-containing complexes.

### Claim 11 (depends on 10)

The method of claim 10 wherein the first reducing agent comprises at least one member selected from the group consisting of 2-mercaptoethanol; 1,4 dithiothreitol; 2,3 dihydroxybutane-1; 4 dithiol; 2-aminoethanethiol HCl; 2-mercaptoethylamine; thioglycolate; cyanide; cysteine; reduced gluthaione; Sn(II); Cu(I); and Ti(II).

### Claim 12 (depends on 10)

The method of claim 10 wherein the first reducing agent is attached to a solid phase.

### Claim 13 (depends on 10)

The method of claim 10 wherein the source of first Sn(II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 to 6.0.

### Claim 14 (depends on 10)

The method of claim 10 wherein the source of first Sn(II) agent comprises a member selected from the group consisting of stannous tartrate, stannous glucoheptonate, stannous gluconate, stannous phosphonate, stannous chloride, and stannous fluoride.

### Claim 15 (depends on 10)

The method of claim 10 wherein the antibody against stage specific embryonic antigen-1 is a product of the MCA-480 hybridoma.

### Claim 16 (depends on 10)

The method of claim 10 wherein following step c), and prior to step d), a second Sn(II) agent is added to the purified reduced intact antibody containing Sn(II)-containing and sulfur-containing complexes in a sufficient amount to completely reduce the radionuclide, whereby the reduced radionuclide and reduced antibody form stable radionuclide-containing and sulfur-containing complexes.

### Claim 17 (depends on 10)

The method of claim 10 wherein following step d), and prior to step e), the frozen purified reduced intact antibody with Sn(II) agent is lyophilized in a vial, whereby the lyophilized purified reduced antibody with Sn(II) agent can be maintained for an indefinite period before radiolabeling in step e) by the addition of the radionuclide to the vial.

### Claim 18 (depends on 18)

The method of claim 18 wherein 85 percent or more of the radionuclide is strongly bonded to the antibody.

### Claim 19 (depends on 10)

The method of claim 10 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate.

### Claim 20 (depends on 10)

The method of claim 10 wherein the radionuclide comprises a member selected from the group consisting of radioisotopes of indium, gold, silver, mercury, technetium, rhenium and copper.