Radiolabeling method
View Patent ↗Antibody binding assays and radiolabeling kits are disclosed for radiolabeling and testing therapeutic antibodies in the commercial setting. In particular, the kits are designed for making and evaluating radiolabeled anti-CD20 conjugates to be used for the treatment and imaging of B cell lymphoma tumors. All kit reagents are sterile and are designed to achieve a high level of antibody radiolabeling and product stability with results which are highly reproducible.
1. A method for radiolabeling a chelator-conjugated antibody with 90 Y, comprising:
(i) mixing chelator-conjugated antibody with a solution containing 90 Y; and
(ii) incubating the mixture for a time between 3 and 10 minutes at appropriate temperature to produce a radiolabeled antibody having sufficient radioincorporation and specific activity of about 3.2 mCi/mg to about 16.7 mCi/mg, without further purification of the radiolabeled antibody from unincorporated radiolabel.
2. The method of claim 1 , wherein the chelator is selected from the group consisting of MX-DTPA, phenyl-DTPA, benzyl-DTPA, CHX-DTPA and DOTA.
3. The method of claim 2 , wherein the chelator is MX-DTPA.
4. The method of claim 2 , wherein the antibody is an anti-CD20 antibody.
5. The method of claim 4 , wherein the antibody is a chimeric anti-CD20 antibody.
6. The method of claim 4 , wherein the anti-CD20 antibody is 2B8.
7. The method of claim 4 , wherein the chelator-conjugated anti-CD20 antibody is 2B8-MX-DTPA.
8. The method of claim 1 , wherein the solution containing the radiolabel is adjusted to a pH of about 3 to 6 before it is mixed with the chelator-conjugated antibody.
9. The method of claim 8 , wherein the pH is adjusted with a sodium acetate solution.
10. The method of claim 9 , wherein the sodium acetate is at a concentration of about 10 to 1000 mM.
11. The method of claim 1 , wherein the radiolabeled antibodies produced in step (ii) have at least 70% immunoreactivity.
12. The method of claim 1 , wherein the radiolabel is 90Y chloride, and the formulation buffer comprises a radioprotectant that inhibits radiolysis of the 90Y-labeled antibody selected from human serum albumin (HSA) and free-radical scavengers.
13. The method of claim 12 , wherein the specific activity of the 90Y -labeled antibodies is in the range of 10-15 mci/mg antibody.
14. The method of claim 12 , wherein the volume quantity of 90Y chloride used is between about 5 to 100 mCi divided by the radioactivity concentration at the time of labeling.
15. The method of claim 14 , wherein the volume quantity of 90Y chloride used is about 45 mCi divided by the radioactivity concentration at the time of labeling.
16. The method of claim 12 , wherein about 1 to 2 ml of MX-DTPA-conjugated antibody at a concentration of about 0.5 to 30 mg/ml is mixed with the radiolabel solution.
17. The method of claim 12 , wherein the chelator is MX-DTPA.
18. The method of claim 1 , wherein the formulation buffer is added in an amount necessary to achieve a total final volume of about 10 ml to about 50 ml.
19. The method of claim 1 , further comprising diluting the radiolabeled antibody to an appropriate concentration in formulation buffer for administration to a human patient.
20. The method of claim 19 , wherein the mixture is incubated for 3 minutes.
21. The method of claim 19 , wherein the mixture is incubated for 5 minutes.
22. The method of claim 19 , wherein the formulation buffer contains physiological saline, a radioprotectant, and unconjugated chelator.
23. The method of claim 22 , wherein the radioprotectant is selected from the group consisting of human serum albumin (HSA), ascorbate, ascorbic acid, phenol, sulfites, glutathione, cysteine, gentisic acid, nicotinic acid, ascorbyl palmitate, HOP(:O)H 2 , glycerol, sodium formaldehyde sulfoxylate, Na 2 S 2 O 5 , Na 2 S 2 O 3 and SO 2 .
24. The method of claim 22 , wherein the unconjugated chelator is DTPA or EDTA.
25. A method for radiolabeling a chelator-conjugated antibody with 90Y, comprising:
(i) mixing chelator-conjugated antibody with a solution containing 90Y; and
(ii) incubating the mixture for 3-10 minutes at an appropriate temperature to produce a radiolabeled antibody having sufficient radioincorporation of at least 95%; and
(iii) diluting the radiolabeled antibody to an appropriate concentration in formulation buffer for administration to a human patient without further purification of the radiolabeled antibody from unincorporated radiolabel.
26. The method of claim 25 , wherein the mixture is incubated for 3 minutes.
27. The method of claim 25 , wherein the mixture is incubated for 5 minutes.