CYSTEINE ENGINEERED ANTIBODIES AND CONJUGATES
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Abstract
Cysteine engineered antibodies comprising a free cysteine amino acid in the heavy chain or light chain are prepared by mutagenizing a nucleic acid sequence of a parent antibody and replacing one or more amino acid residues by cysteine to encode the cysteine engineered antibody; expressing the cysteine engineered antibody; and isolating the cysteine engineered antibody. Certain highly reactive cysteine engineered antibodies were identified by the PHESELECTOR assay. Isolated cysteine engineered antibodies may be covalently attached to a capture label, a detection label, a drug moiety, or a solid support.
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Citations
43 Claims
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1-28. -28. (canceled)
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29. A method of preparing an antibody-drug conjugate comprising reacting at least one free cysteine of a cysteine engineered antibody (Ab) with a linker-drug (L-D) reagent to form an antibody-drug conjugate having Formula I:
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Ab-(L-D)p
Iwherein Ab is the cysteine engineered antibody, L is a linker, D is a drug moiety, and p is 1, 2, 3, or 4; and
wherein the cysteine engineered antibody comprises one or more free cysteine amino acids, wherein at least one free cysteine amino acid is located at a position selected from heavy chain positions 4, 20, 23, 27, 32, 47, 68, 76, 78, 80, 81, 82, 94, 98, 108, 113, 154, 162, and 164 by Kabat numbering and light chain positions 49, 53, 73, 75, 80, 92, 95, 99, 101, 137, 138, 149, and 183 by Kabat numbering.- View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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37. The method of claim 29, wherein D is a maytansinoid having the structure:
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38. The method of claim 29, wherein D is selected from the structures:
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39. The method of claim 29, wherein the antibody drug conjugate has the structure:
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40. The method of claim 29, wherein D is a monomethylauristatin drug moiety MMAE or MMAF having the structures:
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41. The method of claim 29, wherein the antibody-drug conjugate is selected from the structures:
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42. The method of claim 29, wherein the linker-drug (L-D) reagent comprises a thiol-reactive agent.
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43. The method of claim 42, wherein the thiol-reactive agent is selected from a maleimide, an iodoacetamide, and a pyridyl disulfide.
Specification