Cysteine engineered antibodies and conjugates
First Claim
1. A method of screening cysteine engineered antibodies for thiol reactivity comprising:
- (a) introducing one or more cysteine amino acids into a parent antibody by site-directed mutagenesis of a gene encoding a cysteine engineered antibody in a double-stranded (ds) plasmid; and
expressing the cysteine engineered antibody displayed on a viral particle selected from a phage or a phagemid particle;
(b) reacting the cysteine engineered antibody on the viral particle with a thiol-reactive affinity reagent comprising a biotin moiety to generate an affinity labelled, cysteine engineered antibody on the viral particle; and
(c) measuring the binding of the affinity labelled, cysteine engineered antibody on the viral particle to a capture media comprising streptavidin.
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Abstract
Antibodies are engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids. Antibody fragments may also be engineered with one or more cysteine amino acids to form cysteine engineered antibody fragments (ThioFab). Methods of design, preparation, screening, and selection of the cysteine engineered antibodies are provided. Cysteine engineered antibodies (Ab), optionally with an albumin-binding peptide (ABP) sequence, are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered antibody-drug conjugates having Formula I:
Ab-(L-D)p I
where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed.
77 Citations
6 Claims
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1. A method of screening cysteine engineered antibodies for thiol reactivity comprising:
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(a) introducing one or more cysteine amino acids into a parent antibody by site-directed mutagenesis of a gene encoding a cysteine engineered antibody in a double-stranded (ds) plasmid; and
expressing the cysteine engineered antibody displayed on a viral particle selected from a phage or a phagemid particle;(b) reacting the cysteine engineered antibody on the viral particle with a thiol-reactive affinity reagent comprising a biotin moiety to generate an affinity labelled, cysteine engineered antibody on the viral particle; and (c) measuring the binding of the affinity labelled, cysteine engineered antibody on the viral particle to a capture media comprising streptavidin. - View Dependent Claims (2, 3, 4, 5, 6)
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Specification