Cysteine engineered antibodies and conjugates
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.
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.
2. The method of screening of claim 1 wherein the thiol-reactive affinity reagent comprises a maleimide moiety.
3. The method of screening of claim 1 wherein the parent antibody is an antibody fragment Fab.
4. The method of claim 1 wherein the cysteine engineered antibody is displayed on phage.
5. The method of screening of claim 1 wherein the parent antibody is a fusion protein comprising an albumin-binding peptide (ABP).
6. The method of screening of claim 5 wherein the ABP comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.