CYSTEINE ENGINEERED ANTIBODIES AND CONJUGATES

• US 20100003766A1
• Filed: 04/21/2009
• Published: 01/07/2010
• Est. Priority Date: 09/23/2004
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: EIGENBROT, CHARLES W., VANDLEN, RICHARD, LOWMAN, HENRY, RAAB, HELGA E., JUNUTULA, JAGATH REDDY
• Granted Patent: US 8,309,300 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: A61K 47/6889 Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/624 Disulfide-stabilized antibody (dsFv)

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.

Claims

1. A method of screening cysteine engineered antibodies for thiol reactivity comprising:

(a) introducing one or more cysteine amino acids into a parent antibody in order to generate a cysteine engineered antibody;

(b) reacting the cysteine engineered antibody with a thiol-reactive affinity reagent to generate an affinity labelled, cysteine engineered antibody; and

(c) measuring the binding of the affinity labelled, cysteine engineered antibody to a capture media.

2. The method of screening of claim 1 further comprising determining the thiol reactivity of the cysteine engineered antibody with the thiol-reactive reagent.

3. The method of screening of claim 1 wherein step (a) of the process comprises:

(i) site-directed mutagenesis of a gene encoding the cysteine engineered antibody in a double-stranded (ds) plasmid; and

(ii) expressing the cysteine engineered antibody.

4. The method of screening of claim 3 further comprising isolating and purifying the expressed cysteine engineered antibody.

5. The method of screening of claim 1 wherein the thiol-reactive affinity reagent comprises a biotin moiety.

6. The method of screening of claim 1 wherein the thiol-reactive reagent comprises a maleimide moiety.

7. The method of screening of claim 1 wherein the capture media comprises streptavidin.

8. The method of screening of claim 1 wherein the parent antibody is a fusion protein comprising an albumin-binding peptide (ABP).

9. The method of screening of claim 8 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.

10. The method of screening of claim 1 wherein the parent antibody is an antibody fragment Fab.

11. The method of claim 1 wherein the cysteine engineered antibody is displayed on phage.