# US 9,517,276 B2 · Compositions and methods for conjugating activatable antibodies

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- Number: US 9,517,276 B2
- Document ID: US9517276B2
- Status: Granted
- Kind: B2
- Application number: 14/296,207
- Filed: 2014-06-04
- Published: 2015-01-01
- Granted: 2016-12-13
- Earliest claimed: 2013-06-04
- Assignees: CytomX Therapeutics, Inc.
- Applicants: CytomX Therapeutics, Inc.
- Inventors: Henry Bernard Lowman; Luc Roland Desnoyers; Tony W. Liang; Andrei William Konradi; Shweta Singh
- Web page: https://patentgrep.com/patent/US9517276B2

## CPC

- 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}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61K 47/68031: {the drug being an auristatin}
- A61K 47/6851: {the antibody targeting a determinant of a tumour cell}
- A61K 49/00: Preparations for testing in vivo
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/18: against material from animals or humans
- A61K 2039/505: {comprising antibodies}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 2317/21: from primates, e.g. man
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/00: Fusion polypeptide
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention relates generally to compositions and methods for conjugating antibodies and activatable antibodies, and methods of partially reducing antibodies and/or activatable antibodies prior to conjugation, e.g., thiol-based conjugation, with an agent, e.g., a therapeutic and/or diagnostic agent.

## Claims

### Claim 1

A method of partially reducing and conjugating an agent to an activatable antibody resulting in selectivity in the placement of the agent, the method comprising
partially reducing at least one disulfide bond in the activatable antibody with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP), and
conjugating the agent to at least one thiol,
wherein the activatable antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to epidermal growth factor receptor (EGFR),
wherein the AB comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 239, and 241, and a light chain comprising an amino acid sequence of SEQ ID NO: 16, a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 29 to 63 that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, and a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 183-202 and 298-318 coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

### Claim 2 (depends on 1)

The method of claim 1, wherein the at least one disulfide bond is an interchain disulfide bond.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one disulfide bond is a disulfide bond between the activatable antibody and a second molecule.

### Claim 4 (depends on 3)

The method of claim 3, wherein the second molecule is cysteine or glutathione.

### Claim 5 (depends on 1)

The method of claim 1, wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.

### Claim 6 (depends on 1)

The method of claim 1, wherein the agent is a toxin or fragment thereof.

### Claim 7 (depends on 6)

The method of claim 6, wherein the agent is a microtubule inhibitor.

### Claim 8 (depends on 6)

The method of claim 6, wherein the agent is a nucleic acid damaging agent.

### Claim 9 (depends on 6)

The method of claim 6, wherein the agent is a dolastatin or a derivative thereof, an auristatin or derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or derivative thereof, or a calicheamicin or derivative thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the agent is auristatin E or a derivative thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein the agent is monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD).

### Claim 12 (depends on 1)

The method of claim 1, wherein the agent is monomethyl auristatin E (MMAE).

### Claim 13 (depends on 1)

The method of claim 1, wherein the agent is monomethyl auristatin D (MMAD).

### Claim 14 (depends on 1)

The method of claim 1, wherein the agent is DM1 or DM4.

### Claim 15 (depends on 1)

The method of claim 1, wherein the agent is conjugated to the AB via a linker.

### Claim 16 (depends on 15)

The method of claim 15, wherein the linker is a cleavable linker.

### Claim 17 (depends on 1)

The method of claim 1, wherein the agent is a detectable moiety.

### Claim 18 (depends on 17)

The method of claim 17, wherein the detectable moiety is a diagnostic agent.

### Claim 19 (depends on 1)

The method of claim 1, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

### Claim 20 (depends on 1)

The method of claim 1, wherein the MM has an equilibrium dissociation constant for binding to the AB that is greater than the equilibrium dissociation constant of the AB to the target.

### Claim 21 (depends on 1)

The method of claim 1, wherein the MM does not interfere or compete with the AB for binding to the target when the activatable antibody is in a cleaved state.

### Claim 22 (depends on 1)

The method of claim 1, wherein the MM is a polypeptide of no more than 40 amino acids in length.

### Claim 23 (depends on 1)

The method of claim 1, wherein the CM is a polypeptide of up to 15 amino acids in length.

### Claim 24 (depends on 1)

The method of claim 1, wherein the protease is co-localized with the target in a tissue, and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

### Claim 25 (depends on 1)

The method of claim 1, wherein the activatable antibody comprises a linking peptide between the MM and the CM.

### Claim 26 (depends on 1)

The method of claim 1, wherein the activatable antibody comprises a linking peptide between the CM and the AB.

### Claim 27 (depends on 1)

The method of claim 1, wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in an uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.

### Claim 28 (depends on 27)

The method of claim 27, wherein the two linking peptides need not be identical to each other.

### Claim 29 (depends on 27)

The method of claim 27, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

### Claim 30 (depends on 27)

The method of claim 27, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 21), (GGGS)n (SEQ ID NO: 22), GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and GSSSG (SEQ ID NO: 28), where n is an integer of at least one.

### Claim 31 (depends on 1)

The method of claim 1, wherein the activatable antibody in an uncleaved state comprises a spacer, wherein the spacer is joined directly to the MM and has the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.