# US 11,992,528 B2 · Compositions and methods for conjugating activatable antibodies

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,992,528 B2
- Document ID: US11992528B2
- Status: Granted
- Kind: B2
- Application number: 17/227,004
- Filed: 2021-04-09
- Published: 2022-03-24
- Granted: 2024-05-28
- 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/US11992528B2

## 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 an activatable antibody and conjugating an agent thereto resulting in selectivity in the placement of the agent, the method comprising:
reducing at least one interchain disulfide bond in the activatable antibody with a reducing agent without reducing any intrachain disulfide bonds within a masking moiety (MM) in the activatable antibody, thereby partially reducing the activatable antibody,
wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP),
wherein the ratio of the reducing agent to the activatable antibody is less than 5:1, and
wherein the reduction time is 30 minutes to 2 hours; and
conjugating an agent to at least one thiol of the partially reduced activatable antibody,
wherein the activatable antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to a target, the MM coupled to the AB that inhibits the binding of the AB to the target when the activatable antibody is in an uncleaved state, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease,
and
wherein the partial reduction of the activatable antibody and conjugation of the agent thereto does not disturb or negatively affect activation and/or efficacy of the activatable antibody, wherein the activity and/or efficacy of the activatable antibody is selected from the group consisting of: the masking activity of the MM to the AB when the activatable antibody is in an uncleaved state, the activation of the uncleaved activatable antibody by the protease, and the binding activity of the activated activatable antibody to the target of the AB.

### 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, wherein the second molecule is cysteine or glutathione.

### Claim 4 (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 or
wherein the activatable antibody in the uncleaved state comprises a spacer that is joined directly to the MM and has the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

The method of claim 5, wherein the agent is a microtubule inhibitor or a nucleic acid damaging agent.

### Claim 7 (depends on 5)

The method of claim 5, wherein the agent is a dolastatin, an auristatin, a maytansinoid, a duocarmycin, or a calicheamicin.

### Claim 8 (depends on 1)

The method of claim 1, wherein the agent is selected from the group consisting of: auristatin E, monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), DM1, and DM4.

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 9)

The method of claim 9, wherein the linker is a cleavable linker.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 11)

The method of claim 11, wherein the detectable moiety is a diagnostic agent.

### Claim 13 (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 14 (depends on 1)

The method of claim 1, wherein the MM has one or more properties selected from the group consisting of:
(i) 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,
(ii) the MM does not interfere or compete with the AB for binding to the target when the activatable antibody is in a cleaved state, and
(iii) the MM is a polypeptide of no more than 40 amino acids in length.

### Claim 15 (depends on 1)

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

### Claim 16 (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 17 (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 18 (depends on 17)

The method of claim 17, wherein LP1 and LP2 have one or more properties selected from the group consisting of:
(i) LP1 and LP2 are not identical to each other,
(ii) each of LP1 and LP2 is a peptide of 1 to 20 amino acids in length, and
(iii) 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 19 (depends on 1)

The method of claim 1, wherein the CM is a substrate for one or more enzymes selected from the group consisting of: a urokinase plasminogen activator (uPA), a legumain, a matriptase, and a matrix metalloprotease (MMP).

### Claim 20 (depends on 19)

The method of claim 19, wherein the MMP is MMP-9 or MMP-14.