# US 11,028,162 B2 · Methods for manufacturing activatable binding polypeptides comprising matrix metalloprotease cleavable moieties

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- Number: US 11,028,162 B2
- Document ID: US11028162B2
- Status: Granted
- Kind: B2
- Application number: 16/102,531
- Filed: 2018-08-13
- Published: 2019-07-11
- Granted: 2021-06-08
- Earliest claimed: 2007-08-22
- Assignees: The Regents of the University of California; CytomX Therapeutics, Inc.
- Applicants: The Regents of the University of California; CytomX Therapeutics, Inc.
- Inventors: Patrick Sean Daugherty; Nancy Stagliano; Jerry Thomas; Kathryn Kamath; James W. West; Sanjay Khare; Jason Sagert
- Web page: https://patentgrep.com/patent/US11028162B2

## CPC

- C07K 16/22: against growth factors {; against growth regulators}
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/24: against cytokines, lymphokines or interferons
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/2836: {against CD106}
- C07K 16/42: against immunoglobulins
- C12N 15/1034: {Isolating an individual clone by screening libraries}
- C12N 15/1044: {Preparation or screening of libraries displayed on scaffold proteins}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/50: containing protease site
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Activatable binding polypeptides (ABPs), which contain a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM) are provided. Activatable antibody compositions, which contain a TBM containing an antigen binding domain (ABD), a MM and a CM are provided. Furthermore, ABPs which contain a first TBM, a second TBM and a CM are provided. The ABPs exhibit an “activatable” conformation such that at least one of the TBMs is less accessible to target when uncleaved than after cleavage of the CM in the presence of a cleaving agent capable of cleaving the CM. Further provided are libraries of candidate ABPs, methods of screening to identify such ABPs, and methods of use. Further provided are ABPs having TBMs that bind VEGF, CTLA-4, or VCAM, ABPs having a first TBM that binds VEGF and a second TBM that binds FGF, as well as compositions and methods of use.

## Claims

### Claim 1

A method for manufacturing an activatable binding polypeptide (ABP), wherein the ABP comprises a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM), and wherein the TBM specifically binds to a target protein, wherein the target protein is VEGF-A, the method comprising:
a) selecting a MM from a plurality of candidate polypeptides, wherein the selected MM inhibits binding of the TBM to its target protein, and wherein the selected MM does not have an amino acid sequence of a naturally occurring binding partner of the TBM; and
b) constructing the ABP comprising the following structure MM-CM-TBM or TBM-CM-MM, comprising synthesizing a polypeptide construct that includes the ABP,
wherein the VL of the TBM comprises the complementarity determining regions (CDRs) of the VL domain of SEQ ID NO: 30 and the VH of the TBM comprises the complementarity determining regions (CDRs) of the VH domain of SEQ ID NO: 30,
wherein the MM is positioned in the ABP such that it is capable of binding the TBM and is capable of inhibiting binding of the TBM to the target protein when the ABP is in an uncleaved state, and
wherein the CM is a substrate for a protease, wherein the CM is a matrix metalloprotease (MMP) substrate, wherein the MMP substrate is a matrix metalloprotease 1 (MMP-1) substrate, a matrix metalloprotease 2 (MMP-2) substrate, a matrix metalloprotease 9 (MMP-9) substrate, or a matrix metalloprotease 14 (MMP-14) substrate, and wherein said CM is positioned in the ABP such that in a cleaved state in the presence of the target protein, the TBM binds the target protein, and in an uncleaved state in the presence of the target protein, binding of the TBM to the target protein is inhibited by the MM.

### Claim 2 (depends on 1)

The method of claim 1, wherein the TBM comprises an antibody or antigen binding fragment thereof comprising an antigen binding domain (ABD) that binds the target protein.

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

The method of claim 1, wherein the CM is a substrate for a protease that is co-localized in a tissue with the target protein.

### Claim 5 (depends on 1)

The method of claim 1, wherein when the ABP is an uncleaved state the CM is positioned N-terminal to the TBM.

### Claim 6 (depends on 1)

The method of claim 1, wherein when the ABP is an uncleaved state the CM is positioned C-terminal to the TBM.

### Claim 7 (depends on 2)

The method of claim 2, wherein when the ABP is an uncleaved state the CM is positioned N-terminal to a variable light (VL) chain of the ABD.

### Claim 8 (depends on 2)

The method of claim 2, wherein when the ABP is an uncleaved state the CM is positioned N-terminal to a variable heavy (VH) chain of the ABD.

### Claim 9 (depends on 2)

The method of claim 2, wherein the ABD is positioned at the N-terminus of the ABP.

### Claim 10 (depends on 2)

The method of claim 2, wherein the ABD is positioned at the C-terminus of the ABP.

### Claim 11 (depends on 2)

The method of claim 2, wherein the ABD is linked to the CM.

### Claim 12 (depends on 11)

The method of claim 11, wherein the ABD is linked to the CM via a linker peptide.

### Claim 13 (depends on 1)

The method of claim 1, wherein the MM binds the TBM with less affinity and/or avidity than the TBM binds to its target protein.

### Claim 14 (depends on 1)

The method of claim 1, wherein 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 when the ABP is in an uncleaved state the ABP exhibits reduced binding to the target protein relative to when the ABP is in a cleaved state.

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 1)

The method of claim 1, wherein when the ABP is in an uncleaved state the MM is linked to the CM.

### Claim 18 (depends on 16)

The method of claim 16, wherein when the ABP is in an uncleaved state the MM is linked to the CM via a linker peptide.

### Claim 19 (depends on 1)

The method of claim 1, wherein the ABP comprises a first linker peptide (L1) and a second linker peptide (L2), and wherein the ABP has the structural arrangement in the uncleaved state from N-terminus to C-terminus as follows: MM-L1-CM-L2-TBM or TBM-L2-CM-L1-MM.

### Claim 20 (depends on 19)

The method of claim 19, wherein the two linker peptides are not the same.

### Claim 21 (depends on 19)

The method of claim 19, wherein each of L1 and L2 is a peptide of about 1 to 20 amino acids in length.

### Claim 22 (depends on 19)

The method of claim 19, wherein one or both of L1 and L2 comprise a glycine-serine copolymer.

### Claim 23 (depends on 19)

The method of claim 19, wherein at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, GSGGS (SEQ ID NO: 1) and (GGGS)n (SEQ ID NO: 2), where n is an integer of at least one.

### Claim 24 (depends on 19)

The method of claim 19, wherein at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).

### Claim 25 (depends on 2)

The method of claim 2, wherein the ABD is from an antibody selected from the group consisting of bevacizumab and ranibizumab.

### Claim 26 (depends on 1)

The method of claim 1, wherein the VL of the TBM comprises the VL domain of SEQ ID NO: 30 and the VH of the TBM comprises the VH domain of SEQ ID NO: 30.

### Claim 27 (depends on 1)

The method of claim 1, wherein the step of synthesizing a polypeptide construct comprises:
synthesizing a nucleic acid construct that encodes the ABP; and
expressing the ABP in a host cell that includes the nucleic acid construct.