# US 11,028,126 B2 · Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof

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- Number: US 11,028,126 B2
- Document ID: US11028126B2
- Status: Granted
- Kind: B2
- Application number: 16/160,846
- Filed: 2018-10-15
- Published: 2019-05-09
- Granted: 2021-06-08
- Earliest claimed: 2014-01-31
- Assignees: CytomX Therapeutics, Inc.
- Applicants: CytomX Therapeutics, Inc.
- Inventors: Stephen James Moore; Margaret Thy Luu Nguyen; Daniel Robert Hostetter; Olga Vasiljeva
- Web page: https://patentgrep.com/patent/US11028126B2

## CPC

- A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
- A61K 47/6835: {the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site}
- 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 49/0056: {Peptides, proteins, polyamino acids}
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 33/00: Antiparasitic agents
- A61P 35/00: Antineoplastic agents
- C07K 7/06: having 5 to 11 amino acids
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- A61K 2039/505: {comprising antibodies}
- C07K 2317/54: F(ab')2
- C07K 2317/55: Fab or Fab'
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/50: containing protease site
- 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 polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator (uPA), to activatable antibodies and other larger molecules that include the cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator, and to methods of making and using these polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator in a variety of therapeutic, diagnostic and prophylactic indications.

## Claims

### Claim 1

An isolated polypeptide comprising a cleavable moiety (CM) comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 366-368, wherein the cleavable moiety is a substrate for a protease.

### Claim 2 (depends on 1)

The isolated polypeptide of claim 1, wherein the CM comprises the amino acid sequence of SEQ ID NO: 366.

### Claim 3 (depends on 1)

The isolated polypeptide of claim 1, wherein the CM comprises the amino acid sequence of SEQ ID NO: 367.

### Claim 4 (depends on 1)

The isolated polypeptide of claim 1, wherein the CM is a substrate for at least a matriptase protease or a u-plasminogen activator (uPA) protease.

### Claim 5 (depends on 1)

The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises at least one additional moiety (M) selected from the group consisting of a moiety that is located amino (N) terminally to the CM (MN), a moiety that is located carboxyl (C) terminally to the CM (MC), and combinations thereof.

### Claim 6 (depends on 5)

The isolated polypeptide of claim 5, wherein the isolated polypeptide comprises at least one MN and at least one MC.

### Claim 7 (depends on 5)

The isolated polypeptide of claim 5, wherein MN is selected from the group consisting of a masking moiety, an antibody, a protein, a therapeutic agent, an antineoplastic agent, a toxic agent, a drug, a detectable moiety, a diagnostic agent, and an affinity tag.

### Claim 8 (depends on 5)

The isolated polypeptide of claim 5, wherein MC is selected from the group consisting of a masking moiety, an antibody, a protein, a therapeutic agent, an antineoplastic agent, a toxic agent, a drug, a detectable moiety, a diagnostic agent, and an affinity tag.

### Claim 9 (depends on 1)

The isolated polypeptide of claim 1, wherein the polypeptide comprises an antibody or antigen binding fragment thereof (AB) that binds a target.

### Claim 10 (depends on 9)

The isolated polypeptide of claim 9, wherein the CM is a substrate for a protease that is co-localized in a tissue with the target.

### Claim 11 (depends on 9)

The isolated polypeptide of claim 9, 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 12 (depends on 9)

The isolated polypeptide of claim 9, wherein the AB is linked to the CM.

### Claim 13 (depends on 12)

The isolated polypeptide of claim 12, wherein the AB is linked directly to the CM.

### Claim 14 (depends on 12)

The isolated polypeptide of claim 12, wherein the AB is linked to the CM via a linking peptide.

### Claim 15 (depends on 9)

The isolated polypeptide of claim 9, wherein the isolated polypeptide comprises a masking moiety (MM), wherein the MM has an equilibrium dissociation constant for binding to the AB that is greater than the equilibrium dissociation constant of the AB for binding to the target.

### Claim 16 (depends on 15)

The isolated polypeptide of claim 15, wherein the MM is a polypeptide of no more than 40 amino acids in length.

### Claim 17 (depends on 15)

The isolated polypeptide of claim 15, wherein the MM is linked to the CM such that the isolated polypeptide in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM—CM—AB or AB—CM—MM.

### Claim 18 (depends on 17)

The isolated polypeptide of claim 17, wherein the isolated polypeptide comprises a linking peptide between the MM and the CM.

### Claim 19 (depends on 17)

The isolated polypeptide of claim 17, wherein the isolated polypeptide comprises a linking peptide between the CM and the AB.

### Claim 20 (depends on 17)

The isolated polypeptide of claim 17, wherein the isolated polypeptide comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the isolated polypeptide has the structural arrangement from N-terminus to C-terminus as follows in the uncleaved state: MM—LP1—CM—LP2-AB or AB—LP2—CM—LP1—MM.

### Claim 21 (depends on 20)

The isolated polypeptide of claim 20, wherein the two linking peptides need not be identical to each other.

### Claim 22 (depends on 20)

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

### Claim 23 (depends on 15)

The isolated polypeptide of claim 15, wherein the amino acid sequence of the MM is different from that of the target.

### Claim 24 (depends on 15)

The isolated polypeptide of claim 15, wherein the MM does not interfere or compete with the AB for binding to the target in a cleaved state.

### Claim 25 (depends on 9)

The isolated polypeptide of claim 9 comprising a T cell-engaging scFv linked to the AB.

### Claim 26 (depends on 25)

The isolated polypeptide of claim 25, wherein the T cell-engaging scFv comprises a masking moiety.

### Claim 27 (depends on 15)

The isolated polypeptide of claim 15 comprising a T cell-engaging scFv linked to the AB.

### Claim 28 (depends on 27)

The isolated polypeptide of claim 27, wherein the T cell-engaging scFv comprises a masking moiety.

### Claim 29 (depends on 9)

The isolated polypeptide of claim 9 conjugated to an agent.

### Claim 30 (depends on 29)

The isolated polypeptide of claim 29, wherein the agent is a toxin or fragment thereof.

### Claim 31 (depends on 29)

The isolated polypeptide of claim 29, wherein the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, and a calicheamicin or a derivative thereof.

### Claim 32 (depends on 29)

The isolated polypeptide of claim 29, wherein the agent is a detectable moiety.

### Claim 33 (depends on 29)

The isolated polypeptide of claim 29, wherein the agent is conjugated to the polypeptide via a linker.

### Claim 34 (depends on 33)

The isolated polypeptide of claim 33, wherein the linker is a cleavable linker.

### Claim 35 (depends on 15)

The isolated polypeptide of claim 15, wherein the AB is conjugated to an agent.

### Claim 36 (depends on 35)

The isolated polypeptide of claim 35, wherein the agent is a toxin or fragment thereof.

### Claim 37 (depends on 35)

The isolated polypeptide of claim 35, wherein the agent is selected from the group consisting of a dolastatin, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, and a calicheamicin or derivative thereof.

### Claim 38 (depends on 35)

The isolated polypeptide of claim 35, wherein the agent is auristatin E or a derivative thereof.

### Claim 39 (depends on 35)

The isolated polypeptide of claim 35, wherein the agent is monomethyl auristatin E (MMAE).

### Claim 40 (depends on 35)

The isolated polypeptide of claim 35, wherein the agent is monomethyl auristatin D (MMAD).

### Claim 41 (depends on 35)

The isolated polypeptide of claim 35, wherein the agent is DM1 or DM4.

### Claim 42 (depends on 35)

The isolated polypeptide of claim 35, wherein the agent is conjugated to the AB via a linker.

### Claim 43 (depends on 42)

The isolated polypeptide of claim 42, wherein the linker is a cleavable linker.

### Claim 44 (depends on 9)

The isolated polypeptide of claim 9 comprising a detectable moiety.

### Claim 45 (depends on 44)

The isolated polypeptide of claim 44, wherein the detectable moiety is a diagnostic agent.

### Claim 46 (depends on 1)

An isolated nucleic acid molecule encoding the isolated polypeptide of claim 1.

### Claim 47 (depends on 46)

A vector comprising the isolated nucleic acid molecule of claim 46.

### Claim 48 (depends on 9)

An isolated nucleic acid molecule encoding the isolated polypeptide of claim 9.

### Claim 49 (depends on 48)

A vector comprising the isolated nucleic acid molecule of claim 48.

### Claim 50 (depends on 15)

An isolated nucleic acid molecule encoding the isolated polypeptide of claim 15.

### Claim 51 (depends on 50)

A vector comprising the isolated nucleic acid molecule of claim 50.

### Claim 52 (depends on 47)

A method of producing a cleavable moiety (CM) containing polypeptide by culturing a cell under conditions that lead to expression of the polypeptide, wherein the cell comprises the vector of claim 47.

### Claim 53 (depends on 1)

A method of manufacturing a cleavable moiety (CM) containing polypeptide, the method comprising:
(a) culturing a cell comprising a nucleic acid construct that encodes the isolated polypeptide of claim 1 under conditions that lead to expression of the polypeptide, and
(b) recovering the polypeptide.

### Claim 54 (depends on 1)

The isolated polypeptide of claim 1, wherein the CM comprises the amino acid sequence of SEQ ID NO: 368.