# US 12,767,700 B2 · Compositions and methods comprising protease-activated therapeutic agents

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- Number: US 12,767,700 B2
- Document ID: US12767700B2
- Status: Granted
- Kind: B2
- Application number: 18/559,180
- Filed: 2022-05-05
- Published: 2024-07-11
- Granted: 2026-10-20
- Earliest claimed: 2021-05-07
- Assignees: The University of Chicago
- Applicants: The University of Chicago
- Inventors: Jeffrey Hubbell; Jun Ishihara; Juan Mendoza; Aslan Mansurov
- Web page: https://patentgrep.com/patent/US12767700B2

## CPC

- C07K 14/7155: {for interleukins [IL]}
- A61K 38/1709: {from mammals}
- A61K 38/1793: {for cytokines; for lymphokines; for interferons}
- A61K 38/2013: {IL-2}
- A61K 38/208: {IL-12}
- A61K 38/217: {IFN-gamma}
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K 47/643: {Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]}
- A61K 47/6843: {the antibody targeting a material from animals or humans}
- A61P 35/00: Antineoplastic agents
- C07K 14/47: from mammals
- C07K 14/5434: {IL-12}
- C07K 14/55: IL-2
- C07K 14/57: IFN-gamma
- C07K 14/7156: {for interferons [IFN]}
- 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)
- 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

## Abstract

The disclosure relates to the engineering of collagen-binding modification of masked therapeutic agents comprising one or more tumor-associated protease cleavage sites. Upon exposure to tumor-associated proteases in the tumor microenvironment, the polypeptide is cleaved, which unmasks the therapeutic agent, reducing off-target side effects and toxicity associated with systemic administration. Accordingly, aspects of the disclosure relate to a polypeptide comprising a therapeutic agent linked to a masking agent through a linker, wherein the linker comprises one or more tumor-associated protease cleavage sites, and wherein the masking agent blocks the association of the therapeutic agent to its therapeutic target, and further wherein the polypeptide is operatively linked to a collagen binding domain or a tumor-targeting agent.

## Claims

### Claim 1

A polypeptide comprising interleukin-12 (IL-12) linked to a masking agent through a linker, wherein the masking agent comprises IL-12R or an IL-12-binding fragment thereof, and wherein the linker comprises the amino acid sequence of SEQ ID NO: 13.

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the IL12 comprises the p35 and p40 subunits linked through a disulfide bond or through a peptide linker.

### Claim 3 (depends on 1)

The polypeptide of claim 1, wherein the IL12R polypeptide or fragment comprises IL12Rβ1, or a fragment thereof and wherein the IL12Rβ1 polypeptide or fragment comprises human FNI-II domain of IL-12Rβ1.

### Claim 4 (depends on 1)

The polypeptide of claim 1, wherein the masking agent is fused to the N-terminus of the p35 subunit of IL12, and wherein the linker is between the masking agent and the p35 subunit of IL12 or wherein the masking agent is fused to the C-terminus of the p40 subunit of IL12, and wherein the linker is between the masking agent and the p40 subunit of IL12.

### Claim 5 (depends on 1)

The polypeptide of claim 1, wherein the linker comprises the amino acid sequence of SEQ ID NO:219 or SEQ ID NO:220 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:219 or SEQ ID NO:220.

### Claim 6 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide is conjugated to a tumor targeting agent.

### Claim 7 (depends on 6)

The polypeptide of claim 6, wherein the tumor-targeting agent comprises an antibody or antigen-binding fragment thereof, and wherein the antibody or antigen-binding fragment comprises a stroma targeting antibody or stroma-binding fragment thereof.

### Claim 8 (depends on 7)

The polypeptide of claim 7, wherein the tumor targeting agent comprises a Fab that specifically binds to an alternatively spliced domain of fibronectin comprising extra domain A (EDA).

### Claim 9 (depends on 7)

The polypeptide of claim 7, wherein the tumor targeting agent comprises an antibody or antigen binding fragment thereof that specifically binds to a tumor-associated antigen or wherein the tumor targeting agent comprises a collagen binding domain and wherein the collagen binding domain comprises a collagen binding domain from decorin or von Willebrand factor (vWF).

### Claim 10 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide further comprises a serum protein conjugated to the polypeptide.

### Claim 11 (depends on 1)

A pharmaceutical composition comprising the polypeptide of claim 1.

### Claim 12 (depends on 1)

A nucleic acid encoding for the polypeptide of claim 1.

### Claim 13 (depends on 12)

A host cell comprising the nucleic acid of claim 12.

### Claim 14 (depends on 12)

A method for making a polypeptide comprising expressing the nucleic acid of claim 12 in a cell and isolate the expressed polypeptide.

### Claim 15 (depends on 1)

A method for treating cancer comprising administering the polypeptide of claim 1.