# US 10,765,625 B2 · Knottin-drug conjugates and methods of using the same

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- Number: US 10,765,625 B2
- Document ID: US10765625B2
- Status: Granted
- Kind: B2
- Application number: 15/458,852
- Filed: 2017-03-14
- Published: 2017-10-26
- Granted: 2020-09-08
- Earliest claimed: 2016-03-15
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Nicholas Cox; Jennifer R. Cochran; Mark Smith; James R. Kintzing
- Web page: https://patentgrep.com/patent/US10765625B2

## CPC

- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/0053: {Mouth and digestive tract, i.e. intraoral and peroral administration}
- A61K 31/7068: having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
- A61K 47/00: Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
- C07K 14/4702: {Regulators; Modulating activity}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 47/42: Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34)
- 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)
- 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

Provided are knottin-drug conjugates. The conjugates include a knottin peptide that includes an engineered loop that binds to a target on a cancer cell surface, and a drug (e.g., a nucleoside drug) conjugated to the knottin peptide through a linker. Also provided are pharmaceutical compositions and kits that include the knottin-drug conjugates, as well as methods of using the knottin-drug conjugates, e.g., for therapeutic purposes.

## Claims

### Claim 1

A knottin-drug conjugate, comprising:
a knottin peptide comprising an engineered loop that binds to a receptor on a cancer cell surface; and
a chemotherapeutic nucleoside drug conjugated to the knottin peptide through an intracellularly cleavable linker.

### Claim 2 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the knottin peptide is selected from the group consisting of: an EETI-II peptide, an AgRP peptide, a ω-conotoxin peptide, a Kalata B1 peptide, an MCoTI-II peptide, an agatoxin peptide, and a chlorotoxin peptide.

### Claim 3 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the receptor is a cell adhesion receptor.

### Claim 4 (depends on 3)

The knottin-drug conjugate of claim 3, wherein the cell adhesion receptor is an integrin.

### Claim 5 (depends on 4)

The knottin-drug conjugate of claim 4, wherein the integrin is selected from the group consisting of: αvβ1 integrin, αvβ3 integrin, αvβ5 integrin, αvβ6 integrin, α5β1 integrin, and any combination thereof.

### Claim 6 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the receptor is a chemokine receptor.

### Claim 7 (depends on 6)

The knottin-drug conjugate of claim 6, wherein the chemokine receptor is C-X-C chemokine receptor type 4 (CXCR4).

### Claim 8 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the receptor is a growth factor receptor.

### Claim 9 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the receptor is an immune cell receptor.

### Claim 10 (depends on 9)

The knottin-drug conjugate of claim 9, wherein the immune cell receptor is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

### Claim 11 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the receptor is neuropilin-1 (NRP1).

### Claim 12 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the chemotherapeutic nucleoside drug comprises a nucleoside analogue.

### Claim 13 (depends on 12)

The knottin-drug conjugate of claim 12, wherein the nucleoside analogue is selected from the group consisting of: gemcitabine, cytarabine, troxacitabine, decitabine, cladribine, fludarabine, clofarabine, and 2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC).

### Claim 14 (depends on 13)

The knottin-drug conjugate of claim 13, wherein the nucleoside analogue is gemcitabine.

### Claim 15 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the intracellularly cleavable linker is a dipeptide-based intracellularly cleavable linker.

### Claim 16 (depends on 15)

The knottin-drug conjugate of claim 15, wherein the dipeptide-based intracellularly cleavable linker is a valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB)-based intracellularly cleavable linker.

### Claim 17 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the knottin peptide comprises an unnatural amino acid to which the linker is attached.

### Claim 18 (depends on 17)

The knottin-drug conjugate of claim 17, wherein, prior to attachment, the unnatural amino acid comprises a functional group selected from the group consisting of: an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, and iodobenzene.

### Claim 19 (depends on 18)

The knottin-drug conjugate of claim 18, wherein, prior to attachment, the unnatural amino acid comprises an azide functional group.

### Claim 20 (depends on 1)

A pharmaceutical composition comprising:
the knottin-drug conjugate of claim 1; and
a pharmaceutically-acceptable excipient.

### Claim 21 (depends on 20)

The pharmaceutical composition of claim 20, wherein the composition is formulated for parenteral administration.

### Claim 22 (depends on 20)

The pharmaceutical composition of claim 20, wherein the composition is formulated for oral administration.

### Claim 23 (depends on 20)

A kit comprising:
a therapeutically effective amount of the pharmaceutical composition of claim 20; and
instructions for administering the pharmaceutical composition to an individual in need thereof.

### Claim 24 (depends on 23)

The kit of claim 23, wherein the pharmaceutical composition is present in one or more unit dosages.

### Claim 25 (depends on 1)

A method of treating an individual having cancer, comprising:
administering to an individual having cancer a therapeutically effective amount of a knottin-drug conjugate of claim 1.

### Claim 26 (depends on 25)

The method according to claim 25, wherein the individual has a cancer selected from the group consisting of: brain cancer, breast cancer, ovarian cancer, and pancreatic cancer.

### Claim 27

A method of making a knottin-drug conjugate, comprising:
conjugating a chemotherapeutic nucleoside drug to a knottin peptide comprising an engineered loop that binds to a receptor on a cancer cell surface, wherein the chemotherapeutic nucleoside drug is conjugated to the knottin peptide through an intracellularly cleavable linker.

### Claim 28 (depends on 1)

The knottin-drug conjugate of claim 1, wherein the intracellularly cleavable linker is an enzyme-labile linker.

### Claim 29 (depends on 28)

The knottin-drug conjugate of claim 28, wherein the intracellularly cleavable linker is cleavable by a lysosomal protease.

### Claim 30 (depends on 29)

The knottin-drug conjugate of claim 29, wherein the intracellularly cleavable linker is cleavable by cathepsin or plasmin.

### Claim 31 (depends on 25)

The method according to claim 25, wherein the intracellularly cleavable linker is an enzyme-labile linker.

### Claim 32 (depends on 12)

The knottin-drug conjugate of claim 12, wherein the nucleoside analogue is selected from the group consisting of: an adenoside/deoxyadenosine analogue, a guanosine/deoxyguanosine analogue, a thymidine/deoxythymidine analogue, and a deoxyuridine analogue.

### Claim 33 (depends on 15)

The knottin-drug conjugate of claim 15, wherein the dipeptide-based intracellularly cleavable linker is a maleimidocaproyl-valine-citruline-p-aminobenzyl (MC-vc-PAB)-based intracellularly cleavable linker.