# US 11,077,195 B2 · IL-2 conjugates and methods of use thereof

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- Number: US 11,077,195 B2
- Document ID: US11077195B2
- Status: Granted
- Kind: B2
- Application number: 16/918,930
- Filed: 2020-07-01
- Published: 2020-10-22
- Granted: 2021-08-03
- Earliest claimed: 2019-02-06
- Assignees: Synthorx, Inc.
- Applicants: Synthorx, Inc.
- Inventors: Jerod Ptacin; Carolina E. Caffaro; Marcos Milla
- Web page: https://patentgrep.com/patent/US11077195B2

## CPC

- A61K 47/34: Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers (A61K47/10 takes precedence)
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61P 35/00: Antineoplastic agents
- A61P 37/04: Immunostimulants
- C07K 14/55: IL-2
- 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: 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

Disclosed herein are compositions, kits, and methods comprising interleukin (IL) conjugates (e.g., IL-2 conjugates) useful for the treatment of one or more indications. Also described herein are pharmaceutical compositions and kits comprising one or more of the interleukin conjugates (e.g., IL-2 conjugates).

## Claims

### Claim 1

An IL-2 conjugate comprising: an IL-2 polypeptide comprising a substituted lysine comprising an axidoethoxy moiety covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein:
the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1; and
the substituted lysine substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO: 1.

### Claim 2 (depends on 1)

The IL-2 conjugate of claim 1, which is a pharmaceutically acceptable salt, solvate, or hydrate.

### Claim 3 (depends on 1)

The IL-2 conjugate of claim 1, wherein the PEG has a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, or 50 kDa.

### Claim 4 (depends on 1)

The IL-2 conjugate of claim 1, wherein the substituted lysine substitutes for the amino acid at position P65 in reference to the amino acid positions within SEQ ID NO: 1.

### Claim 5 (depends on 1)

The IL-2 conjugate of claim 1, wherein the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO: 1.

### Claim 6 (depends on 4)

The IL-2 conjugate of claim 4, wherein the PEG has a molecular weight of about 30 kDa.

### Claim 7 (depends on 1)

The IL-2 conjugate of claim 1, wherein the substituted lysine is N6-((2-azidoethoxy)-carbonyl)-L-lysine.

### Claim 8

An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG30 kD] is an L-amino acid having the structure of Formula (XVI) or Formula (XVII):
wherein:
m is 2;
n is an integer such that —(OCH2CH2)n—OCH3 has a molecular weight of about 30 kDa;
and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 50 that are not replaced.

### Claim 9 (depends on 8)

The IL-2 conjugate of claim 8, which is a pharmaceutically acceptable salt, solvate, or hydrate.

### Claim 10 (depends on 1)

A pharmaceutical composition comprising the IL-2 conjugate of claim 1 and a pharmaceutically acceptable excipient.

### Claim 11 (depends on 8)

A pharmaceutical composition comprising a mixture of IL-2 conjugates of claim 8 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which the [AzK_L1_PEG30 kD] is an L-amino acid having the structure of Formula (XVI) and IL-2 conjugates in which the [AzK_L1_PEG30 kD] is an L-amino acid having the structure of Formula (XVII).

### Claim 12

An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid at position K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, or L71 is replaced by the structure of Formula (I):
wherein:
Z is CH2 and Y is
Y is CH2 and Z is
Z is CH2 and Y is
or
Y is CH2 and Z is
W is a PEG group having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa;
X is an L-amino acid having the structure:
X−1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.

### Claim 13 (depends on 12)

The IL-2 conjugate of claim 12, which is a pharmaceutically acceptable salt, solvate, or hydrate.

### Claim 14 (depends on 12)

The IL-2 conjugate of claim 12, wherein the PEG group has a molecular weight of about 30 kDa.

### Claim 15 (depends on 12)

The IL-2 conjugate of claim 12, wherein the amino acid at position P64 is replaced by the structure of Formula (I).

### Claim 16 (depends on 15)

The IL-2 conjugate of claim 15, wherein the PEG group has a molecular weight of about 30 kDa.

### Claim 17 (depends on 12)

The IL-2 conjugate of claim 12, wherein the structure of Formula (I) has the structure of Formula (IV) or Formula (V):
wherein:
W is a PEG group having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa.

### Claim 18 (depends on 17)

The IL-2 conjugate of claim 17, wherein W is a PEG group having a molecular weight of about 30 kDa.

### Claim 19 (depends on 12)

A pharmaceutical composition comprising the IL-2 conjugate of claim 12 and a pharmaceutically acceptable excipient.

### Claim 20 (depends on 17)

A pharmaceutical composition comprising a mixture of IL-2 conjugates of claim 17 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (IV) and IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (V).

### Claim 21 (depends on 18)

A pharmaceutical composition comprising a mixture of IL-2 conjugates of claim 18 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (IV) and IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (V).

### Claim 22 (depends on 12)

The IL-2 conjugate of claim 12, wherein amino acid P64 is replaced by the structure of Formula (XII) or (XIII):
wherein:
n is an integer such that —(OCH2CH2)n—OCH3 has a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 3 that are not replaced.

### Claim 23 (depends on 22)

The IL-2 conjugate of claim 22, which is a pharmaceutically acceptable salt, solvate, or hydrate.

### Claim 24 (depends on 22)

A pharmaceutical composition comprising the IL-2 conjugate of claim 22 and a pharmaceutically acceptable excipient.

### Claim 25 (depends on 22)

A pharmaceutical composition comprising a mixture of IL-2 conjugates of claim 22 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which amino acid P64 is replaced by the structure of Formula (XII) and IL-2 conjugates in which amino acid P64 is replaced by the structure of Formula (XIII).

### Claim 26

An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein the amino acid at position P64 is replaced by the structure of Formula (I):
wherein:
Z is CH2 and Y is
or
Y is CH2 and Z is
W is a PEG group having a molecular weight of about 30 kDa;
X is an L-amino acid having the structure:
X−1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.

### Claim 27 (depends on 26)

The IL-2 conjugate of claim 26, which is a pharmaceutically acceptable salt, solvate, or hydrate.

### Claim 28 (depends on 1)

The IL-2 conjugate of claim 1, wherein the PEG has a molecular weight of about 30 kDa.

### Claim 29 (depends on 1)

The IL-2 conjugate of claim 1, wherein the IL-2 comprises an amino acid sequence having about 95% sequence identity to SEQ ID NO: 1.

### Claim 30 (depends on 29)

The IL-2 conjugate of claim 29, wherein the IL-2 comprises an amino acid sequence having about 97% sequence identity to SEQ ID NO: 1.

### Claim 31 (depends on 1)

The IL-2 conjugate of claim 1, wherein the IL-2 comprises an amino acid sequence having about 99% sequence identity to SEQ ID NO: 50.

### Claim 32 (depends on 24)

The pharmaceutical composition of claim 24, wherein the average molecular weight of —(OCH2CH2)n—OCH3 in the IL-2 conjugates in the pharmaceutical composition is about 30 kDa.

### Claim 33 (depends on 25)

The pharmaceutical composition of claim 25, wherein the average molecular weight of —(OCH2CH2)n—OCH3 in the IL-2 conjugates in the pharmaceutical composition is about 30 kDa.