JPWO2020163532A5

JPWO2020163532A5 -

Info

Publication number: JPWO2020163532A5
Application number: JP2021545374A
Authority: JP
Publication date: 2023-02-14

Claims

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

2. The IL-2 conjugate of claim 1, wherein 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.

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

4. The IL-2 conjugate of any one of claims 1-3, wherein the substituted lysine replaces an amino acid at position P65 with reference to an amino acid position within SEQ ID NO:1.

5. The IL-2 conjugate of any one of claims 1-4, wherein the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:1.

IL-2 conjugate according to any one of claims 1 to 5, wherein the substituted lysine is N6-((2-azidoethoxy)-carbonyl)-L-lysine.

7. The IL-2 conjugate of any one of claims 1-6, wherein the IL-2 polypeptide comprises an amino acid sequence having about 95% sequence identity with SEQ ID NO:1.

8. The IL-2 conjugate of any one of claims 1-7, wherein the IL-2 polypeptide comprises an amino acid sequence having about 97% sequence identity with SEQ ID NO:1.

3. The IL-2 conjugate of claim 1, comprising the amino acid sequence of SEQ ID NO: 3, selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 or L71. at least one amino acid residue in the IL-2 conjugate at the amino acid position of formula (I):

(in the formula:
Z is _CH2 and Y is

is;
Y is _CH2 and Z is

is;
Z is _CH2 and Y is

or Y is _CH2 and Z is

is;
W is about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa,
a PEG group having a molecular weight selected from 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa;
X is the structure:

is L-lysine with
X-1 indicates the point of attachment to the previous amino acid residue; and X+1 indicates the point of attachment to the subsequent amino acid residue).
said IL-2 conjugate, wherein said IL-2 conjugate is replaced with

IL-2 conjugate according to claim 9, wherein the amino acid at position P64 is replaced by the structure of formula (I).

The structure of formula (I) is represented by formula (IV) or formula (V):

The IL-2 conjugate of claim 9 or 10, having the structure of

The IL-2 conjugate of any one of claims 9-11, wherein W is a PEG group having a molecular weight of about 30 kDa.

comprising the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG30kD] is of formula (XVI) or formula (XVII):

(in the formula:
m is 2;
n is an integer such that —(OCH ₂ CH ₂ ) _n —OCH ₃ has a molecular weight of about 30 kDa;
is an L-amino acid having the structure of
An IL-2 conjugate according to claim 9.

Amino acid P64 is of formula (XII) or formula (XIII):

(in the formula:
n is an integer such that —(OCH ₂ CH ₂ ) _n —OCH ₃ 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 Wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO:3 that are not substituted)
replaced by the structure of
An IL-2 conjugate according to claim 9.

IL-2 conjugate according to any one of claims 1 to 14, which is a pharmaceutically acceptable salt, solvate or hydrate.

A pharmaceutical composition comprising an IL-2 conjugate according to any one of claims 1-15 and a pharmaceutically acceptable excipient.

A pharmaceutical composition comprising a mixture of the IL-2 conjugate of claim 13 and a pharmaceutically acceptable excipient, said mixture comprising L- Said pharmaceutical composition comprising an IL-2 conjugate that is an amino acid and an IL-2 conjugate in which [AzK_L1_PEG30kD] is an L-amino acid having the structure of formula (XVII).

13. A pharmaceutical composition comprising a mixture of an IL-2 conjugate according to claim 11 or 12 and a pharmaceutically acceptable excipient, said mixture wherein the structure of formula (I) is of formula (IV) Said pharmaceutical composition comprising an IL-2 conjugate having the structure of formula (I) and an IL-2 conjugate in which the structure of formula (I) has the structure of formula (V).

A pharmaceutical composition comprising a mixture of an IL-2 conjugate according to claim 14 and a pharmaceutically acceptable excipient, said mixture wherein amino acid P64 is replaced by the structure of formula (XII) Said pharmaceutical composition comprising an IL-2 conjugate and an IL-2 conjugate in which amino acid P64 is replaced by the structure of formula (XIII).

20. A pharmaceutical composition according to claims 16-19 for use in a method of treating cancer in a subject in need thereof, said method comprising administering an effective amount of said pharmaceutical composition to said subject The pharmaceutical composition, comprising:

Cancers include renal cell carcinoma (RCC), melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), urothelial carcinoma (UC), Merkel cell carcinoma (MCC), ovarian cancer , gastric cancer, breast cancer, bladder cancer, bone cancer, brain cancer, colon cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, prostate cancer, flat skin 21. The pharmaceutical composition according to claim 20, selected from epithelial carcinoma (CSCC), and classical Hodgkin's lymphoma (cHL).

A method for producing an IL-2 conjugate, comprising:
formula:

or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein said IL-2 polypeptide comprises SEQ ID NO: 1 and at least 80% of containing amino acid sequences having sequence identity, position X-1 indicating the point of attachment to the previous amino acid residue, position X+1 indicating the point of attachment to the subsequent amino acid residue, and position X indicating the non-natural Indicates the position of the amino acid substituted with an amino acid, and the position X is K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or said IL-2 polypeptide that is L72, or a pharmaceutically acceptable salt, solvate or hydrate thereof;
formula:

(where n is an integer such that mPEG-DBCO comprises PEG with 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).
with mPEG-DBCO to form an IL-2 conjugate.

A method for producing an IL-2 conjugate, comprising:
formula;:

or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein said IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:3; position X is P64 with reference to the amino acid position in SEQ ID NO:3;
said IL-2 polypeptide, or a pharmaceutically acceptable salt thereof, wherein position X-1 represents the point of attachment to the previous amino acid residue and position X+1 represents the point of attachment to the subsequent amino acid residue , a solvate or hydrate, and
formula:

(Where n is an integer such that mPEG-DBCO contains PEG with a molecular weight of about 30 kDa)
mPEG-DBCO of
to form an IL-2 conjugate, comprising:
Said IL-2 conjugate has the formula (XVI) or formula (XVII):

(in the formula:
m is 2; and the wavy line indicates a covalent bond to an amino acid residue within SEQ ID NO:3 that is not substituted)
the aforementioned method.