JPWO2021185361A5

JPWO2021185361A5 -

Info

Publication number: JPWO2021185361A5
Application number: JP2022556114A
Authority: JP
Publication date: 2024-04-02

Claims

IL-2 muteins,
The mutein has the following mutations compared to wild-type human IL-2 :
(a) a truncated B'C' loop region and mutations in the binding interface between IL-2 and IL-2Rβγ;
(b) a truncated B'C' loop region, a mutation in the binding interface between IL-2 and IL-2Rβγ, and a mutation in the binding interface between IL-2 and IL-2Rα; or
(c) a mutation in the binding interface between IL-2 and IL-2Rβγ, and a mutation in the binding interface between IL-2 and IL-2Rα,
the truncated B'C' loop region has a sequence having a length of less than 10 amino acids located between amino acid residues aa72 and aa84; and
(i) at positions aa74 to aa83, the sequence GDASIH;
(ii) at positions aa74 to aa83, the sequence: (Q/G)S(K/A/D)N(F/I)H;
(iii) replacement of the sequence at aa 74 to aa 83 with the B'C' loop sequence from human IL-15; or
(iv) a C-terminal truncation of the sequence at positions aa74 to aa83, wherein 1, 2, 3 or 4 amino acids are truncated from the C-terminus;
The mutation comprising:
wherein the amino acid positions are numbered according to SEQ ID NO:1;
The IL-2 mutein comprising :

The IL-2 mutein according to claim 1, comprising:
The mutation at the IL-2Rβγ binding interface includes a mutation of N88R, or
The mutation at the IL-2Rβγ binding interface is
L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16T, H16Y, H16A, H16E, H16D, H16R, L19D, L19E, L19R, L19S, D20N, D20Q, D20E, D20A, D20R, D20 S, D84N, D84E, D84Q, D84T, D84S, D84R, D84G, D84M, D84F, D84L, D84K, D84H, S87T, S87R, S87K, S87L, S87M, S87H, N88D, N88T, N88Q, N88E, N88K, N88H, N88 M, N88S, N88L, V91I, V91L, V91D, V91E, V91N, V91Q, V91S, V91H, I92E, I92T, I92K, I92R, I92L, E95Q, E95G, E95D, E95N, Q126E, Q126D, Q126A, Q126S, D84N +E95Q, D84E+E95Q, comprising a mutation selected from the group consisting of D84T+E95Q, D84Q+E95Q, D84T+H16T, D84N+V91I, D84T+Q126E, D84N+Q126E, H16T+D84Q, H16T+V91I,
Preferably, the mutation at the IL-2Rβγ binding interface is a mutation of N88R, or
D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H Select from the group consisting of 16T+V91I, N88D, N88Q, N88T, Q126E, V91I including mutations caused by
Said IL-2 mutein.

The IL-2 mutein of claim 1, wherein the mutation in the IL-2Rβγ binding interface comprises N88R.

The mutation at the IL-2Rα binding interface includes a combination of K35E+T37E+R38E+F42A, a combination of K35E+T37E+R38E, or mutation F42A,
The mutein according to any one of claims 1 to 3 .

5. The IL-2 mutant protein comprises a loop region having a sequence from aa 74 to aa 83 selected from the group consisting of GDASIH, QSKNFH, GSKNFH, QSANFH, and QSANIH . mutant protein.

The IL-2 mutant protein is
(i) B'C' loop region sequence located between amino acid residues aa72 and aa84 : AGDASIH, AQSKNFH or SGDASIH ; and
(ii) a mutation in the IL-2Rβγ binding interface selected from the group consisting of N88R, D20N, and N88D;
The mutant protein according to any one of claims 1 to 5 , comprising:

The IL-2 mutein is
(i) the B'C' loop region sequence of AGDASIH located between amino acid residues aa72 and aa84; and
(ii) Mutation N88R in the IL-2Rβγ binding interface.
The mutant protein according to any one of claims 1 to 6, comprising:

The IL-2 mutant protein is
(i) the mutation combination K35E+T37E+R38E+F42A;
(ii) B'C' loop region sequence located between amino acid residues aa72 and aa84: AGDASIH, AQSKNFH or SGDASIH; and
(iii) a mutation in the IL-2Rβγ binding interface selected from the group consisting of N88R, D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88Q, N88T, Q126E, and V91I;
The IL-2 mutein according to any one of claims 1 to 7, comprising:

The IL-2 mutein is
(i) mutation combination K35E+T37E+R38E+F42A; and
(ii) a mutation in the IL-2Rβγ binding interface selected from the group consisting of D84N, D84Q, E95N, H16N, and H16N+D84N;
The IL-2 mutein of claim 1, comprising:

10. The IL-2 mutein of any one of claims 1 to 9, wherein the IL-2 mutein further comprises a mutant T3A.

IL -2 according to any one of claims 1 to 10 , further comprising no more than 0 to 5 amino acid mutations, preferably the mutant protein comprises a C125S or C125A mutation, compared to wild-type IL-2. -2 mutant protein.

Claims 1-11, wherein the wild-type IL-2 comprises SEQ ID NO: 1, 2 or 3, or a sequence having 95%, 96%, 97%, 98% or 99% homology thereto. IL-2 mutein according to any one of the above.

(a). the amino acid sequence of SEQ ID NO:516;
(b) the amino acid sequence of any one of SEQ ID NOs: 148, 197, 489, and 513;
(c) an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-147, 149-196, 198-487, 490-512, 514-515, and 517-638; or
(d) an amino acid sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homology to any one of the amino acid sequences of (a) to (c);
The IL-2 mutein according to claim 1 ,

reduced binding affinity for the IL-2Rβγ receptor and improved expression levels when expressed in the form of an Fc fusion protein in mammalian cells compared to wild-type IL-2 protein 14. A mutein according to any one of claims 1 to 13 , having a purity, or both .

A fusion protein comprising an IL-2 mutein according to any one of claims 1 to 14 .

16. The fusion protein of claim 15, wherein the IL-2 mutein is fused to an Fc antibody fragment via a linker.

(a) the linker is GSGS or ( _GS );
(b) the Fc fragment comprises a human IgG1 Fc, or a mutation that reduces or eliminates binding of the Fc to an FcγR;
(c) the Fc fragment comprises the mutations L234A+L235A;
(d) the Fc fragment has an amino acid sequence having at least 85%, at least 95%, at least 96% or 100% identity to SEQ ID NO: 12; and/or
(e) the Fc fragment comprises Knob mutations or mutations T366W and S354C ; or the Fc fragment comprises Hole mutations or mutations Y349C, T366S, L368A, and Y407V;
The fusion protein of claim 16 .

18. An IL-2-Fc dimeric protein comprising the fusion protein of claim 16 or 17 .

A homodimer comprising a first monomer and a second monomer , each monomer carrying from its N-terminus to its C-terminus i) an IL-2 mutein, ii) a linker, and iii) an Fc fragment. IL-2-Fc dimeric protein according to claim 18, comprising:

20. The IL-2-Fc dimeric protein of claim 19, wherein the linker is (G4S)2 and the Fc fragment has the amino acid sequence of SEQ ID NO: 12.

21. The IL-2-Fc dimeric protein of claim 20 , wherein each monomer comprises an IL-2 mutein selected from SEQ ID NOs: 516, 148, 197, 489 and 513 linked at its C-terminus to the amino acid sequence of SEQ ID NO: 12 via a linker (G4S)2.

A heterodimer comprising a first monomer and a second monomer ,
the first monomer comprises, from N-terminus to C-terminus, i) an IL-2 mutein, ii) a linker, and iii) a first Fc fragment; and
the second monomer comprises a second Fc fragment;
The IL-2-Fc dimeric protein according to claim 18.

23. The IL-2-Fc dimeric protein of claim 22, wherein the first Fc fragment and the second Fc fragment comprise a first and a second heterodimerization mutation , respectively, that promotes the first and second monomers to form the heterodimer.

The IL-2-Fc dimeric protein of claim 23, wherein the first heterodimer mutation in the first Fc fragment comprises a Knob mutation and the second heterodimer mutation in the second Fc fragment comprises a Hole mutation; or, the first heterodimer mutation in the first Fc fragment comprises a Hole mutation and the second heterodimer mutation in the second Fc fragment comprises a Knob mutation .

The IL-2-Fc dimeric protein of claim 24, wherein the knob mutation is T366W/S354C and the hole mutation is Y349C/T366S/L368A/Y407V.

IL-2-Fc dimeric protein according to any one of claims 22 to 25, wherein the linker is (G4S)2 and the Fc fragment has the amino acid sequence of SEQ ID NO: 12.

27. The IL-2-Fc dimeric protein according to any one of claims 22 to 26, wherein the first monomer comprises an IL-2 mutein selected from SEQ ID NOs: 516, 148, 197, 489 and 513 linked at its C-terminus via a linker (G4S)2 to the amino acid sequence of SEQ ID NO : 9 ; and the second monomer comprises the amino acid sequence of SEQ ID NO: 10.

An immune complex comprising the IL-2 mutein according to any one of claims 1 to 14 and an antigen-binding molecule, wherein the antigen-binding molecule is an antibody or an antibody fragment.

An IL-2 mutein according to any one of claims 1 to 14 , or a fusion protein according to any one of claims 15 to 17 , or a fusion protein according to any one of claims 18 to 27. An isolated polynucleotide encoding an IL-2-Fc dimeric protein or an immune complex according to claim 28 .

An expression vector comprising the polynucleotide of claim 29 .

A host cell comprising a polynucleotide according to claim 29 or a vector according to claim 30 .

An IL-2 mutein according to any one of claims 1 to 14 , or a fusion protein according to any one of claims 15 to 17 , or a fusion protein according to any one of claims 18 to 27. A pharmaceutical composition comprising an IL-2-Fc dimeric protein, or an immunoconjugate according to claim 28 , and a pharmaceutically acceptable carrier.

A pharmaceutical composition for use in therapy comprising an IL-2 mutein according to any one of claims 1 to 14 , or a fusion protein according to any one of claims 15 to 17 , or an IL-2-Fc dimeric protein according to any one of claims 18 to 27 , or an immunoconjugate according to claim 28 .

34. A pharmaceutical composition for use according to claim 33 for use in the treatment of cancer or an autoimmune disease or for use in stimulating the immune system of a subject.

1. A method for obtaining an IL-2 mutein, comprising:
(a). the mutation shortens the B'C' loop region sequence of wild-type IL-2, and introduces one or more mutations in the binding interface of IL-2 with IL-2Rβγ, and optionally A step of introducing one or more mutations into the binding interface with IL-2Ra,
The shortened B'C' loop region has a sequence of less than 10 amino acids located between amino acid residues aa72 and aa84, and
(i) Sequence of GDASIH from aa74 to aa83;
(ii) sequence of (Q/G)S(K/A/D)N(F/I)H from aa74 to aa83;
(iii) substitution of the B'C' loop sequence derived from human IL-15 in the sequence from aa position 74 to aa position 83; or
(iv) a C-terminal truncation of the sequence at positions aa73 to aa83, wherein 1, 2, 3 or 4 amino acids are cleaved from the C-terminus;
wherein the amino acid positions are numbered according to SEQ ID NO: 1,
Said step;
(b). expressing the IL-2 mutant protein obtained from (a) in a mammalian cell in the form of a fusion to Fc;
(c). (i) improved expression level and/or protein purity after one-step affinity chromatography as measured by SEC-HPLC ; (ii) improved attenuated IL2Rβ binding and/or altered IL-2Ra binding; identifying an IL-2 mutant protein having one or any combination of the properties determined;
The method described above .

The mutation in the binding interface of IL-2 with IL-2Rβγ comprises an N88R mutation, or the mutation in the binding interface of IL-2Rβγ comprises
L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16T, H16Y, H16A, H16E, H16D, H16R, L19D, L19E, L19R, L19S, D20N, D20Q, D20E, D20A, D20R, D20 S, D84N, D84E, D84Q, D84T, D84S, D84R, D84G, D84M, D84F, D84L, D84K, D84H, S87T, S87R, S87K, S87L, S87M, S87H, N88D, N88T, N88Q, N88E, N88K, N88H, N88 M, N88S, N88L, V91I, V91L, V91D, V91E, V91N, V91Q, V91S, V91H, I92E, I92T, I92K, I92R, I92L, E95Q, E95G, E95D, E95N, Q126E, Q126D, Q126A, Q126S, D84N +E95Q, D84E+E95Q, comprising a mutation selected from the group consisting of D84T+E95Q, D84Q+E95Q, D84T+H16T, D84N+V91I, D84T+Q126E, D84N+Q126E, H16T+D84Q, H16T+V91I,
36. The method of claim 35.