Interleukin-2 variants and their fusion proteins

Abstract

This invention provides a novel IL-2 molecule with improved properties, particularly an immune complex with a PD-1 antibody. [Solution] The present invention relates to a novel interleukin-2 (IL-2) mutant protein and its use. Specifically, the present invention relates to an IL-2 mutant protein having improved properties compared to wild-type IL-2, such as improved IL-2 receptor binding properties and improved drug potential. The present invention further provides fusion proteins, dimers, immune complexes containing the IL-2 mutant protein, and nucleic acids encoding the IL-2 mutant protein, dimers, and immune complexes, vectors containing the nucleic acids, and host cells. More specifically, the present invention provides an immune complex particularly containing the IL-2 mutant protein and an anti-PD-1 antibody. The present invention further provides methods for preparing the IL-2 mutant protein, fusion proteins, dimers, and immune complexes, pharmaceutical compositions containing them, and therapeutic uses.

Claims

1. (i) an antibody that binds to PD-1, and (ii) an immunoglobulin containing IL-2 mutant proteins. The fusion is such that the mutant protein is wild-type IL-2 (preferably human IL-2). Preferably, compared to IL-2) containing the sequence of SEQ ID NO: 3, it contains the following mutations: (i) At the interface between IL-2 and IL-2Rα, particularly at positions 35 and / or 42 , mutations that eliminate or reduce binding affinity to the IL-2Rα receptor, and / or (ii) At the interface between IL-2 and IL-2Rβγ, particularly at positions 88, 127 and / Alternatively, at least one position selected from 130, a bond to the IL-2Rβγ receptor. Mutations that weaken the combination Furthermore (iii) Shortened B'C' loop region (i.e., amino acid residues aa72 and aa8) (An array connecting 4), preferably the shortened loop region is 10, 9, Having an amino acid length of 8, 7, 6, or less than 5 amino acids, and preferably 7 amino acids The shortened B'C' loop region is long, and preferably, the shortened B'C' loop region is related to the protein expression level and / or it brings about an improvement in purity, The positions of the amino acids are numbered according to Sequence ID No.

3. Immune complex.

2. The aforementioned mutant protein, compared to wild-type IL-2, (i) N88R+S130R, N88D, N88R, F42A + N88R + S127E, or K35E + N88R + S127E, and (ii) B'C' loop region array AGDASIH or AQSKNFH, In addition, optionally including (iii) T3A, The immune complex according to claim 1.

3. The aforementioned IL-2 mutant protein is represented by SEQ ID NO: 4, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:

27. The amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 31 or at least 90 of them %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% It contains, or consists of, an amino acid sequence having the same identity as, The immune complex according to claim 1.

4. The aforementioned immune complex A first monomer containing an IL-2 mutant protein fused with an Fc fragment, and A second monomer comprising an antibody or fragment thereof that specifically binds to PD-1, preferably Alternatively, the fragment comprises a second heavy chain and a light chain of the anti-PD-1 antibody. containing monomers The immune complex according to any one of claims 1 to 3.

5. The first monomer contains a Knob mutation in the Fc fragment, and the second monomer has a mutation in the antibody heavy chain. containing a hole mutation, or containing a hole mutation in the Fc fragment of the first monomer. Furthermore, the antibody heavy chain of the second monomer contains a knob mutation. The immune complex according to claim 4.

6. The Fc fragment in the first monomer is IgG1, IgG2, IgG3, or IgG4. It is an Fc fragment, preferably the amino acid of SEQ ID NO: 6, SEQ ID NO: 42, or SEQ ID NO:

43. Containing or consisting of acid sequences, The immune complex according to claim 4 or 5.

7. The IL-2 mutant protein fused with the aforementioned Fc fragment is sequence number 7, sequence number 24, The amino acid sequence of sequence number 26, sequence number 28, sequence number 30, or sequence number 32 or These at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It contains, or consists of, amino acid sequences that have 98% or 99% identity. 、 The immune complex according to any one of claims 4 to 6.

8. The PD-1 antibody or its antigen-binding fragment comprises a heavy chain including a heavy chain variable region, and the heavy The chain variable regions correspond to the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. Including HCDR1, HCDR2, and HCDR3 as shown, The immune complex according to any one of claims 4 to 7.

9. The PD-1 antibody or its antigen-binding fragment comprises a light chain including a light chain variable region, and the light The chain variable region corresponds to the amino acid arrangement of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The columns include LCDR1, LCDR2, and LCDR3, The immune complex according to any one of claims 4 to 8.

10. The anti-PD-1 antibody or its antigen-binding fragment is The amino acid sequence shown in Sequence ID No. 8 or at least 90%, 91%, 92% thereof, Ami with 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity A heavy chain variable region containing or consisting of an acid sequence, and The amino acid sequence shown in Sequence ID No. 15 or at least 90%, 91%, and 92% thereof A that has 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Including a light chain variable region containing a mino acid sequence, The immune complex according to any one of claims 4 to 9.

11. The anti-PD-1 antibody or its antigen-binding fragment is The amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 22, or at least the same. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and 98% Or it contains an amino acid sequence with 99% identity, or a heavy chain consisting of such sequences. call The amino acid sequence shown in Sequence ID No. 20 or at least 85%, 90%, and 91% thereof , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity It contains an amino acid sequence, or a light chain composed of such sequences. The immune complex according to any one of claims 4 to 9.

12. The IL-2 mutant protein and Fc are linked via a linker, or the IL-2 The mutant protein and the anti-PD-1 antibody are linked via a linker, preferably the The 'nka' is (GGGGS) ｎ Selected from among, n = 1, 2, 3, or 4, For example, the linker is sequence number 5. The immune complex according to any one of claims 1 to 11.

13. One or more chains of the immune complex according to any one of claims 1 to 12, or Encoding one monomer and / or a second monomer, Isolated polynucleotides.

14. A polynucleotide comprising the polynucleotide described in claim 13, Expression vector.

15. Host cells comprising the polynucleotide described in claim 13 or the vector described in claim 14 A cell, preferably the host cell being a yeast cell or a mammalian cell, particularly HEK2 These are 93 cells or CHO cells. host cell.

16. A method for producing an immune complex according to any one of claims 1 to 12, wherein This includes culturing the host cells described in claim 15 under conditions suitable for the expression of the immune complex. 、 method.

17. The immune complex according to any one of claims 1 to 12, and optionally comprising a pharmaceutical excipient, Pharmaceutical composition.

18. The immune complex according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 17 The use of a substance in the preparation of drugs for the prevention and / or treatment of cancer, preferably The cancer in question is a solid tumor or a hematological malignancy, such as a gastrointestinal tumor or a melanoma, such as a colon tumor. Rectal cancer or colon cancer, for example, said cancer is resistant to PD-1 antibody treatment. use.

19. The pharmaceutical composition further comprises a second therapeutic agent. The use described in claim 18.

20. A method for preventing and / or treating cancer in a subject, the method according to claims 1 to 12 The immune complex described in any one of the claims or the pharmaceutical composition described in claim 17 is administered to the subject. This includes, preferably, that the cancer be a solid tumor or a hematological tumor, such as a gastrointestinal tumor. or melanoma, for example, colorectal cancer or colon cancer, for example, said cancer is PD-1 antibody It is a treatment-resistant cancer. method.

21. The mutated protein, the fusion protein, or the pharmaceutical composition may be used in combination with the second therapeutic agent. It is administered by oral therapy. The method according to claim 20.

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