Interleukin 2 variants and their fusion proteins

Abstract

The present invention relates to novel interleukin 2 (IL-2) muteins and their uses. In particular, the present invention relates to IL-2 muteins having improved properties, such as improved IL-2 receptor binding properties and improved druggability, compared to wild-type IL-2. The present invention further provides fusion proteins, dimers, and immunoconjugates comprising said IL-2 muteins, as well as nucleic acids encoding said IL-2 muteins, dimers, and immunoconjugates, vectors and host cells comprising said nucleic acids. More particularly, the present invention provides immunoconjugates, particularly comprising an IL-2 mutein and an anti-PD-1 antibody. The present invention further provides methods for preparing said IL-2 muteins, fusion proteins, dimers, and immunoconjugates, pharmaceutical compositions comprising them, and therapeutic uses.

Description

[Technical field]

[0001] The present invention relates to novel interleukin 2 (IL-2) muteins and their uses. In particular, the present invention relates to IL-2 muteins having improved properties, such as improved IL-2 receptor binding properties and improved druggability, compared to wild-type IL-2. The present invention further provides fusion proteins, dimers, and immunoconjugates comprising said IL-2 muteins, as well as nucleic acids encoding said IL-2 muteins, dimers, and immunoconjugates, vectors and host cells comprising said nucleic acids. More particularly, the present invention provides immunoconjugates, particularly comprising an IL-2 mutein and an anti-PD-1 antibody. The present invention further provides methods for preparing said IL-2 muteins, fusion proteins, dimers, and immunoconjugates, pharmaceutical compositions comprising them, and therapeutic uses. [Background technology]

[0002] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a cytokine that primarily stimulates activated T cells, especially CD4 + It is a pluripotent cytokine produced by T helper cells. In eukaryotic cells, human IL-2 (uniprot:P60568) is synthesized as a precursor polypeptide of 153 amino acids, which produces mature secretory IL-2 after removal of the N-terminal 20 amino acids. The sequences of IL-2 of other species are also disclosed, see NCBI Ref Seq No. NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).

[0003] Interleukin-2 has four antiparallel amphipathic α-helices that form a quaternary structure essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). In most cases, IL-2 acts through three different receptors: interleukin-2 receptor α (IL-2Rα, CD25), interleukin-2 receptor β (IL-2Rβ, CD122), and interleukin-2 receptor γ (IL-2Rγ, CD132). IL-2Rβ and IL-2Rγ are crucial for IL-2 signaling, whereas IL-2Rα (CD25) is not essential for signaling but can confer high affinity binding of IL-2 to the receptor (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). The trimeric receptor formed by combining IL-2Rα, β, and γ (IL-2αβγ) is a high-affinity receptor for IL-2 (KD approximately 10 pM), the dimeric receptor consisting of β and γ (IL-2βγ) is an intermediate-affinity receptor (KD approximately 1 nM), and the IL-2 receptor formed only from the α subunit is a low-affinity receptor.

[0004] Immune cells express dimeric or trimeric IL-2 receptors. The dimeric receptor binds the cytotoxic CD8 + Expressed on T cells and natural killer (NK) cells, the trimeric receptor binds primarily to activated lymphocytes and CD4 + CD25 + FoxP3 + It is expressed on suppressive regulatory T cells (Treg) (Byman, O. and Sprent. J. Nat. Rev. Immunol. 12, 180-190 (2012)). Resting effector T cells and NK cells do not have CD25 on their cell surface and are therefore relatively insensitive to IL-2. On the other hand, Treg cells consistently express the highest levels of CD25 in vivo, and therefore IL-2 normally preferentially stimulates the proliferation of Treg cells.

[0005] IL-2 mediates multiple actions in the immune response through binding to IL-2 receptors on different cells. On the one hand, IL-2 has immune system stimulating properties and can stimulate the proliferation and differentiation of T cells and natural killer (NK) cells. Therefore, IL-2 has been approved for use as an immunotherapeutic agent in the treatment of cancer and chronic viral infections. On the other hand, IL-2 inhibits the immunosuppressive CD4 + CD25 + It can also promote the maintenance of regulatory T cells (i.e., Treg cells) (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)), leading to immune suppression by activated Treg cells in patients.

[0006] It has been found through years of clinical practice that high doses of IL-2 can produce remarkable clinical effects in the treatment of cancers such as melanoma and renal cancer, but at the same time, it can also cause severe drug-related toxicities and side effects, including cardiovascular toxicity such as vascular leak syndrome and hypotension. Studies have shown that these toxicities are likely due to the excessive activation of IL-2 on lymphocytes (especially T cells and NK cells) to stimulate the release of inflammatory factors. For example, this can cause vascular endothelial cells to shrink, enlarging the gap between cells and causing tissue fluid outflow, thereby causing the side effect of vascular leakage.

[0007] Another limiting issue for the clinical use of IL-2 is its very short half-life, which makes it difficult to administer. As the molecular weight of IL-2 is only 15KDa, it is mainly removed by glomerular filtration, and its human half-life is only about 1 hour. To achieve a sufficiently high human exposure, clinically, high doses of IL-2 need to be infused every 8 hours. However, frequent administration not only imposes a large burden on patients, but more importantly, high-dose infusion of IL-2 causes very high peak blood concentrations (Cmax), which is likely another factor in causing drug toxicity.

[0008] To overcome these problems associated with IL-2 immunotherapy, several approaches have been taken. For example, the combination of IL-2 with several anti-IL-2 monoclonal antibodies has been found to enhance the therapeutic effect of IL-2 in vivo (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)). Several methods of engineering the IL-2 molecule have also been proposed. For example, Helen R. Mott et al. have disclosed a mutant protein F42A of human IL-2 with eliminated IL-2Rα binding capacity. Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8:15373, DOI:10.1038 / ncomms15373) have disclosed a triple mutant human IL-2 mutant protein IL-2 with eliminated IL-2Rα binding capacity. 3X also proposed a novel IL-2 mutant protein with residue mutations R38D+K43E+E61R at amino acid residue positions 38, 43 and 61, respectively. CN1309705A discloses mutations at positions D20, N88 and Q126, which result in reduced binding of IL-2 to IL-2Rβγ. These mutant proteins still have defective pharmacokinetic and / or pharmacodynamic properties, and also suffer from low expression levels and / or relatively poor molecular stability when expressed in mammalian cells.

[0009] Programmed cell death protein 1 (PD-1 or CD279) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is a cell surface receptor and is expressed on activated B cells, T cells, and myeloid cells. The structure of PD-1 is a monomeric type 1 transmembrane protein consisting of an immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing a tyrosine-based immunoreceptor inhibitory motif (ITIM) and a tyrosine-based immunoreceptor switch motif (ITSM). Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to downregulate T cell activation after binding to PD-1. Both PD-L1 and PD-L2 are B7 homologs and bind to PD-1 but not other CD28 family members. One ligand for PD-1, PD-L1, is abundant in many human cancers. The interaction between PD-1 and PD-L1 results in a reduction in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune escape of cancer cells.

[0010] Various antibodies that bind to PD-1 are known in the art, for example, the PD-1 antibodies disclosed in WO2017024465A1.

[0011] Thus, there is a need in the art to further develop novel IL-2 molecules, particularly immunoconjugates with PD-1 antibodies, that have improved properties (e.g., reduced binding to its receptor, improved druggability, etc.). Summary of the Invention

[0012] The present invention relates to the following embodiments. 1. An immunoconjugate comprising: (i) an antibody that binds to PD-1; and (ii) an IL-2 mutein, said mutein comprising the following mutations compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:3): (i) mutations at the IL-2 and IL-2Rα binding interface, particularly at positions 35 and / or 42, that eliminate or reduce binding affinity to the IL-2Rα receptor; and / or (ii) a mutation at the IL-2 and IL-2Rβγ binding interface, particularly at at least one position selected from positions 88, 127 and / or 130, that weakens binding to the IL-2Rβγ receptor; And (iii) a shortened B'C' loop region (i.e. the sequence linking amino acid residues aa72 and aa84), preferably said shortened loop region having a length of less than 10, 9, 8, 7, 6, or 5 amino acids, and preferably 7 amino acids in length, preferably said shortened B'C' loop region results in improved protein expression and / or purity; and and optionally (iv) a mutation at the N-terminus of IL-2, particularly at position 3, which removes the O-glycosylation at the N-terminus of IL2; wherein the amino acid positions are numbered according to SEQ ID NO:3. immune complex. 2. The mutant protein has, compared to wild-type IL-2: (i) N88D, N88R, N88R+S130R, F42A+N88R+S127E, F42A+N88R+S127E, or K35E+N88R+S127E, and (ii) the B'C' loop region sequence AGDASIH or AQSKNFH; and optionally (iii) T3A. 3. The immune complex of embodiment 1, wherein the IL-2 mutein comprises or consists of the amino acid sequence of SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. 4. The immune complex comprises: a first monomer comprising an IL-2 mutein fused to an Fc fragment, and The immune complex of any one of embodiments 1 to 3, comprising a second monomer comprising an antibody or a fragment thereof that specifically binds to PD-1, preferably wherein said fragment comprises one heavy chain and one light chain of said anti-PD-1 antibody. 5. The immune complex of embodiment 4, comprising a Knob mutation in the Fc fragment of the first monomer and a hole mutation in the antibody heavy chain of the second monomer, or comprising a hole mutation in the Fc fragment of the first monomer and a knob mutation in the antibody heavy chain of the second monomer. 6. The immune complex according to embodiment 4 or 5, wherein the Fc fragment in said first monomer is an Fc fragment of IgG1, IgG2, IgG3 or IgG4, and preferably comprises or consists of the amino acid sequence of SEQ ID NO:6, SEQ ID NO:42 or SEQ ID NO:43. 7. An immune complex according to any one of embodiments 4 to 6, wherein the IL-2 mutein fused to the Fc fragment comprises or consists of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. 8. The immune complex of any one of embodiments 4 to 7, wherein the PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain that comprises a heavy chain variable region, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. 9. The immune complex of any one of embodiments 4 to 8, wherein the PD-1 antibody or antigen-binding fragment thereof comprises a light chain that comprises a light chain variable region, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. 10. The anti-PD-1 antibody or antigen-binding fragment thereof a heavy chain variable region comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and The immune complex according to any one of embodiments 4 to 9, comprising a light chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. 11. The anti-PD-1 antibody or antigen-binding fragment thereof A heavy chain comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 22, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and The immune complex according to any one of embodiments 4 to 9, comprising a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. 12. The IL-2 mutein and Fc are linked via a linker, or the IL-2 mutein and the anti-PD-1 antibody are linked via a linker, preferably the linker is (GGGGS) n The immune complex according to any one of embodiments 1 to 11, wherein n=1, 2, 3 or 4, for example, the linker being SEQ ID NO:5. 13. An isolated polynucleotide encoding one or more chains of an immunoconjugate according to any one of embodiments 1 to 12, or the first monomer and / or the second monomer. 14. An expression vector comprising the polynucleotide of embodiment 13. 15. A host cell comprising a polynucleotide according to embodiment 13 or a vector according to embodiment 14, preferably said host cell being a yeast cell or a mammalian cell, in particular a HEK293 cell or a CHO cell. 16. A method for producing an immune complex according to any one of embodiments 1 to 12, comprising culturing a host cell according to embodiment 15 under conditions suitable for the expression of said immune complex. 17. A pharmaceutical composition comprising an immunoconjugate according to any one of embodiments 1 to 12, and optionally a pharmaceutical excipient. 18. Use of an immunoconjugate according to any one of embodiments 1 to 12 or a pharmaceutical composition according to embodiment 17 in the preparation of a medicament for the prevention and / or treatment of cancer, preferably wherein said cancer is a solid tumor or a hematological tumor, such as a gastrointestinal tumor or a melanoma, such as colorectal cancer or colon cancer, for example wherein said cancer is a cancer resistant to PD-1 antibody treatment. 19. The use according to embodiment 18, wherein the pharmaceutical composition further comprises a second therapeutic agent. 20. A method for preventing and / or treating cancer in a subject, comprising administering to said subject an immunoconjugate according to any one of embodiments 1 to 12 or a pharmaceutical composition according to embodiment 17, preferably wherein said cancer is a solid tumor or a hematological tumor, such as a gastrointestinal tumor or a melanoma, such as colorectal cancer or colon cancer, for example wherein said cancer is a cancer resistant to PD-1 antibody treatment. 21. The method of embodiment 20, wherein the mutant protein, the fusion protein or the pharmaceutical composition is administered in combination therapy with a second therapeutic agent. [Brief description of the drawings]

[0013] [Figure 1A]1 shows the molecular structure of the anti-PD-1 and IL-2 variant immune complex of the present invention; [Figure 1B] 1 shows the molecular structures of IL-2-Fc fusion proteins, molecule 2124 and molecule 3010. [Diagram 2] This is the crystal structure of IL-2 bound to its receptor (PDB:2ERJ). [Diagram 3] Binding curves of immune complexes and control molecules to IL-2Rβγ. [Figure 4] Binding curves of immune complexes or control molecules to IL-2Rα. [Diagram 5] Binding curves of immune complexes or control molecules to human PD1. [Figure 6] Activity assay of immune complexes or control molecules in CTLL2WT (huPD1-) and CTLL2-hPD-1 (huPD1+). [Figure 7] activity of immune complexes or control molecules in PD-1- and PD-1+ T cell populations (CD4 or CD8), respectively. [Figure 8] The activity of the immune complexes in HEK-Blue™ IL-2 Cells (huPD-1- cells) and cells overexpressing PD-1 (HEK293+hIL2R+hPD-1 / SEAP stably transformed cell line (huPD-1+ cells)) is shown. [Figure 9A] The effects of 2132 and 2063 on mouse antitumor efficacy were shown. [Figure 9B] The effects of 2132 and 2063 on mouse body weight are shown. [Figure 10A] The effect of 2063 on the antitumor activity of mouse MC38 tumors was shown. [Figure 10B] The effect of 2063 on mouse body weight is shown. [Figure 11A] The effect of 2063 on the antitumor effect of mouse B16F10 tumors was shown. [Figure 11B] The effect of 2063 on the antitumor effect of mouse B16F10 tumors - individual tumor values ​​shown, [Figure 11C]The effect of 2063 on mouse body weight is shown. [Figure 12A] The effect of 2149 on the antitumor activity of mouse MC38 tumors was shown. [Figure 12B] The effect of 2149 on the antitumor effect of mouse MC38 tumors-survival curves are shown. [Figure 12C] The effect of 2149 on mouse body weight is shown. [Figure 13A] The effect of 2149 on the antitumor activity of mouse B16F10 tumors was shown. [Figure 13B] The effect of 2149 on the antitumor effect of mouse B16F10 tumors - individual tumor values ​​shown, [Figure 13C] The effect of 2149 on the antitumor effect of mouse B16F10 tumors-survival curves are shown. [Figure 13D] The effect of 2149 on mouse body weight is shown. [Figure 14A] Effects of 2061 and 2149 on antitumor activity in mouse B16F10 tumors - Individual tumor values ​​shown [Figure 14B] The effect of 2061 and 2149 on the antitumor efficacy of mouse B16F10 tumors-survival curve plots are shown. [Figure 14C] The effects of 2061 and 2149 on mouse body weight are shown. [Figure 15A] The effect of 2214 on mouse antitumor activity was shown. [Figure 15B] The effect of 2214 on mouse antitumor efficacy-survival curves are shown. [Figure 15C] The effect of 2214 on mouse body weight is shown. [Figure 16A] The effect of 2214 on the antitumor activity of mouse B16F10 tumors was shown. [Figure 16B] The effect of 2214 on the antitumor effect of mouse B16F10 tumors-survival curves are shown. [Figure 16C] The effect of 2214 on mouse body weight is shown. Detailed Description of the Invention

[0014] I. Definition Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, forms, or reagents described herein, as they may be modified. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the claims. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0015] The following definitions will be used to interpret the specification, and where appropriate, terms used in the singular may also include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0016] The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​from 5% less than the number or numerical value specified as the lower limit to 5% more than the number or numerical value specified as the upper limit.

[0017] As used herein, the term "and / or" refers to any one of possible options or two or more of possible options.

[0018] As used herein, the term "comprises" or "comprises" means including the recited elements, integers, or steps, but not excluding any other elements, integers, or steps. When the term "comprises" or "comprises" is used herein, unless otherwise specified, it also includes consisting of the recited elements, integers, or steps. For example, when referring to an IL-2 mutein that "comprises" or "comprises" a mutation or combination of mutations, it is also intended to include IL-2 muteins that have only the mutation or combination of mutations.

[0019] As used herein, wild-type "interleukin-2" or "IL-2" refers to a parent IL-2 protein, preferably a naturally occurring IL-2 protein, such as native IL-2 protein from human, mouse, rat, non-human primate, including unprocessed (e.g., signal peptide not removed) and processed (e.g., signal peptide removed) forms, as a template into which a mutation or combination of mutations of the invention is introduced. The full-length native human IL-2 sequence, including the signal peptide, is set forth in SEQ ID NO:1, and the sequence of the mature protein is set forth in SEQ ID NO:2. It should be noted that this term also includes naturally occurring IL-2 allelic and splice variants, isotypes, homologs, and species homologs. This expression also includes variants of native IL-2, for example, said variants may have at least 95%-99% or more identity with native IL-2, or may have 1-10 or less or 1-5 or less amino acid mutations (e.g., conservative substitutions), and preferably have essentially the same IL-2Rα and / or IL2Rβγ binding affinity as the native IL-2 protein. Thus, in some embodiments, wild-type IL-2 may contain amino acid mutations that do not affect its binding to the IL-2 receptor, as compared to the native IL-2 protein, for example, the native human IL-2 protein with the mutation C125S introduced at position 125 (uniprot: P60568) belongs to the wild-type IL-2 of the present invention. An example of a wild-type human IL-2 protein containing the C125S mutation is shown in SEQ ID NO:3. In some embodiments, the wild-type IL-2 sequence has at least 85%, 95%, or even at least 96%, 97%, 98% or 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3.

[0020] As used herein, amino acid mutations may be amino acid substitutions, deletions, insertions and additions. Any combination of substitutions, deletions, insertions and additions may be performed to obtain a final mutein construct with desired properties (e.g., reduced IL-2Rα binding affinity and / or improved druggability and / or weakened IL-2Rβγ). Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy termini of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. For example, an alanine residue may be deleted at full-length human IL-2 position 1, or one or more amino acids may be deleted in the B'C' loop region to shorten the length of the loop region. In some embodiments, preferred amino acid mutations are amino acid substitutions, such as combinations of single amino acid substitutions or substitutions of amino acid sequence segments. For example, the whole or part of the B'C' loop region sequence of wild-type IL-2 may be replaced with a different sequence (e.g., the B'C' loop of IL-15), preferably resulting in a shortened length B'C' loop region sequence.

[0021] In the present invention, when referring to an amino acid position in the IL-2 protein or an IL-2 sequence segment, the wild-type human IL-2 protein (IL-2 WTThe amino acid position of an IL-2 protein or polypeptide is determined by reference to the amino acid sequence of SEQ ID NO:3 of SEQ ID NO:3 (also referred to as "IL-2 polypeptide sequence"). Corresponding amino acid positions on other IL-2 proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO:3. Thus, in the present invention, unless otherwise specified, an amino acid position of an IL-2 protein or polypeptide is an amino acid position numbered according to SEQ ID NO:3. For example, reference to "F42" refers to the phenylalanine residue F at position 42 of SEQ ID NO:3, or the amino acid residue at the corresponding position aligned on other IL-2 polypeptide sequences. At the same time, for ease of understanding and comparison, when the mutations of the present invention relate to the truncation or deletion of some specific segment sites (for example, the sequence of the B'C' loop region, i.e., a total of 11 amino acid residues at positions 73-83 of SEQ ID NO: 3), given a specific mutation region and its mutation scheme, the numbering of amino acid residues outside this region remains unchanged, for example, after the sequence of the B'C' loop region, i.e., a total of 11 amino acid residues at positions 73-83 of SEQ ID NO: 3, is truncated to 7 amino acid residues, 80-83 in the numbering is no longer assigned, and the position numbering of the amino acid residue immediately next to the B'C' loop region is still 84. The sequence alignment performed for amino acid position determination can be performed using the Basic Local Alignment Search Tool, which can be obtained from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameters.

[0022] In the present specification, when referring to IL-2 muteins, single amino acid substitutions are described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, the substitution of lysine at position 35 with glutamic acid can be represented as K35E. When there are multiple selectable amino acid substitution patterns (e.g., D, E) at one given position (e.g., K35 position), the amino acid substitution can be represented as K35D / E. Correspondingly, individual single amino acid substitutions can be linked by a plus sign "+" or "-" to represent combination mutations at multiple given positions. For example, combination mutations at positions F42A, N88R, and S127E can be represented as F42A+N88R+S127E, or F42A-N88R-S127E.

[0023] As used herein, "percent sequence identity" can be determined by comparing two optimally aligned sequences within a comparison window. Preferably, sequence identity is determined over the entire length of the reference sequence (e.g., SEQ ID NO: 3). Sequence alignment methods for comparison are well known in the art. Suitable algorithms for determining percent sequence identity include, for example, BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25:3389-402, 1977 and Altschul et al. J. Mol. Biol. 215:403-10, 1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. For purposes of this application, percent identity can be determined using the Basic Local Alignment Search Tool, which can be obtained from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using default parameters.

[0024] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the biological function of the protein / polypeptide that contains the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A typical conservative amino acid substitution refers to the replacement of one amino acid with another amino acid that has similar chemical properties (e.g., charge or hydrophobicity). Conservative substitution tables of functionally similar amino acids are well known in the art. In the present invention, the conservative substitution residue is derived from the following conservative substitution table X, particularly the preferred conservative amino acid substitution residues in table X.

[0025] [Table 1]

[0026] For example, a wild-type IL-2 protein may have conservative amino acid substitutions with one of SEQ ID NOs: 1-3, or may have only conservative amino acid substitutions, and in one preferred embodiment, the conservative substitutions have 10 or fewer amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 residues. Further for example, a mutant IL-2 protein of the invention may have conservative amino acid substitutions with an IL-2 mutant protein sequence specifically set forth herein (e.g., any one of SEQ ID NO: 4, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31), or may have only conservative amino acid substitutions, and in one preferred embodiment, the conservative substitutions have 10 or fewer amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues.

[0027] "Affinity" or "binding affinity" can be used to reflect the interbinding capacity of an interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y is expressed as the equilibrium dissociation constant (K D), and the equilibrium dissociation constant can be expressed as the dissociation rate constant and the association rate constant (k dis and k on (wherein A is the ratio of A to B). The binding affinity may be measured by routine methods known in the art. One specific method for measuring affinity is the SPR affinity measurement technique or the BLI measurement technique described herein.

[0028] In this specification, the antigen-binding molecule is a polypeptide molecule that can specifically bind to an antigen, such as an immunoglobulin molecule, an antibody or an antibody fragment, such as a Fab fragment and an scFv fragment.In one embodiment, the antigen-binding molecule of the present invention is a binding molecule, such as an antibody, such as a monoclonal antibody, that is antigenic to an immune checkpoint molecule.In one embodiment, the immune checkpoint molecule is PD-1 or PD-L1 or PD-L2.

[0029] As used herein, an antibody Fc fragment refers to a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region, and can include native sequence Fc fragments and variant Fc fragments. Native sequence Fc fragments include various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In one embodiment, the human IgG heavy chain Fc fragment extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. In other embodiments, the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present. In some other embodiments, the Fc fragment is a variant Fc fragment that includes a mutation, for example, a L234A-L235A mutation. Unless otherwise specified herein, the numbering of amino acid residues in the Fc fragment is according to the EU numbering system, also referred to as the EU index, as described, for example, in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. In some embodiments, the antibody Fc fragment can have an IgG1 hinge sequence or a part of an IgG1 hinge sequence at the N-terminus, such as the sequence E216 to T225 or the sequence D221 to T225 according to EU numbering. The hinge sequence can include mutations.

[0030] IL-2 protein belongs to the family of short-chain type I cytokines, which has a four alpha-helical bundle (A, B, C, D) structure. In this specification, the terms "B'C'Loop" or "B'C'Loop region" or "B'C'Loop sequence" may be used interchangeably and refer to the linking sequence between the B and C helices of IL-2 protein. The B'C'Loop sequence of an IL-2 protein can be determined by analyzing the crystal structure of IL-2 (e.g., PDB:2ERJ). For the purposes of the present invention, according to the numbering of SEQ ID NO:3, the B'C'Loop sequence refers to the sequence linking the residue at position 72 and the residue at position 84 in the IL-2 polypeptide. In the wild-type IL-2 proteins of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, the linking sequence comprises a total of 11 amino acids, A73-R83. Correspondingly, the term "truncated loop region" or "truncated B'C' loop region" as used herein refers to a mutant protein having a B'C' loop sequence that is shortened in length compared to the wild-type IL-2 protein, i.e., the length of the linking sequence between amino acid residues aa72 and aa84 according to the numbering of SEQ ID NO: 3 is shortened. A "truncated loop region" can be achieved by replacement or truncation of the loop sequence. The replacement or truncation can occur in any region or part of the B'C' loop sequence. For example, the replacement or truncation can be a replacement of the loop region A73-R83 sequence (e.g., replacing the B'C loop region of IL-15) or a truncation from one or more amino acid residues at the C-terminus of said sequence. For further example, the replacement or truncation can be a replacement of the loop region Q74-R83 sequence or a truncation from one or more amino acid residues at the C-terminus of said sequence. After the above substitution or truncation, if necessary, single amino acid substitutions, such as amino acid substitutions to eliminate glycosylation, and / or back mutations can be further introduced into the loop region sequence to further improve the properties of the mutant protein, such as druggability, etc. Thus, in this specification, the shortened B'C' loop region after mutation can be described by linking the sequence between the residue at position 72 and the residue at position 84 after mutation.

[0031] As used herein, "IL-2Rα binding interface" mutations refer to mutations occurring at amino acid sites where IL-2 and IL-2Rα (i.e., CD25) interact. These interaction sites can be determined by analyzing the crystal structure (e.g., PDB:1Z92) of IL-2 and its receptor complex. In some embodiments, the mutations specifically refer to mutations in the region of amino acid residues 35-72 of IL-2, particularly at amino acid sites 35, 37, 38, 41, 42, 43, 45, 61, 62, 68, 72. Preferably, IL-2 proteins containing the mutations have reduced or eliminated IL-2Rα binding compared to the corresponding protein prior to introduction of the mutations.

[0032] As used herein, "IL-2βγ binding interface" mutations refer to mutations occurring at amino acid sites where IL-2 and IL-2Rβγ (i.e., CD122 and CD132) interact. These interacting amino acid sites can be determined by analyzing the crystal structure (e.g., PDB:2ERJ) of IL-2 and its receptor complex. In some embodiments, the mutations refer specifically to mutations in the amino acid residues 12-20, amino acid residues 84-95, and amino acid residues 126-130 regions of IL-2, particularly to mutations at amino acid sites 12, 15, 16, 19, 20, 84, 87, 88, 91, 92, 95, 126, 127, 130. Preferably, the IL-2 protein containing the mutations has weakened IL-2Rβγ binding compared to the corresponding protein before the mutations are introduced.

[0033] As used herein, with respect to IL-2Rβγ receptor binding, "weakening" an IL-2 protein molecule refers to introducing a mutation into the IL-2Rβγ binding interface, where the mutation results in a reduced binding affinity to the IL-2Rβγ receptor, compared to the corresponding IL-2 protein before the introduction of the mutation. More preferably, compared to the corresponding protein, the weakened molecule has a reduced activity of activating T cells (e.g., CD8+ T cells or CD4+ T cells) and / or NK cells. For example, by detecting the ratio of the EC50 values ​​of the weakened molecule and the corresponding protein for activating T cell pSTAT5 signaling, the fold reduction can reach, for example, 5-fold or more, for example, 10-fold or more, or 50-fold or more, or 100-fold or more, or even 1000-fold or more. For example, compared to the corresponding protein, the activity of the weakened molecule to activate T cells can be reduced by 10-fold to 50-fold, or 50-fold to 100-fold, or 100-fold to 1000-fold, or more. Thus, in some embodiments of the present invention, the attenuated molecules of the present invention have "attenuated" binding affinity to the IL-2Rβγ receptor and "attenuated" T cell activation activity.

[0034] "Antigen-binding fragment" refers to a molecule that includes a portion of an intact antibody and binds to an antigen bound by the intact antibody, but is distinct from the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single chain antibody (e.g., scFv), single domain antibody (e.g., VHH), bivalent antibody or fragment thereof, or camelid antibody.

[0035] The term "antigen" refers to a molecule that elicits an immune response. Such immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that almost any macromolecule, including proteins or peptides, may serve as an antigen. Additionally, antigens may be derived from recombinant or genomic DNA. In some embodiments, the antigen described in the present invention is a tumor-associated antigen, i.e., an antigen associated with the development, development or progression of a tumor, such as PD-1, PD-L1, or PD-L2.

[0036] "Complementarity determining regions", "CDR regions" or "CDRs" are the regions in an antibody variable domain that are hypervariable in sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2 and CDR3 and are numbered sequentially from the N-terminus. The CDRs in the heavy chain variable domain of an antibody are referred to as HCDR1, HCDR2 and HCDR3, and the CDRs in the light chain variable domain of an antibody are referred to as LCDR1, LCDR2 and LCDR3. For a given light or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined by any one or a combination of a number of known antibody CDR assignment systems, including, for example, the Chothia system, which is based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), which is based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics (International The database (IMGT) (available on the World Wide Web at imgt.cines.fr / ) and a North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures.

[0037] For example, according to different CDR determination methods, the residues of each CDR are as follows:

[0038] [Table 2]

[0039] A CDR may be determined by having the same Kabat numbering position as the sequence of a reference CDR (eg, any one of the exemplary CDRs of the invention).

[0040] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" includes a CDR sequence determined by any one of the above methods.

[0041] Unless otherwise specified, in the present invention, references to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues) refer to numbered positions based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0042] In one embodiment, the heavy and light chain variable region CDRs of the antibodies of the invention are CDR sequences defined according to the North numbering scheme.

[0043] The term "linker" as used herein refers to any molecule that can be directly linked to different portions of a fusion protein. Examples of linkers that establish a covalent bond between different portions of a fusion protein include peptide linkers and non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The term "peptide linker" according to the present invention refers to a sequence of amino acids that links the amino acid sequence of a first portion of a fusion protein to a second portion of the fusion protein. For example, a peptide linker can link the IL-2 portion of a fusion protein to an Fc domain or a fragment thereof. For example, a peptide linker can also link an antibody to IL-2, such as linking the C-terminus of an antibody heavy chain to IL-2. Preferably, said peptide linker has a length sufficient to link two entities such that they maintain a conformation relative to each other without interfering with the desired activity. The peptide linker may comprise primarily, but not necessarily, the amino acid residues Gly, Ser, Ala, or Thr. Useful linkers include, for example, (GS) n , (GSGGS) n , (GGGGS) n , (GGGS) n and (GGGGS) n G, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10). Useful linkers further include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Preferably, the linker of the invention is (GGGGS) n where n=1, 2, 3, 4 or 5, preferably 2. Preferably, the linker of the present invention is SEQ ID NO:5.

[0044] "IgG-type antibody" refers to the IgG type to which the heavy chain constant region of the antibody belongs. All antibodies of the same type have the same heavy chain constant region, and antibodies of different types have different heavy chain constant regions. For example, an antibody of IgG4 type refers to an antibody whose heavy chain constant region is derived from IgG4, or an antibody of IgG1 type refers to an antibody whose heavy chain constant region is derived from IgG1.

[0045] A "humanized" antibody refers to an antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody comprises at least one, and typically two, of substantially all of the variable domains, in which all or substantially all of the CDRs (e.g., CDRs) correspond to portions derived from a non-human antibody, and all or substantially all of the FRs correspond to portions derived from a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0046] "Human antibody" or "fully human antibody" or "fully humanized antibody," which may be used interchangeably, refer to an antibody produced from a human or human cell, or derived from a non-human source, and having an amino acid sequence that corresponds to that of an antibody encoded in a human antibody library or another human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0047] The antibody moiety in the immunoconjugates of the invention may be a humanized antibody, a human antibody, or a chimeric antibody.

[0048] The term "fusion" as used herein refers to a fusion formed by linking two or more initially separate proteins / genes / compounds. When the entity that constitutes the fusion is a protein, it is called a fusion protein. Fusion proteins are included within the scope of fusions in this application. For example, the linkage of IL-2 with an Fc dimer may constitute an IL-2 fusion protein. The linkage between the two entities that constitute the fusion may or may not be achieved via a linker.

[0049] The term "immunoconjugate" as used herein refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody or antibody fragment. As described herein, the IL-2 molecule may be linked to the antibody through various interactions and various structures. For example, a fusion protein of IL-2 and Fc, and a fragment of an antibody molecule comprising a heavy chain and a light chain may constitute an immune complex through dimerization. Preferably, the immune complex of the present invention has the structure shown in FIG. 1A, or the structure shown in FIG. 1A with the IL-2 portion and the PD-1 antibody portion swapped.

[0050] As used herein, the terms "first" and "second," such as with respect to Fc domains or monomers, are used to facilitate distinction when there is more than one of each type of module. Unless expressly stated as such, the use of these terms is not intended to confer a particular order or orientation on the immune complex.

[0051] The term "therapeutic agent" as used herein includes any substance effective in the treatment or prevention of a tumor, such as a cancer, including chemotherapeutic agents, cytokines, antiangiogenic agents, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0052] The term "effective amount" refers to an amount or dosage that provides the desired effect in a patient in need of treatment or prevention after administration of one or more doses of an antibody or fragment or composition or combination of the invention to the patient. An "effective amount" can include a "therapeutically effective amount" or a "prophylactically effective amount."

[0053] A "therapeutically effective amount" refers to an amount that effectively achieves a desired therapeutic result at a required dose for a required period of time. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody fragment or composition or combination are not overwhelmed by the beneficial effects of the treatment. Compared to untreated subjects, a "therapeutically effective amount" preferably suppresses a measurable parameter (e.g., tumor volume) by at least about 40%, and more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%. A "prophylactically effective amount" refers to an amount that effectively achieves a desired prophylactic result at a required dose for a required period of time. Generally, the prophylactically effective amount is less than the therapeutically effective amount, since the prophylactic dose is applied before or at an early stage of disease in the subject.

[0054] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and progeny derived therefrom regardless of the number of passages. The progeny may not be identical to the parent cell in nucleic acid content and may contain mutations. As used herein, includes mutant progeny having the same function or biological activity as screened or selected from the primary transformed cell.

[0055] The term "label" as used herein refers to a compound or composition that is directly or indirectly attached or fused to a reagent (e.g., a polynucleotide probe or an antibody) and facilitates detection by the reagent to which it is attached or fused. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, when labeled by enzyme catalysis, may catalyze the chemical alteration of a substrate compound or composition that is detectable. The term is intended to include direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled.

[0056] An "individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, goats, cats, dogs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0057] The term "anti-tumor effect" refers to a biological effect that can be displayed by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell viability. In some embodiments, the anti-tumor effect also refers to an anti-tumor effect that does not reduce the subject's body weight.

[0058] The terms "tumor" and "cancer" are used interchangeably herein and include solid tumors and hematological tumors.

[0059] The term "cancer" refers to or describes a mammalian physiological disorder typically characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the invention include solid or hematological tumors, including metastatic forms of cancer. The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells or tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0060] The term "auxiliary pharmaceutical material" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete or incomplete)), excipient, vector, stabilizer, etc., administered with an active substance.

[0061] The term "pharmaceutical composition" refers to a composition that contains an active ingredient in a form that is effective for the biological activity of the active ingredient, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0062] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including, but not limited to, a drug kit, a pharmaceutical composition. The term "non-fixed combination" refers to active ingredients (e.g., (i) the mutein or fusion of the present invention, and (ii) other therapeutic agents) being administered to a patient in separate entities simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, where such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient's body. The term "fixed combination" refers to two or more active agents being administered to a patient simultaneously in the form of a single entity. Preferably, by selecting the doses and / or time intervals of the two or more active agents, the combination of each component can achieve a better effect in treating a disease or condition than any one component used alone. Each component may be in a single formulation, which may be the same or different.

[0063] The term "combination therapy" refers to the administration of two or more therapeutic agents or forms of treatment (e.g., radiation therapy or surgery) to treat a disease as described herein. Such administration includes co-administration of these therapeutic agents at approximately the same time, e.g., in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in multiple or separate containers (e.g., tablets, capsules, powders and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. It should be noted that such administration includes the sequential use of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment program provides the beneficial effect of the pharmaceutical combination in treating the disease or condition as described herein.

[0064] As used herein, "treatment" refers to relieving, interrupting, slowing, ameliorating, arresting, reducing, or reversing the progression or severity of an existing symptom, condition, medical condition, or disease.

[0065] As used herein, "prevention" includes the inhibition of the onset or progression of a disease, condition, or symptom associated with a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a prevention program. In the context of cancer generally, the term "prevention" refers to the administration of a drug before symptoms or symptoms of cancer arise, particularly before cancer occurs in a subject at risk of suffering from cancer.

[0066] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0067] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, such as fresh, frozen and / or preserved organ or tissue samples, biopsy or aspirate samples, blood or any blood component, bodily fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites) or interstitial fluid, or cells from any stage of pregnancy or development in a subject. Tissue samples may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. II. IL-2 Muteins of the Invention Advantageous Biological Properties of the IL-2 Muteins of the Invention Through long-term research, the inventors have discovered that the following molecular mutations and modifications can be carried out as follows, or one or more of the above mutations and modifications can be combined to simultaneously improve the efficacy of IL-2, reduce the toxicity and side effects of IL-2, and achieve good production performance:

[0068] (i) Specific residue mutations are introduced into the binding interface between IL-2 and IL-2Rβγ receptor to weaken the binding to IL-2Rβγ receptor and to some extent downregulate the activity of IL-2. By including such a mutation that weakens IL-2Rβγ receptor binding, the IL-2 mutein of the present invention can activate lymphocytes to kill tumors while avoiding the release of a large amount of inflammatory factors caused by excessive activation of lymphocytes and the resulting drug-related toxicity. The weakening mutation can reduce the binding affinity of the IL-2 mutein of the present invention to the IL-2 receptor, which is widely present in lymphocytes, thereby reducing the clearance of the IL-2 mutein mediated by the IL-2 receptor and prolonging the duration of action of the IL-2 mutein.

[0069] (ii) The mutant IL-2 protein of the present invention is constructed into an IL-2-Fc dimer, which increases the molecular weight of the IL-2 mutant protein of the present invention, significantly reduces renal clearance, and can further extend the half-life of the IL2-Fc fusion protein through FcRn-mediated recycling in vivo, thereby overcoming the problems of high peak blood concentration caused by the short half-life and frequent and high-dose administration of IL-2.

[0070] (iii) Modifying the B'C' loop structure of IL-2, for example by replacing it with the loop of an IL-15 molecule or truncating the B'C' loop of an IL-2 molecule, which can significantly enhance the stability of the B'C' loop structure in the IL-2 mutein of the present invention and significantly improve the productivity of the IL-2 mutein and the IL-2-Fc dimer molecule constructed therewith, such as significantly improved expression level and purity.

[0071] (iv) retain substantially the same IL-2Rα binding activity as wild-type IL-2; or (v) combine one or more specific mutations at the binding interface between IL-2 and IL-2Rα receptor to change the binding ability of the IL-2 mutein to IL-2Rα. In addition, the present inventors have found that, while maintaining the above-mentioned excellent properties in the mutein of the present invention, the binding activity of the IL-2 mutein to IL-2Rα can also be adjusted as necessary to meet different drug formulation requirements of IL-2 in many aspects such as anti-tumor or autoimmune disease treatment, thereby further imparting excellent pharmacodynamic properties to the mutein of the present invention.

[0072] As a result, the IL2-Fc series molecules of the present invention have weakened binding affinity to the receptor IL2Rβ / γ by modifying the sequence, achieving better pharmacokinetic and efficacy results, while significantly improving the drug discovery potential, such as protein expression level and purity.

[0073] Therefore, the present invention provides an IL-2 mutant protein with improved druggability and improved IL-2 receptor binding properties. The IL-2-Fc molecule containing the IL-2 mutant protein of the present invention can effectively avoid the excessive release of inflammatory factors caused by strong stimulation of lymphocytes, and has more stable and long-acting pharmacokinetic properties. Therefore, a relatively low single dose can achieve a sufficiently high human drug exposure to avoid drug-related toxicity due to too high Cmax. More meaningfully, the long-acting IL-2-Fc molecule of the present invention has a weakened lymphocyte immune-stimulating activity compared to natural IL-2, but has significantly improved pharmacokinetic properties, so that the in vivo effective drug concentration of the molecule of the present invention can last longer, exert a relatively long continuous stimulation on lymphocytes, achieve pharmacodynamic effects comparable to or better than those of natural IL-2 molecules, and achieve better antitumor effects and tolerance in animals.

[0074] Furthermore, the mutant IL-2 proteins having advantageous biological properties of the present invention may further form immune complexes with antigen-binding molecules (e.g., antibodies or fragments thereof), activating and proliferating T cells or NK cells while enhancing the immune or immunotherapeutic effects of the antigen-binding molecules.

[0075] Improved drug discoverability In some embodiments, the IL-2 muteins of the invention have improved druggability, e.g., when expressed in mammalian cells such as HEK293 or CHO cells, particularly when expressed as an Fc fusion protein, have one or more properties selected from the following: (i) superior expression yields over wild-type IL-2 protein, and (ii) amenable to purification to higher protein purity.

[0076] In some embodiments of the invention, the IL-2 muteins of the invention show increased expression levels compared to wild-type IL-2. In some embodiments of the invention, the increased expression occurs in a mammalian cell expression system. The expression level can be measured by any suitable method that allows quantitatively or semi-quantitatively analyzing the amount of recombinant IL-2 protein in the cell culture supernatant (preferably the supernatant after one-step affinity chromatography purification). For example, the amount of recombinant IL-2 protein in the sample can be evaluated by Western blot or ELISA. In some embodiments, the IL-2 muteins of the invention show increased expression in mammalian cells by at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold or 4-fold or more, or at least 5-, 6-, 7-, 8- or 9-fold, or even about 10-, 15-, 20-, 25-, 30- and 35-fold, or more.

[0077] In some embodiments, the IL-2 mutein-Fc fusions of the invention exhibit higher purity compared to wild-type IL-2 protein fusions, as shown by measuring the purity of the purified protein after Protein A affinity chromatography. In some embodiments, the purity of the protein is detected by SEC-HPLC technique. In some preferred embodiments, after one-step Protein A affinity chromatography purification, the purity of the IL-2 mutein-Fc fusions of the invention can reach 70%, or 80%, or 90% or more, preferably 92%, 93%, 94%, 95%, 98% or 99% or more.

[0078] In some embodiments, the IL-2-Fc dimeric protein of the present invention exhibits higher purity compared to the corresponding IL-2-Fc dimeric protein formed by wild-type IL-2 protein, as shown by measuring the purity of the purified protein after protein A affinity chromatography. In some embodiments, the purity of the protein is detected by SEC-HPLC technique. In some preferred embodiments, the purity of the IL-2-Fc dimeric protein of the present invention after one-step protein A affinity chromatography purification can reach 70%, or 80%, or 90% or more, preferably 92%, 93%, 94%, 95%, 98% or 99% or more.

[0079] Attenuated IL-2βγ receptor binding By introducing mutations into the IL-2Rβγ binding interface, in some embodiments, the IL-2 muteins of the present invention have weakened IL-2βγ binding affinity compared to the corresponding protein prior to the introduction of said mutations.

[0080] In some embodiments, the IL-2 mutein of the invention has reduced binding affinity to the IL-2Rβ and / or IL-2Rβγ receptor, compared to before weakening by introducing a mutation in the IL-2Rβγ binding interface. In some embodiments, the IL-2 mutein of the invention has reduced binding affinity to the IL-2Rβ receptor, for example, 1-fold to 20-fold or more, including in some embodiments, elimination of binding to the IL-2Rβ receptor, compared to before weakening. In some embodiments, the IL-2 mutein of the invention has reduced binding affinity to the IL-2Rβγ receptor, for example, 1-fold to 100-fold or more, compared to before weakening. In some embodiments, the IL-2 mutein of the invention does not bind to the IL-2Rβ receptor, but can still bind to the IL-2Rβγ receptor, and preferably, the binding to the IL-2Rβγ receptor can be reduced 1-fold to 100-fold, for example, about 20-fold to 80-fold, compared to before weakening. The SPR affinity measurement technique can be used to determine the equilibrium dissociation constant (K D ) can be used to determine binding affinity.

[0081] By introducing a mutation into the IL-2Rβγ binding interface, in some embodiments, the IL-2 mutant protein of the present invention has reduced IL-2 activity, e.g., an IL-2 activity selected from at least one of the following, compared to the corresponding protein before the introduction of the mutation:

[0082] - reduced activation of T cells (e.g. CD4+ and CD8+ T cells, e.g. CD4+ / CD8+CD25- T cells, CD4+CD25+ T cells) compared to before attenuation, - Reduced activation of NK cells compared to before attenuation, -Reduction in the release of inflammatory factors from T cells / NK cells stimulated by IL-2 compared to before attenuation.

[0083] In one embodiment, the IL-2 mutein of the invention results in a decrease in IL-2 mediated activation and / or proliferation of lymphocytes (e.g., T cells and / or NK cells) compared to before attenuation. In one embodiment, lymphocytes are CD25 - CD4+ and CD8+ T cells, such as T cells. In one embodiment, in the STAT5 phosphorylation measurement test, the ability of the IL-2 mutant protein to activate CD4+ and CD8+ T cells is identified by detecting the activation of STAT5 phosphorylation signal in lymphocytes, such as T cells or NK cells, by the IL-2 mutant protein. For example, the half-maximal effective concentration (EC50) can be determined by analyzing STAT5 phosphorylation in cells by flow cytometry, as described in the examples of the present application. For example, by detecting the ratio of the EC50 values ​​of the above-mentioned IL-2 weakening molecule of the present invention and the corresponding protein to activate the STAT5 phosphorylation signal in T cells, the IL-2 mutant molecule of the present invention has a "weakened" T cell activation activity. Based on the ratio, the T cell activation activity of the IL-2 mutant molecule of the present invention can be reduced, for example, by 5 times or more, for example, 10 times or more, or 50 times or more, or 100 times or more, or even 1000 times or more. For example, compared to the corresponding protein, the T cell activation activity of the IL-2 mutant molecules of the invention can be reduced by 10-fold to 50-fold, or 50-fold to 100-fold, or 100-fold to 1000-fold, or more. In some preferred embodiments, the IL-2 mutant proteins of the invention have reduced cell surface IL-2 receptor-mediated clearance of IL-2 and exhibit increased in vivo half-life compared to wild-type IL-2.

[0084] In some preferred embodiments, the IL-2 muteins of the invention have reduced IL-2 and its receptor-mediated in vivo toxicity compared to wild-type IL-2.

[0085] Maintained or altered (preferably attenuated) IL-2Rα receptor binding IL-2 protein initiates signal transduction and exerts its function by interacting with the IL-2 receptor. Wild-type IL-2 exhibits different affinities for different IL-2 receptors. IL-2β and γ receptors, which have low affinity for wild-type IL-2, bind to resting effector cells (CD8 + IL-2Rα, which has a high affinity for wild-type IL-2, is expressed on regulatory T cells (Treg) cells and activated effector cells. Due to its high affinity, wild-type IL-2 preferentially binds to IL-2Rα on the cell surface, further recruits IL-2Rβγ, and releases downstream p-STAT5 signals via IL-2Rβγ to stimulate Treg cells and activated effector cells. Without being bound by theory, altering the affinity of IL-2 for the IL-2Rα receptor may result in increased expression of CD25 + It alters the bias of IL-2 to preferentially activate cells and alters the immune downregulatory effects of Treg cells mediated by IL-2.

[0086] In some embodiments, the IL-2 muteins of the invention have maintained or altered IL-2Rα receptor binding ability compared to wild-type IL-2.

[0087] In some embodiments, the IL-2 mutein of the present invention maintains binding to the IL-2Rα receptor compared to wild-type IL-2. As used herein, the phrase "maintains binding to the IL-2Rα receptor" refers to the IL-2 mutein having equivalent binding activity to the IL-2Rα receptor compared to the wild-type IL-2 protein. Preferably, "equivalent binding activity" refers to the value of the binding activity (e.g., binding affinity K D ) is between 1:20 and 20:1, preferably between 1:10 and 10:1. Preferably, the IL-2 mutein has no mutation in the IL-2Rα binding interface compared to wild-type IL-2.

[0088] In some embodiments, the IL-2 muteins of the invention are attenuated IL-2 muteins and maintain binding to the IL-2Rα receptor. In some further embodiments, the attenuated IL-2 muteins of the invention do not have a mutation in the IL-2Rα binding interface compared to wild-type IL-2. Preferably, the attenuated IL-2 muteins have improved Treg selectivity and / or improved NK cell (e.g., CD3 - CD56 + In one embodiment, in a STAT5 phosphorylation measurement test, the selectivity of the IL-2 mutein for lymphocytes is identified by detecting activation of STAT5 phosphorylation signals in different lymphocytes, such as Treg cells, NK cells, CD4+ and CD8+ effector T cells, by the IL-2 mutein. In one embodiment, in a STAT5 phosphorylation measurement test, the selectivity of the IL-2 mutein can be reflected by an IL-2 mutein dose window that selectively activates a specific lymphocyte (one or more types) without substantially activating other lymphocytes. For example, in some embodiments, the attenuated IL-2 mutein of the present invention selectively activates CD25 - / low Improved Treg selectivity and / or improved NK cell (CD3 - CD56 + NK cell) selectivity. In some further embodiments, the improved selectivity may be reflected by lower drug-associated toxicity of the IL-2 muteins.

[0089] In some other embodiments, the IL-2 muteins of the invention introduce mutations in the IL-2Rα binding interface such that the IL-2 mutein reduces or eliminates IL-2Rα receptor binding compared to wild-type IL-2.

[0090] In some further embodiments, the IL-2 muteins of the invention have a CD25 +In some further embodiments, the IL-2 muteins of the invention reduce the bias of IL-2 to preferentially activate Treg cells. In some further embodiments, the IL-2 muteins of the invention reduce the immune downregulation effect of IL-2 mediated by Treg cells compared to wild-type IL-2.

[0091] In some other embodiments, the IL-2 muteins of the invention have an immune downregulating effect. In some further embodiments, the IL-2 muteins of the invention can be used to treat autoimmune diseases.

[0092] Thus, in some embodiments, the IL-2 muteins of the invention have improved properties, for example selected from one or more of the following: improved ability to bind to IL-2 receptors;

[0093] - maintaining or altering (e.g. reducing or increasing, preferably reducing) binding affinity to the IL-2R receptor (IL-2Rαβγ, IL-2Rα and / or IL2Rαβγ) compared to wild-type IL-2; - maintaining or altering (e.g., reducing or increasing) activation of CD25+ cells (e.g., CD8+ T cells and Treg cells) compared to wild-type IL-2; - maintaining or altering (e.g., removing or reducing, or increasing) the bias of IL-2 to preferentially activate CD25+ cells (e.g., Treg cells) compared to wild-type IL-2; - Maintenance or alteration (eg reduction or increase) of the downregulation of immune responses by Treg cells induced by IL-2 compared to wild-type IL-2.

[0094] In some embodiments, wild-type IL-2 (e.g., IL-2 as set forth in SEQ ID NO:1 or SEQ ID NO:3) is administered. WT), the IL-2 muteins of the invention have a reduced binding affinity to the IL-2Rα receptor by at least 5-fold, at least 10-fold, or at least 25-fold, in particular at least 30-fold, 50-fold or 100-fold or more. In a preferred embodiment, the muteins of the invention do not bind to the IL-2 receptor α. The SPR affinity measurement technique allows the determination of the equilibrium dissociation constant (K D ) can be used to determine binding affinity.

[0095] In one embodiment, the IL-2 muteins of the invention inhibit IL-2-mediated CD25 activation as compared to wild-type IL-2. + This results in a decrease in cell activation and / or proliferation. In one embodiment, CD25 + The cells are CD25 + CD8 + In another embodiment, the CD25 + The cells are Treg cells. In one embodiment, the STAT5 phosphorylation assay measures CD25 + The ability of IL-2 muteins to activate cells was determined by the CD25 + It is identified by detecting the activation of STAT5 phosphorylation signal in cells.For example, the half-maximal effective concentration (EC50) can be determined by analyzing STAT5 phosphorylation in cells by flow cytometry, as described in the examples of this application.

[0096] In one embodiment, compared to wild-type IL-2, the IL-2 muteins of the invention inhibit CD25 + In one embodiment, the bias of IL-2 to preferentially activate CD25 cells is eliminated or reduced. + The cells are CD25 + CD8 + In another embodiment, the CD25 +The cells are Treg cells. In one embodiment, the STAT5 phosphorylation assay measures CD25 - The ability of IL-2 mutant proteins to activate CD25 cells was - Cells and CD25 + The IL-2 mutant protein is identified by detecting the EC50 value of the IL-2 mutant protein that activates the STAT5 phosphorylation signal in CD25 cells. + Activation bias of IL-2 mutant proteins against CD25 cells - and CD25 + The ratio of EC50 values ​​that activate STAT5 phosphorylation signals in T cells is determined. Preferably, the CD25 + The bias of the mutant protein towards is reduced by at least 10-fold, preferably at least 100-fold, 150-fold, 200-fold, 300-fold or more.

[0097] In some embodiments, the IL-2 muteins of the invention have the properties of the muteins shown in PCT / CN2021 / 081840, which is incorporated herein in its entirety. Mutant Proteins of the Present Invention In one aspect, the present invention provides an IL-2 mutein comprising the following mutations compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:3):

[0098] (i) mutations at the IL-2 and IL-2Rα binding interface, particularly at positions 35 and / or 42, that eliminate or reduce binding affinity to the IL-2Rα receptor; and / or (ii) a mutation in the IL-2 and IL-2Rβγ binding interface, particularly at at least one position selected from positions 88, 127 and / or 130, that weakens / reduces binding to the IL-2Rβγ receptor; and (iii) a truncated B'C' loop region (i.e. the sequence linking amino acid residues aa72 and aa84), preferably said truncated loop region having a length of less than 10, 9, 8, 7, 6, or 5 amino acids, and preferably 7 amino acids in length, preferably said truncated B'C' loop region resulting in improved protein expression and / or purity; Here, the amino acid positions are numbered according to SEQ ID NO:3.

[0099] In one embodiment, the mutant protein comprises mutations (i) and (iii), or comprises mutations (ii) and (iii), or comprises (i), (ii) and (iii).

[0100] Mutations in the IL-2Rβγ binding interface The mutation in the IL-2Rβγ binding interface applied to the mutant proteins of the present invention may be any mutation that, in combination with other mutations of the present invention, can result in weakened or reduced IL-2Rβγ binding affinity and / or weakened activity to activate lymphocytes (e.g., T cells / NK cells).

[0101] Examples of such mutations include, but are not limited to, mutations in the IL-2 and IL-2Rβγ binding interface, particularly at at least one position selected from positions 88, 127 and 130, that result in weakened or reduced IL-2Rβγ receptor binding.

[0102] In some embodiments, the mutations in the IL-2Rβγ binding interface include one or more of the following mutations or a combination of the following mutations.

[0103] N88D, N88R, S127E, S130R, N88R+S130R and N88R+S127E.

[0104] In some other embodiments, the IL-2 muteins of the present invention comprising a mutation in the IL-2Rβγ binding interface of the present invention have weakened or reduced IL-2Rβγ binding, e.g., have weakened or reduced IL-2Rβγ binding affinity by SPR affinity measurements.

[0105] Mutations in the B'C' loop region In one aspect, the IL-2 mutant protein of the present invention comprises mutations in the B'C' loop region compared to wild-type IL-2, preferably said mutations result in improved stability of the B'C' loop region, and more preferably said mutations result in the IL-2 mutant protein of the present invention having improved druggability, such as increased expression level and / or purity.

[0106] In some embodiments, the introduced mutations result in the mutant protein comprising a shortened B'C' loop region (i.e., the length of the linking sequence between amino acid residues aa72 and aa84 is shortened) compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:3).

[0107] Preferably, the shortened loop region has a length of less than 10, 9, 8, 7, 6 or 5 amino acids, and is preferably 7 amino acids in length, where the amino acid residues are numbered according to SEQ ID NO:3.

[0108] As used herein, the mutations in the B'C' loop region as applied to the present invention include truncations and substitutions in the B'C' loop region. In one embodiment, the mutations include truncations (e.g., truncations of 1, 2, 3, or 4 amino acids in the B'C' loop region) or substitutions of amino acid residues aa73 to aa83 in the B'C' loop region, e.g., truncations to A(Q / G)SKN(F / I)H, preferably to AQSKNFH, or substitutions to SGDASIH. In another embodiment, the mutations include truncations or substitutions of amino acid residues aa74 to aa83 in the B'C' loop region, e.g., truncations to (Q / G)SKN(F / I)H, or substitutions to GDASIH or AGDASIH.

[0109] In some embodiments, the IL-2 muteins of the invention comprise a B'C' loop chimeric mutation. Compared to wild-type IL-2, the muteins comprise a substitution for all or part of the sequence linking aa72 to aa84, such as a substitution with a short B'C' loop sequence from another four-helix short-chain cytokine family member. From other four-helix short-chain cytokine IL family members, such as IL-15, IL-4, IL-21, or from IL family members from non-human species (e.g., mouse), a short B'C' loop can be identified by superposing the crystal structure. In one embodiment, the sequence for substitution is the B'C' loop sequence from interleukin IL-15, particularly human IL-15. In one embodiment, the substitution comprises substitution of amino acid residues aa73 to aa83 in the B'C' loop region. In another embodiment, the substitution comprises substitution of amino acid residues aa74 to aa83 in the B'C' loop region. Preferably, after substitution, the IL-2 muteins of the invention have a B'C' loop sequence (ie, the sequence linking aa72 to aa84) selected from SGDASIH or AGDASIH, with AGDASIH being preferred.

[0110] In some embodiments, the IL-2 muteins of the invention comprise a B'C' loop truncation mutation. Compared to wild type IL-2, the muteins comprise a truncation of the sequence linking aa72 to aa84. In one embodiment, the truncation comprises truncation of amino acid residues aa73 to aa83 in the B'C' loop region. In another embodiment, the truncation comprises truncation of amino acid residues aa74 to aa83 in the B'C' loop region. For example, one, two, three or four amino acids can be truncated from the C-terminus. Preferably, after truncation, the B'C' loop region of the IL-2 muteins of the invention has the sequence A(Q / G)SKN(F / I)H, preferably AQSKNFH. Preferably, after truncation, the IL-2 muteins of the invention have a B'C' loop sequence (i.e., the sequence linking aa72 to aa84) selected from the following:

[0111] [Table 3] In one preferred embodiment, an IL-2 mutein of the invention comprises a B'C' loop region sequence (ie, the sequence linking aa72 to aa84) selected from AQSKNFH or AGDASIH.

[0112] Mutations in the IL-2Rα binding interface In one embodiment, the IL-2 mutein of the invention comprises one or more mutations in the IL-2Rα binding interface compared to wild-type IL-2, preferably at positions 35 and / or 42. Preferably, said mutations eliminate or reduce binding affinity to the IL-2Rα receptor.

[0113] In some preferred embodiments, the IL-2Rα binding interface mutation of the present invention comprises the mutation K35E and / or F42A.

[0114] In some other embodiments, the IL-2 muteins of the present invention comprising mutations in the IL-2Rα binding interface of the present invention have altered IL-2Rα binding, e.g., altered (preferably reduced or eliminated) IL-2Rα binding by SPR affinity measurements.

[0115] Other mutations Except for the above "mutations in the IL-2Rβγ binding interface", "mutations in the B'C' loop region" and "mutations in the IL-2Rα binding interface", the IL-2 muteins of the present invention may further have one or more mutations in other regions or positions, so long as they retain one or more of the above beneficial properties of the IL-2 muteins of the present invention. For example, the IL-2 muteins of the present invention may further include a substitution at position 125, such as C125S, C125A, C125T, or C125V, to provide additional advantages such as improved expression or homogeneity or stability (see, for example, U.S. Pat. No. 4,518,584). Furthermore, for example, the IL-2 muteins of the present invention may further include a substitution at position 3, such as T3A, to remove the O-glycosylation at the N-terminus of IL2. Those skilled in the art will know how to determine additional mutations that can be incorporated into the IL-2 muteins of the present invention.

[0116] Preferred Exemplary Mutation Combinations In some preferred embodiments, the IL-2 muteins of the invention have attenuated IL-2Rβγ binding and have improved properties selected from one or all two of: (i) reduced (or eliminated) IL-2Rα binding, and (ii) improved expression levels and purity. In some embodiments, the IL-2 muteins maintain IL-2Rα receptor binding compared to wild-type IL-2 protein.

[0117] In some embodiments, the present invention provides an IL-2 mutein that, compared to wild-type IL-2, comprises:

[0118] (i) N88D, N88R, N88R+S130R, F42A+N88R+S127E, or K35E+N88R+S127E, and (ii) the B'C' loop region sequence AGDASIH or AQSKNFH; and, optionally, (iii) T3A.

[0119] In some embodiments, the present invention provides an IL-2 mutein that, compared to wild-type IL-2, comprises:

[0120] (i) N88D, N88R, N88R+S130R, F42A+N88R+S127E, or K35E+N88R+S127E, and (ii) the B'C' loop region sequence AGDASIH or AQSKNFH; and (iii) T3A.

[0121] In some embodiments, the IL-2 muteins of the invention comprise: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:4; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:4; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 4; Preferably, the mutein comprises an N88D substitution and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0122] In some embodiments, the IL-2 muteins of the invention comprise: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 23; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 23; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 23; Preferably, the mutein comprises an N88R substitution and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0123] In some embodiments, the IL-2 muteins of the invention comprise: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 25; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 25; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 25; Preferably, said mutein comprises the N88R+S130R substitutions and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0124] In some embodiments, the IL-2 muteins of the invention comprise: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 27; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 27; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 27; Preferably, said mutein comprises the F42A+N88R+S127E substitutions and the B'C' loop region sequence AQSKNFH, and optionally T3A.

[0125] In some embodiments, the IL-2 muteins of the invention comprise: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 29; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 29; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 29; Preferably, said mutein comprises the F42A+N88R+S127E substitutions and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0126] In some embodiments, the IL-2 muteins of the invention comprise: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 31; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 31; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 31; Preferably, said mutein comprises the K35E+N88R+S127E substitutions and the B'C' loop region sequence AQSKNFH, and optionally T3A.

[0127] For mutations and combinations of mutations applicable to the present invention, reference may also be made to the applicant's co-pending application PCT / CN2021 / 081840, which is incorporated herein by reference in its entirety.

[0128] The difference in sequence between the IL-2 mutein and the wild-type protein may be expressed by sequence identity or by the number of different amino acids between them. In one embodiment, the IL-2 mutein and the wild-type protein have at least 85%, 86%, 87%, 88%, 89% identity, preferably 90% or more identity, preferably 95%, but preferably 97% or less, more preferably 96% or less identity. In another embodiment, excluding the above mutations of the present invention, the IL-2 mutein and the wild-type protein may have 15 or less mutations, for example 1 to 10, or 1 to 5 mutations, for example 0, 1, 2, 3, 4 mutations. In one embodiment, the remaining mutations may be conservative substitutions. III. Fusion proteins and IL-2-Fc dimeric proteins In one aspect, the present invention further provides a fusion protein comprising an IL-2 mutein of the invention. In one preferred embodiment, the IL-2 mutein of the invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment.

[0129] In one embodiment, the invention provides an IL-2 mutein fusion protein comprising an IL-2 mutein of the invention fused to an antibody Fc fragment.

[0130] The Fc fragment used in the present invention can include mutations that reduce or eliminate effector function. In one preferred embodiment, the Fc fragment has reduced Fc region-mediated effector function, for example, reduced or eliminated ADCC and / or ADCP and / or CDC effector function. For example, in some specific embodiments, the Fc fragment used in the present invention has L234A / L235A mutations or L234A / L235E / G237A that reduce binding to Fcγ receptor.

[0131] In a further preferred embodiment, the Fc fragment may have a mutation that increases serum half-life, for example a mutation that improves binding of the Fc fragment to FcRn.

[0132] In some embodiments, the Fc fragment fused to the IL-2 mutein is a human IgG Fc, such as human IgG1 Fc, human IgG2 Fc or human IgG4 Fc. In one embodiment, the Fc fragment comprises or consists of the amino acid sequence of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:42 or SEQ ID NO:43 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto.

[0133] In some embodiments, the IL-2 mutein is fused to Fc, either directly or via a linker. - Linkers can be selected to improve the activation effect of the Fc fusion protein on T cells. In one embodiment, the linker is (GGGGS) n Or GSGS, more preferably (GGGGS)2.

[0134] In a further embodiment, the present invention also provides a dimeric molecule comprising an IL-2 mutein of the present invention fused to an Fc fragment, which increases the molecular weight to 60-80 KDa, significantly reduces renal clearance, and can further extend the half-life of the IL2-Fc fusion protein through FcRn-mediated recycling in vivo.

[0135] In some embodiments, the IL-2-Fc dimeric protein provided by the present invention is a homodimer, wherein the first monomer and the second monomer each comprise, from N-terminus to C-terminus, i) an IL-2 mutein, ii) a linker, and iii) an Fc fragment.

[0136] In some other embodiments, the IL-2-Fc dimeric protein provided by the present invention is a heterodimer, a) a first monomer comprising, from N-terminus to C-terminus, i) an IL-2 mutein, ii) a linker, and iii) a first Fc fragment; b) a second monomer comprising a second Fc fragment.

[0137] In some embodiments, the first Fc fragment and the second Fc fragment each comprise a first and second heterodimerization mutation that promotes the formation of a heterodimer between the first and second monomers. In some preferred embodiments, the first and second heterodimerization mutations comprise a combination of Knob:Hole mutations, such as the mutation combination T366W / S354C:Y349C / T366S / L368A / Y407V.

[0138] In some preferred embodiments, 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 alternatively, 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.

[0139] As will be appreciated by those skilled in the art, the Fc fragment of the fusion proteins and dimeric molecules applied in the present invention may be any antibody Fc fragment. In one embodiment, the Fc fragment of the present invention is effector function silent.

[0140] In one embodiment, the Fc fragment is modified with one or more properties selected from an effector function of the Fc region and a complement activation function of the Fc region. In one embodiment, the effector function or complement activation function is reduced or eliminated compared to a wild-type Fc region of the same isotype. In one embodiment, the effector function is reduced or eliminated by methods such as reducing glycosylation of the Fc region, using an Fc isotype that naturally has a reduced or eliminated effector function, and modifying the Fc region.

[0141] In one embodiment, effector function is reduced or eliminated by reducing glycosylation of the Fc region. In one embodiment, glycosylation of the Fc region is reduced by methods such as producing the fusion protein or dimeric molecule of the invention in an environment that does not permit wild-type glycosylation, removing carbohydrate groups already present in the Fc region, and modifying the Fc region to prevent wild-type glycosylation. In one embodiment, glycosylation of the Fc region is reduced by methods that modify the Fc region to prevent wild-type glycosylation, for example, comprising a mutation at position 297 of the Fc region, e.g., an N297A mutation, such that the wild-type asparagine residue at the following position is replaced with another amino acid that interferes with glycosylation at that position:

[0142] In one embodiment, at least one Fc region modification reduces or eliminates effector function. In one embodiment, at least one Fc region modification reduces or eliminates effector function. In one embodiment, at least one Fc region modification reduces or eliminates effector function. The modification is selected from a point mutation in the Fc region selected from positions 82, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439 that disrupts binding to one or more Fc receptors, a point mutation in the Fc region selected from positions 270, 322, 329 and 321 that disrupts binding to C1q, and a point mutation at position 132 in the CH1 structural domain. In one embodiment, the effector function is reduced or eliminated by point mutations L234A&L235A (i.e., LALA mutations) in the Fc region. In one embodiment, the modification is a point mutation in the Fc region selected from positions 270, 322, 329 and 321 that disrupts binding to C1q. In another embodiment, the modification is to eliminate some Fc regions.

[0143] As will be understood by those skilled in the art, in order to promote the formation of the heterodimer of the present invention, the Fc fragment of the dimeric molecule of the present invention can include a mutation that favors the dimerization of the first monomer and the second monomer. Preferably, the corresponding knob mutation and hole mutation are introduced into the first monomer and the second monomer based on the knob-in-hole technology.

[0144] Thus, in one embodiment, the dimeric molecule of the present invention comprises: i) a homodimeric Fc-region of the human IgG1 subclass, optionally with the mutations P329G, L234A and L235A or with the mutations L234A and L235A, or ii) a homodimeric Fc-region of the human IgG4 subclass, optionally bearing the mutations P329G, S228P and L235E, or iii) a heterodimeric Fc-region, wherein a) one Fc-region polypeptide comprises the mutation T366W and another Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C; or c) a heterodimeric Fc-region, wherein one Fc-region polypeptide comprises the mutations T366W and S354C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C; or iv) a heterodimeric Fc-region of the human IgG4 subclass, wherein both Fc-region polypeptides contain the mutations P329G, L234A and L235A; and a) one Fc-region polypeptide comprises the mutation T366W and another Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C; or c) a heterodimeric Fc-region of the human IgG4 subclass, wherein one Fc-region polypeptide comprises the mutations T366W and S354C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C; or v) a heterodimeric Fc-region of the human IgG4 subclass, wherein both Fc-region polypeptides contain the mutations P329G, S228P and L235E; and a) one Fc-region polypeptide comprises the mutation T366W and another Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C; or c) a heterodimeric Fc-region of the human IgG4 subclass, wherein one Fc-region polypeptide comprises the mutations T366W and S354C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

[0145] In some embodiments, the Fc region further comprises other mutations that are advantageous for the purification of the heterodimer. For example, to facilitate the purification of the heterodimer using protein A, the H435R mutation (Eric J. Smith, Scientific Reports | 5:17943 | DOI:10.1038 / srep17943) can be introduced into one of the Fc regions of the heterodimer (e.g., the Fc region with the Hole mutation). In other embodiments, for heterodimer monomers that include a hinge region, a mutation such as C220S can also be introduced into the hinge region to facilitate the formation of the heterodimer.

[0146] The Fc region applied to the fusion protein of the present invention can also be used as the Fc portion in the immune complex of the present invention.

[0147] In some embodiments, the IL-2 mutein fused to the Fc region comprises (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32; wherein said IL-2 mutein comprises a mutation as described herein.

[0148] In some embodiments, the IL-2 mutein fused to Fc of the present invention comprises (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 7; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 7; Preferably, the mutein comprises an N88D substitution and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0149] In some embodiments, the IL-2 mutein fused to Fc of the present invention comprises (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 24; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 24; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 24; Preferably, the mutein comprises an N88R substitution and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0150] In some embodiments, the IL-2 mutein fused to Fc of the present invention comprises (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 26; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 26; Preferably, said mutein comprises the N88R+S130R substitutions and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0151] In some embodiments, the IL-2 mutein fused to Fc of the present invention comprises (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 28; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 28; Preferably, said mutein comprises the F42A+N88R+S127E substitutions and the B'C' loop region sequence AQSKNFH, and optionally T3A.

[0152] In some embodiments, the IL-2 mutein fused to Fc of the present invention comprises (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 30; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 30; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 30; Preferably, said mutein comprises the F42A+N88R+S127E substitutions and the B'C' loop region sequence AGDASIH, and optionally T3A.

[0153] In some embodiments, the IL-2 mutein fused to Fc of the present invention comprises (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 32; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 32; or (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 32; Preferably, said mutein comprises the K35E+N88R+S127E substitutions and the B'C' loop region sequence AQSKNFH, and optionally T3A.

[0154] As will be apparent to those skilled in the art, the linker linking the IL-2 mutein and the Fc fragment in the fusion proteins and dimeric molecules applied in the present invention may be any linker known in the art. In some embodiments, the linker may comprise an IgG1 hinge or a linker sequence selected from (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1. Preferably, the linker comprises (G4S)2, i.e., GGGGSGGGGS (SEQ ID NO: 5). IV. Immune complexes The present invention further provides an immune complex comprising an IL2 mutein or IL-2 mutein fusion protein of the invention (e.g., fused to an Fc fragment) and an antigen-binding molecule. Preferably, the antigen-binding molecule is an immunoglobulin molecule, in particular an IgG molecule, or an antibody or antibody fragment, in particular an Fab molecule and an scFv molecule, or a half antibody (comprising or composed of one heavy and one light chain).

[0155] In some embodiments, the antigen-binding molecule specifically binds to an antigen presented on tumor cells or in the tumor environment, and is particularly preferably PD-1, PD-L1 and / or PD-L2. Thus, the immunoconjugate of the present invention can target tumor cells or the tumor environment after administration to a subject, providing additional therapeutic advantages, such as the possibility of treatment at a lower dose and thus reducing side effects, enhancing immunotherapeutic or antitumor effects, etc.

[0156] In the immunoconjugates of the invention, the IL-2 muteins of the invention can be linked to another molecule or antigen-binding molecule directly or via a linker, and in some embodiments, a proteolytic cleavage site is included between the two. In the immunoconjugates of the invention, the IL-2 muteins of the invention or fusion proteins thereof may be linked to other molecules or antigen-binding molecules via dimerization.

[0157] In some embodiments, the antibody is an antibody to an antigen associated with a tumor, such as PD-1, PD-L1, or PD-L2.

[0158] The antibody suitable for linking to the IL-2 mutein may be a complete antibody or an antigen-binding fragment thereof. In some embodiments, the antibody of the invention is an IgG1, IgG2, IgG3 or IgG4 format antibody, preferably an IgG1 format antibody. In some embodiments, the antibody of the invention is a monoclonal antibody. In some embodiments, the antibody of the invention is humanized. In some embodiments, the antibody of the invention is a human antibody. In some embodiments, the antibody of the invention is a chimeric antibody. In one embodiment, the antigen-binding fragment of the antibody of the invention is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g., scFv), (Fab')2, single domain antibody (e.g., VHH), dAb (domain antibody), linear antibody or half antibody.

[0159] In one embodiment of the invention, the immunoconjugate of the invention comprises an IL-2 mutein or a fusion protein thereof, and an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0160] In one embodiment of the invention, the immunoconjugate of the invention comprises a first monomer comprising an IL-2 mutein fused to an Fc fragment, and and a second monomer comprising an antibody or fragment thereof that specifically binds to PD-1, preferably a fragment comprising one heavy chain and one light chain of the anti-PD-1 antibody.

[0161] In some embodiments, the Fc fragment of a first monomer comprises a Knob mutation and the antibody heavy chain of a second monomer comprises a hole mutation, or vice versa. In some embodiments, the Knob mutation is Knob:S354C&T366W and / or the Hole mutation is Y349C&T366S&L368A&Y407V.

[0162] In one specific embodiment, an IL-2 mutein fusion protein of the invention has the format shown in Format 1 of FIG. 1A.

[0163] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof against PD-1 is an anti-PD-1 antibody or antigen-binding fragment thereof disclosed in WO2017024465A1. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably three CDRs, i.e., HCDR1, HCDR2H and HCDR3, or LCDR1, LCDR2 and LCDR3, more preferably six CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of the anti-PD-1 antibody or antigen-binding fragment thereof disclosed in WO2017024465A1, or comprises the VH and / or VL of the anti-PD-1 antibody or antigen-binding fragment thereof disclosed in WO2017024465A1, or comprises the heavy chain and / or light chain of the above antibody.

[0164] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from a heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (LCDRs) from a light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0165] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain variable region (VH). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0166] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises an antibody heavy chain. In some embodiments, the anti-PD-1 antibody heavy chain comprises a heavy chain variable region and a heavy chain constant region. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises an antibody light chain. In some embodiments, the light chain of the anti-PD-1 antibody of the invention comprises a light chain variable region and a light chain constant region. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof further comprises a heavy chain and a light chain.

[0167] In some embodiments, the heavy chain variable region VH is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:8; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:8; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 8, and preferably said amino acid changes do not occur in the CDR regions.

[0168] In some embodiments, the light chain variable region V is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 15; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 15; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 15, and preferably said amino acid changes do not occur in the CDR regions.

[0169] In some embodiments, the three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region are (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 8; or (ii) A sequence containing at least one amino acid change in total in the three HCDR regions relative to the sequence of (i), and containing no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

[0170] In some embodiments, the three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 from the light chain variable region are (i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 15; or (ii) A sequence containing at least one amino acid change in total in the three LCDR regions relative to the sequence of (i), and containing no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

[0171] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:9, or alternatively, HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:9.

[0172] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10, or alternatively, HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10.

[0173] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:11, or alternatively, the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:11.

[0174] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16.

[0175] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 17.

[0176] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 18, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 18.

[0177] In some embodiments, the heavy chain constant region HC of the anti-PD-1 antibody or antigen-binding fragment thereof is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, such as an IgG1 heavy chain constant region having L234A&L235A(LALA) mutations. In some embodiments, a knob-into-hole mutation is introduced into the heavy chain constant region, such as by introducing S354C and T366W mutations to obtain an antibody heavy chain having knob mutations, and / or by introducing Y349C&T366S&L368A&Y407V mutations to obtain an antibody heavy chain having hole mutations. In some embodiments, the light chain constant region of the anti-PD-1 antibody or antigen-binding fragment thereof is a lambda or Kappa light chain constant region.

[0178] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment thereof comprises: (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 21; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 21; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 21.

[0179] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment thereof comprising a hole mutation comprises (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 13; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 13; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 13.

[0180] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof that comprises a hole mutation comprises (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 14; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 14; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 14.

[0181] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises: (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 22; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 22; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 22.

[0182] In some embodiments, the light chain constant region of the antibody or antigen-binding fragment thereof comprises: (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 19; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 19; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 19.

[0183] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises: (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 20; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 20; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 20.

[0184] In some specific embodiments of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof is It comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO:8, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO:15.

[0185] In some specific embodiments of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof is These include HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

[0186] In some specific embodiments of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof is and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0187] In some specific embodiments of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof is and a light chain comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0188] In some preferred embodiments, the anti-PD-1 antibody fragment in the immunoconjugate comprises or consists of one heavy chain and one light chain.

[0189] In some specific embodiments of the invention, the anti-PD-1 antibody fragment comprises: a heavy chain comprising a hole mutation, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and The light chain comprises or consists of an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0190] The immunoconjugates of the present invention preferably have one or more or all of the following properties compared to an anti-PD-1 antibody, compared to an IL-2 mutein or a fusion protein thereof with Fc, and / or compared to known immunoconjugates comprising an anti-PD-1 antibody and an IL-2 mutein: (1) has reduced or eliminated binding affinity to IL-2Rα, particularly compared to wild-type IL-2 or a fusion protein thereof; (2) has a reduced binding affinity to IL-2Rβγ, particularly compared to an IL-2 mutein or a fusion protein thereof with its Fc, and / or compared to known immune complexes comprising an anti-PD-1 antibody and an IL-2 mutein; (3) It can selectively activate cells that express PD-1, i.e., it has high selectivity for PD-1; (4) It has low activity against T cells that do not express PD-1 (e.g., CD8+ or CD4+ T cells) and high activity against T cells that express PD-1 (e.g., CD8+ or CD4+ T cells), and has high selectivity for PD-1, particularly compared to known immune complexes containing anti-PD-1 antibodies and IL-2 mutant proteins; (5) the activity of IL-2 in cells expressing (e.g., overexpressing) the IL-2 receptor is weaker, particularly compared to known immune complexes that include anti-PD-1 antibodies and IL-2 mutant proteins; (6) In cells expressing IL-2 receptor and PD-1, it has stronger IL-2 activity than cells expressing IL-2 receptor but not PD-1, and shows selectivity for PD-1 positive cells; (7) Preferably, it has a more potent anti-tumor effect and / or has lower toxicity (e.g., no effect or relatively little effect on the body weight of the treated subject) compared to an anti-PD-1 antibody, compared to an IL-2 mutein or a fusion protein thereof with Fc, compared to a combination of an anti-PD-1 antibody and an IL-2 mutein or a fusion protein thereof, and / or compared to a known immunoconjugate comprising an anti-PD-1 antibody and an IL-2 mutein. V. Polynucleotides, Vectors and Hosts The present invention provides nucleic acids encoding any chain or any monomer or domain of the above IL-2 muteins or fusion proteins or dimeric molecules or complexes. Polynucleotide sequences encoding the muteins of the present invention can be produced by de novo solid-phase DNA synthesis or PCR mutagenesis of existing sequences encoding wild-type IL-2, by methods well known in the art. It should be noted that the polynucleotides and nucleic acids of the present invention may also include a segment encoding a secretory signal peptide, operably linked to the segment encoding the mutein of the present invention, to direct the secretory expression of the mutein of the present invention.

[0191] The present invention also provides a vector comprising the nucleic acid of the present invention.In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector.Vector includes, but is not limited to, virus, plasmid, cosmid, lambda phage or yeast artificial chromosome (YAC).In a preferred embodiment, the expression vector of the present invention is pYDO_017 expression vector.

[0192] The present invention also provides a host cell comprising said nucleic acid or said vector. Host cells adapted to replicate and support the expression of mutant IL-2 proteins or fusions or dimers or immunoconjugates are well known in the art. Such cells can be transfected or transduced with a particular expression vector, and many cells containing the vector can be grown to inoculate large-scale fermenters, resulting in sufficient quantities of IL-2 mutants or fusions or dimers or immunoconjugates for clinical use. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney lines (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (C V1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (described, for example, in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)), and myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocyte (eg, Y0, NS0, Sp20 cell). VI. Preparation method In a further aspect, the present invention provides a method for preparing an IL-2 mutein or fusion or dimer or complex of the present invention, said method comprising culturing a host cell as provided above comprising a nucleic acid encoding said protein or fusion or dimer or complex under conditions suitable for expression of the IL-2 mutein or fusion or dimer or complex, and optionally recovering said protein or fusion or dimer or complex from said host cell (or host cell medium).

[0193] In one embodiment, a vector comprising a nucleic acid encoding an IL-2 mutein is transfected into cells to allow expression, and then the cells (or cell culture supernatant) are harvested and the IL-2 mutein is extracted and purified to obtain the IL-2 mutein. In one specific embodiment, the purification method is an affinity purification method. In another specific embodiment, the purification method is an ion exchange purification method.

[0194] In one embodiment, a vector comprising a nucleic acid encoding an IL-2 mutein fused to an Fc is transfected into cells to be expressed, and then the cells (or cell culture supernatant) are harvested and the IL-2 mutein fused to said Fc is extracted and purified to obtain said IL-2 mutein fused to said Fc. In one specific embodiment, said purification method is an affinity purification method. In another specific embodiment, said purification method is an ion exchange purification.

[0195] In one embodiment, a vector comprising a nucleic acid encoding an IL-2 mutant protein fused to Fc, a nucleic acid encoding a PD-1 antibody heavy chain, and a nucleic acid encoding a PD-1 antibody light chain is transfected into cells, and each is expressed and assembled into an immune complex, and then the cells (or cell culture supernatant) are harvested, the immune complex is extracted, and purified to obtain the immune complex. In one specific embodiment, the purification method is an affinity purification method. In another specific embodiment, the purification method is an ion exchange purification method. VII.Measurement method A variety of assays known in the art can be used to identify, screen, or characterize the physical / chemical properties and / or biological activities of the IL-2 muteins provided herein.

[0196] In one embodiment, the binding activity of the IL-2 mutein of the present invention to the IL-2 receptor can be measured. For example, binding to human IL-2Rα or β protein or IL-2Rβγ or IL-2Rαβγ can be measured by methods known in the art, such as ELISA, Western blot, etc., or the exemplary methods disclosed in the Examples herein. For example, cells, such as yeast display cells, transfected to express the mutein on the cell surface can be reacted with labeled (e.g., biotin-labeled) IL-2Rα or β protein or IL-2Rβγ or IL-2Rαβγ complexes, and measured by flow cytometry. Alternatively, binding dynamics (e.g., K D Binding of the mutant proteins to the receptor, including the binding values, can be measured in SPR assays using IL-2-Fc fusion or dimeric molecular forms.

[0197] In a further embodiment, the ability of an IL-2 mutein to bind to the IL-2 receptor can be measured indirectly, by measuring signaling and / or immunostimulatory effects that occur downstream of receptor binding.

[0198] Thus, in some embodiments, an assay is provided for identifying mutant IL-2 proteins having biological activity. Biological activity may include, for example, the ability to induce proliferation of IL-2 receptor-bearing T and / or NK cells and / or Treg cells, the ability to induce IL-2 signaling in IL-2 receptor-bearing T and / or NK cells and / or Treg cells, the ability to induce apoptosis in T cells, the ability to induce tumor regression and / or improve survival, and reduced in vivo toxicity properties such as reduced vascular permeability. The present invention also provides mutant IL-2 proteins, Fc fusions thereof and dimeric molecules comprising the same having such biological activity in vivo and / or in vitro.

[0199] Methods such as those well known in the art can be used to measure the biological activity of IL-2. For example, a suitable assay for testing the ability of an IL-2 mutein of the invention (e.g., in the form of a dimeric molecule) to stimulate the production of IFN-γ by NK cells can include incubating cultured NK cells with a mutated IL-2 protein of the invention, followed by measuring the concentration of IFN-γ in the medium by ELISA. IL-2 signaling induces several signaling pathways, involving JAK (Janus kinase) and STAT (signal transducer and activator of transcription) signaling molecules.

[0200] Interaction of IL-2 with the receptor β and γ subunits results in phosphorylation of the receptor and JAK1 and JAK3 (which bind to the β and γ subunits, respectively). STAT5 then binds to the phosphorylated receptor and is itself phosphorylated on critical tyrosine residues. This causes STAT5 to dissociate from the receptor, STAT5 dimerization, and the STAT5 dimers to translocate to the cell nucleus, where they promote the transcription of target genes. Thus, the ability of mutant IL-2 polypeptides to induce signaling through the IL-2 receptor can be assessed, for example, by measuring the phosphorylation of STAT5. Details of this method are disclosed in the Examples. For example, PBMCs can be treated with mutant IL-2 polypeptides or fusions or dimers or immune complexes of the invention, and the level of phosphorylated STAT5 can be measured by flow cytometry.

[0201] In the case of immune complexes with antibodies against the antigens of the present invention, the above-mentioned activity or levels of IL-2 can be determined by assaying in cells expressing the antigen.

[0202] Furthermore, the effect of mutant IL-2 or its fusion or dimer or immunoconjugate on tumor growth and survival can be evaluated in various animal tumor models known in the art. For example, xenografts of cancer cell lines can be implanted into immunodeficient mice and treated with mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the present invention. Based on tumor inhibition rate (e.g., calculated in comparison with isotype control antibody), the in vivo antitumor effect of mutant IL-2 polypeptides, fusions, dimers and immunoconjugates of the present invention can be detected. In addition, based on the body weight change of animals (e.g., absolute body weight change or body weight change percentage compared with before administration), the in vivo toxicity of mutant IL-2 polypeptides, fusions, dimers and immunoconjugates of the present invention can be measured. The in vivo toxicity can also be measured based on mortality, survival observation (visible symptoms of adverse effects such as behavior, body weight, body temperature, etc.) and clinical and anatomical pathology (e.g., blood chemistry value measurement and / or histopathological analysis).

[0203] In a further aspect, the druggability of the mutant protein of the present invention, such as expression level and product purity, can be characterized by methods known in the art. For measuring the expression level, if the mutant protein is secreted from cultured cells and expressed in the culture supernatant, the protein content of the cell culture fluid collected by centrifugation can be measured. Alternatively, it can be measured after one-step purification of the collected cell culture fluid, for example after one-step affinity chromatography purification. For measuring the product purity, the purity can be measured after one-step affinity chromatography purification is performed on the culture supernatant of the obtained production cells to detect the purification performance of the mutant protein. Preferably, the mutant protein of the present invention has a significantly better purity than the wild-type protein after this one-step affinity chromatography purification, indicating that the mutant protein of the present invention has better purification performance. The purity measurement method can be any conventional method known in the art, including but not limited to the SEC-HPLC method.

[0204] In yet another embodiment, the pharmacokinetic properties, such as half-life, of the mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the invention can be characterized by methods known in the art. VIII. Pharmaceutical Compositions and Formulations The present invention further includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising mutant IL-2 polypeptides or fusions or dimers or immunoconjugates, and compositions comprising polynucleotides encoding mutant IL-2 polypeptides or fusions or dimers or immunoconjugates. These compositions may also optionally contain suitable pharmaceutical auxiliary materials, such as pharma- ceutically acceptable vectors, pharmaceutical excipients including buffers, as known in the art.

[0205] Pharmaceutical compositions containing the mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapment or lyophilization processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable vectors, diluents, excipients or auxiliaries that help process the protein into a pharma-ceutically usable preparation. The appropriate formulation depends on the chosen route of administration.

[0206] The immunoconjugates can be formulated into the composition in free acid or free base, neutral or salt form. Pharmaceutically acceptable salts are those that substantially retain the biological activity of the free acid or free base. These include acid addition salts, such as those formed between the free amino groups of the proteinaceous composition, inorganic acids (e.g., hydrochloric acid, phosphoric acid, etc.), or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or iron, or organic bases, such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms. IX. Combination Products In one embodiment, the present invention further provides a combination product comprising a mutant IL-2 polypeptide or fusion or dimer or immunoconjugate of the present invention and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, etc.). The combination products of the present invention can be used in the treatment methods of the present invention.

[0207] In some embodiments, the combination product is used to prevent or treat cancer. X. Treatment Methods and Uses In one aspect, the invention relates to a method of preventing or treating a disease, such as cancer, in a subject, comprising administering to the subject an effective amount of any mutant IL-2 polypeptide or fusion or dimer or immunoconjugate described herein. The cancer may be an early, intermediate or late stage cancer or a metastatic cancer. In some embodiments, the cancer may be a solid tumor or a hematological tumor. In some embodiments, the cancer is a gastrointestinal tumor or melanoma, e.g., colon cancer or colorectal cancer. In some embodiments, the tumor is a tumor or cancer that is resistant to a known drug, e.g., a known anti-PD-1 antibody, e.g., a refractory tumor or cancer.

[0208] In some embodiments, the cancer is one characterized by having elevated PD-1, PD-L1, and / or PD-L2 protein and / or nucleic acid levels (e.g., increased expression). In some embodiments, the mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the invention may be used to stimulate the host's immune system, e.g., to enhance a cellular immune response. "Stimulating the immune system" according to any of the above embodiments may include one or more of an overall increase in immune function, an increase in T cell function, an increase in B cell function, restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0209] The mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the invention (and pharmaceutical compositions containing them, optionally other therapeutic agents) may be administered by any suitable method, including parenteral, pulmonary and intranasal administration, and may be administered intralesionally if required for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending in part on whether administration is short-term or long-term, administration may be by any suitable route, for example, injections, such as intravenous or subcutaneous injections. A variety of administration schedules are included herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration and pulse infusion.

[0210] The appropriate dose of the mutant IL-2 polypeptide or fusion or dimer or immunoconjugate of the invention to prevent or treat a disease (whether administered alone or in combination with one or more other therapeutic agents) is determined by the type of disease being treated, the type of antibody, the severity and progression of the disease, whether the administration is prophylactic or therapeutic, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient in a single treatment or over a series of treatments. In some embodiments, the mutant IL-2 polypeptide or fusion protein or dimer or immunoconjugate of the invention can be administered to the patient at higher doses without causing toxicity.

[0211] In yet another aspect, the present invention further provides the use of a mutant IL-2 polypeptide or fusion or dimer or immunoconjugate of the invention in the preparation of a medicament for use in the aforementioned methods (e.g., for treatment).

[0212] The following examples are presented to aid in the understanding of the present invention, and are not intended, nor should they be construed, as limiting the scope of the invention in any manner whatsoever.

[0213] These and other aspects and embodiments of the present invention are described in the drawings (followed by a brief description of the drawings) and the following detailed description of the invention, and are illustrated in the following examples. Any or all of the features described above and throughout this application can be combined in each embodiment of the present invention. The present invention is further described using the following examples, which are given by way of illustration and not by way of limitation, and it should be understood that various modifications may be made by those skilled in the art.

[0214] Example 1. Design of immunoconjugates of anti-hPD-1 and IL-2 mutants The present invention has designed an immune complex molecule (αPD-1 / IL-2m immune complex) that can specifically bind to human PD-1, block the combination of PD-1 and PD-L1, and release the immune brake mechanism, while also specifically bind to the IL-2 receptor on T cells or NK cells, thereby activating and proliferating T cells or NK cells. The immune complex molecule contains an anti-PD-1 antibody and an IL-2 mutant, and can enhance the immune effect of the PD-1 antibody.

[0215] As shown in FIG. 1A, the molecular form of the immune complex of the present invention includes two parts: 1) a second monomer, which is derived from an antibody that binds to PD-1, and the PD-1-binding antibody sequence is derived from WO2017024465A1; and 2) a first monomer, which is an IL-2 mutant (IL-2 mutein or IL-2m), which is selected as a mutation site at the binding interface of the IL-2 and receptor complex according to the crystal structure of IL-2 and receptor complex 2ERJ (FIG. 2), and which is capable of selectively binding IL-2 to the receptor. The IL-2 B'C' loop region sequence (A73-R83, wild type is SEQ ID NO: 40) was engineered to reduce binding of IL-2 and improve the druggability of IL-2, and the loop region was optimized by replacing the IL-2 B'C' loop region with the human IL-15 B'C' loop region (AGDASIH, SEQ ID NO: 39) or by deleting the last 4 amino acids of the IL-2 B'C' loop region to obtain a truncated IL-2 B'C' loop region (AQSKNFH, SEQ ID NO: 41).

[0216] The sequence information of the control molecule, IL-2-Fc fusion protein, and IL-2 mutant-anti-PD-1 antibody immune complex used in the examples is shown in the sequence listing, and the IL-2 mutation information is shown in the table below.

[0217] [Table 4]

[0218] Example 2. Preparation of IL-2 receptor and immune complexes Expression and purification of IL-2 receptor Vector construction The IL-2 receptor IL-2Rα (Uiprot:P01589, aa22-217) was constructed in a pTT5 vector (Addgene) by adding an avi tag (GLNDIFEAQKIEWHE, this tag peptide is biotinylated by the BirA enzyme) and six histidine tags (HHHHHH) to the C-terminus of the sequence, and then transfected and expressed in HEK293 cells, and affinity purified through a nickel column (Histrap excel, GE, 17-3712-06) to obtain IL-2Rα.

[0219] The IL-2Rβγ complex is an Fc heterodimer based on Knobs in holes, in which the sequence of IL-2Rβ was constructed at the N-terminus of the Fc-Knob (SEQ ID NO:37) and the sequence of IL-2Rγ was constructed at the N-terminus of the Fc-Hole (SEQ ID NO:38), each constructed in the pcDNA3.1 vector, and then co-expressed in cells. The vector containing IL-2Rβ and the vector containing IL-2Rγ were co-transfected and expressed in HEK293 cells using the transient transfection method. First, plasmid DNA and transfection reagent PEI (Polysciences, 23966) were prepared in an ultra-clean bench, 3 mL of Opti-MEM medium (Gibco product number: 31985-070) was placed in a 50 mL centrifuge tube, 30 μg of DNA of the corresponding plasmid was added, the Opti-MEM medium containing the plasmid was filtered through a 0.22 μm filter head, then 90 μg of PEI (1 g / L) was added and left to stand for 20 min. The DNA / PEI mixture was gently poured into 27 mL of HEK293 cells, mixed uniformly, and continued to be cultured at 37 °C, 8% CO2 for 6 days. The cell supernatant was obtained for purification to obtain IL-2Rβγ complex. For nickel column affinity purification, the nickel column (5mL Histrap excel, GE, 17-3712-06) used for purification was soaked in 0.1M NaOH for 2h, then rinsed with 5-10x column volumes of ultrapure water to remove the alkaline solution. Before purification, the purification column was equilibrated with 5x column volumes of binding buffer (20mM Tris, pH 7.4, 300mM NaCl), the cell supernatant was passed through the equilibrated column, 10x column volumes of washing buffer (20mM Tris 7.4, 300mM NaCl, 10mM imidazole) was passed through the column to remove nonspecifically binding heteroproteins, and then the target protein was eluted with 3x-5x column volumes of elution solution (20mM Tris 7.4, 300mM NaCl, 100mM imidazole). The collected protein was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), then further isolated and purified with superdex200 increase (GE, 10 / 300GL, 10245605), the elution peak of the monomer was collected, and the column equilibration and elution buffer was PBS.

[0220] Mabselect affinity purification was centrifuged at 13000 rpm for 20 min, the supernatant was collected, and the supernatant was purified using a pre-packed column Hitrap Mabselect Sure. The operation was as follows: before purification, the packed column was equilibrated with 5 column volumes of equilibration solution (0.2 M Tris, 0.15 M NaCl, pH 7.2), the collected supernatant was passed through the column, the packed column was washed with 10 column volumes of equilibration solution to remove non-specifically bound proteins, the packing was rinsed with 5 column volumes of elution buffer (0.1 M sodium citrate, pH 3.5), the eluate was collected, 80 μL of Tris (2 M Tris) was added per mL of eluate, and the eluate was exchanged into PBS buffer using an ultrafiltration concentration tube, and the concentration and purity were measured.

[0221] Ion exchange purification used ion exchange chromatography to separate heterodimeric molecules in the bispecific molecules and remove homodimeric impurities.

[0222] Preparation of immune complexes The heavy chain hole and light chain of the anti-PD-1 antibody were each constructed in a pcDNA3.1 vector, and the IL-2 protein was connected to the N-terminus of IgG1 Fc Knob via a linker and constructed in a pcDNA3.1 vector. The above three plasmids were co-transfected and expressed into HEK293 cells with 3 times the amount of PEI (for example, to transfect 3 mL of HEK293, 1 ug heavy chain hole + 1 ug light chain + 1 ug IL-2m-linker-Fc-knob + 9 μg PEI) to prepare each immune complex molecule used in this example.

[0223] The cell preparation and sample collection and purification were similar to the receptor preparation method described above.

[0224] Example 3. Measurement of affinity between IL-2 and receptor Surface plasmon resonance (SPR) is a method for determining the equilibrium dissociation constant (K D) was used to determine the surface plasmon resonance (SPR) angle. According to the principle of SPR, when a polarized light beam is incident on the end face of a prism at a certain angle, a surface plasmon wave is generated at the interface between the prism and the gold film, which causes the resonance of the free electrons in the metal film, i.e., surface plasmon resonance. During analysis, a layer of protein is first immobilized on the surface of the sensing chip, and then the sample to be measured flows over the surface of the chip. If there are molecules in the sample that can interact with the proteins on the chip surface, this will cause a change in the refractive index of the gold film surface, which will ultimately lead to a change in the SPR angle. By detecting the change in the SPR angle, information such as the affinity and kinetic constants of the analyte can be obtained.

[0225] In this example, the K of immune complexes and IL-2 receptors was measured using Biacore T200 (Cytiva). D The specific method was as follows: IL2Rα and IL2Rβγ proteins containing biotin tags were captured on a chip surface coupled with SA (streptavidin), respectively, and then the affinity and rate constants were obtained by detecting the binding and dissociation between the chip surface proteins and the examined immune complexes and control molecules in the mobile phase.

[0226] The method included chip preparation and affinity detection. The measurement process used 10x HBS-EP+ (BR-1006-69, Cytiva) diluted 10 times as the experiment buffer. The chip preparation process used an amino coupling kit (BR-1006-33, Cytiva) to couple SA to the surface of a CM5 chip (29-1496-03, Cytiva), and after coupling, 1M ethanolamine was injected to block the remaining activation sites. As for affinity detection, each cycle included receptor capture, binding to a specific concentration of the molecule under study, and regeneration of the chip. Gradient-diluted molecules (molecule concentration gradient was 0 nM to 400 nM) flowed over the chip surface in order from low to high concentration at a flow rate of 30 μL / min, with a binding time of 180 s and a dissociation time of 300 s. Finally, 5 mM NaOH (BR-1003-58, Cytiva) was used to regenerate the chip. Data results were analyzed using Biacore T200 analytical software (version 3.1) using analytical 1:1 binding or steady state analytical models.

[0227] Biacore T200 (Cytiva, T200) measured the affinity of the studied immune complex or control molecule with human PD1 (product number: PD1-H5221, ACROBiosystem), and the specific method was as follows: After capturing the studied molecule on a surface coupled with Protein A (29127555, Cytiva), the affinity and rate constant were obtained by detecting the binding and dissociation between the chip surface molecule and the antigen in the mobile phase. The measurement process used 10x HBS-EP+ (BR-1006-69, Cytiva) diluted 10 times as the experimental buffer. As for affinity detection, each cycle included the capture of the studied molecule, binding to a specific concentration of antigen, and regeneration of the chip. Gradient-diluted antigen (when binding with the studied molecule, the concentration gradient of the antigen is 0 nM to 40 nM) flowed over the chip surface in the order of low concentration to high concentration at a flow rate of 30 μL / min, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated with 10 mM Glycine-HCl, pH 1.5 (BR-1003-54, Cytiva). Data results were analyzed using the Biacore T200 analysis software (version 3.1) using a 1:1 binding model.

[0228] Table 2 and Figure 3 show the binding constants and binding curves of the immune complex or control molecule to IL-2Rβγ, respectively. 3010 is the wild-type IL-2 sequence fused to Fc, the sequence is shown in the sequence listing, and its affinity is 1.09 nM. 2061 (derived from US20180326010A1) is the control molecule, and the affinity of 2061 to IL-2Rβγ is 1.48 nM. The affinities of the IL-2 immune complexes in this study were both weaker than 3010 and 2061.

[0229] Table 3 and Figure 4 show the affinity and binding curves of the immune complex or control molecule for IL-2Rα, respectively. The affinity of 3010 for IL-2Rα was 4.38E-08M, while 2061 was non-binding. The bispecific molecule in this study had weaker binding to IL-2Rα than wild-type IL-2, but stronger binding than the control molecule 2061.

[0230] Table 4 and Figure 5 show the affinity and binding curves of the immune complex or control molecule to human PD1, respectively, and both the control molecule and the molecule in this study had strong affinity to human PD1.

[0231] [Table 5]

[0232] [Table 6]

[0233] [Table 7]

[0234] Example 4. In vitro activity assay of immune complexes 1. Detection of PD-1 activity in CTLL2 (huPD1-) and CTLL2-hPD-1 (huPD1+) IL-2 bound to the IL-2 receptor on the surface of CTLL2 cells, activated the CTLL2 JAK-STAT signaling pathway, and induced reporter gene signaling. Human PD-1 (hPD-1, unprot:Q15116) was overexpressed on the surface of CTLL2 cell lines, which could further enhance the CTLL2 JAK-STAT signaling pathway under the enrichment effect of hPD-1.

[0235] Construction of CTLL2-hPD-1 cell line: Construction and packaging of Lentvirus+hPD-1 lentivirus: 1.6x10^6 293T cells were plated in a T75 culture flask to reach a confluence rate of 75%-80%.

[0236] 2. As shown in the table below, the components were mixed uniformly to form a package system, and the system was allowed to stand at room temperature for 15 minutes.

[0237] [Table 8] 3. Discard the medium in the Petri dish and add 6 mL of fresh 10% FBS (PEAK) DMEM (ATCC) medium.

[0238] 4. Steps <3> After replacing the medium with <2> The medium package system prepared in step 1 was added, and the mixture was left to stand in a 37°C, 5% CO2 incubator for 4 to 6 hours.

[0239] 5. After placing the cells in a 37℃, 5% CO2 incubator for 4 to 6 hours, the medium was replaced with 2% serum-free DMEM medium and cultured, and the viruses were collected at 48 and 72 hours, respectively.

[0240] 6. For virus concentration, the collected virus was centrifuged and filtered through a 0.45 μm filter membrane. The volume ratio of each component was 87:10:3 (virus supernatant: 50% PEG8000: 5M NaCl), which was mixed homogeneously and concentrated overnight at 4°C. After centrifugation at 3000g for 20 min at 4°C, the virus was resuspended in 1 mL of CTS medium (Gibco, A3021002), dissolved at 4°C, and stored at -80°C.

[0241] CTLL2 (Promega, CS2028B04) was infected with Lentvirus + hPD-1, and CTLL2-hPD-1 stable transformed cell lines were obtained through pressure screening and sorting.

[0242] Testing Method: 1. Assay Medium: 1% MEM NEAA (Gibco, 11140-050), 10% FBS (PEAK, PS-FB1) and 89% IMDM (Gibco, 12440-053) were placed.

[0243] 2. CTLL2 or CTLL2-hPD-1 cells were washed twice with Assay Medium.

[0244] The density of CTLL2 or CTLL2-hPD-1 cells was adjusted using Assay Medium containing 3.0.4 ng / mL rhIL2 (R&D, 202-IL) and plated in the center of a 96-well white cell culture plate (NUNC), 50,000 cells per well.

[0245] 4. An equal volume of Assay Medium was plated onto the edge wells and the cells were starved for 18 to 20 hours at 37°C in 5% CO2.

[0246] 5. The diluted test immune complex molecules were added to the cell plates, and incubated at 37°C, 5% CO2 for 6 hours.

[0247] 6. The culture plate was removed and equilibrated at room temperature for 15-20 min. An equal volume of Luciferase assay system reagent (Bio-Glo) was added to each well, incubated at room temperature for 5-15 min, and read on a microplate reader (Molecular Devices).

[0248] The results are shown in Figure 6. The results in Figure 6 show that the αPD-1 / IL-2m immunoconjugate in this study has stronger activity on PD-1 positive CTLL2 cells than on PD-1 negative CTLL2 cells, where the activity (EC50) between the two cells of 2132 has 24-fold selectivity, 2063 has 42-fold selectivity, 2149 has 115-fold selectivity, 2219 has 500-fold selectivity, and 2213 and 2214 have >10,000-fold selectivity. This result indicates that the immunoconjugate molecule of the present invention can selectively activate PD-1 positive CTLL2 cells.

[0249] 2. Detection of pSTAT5 signaling of αPD-1 / IL2m immune complex molecules in PBMC cells The binding of IL-2 to the IL-2 receptor on the surface of T cells activated the JAK-STAT signal pathway in T lymphocytes, and the level of STAT5 phosphorylation was an important indicator for evaluating the activation level of the signal pathway.

[0250] Testing Method: 1. Resuscitation of PBMC Cells (1) PBMC cells were frozen in liquid nitrogen (MiaotongBiotech, product no. PB100C) and thawed by quick shaking at 37°C.

[0251] (2) The cells were slowly added to 10 mL of CTS medium (Gibco), the medium was pre-warmed to 37°C, and 100 μL of DNA enzyme (STRMCELL, product number 07900) was added.

[0252] (3) The mixture was centrifuged at 300 g / 8 min, and the supernatant was removed.

[0253] (4), resuspended in 10 mL of CTS, transferred to a T75 culture flask, and stabilized overnight in an incubator at 37°C and 5%.

[0254] 2. pSTAT5 Experiments (1) Alexa Fluor TM PD-1 mAb (Innovent, ADI-11416) was labeled using the 488Antibody Labeling Kit (ThermoFisher, A20181), AF488-anti human PD-1 fluorescent antibody was prepared, and PBMC floating cells cultured overnight with the fluorescent antibody were labeled.

[0255] (2) 5x10 labeled PBMC suspension cells 5 The cells were plated in a 96-well U-shaped plate at a cell number of 100 cells / well.

[0256] (3) Different dilutions of the test immune complex were added to a 96-well plate, and the test sample and cells were incubated at 37° C. for 30 min.

[0257] (4) The mixture was centrifuged at 400 g / 5 min, and the supernatant was removed.

[0258] (5) 200 μL / well of 4% tissue cell fixative (Solarbio, P1110) was added and centrifuged at 400 g / 30 min at room temperature.

[0259] (6) The membrane rupture solution was added at 200 μL / well, and the plate was left to stand at 4° C. for 30 minutes and centrifuged at 400 g / 10 minutes.

[0260] (7) Perm / wash Buffer (BD) was added at 200 μL / well, and the cells were washed twice.

[0261] (8) Place the antibody staining solution, the amount of AF647-pSTAT5 antibody was 3 μL / 100 μL perm / wash buffer / well, and the remaining staining antibody was 1 μL / 100 μL perm / wash buffer / well, incubated at room temperature for 1.5 h, and washed twice with perm / wash buffer.

[0262] [Table 9]

[0263] (9) The cells were resuspended in 150 μL of perm / wash buffer / well and subjected to flow cytometry. 3. Detection of pSTAT5 signaling of immune complex molecules in activated PBMC cells After T lymphocytes were activated, the effect of immune complexes on pSTAT5 signaling in activated T lymphocytes under the action of PD-1 was investigated and verified.

[0264] 1. Resuscitation of PBMC Cells (1) PBMC cells were frozen in liquid nitrogen and thawed by rapid shaking at 37°C.

[0265] (2) The cells were slowly added to 10 mL of CTS medium (pre-warmed to 37°C and containing 100 μL of DNA enzyme).

[0266] (3) The mixture was centrifuged at 300 g / 8 min, and the supernatant was removed.

[0267] (4), resuspended in 10 mL of CTS, transferred to a T75 culture flask, and stabilized overnight in an incubator at 37°C and 5%.

[0268] 2. Activation and quiescence of T lymphocytes (1) Suspension cells were removed from PBMCs cultured overnight, counted, and CD3 / CD28 beads were added in an amount equivalent to the number of cells, and the cells were activated and stimulated for 48 hours.

[0269] (2) The beads and medium were removed, and the activated cells were washed.

[0270] (3) The activated cells were transferred to a T75 culture flask and rested at 37°C and 5% for 48 h.

[0271] 3. pSTAT5 Experiments (1) Alexa Fluor TM PD-1 mAb (Innovent, ADI-11416) was labeled using the 488Antibody Labeling Kit (ThermoFisher, A20181), AF488-anti human PD-1 fluorescent antibody was prepared, and activated and resting T cells were labeled with the fluorescent antibody.

[0272] (2), 5x10 5 The cells were plated in a 96-well U-shaped plate at a cell number of 100 cells / well.

[0273] (3) Different dilutions of the detection molecules were added to a 96-well plate, and the detection molecules were incubated with the cells at 37°C for 30 min.

[0274] (4) The mixture was centrifuged at 400 g / 5 min, and the supernatant was removed.

[0275] (5) 200 μL / well of 4% tissue cell fixative was added, and the plates were centrifuged at 400 g / 30 min at room temperature.

[0276] (6) The membrane rupture solution was added at 200 μL / well, and the plate was left to stand at 4° C. for 30 minutes and centrifuged at 400 g / 10 minutes.

[0277] (7) Perm / wash Buffer was added at 200 μL / well, and the cells were washed twice.

[0278] (8) Place the antibody staining solution, the amount of pSTAT5 antibody (BD) was 3 μL / 100 μL perm / wash buffer / well, and the remaining staining antibody was 1 μL / 100 μL perm / wash buffer / well, incubated at room temperature for 1.5 h, and washed twice with perm / wash buffer.

[0279] (9) The cells were resuspended in 150 μL of perm / wash buffer / well and subjected to flow cytometry.

[0280] The results in Figure 7 show that in PD-1 negative (PD-1-) T cells (CD4+PD1-T or CD8+PD1-T), the activity of all of the molecules in this study was weaker than that of the control molecule 2061, while in PD-1 positive (PD-1+) T cells (CD4+PD1+T or CD8+PD1+T), the activity of 2063 was superior to that of the control molecule 2061, and the selectivity of 2063 against PD-1 was greater than that of 2061. Some other molecules in this study also had weaker activity than 2061 in PD-1+ T cells, and the molecules in this study were less toxic due to highly active IL-2 than 2061, and the tolerable dose in vivo was also higher than that of 2061. 4. HEK-Blue TM Detection of the activity of the immune complex of the present invention by IL-2 cell reporter assay We overexpressed IL2R (CD25, CD122, CD132), JAK3 and STAT5 genes in HEK293 cells to develop HEK293+hIL2R / SEAP cell lines (huPD-1- cells, HEK-Blue TMWe constructed a HEK293+hIL2R / SEAP cell reporter (IL-2 Cells, Invivogen, hkb-il2) and activated the reporter gene in the HEK293+hIL2R / SEAP cell reporter under the action of IL2.

[0281] [Table 10]

[0282] Construction of HEK293+hIL2R+hPD-1 / SEAP cell line: For the following experiments, Lentvirus+hPD-1 lentivirus was constructed and packaged as described above, HEK293+hIL2R / SEAP (Invivogen, hkb-il2) was infected with Lentvirus+hPD-1, and HEK293+hIL2R+hPD-1 / SEAP stable transformed cell line (huPD-1+ cells) was pressure screened and sorted.

[0283] Testing Method: 1. Cells were digested, cell density was adjusted, and plated in the center of a 60-well plate, 50,000 cells per well.

[0284] 2. As shown in the figure, diluted immune complexes and control molecules were added to the corresponding cell well plates and incubated at 37°C for 20h to 24h.

[0285] 4.20 μL of cell culture supernatant was added to 180 μL of QUANTI-Blue, left at room temperature for 15 minutes, and OD630 was measured.

[0286] HEK-Blue TM IL-2 Cells (huPD-1-cells) were HEK293 cells that overexpressed the IL-2 receptor. As can be seen from Figure 8 and Table 4, the IL-2 activity of the immune complex obtained in this study was at least 3.14 times weaker than 2061, and at most 2.21E+08 times weaker.

[0287] In cells overexpressing PD-1 (HEK293+hIL2R+hPD-1 / SEAP stably transfected cell line (huPD-1+ cells)), the immunoconjugates could reach potent IL-2 activity and maintained high selectivity in the two cells, with 2063's selectivity reaching 3.52-fold, 2132's selectivity reaching 53.45-fold, 2149's selectivity reaching 96.04-fold, 2219's selectivity reaching 606.11-fold, 2214's selectivity reaching 5119.78-fold, and 2213's selectivity reaching 1.57E+07-fold.

[0288] [Table 11] Example 5. In vivo efficacy studies of immune complexes To prove the efficacy of αPD-1 / IL2m immunoconjugate in vivo, MC38 cells (mouse colon cancer cell line, Shanghai Hegen Bio) were used to inoculate hPD-1 knock-in mice to measure the antitumor efficacy of the bifunctional PD-1 antibody and IL-2 mutant molecule immunoconjugate of the present invention (2063, 2132). The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010005748.

[0289] MC38 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS (1x) to a cell concentration of 5x10 6 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank area of ​​hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.

[0290] Six days after tumor cell inoculation, the tumor volume of each mouse was detected and divided into groups (8 mice per group), with the dosage and administration method shown in Table 5.

[0291] [Table 12]

[0292] h-IgG, 2063 and 2132 were used at concentrations of 2 mg / mL, 1 mg / mL and 1 mg / mL, respectively, and were administered once a week for a total of three times (QWx3) on days 6, 13 and 20 after MC38 cell inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week, with monitoring ending after 24 days, as shown in Figure 9A.

[0293] On the 24th day after inoculation, the relative tumor inhibition rate (TGI%) was calculated using the following formula: TGI% = 100% x (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group). Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated using the following formula: V = L x W 2 Body weight was measured using an electronic balance.

[0294] The results of tumor inhibition rate are shown in Table 6. On the 24th day after inoculation, the tumor inhibition rates of 2063 and 2132 were 98% and 96%, respectively, compared with the h-IgG, 20 mg / kg group. At the same time, the detection results of mouse body weight (Figure 9B) showed that on the 24th day after inoculation, there was no significant difference in mouse body weight.

[0295] [Table 13] To further prove that the in vivo efficacy of the αPD-1 / IL2m immunoconjugate is superior to the parent anti-PD-1 monoclonal antibody (Sintilimab, also called IBI308), MC38 cells (mouse colon cancer cell line, Shanghai Hegen Bio) were used to inoculate hPD-1 knock-in mice to measure the antitumor efficacy of the immunoconjugate of the present invention (2063). The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010004237.

[0296] MC38 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS (1x) to a cell concentration of 5x10 6 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank area of ​​hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.

[0297] Twelve days after tumor cell inoculation, the tumor volume of each mouse was detected and grouped (8 mice per group), with the dosage and administration method shown in Table 7.

[0298] [Table 14]

[0299] h-IgG, 2063 and IBI308 were all used at a concentration of 1 mg / mL and administered once a week for a total of three times (QWx3). They were administered on the 12th, 19th and 26th days after MC38 cell inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 10A, and the monitoring was terminated after 29 days. On the 29th day after inoculation, the relative tumor inhibition rate (TGI%) was calculated, and the calculation formula was TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group). Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 Body weight was measured using an electronic balance.

[0300] The results of tumor inhibition rate are shown in Table 8. On the 29th day after inoculation, the tumor inhibition rates of 2063 and IBI308 were 112% and 56%, respectively, compared with the h-IgG, 20 mg / kg group. At the same time, the detection results of mouse body weight (Figure 10B) showed that on the 29th day after inoculation, the mouse body weight had no significant difference.

[0301] [Table 15]

[0302] To further prove the in vivo efficacy of αPD-1 / IL2m immune complex, PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) were used to inoculate hPD-1 knock-in mice, and the antitumor efficacy of the αPD-1 / IL2m immune complex of the present invention (2063), parent anti-PD-1 monoclonal antibody (IBI308), and PD-1 monoclonal antibody and IL2m-Fc fusion protein (2124, IL-2 sequence is the same as 2063, see sequence listing) was measured. The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010004768.

[0303] B16F10 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS (1x) until the cell concentration reached 2.5x10 6 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0304] Seven days after tumor cell inoculation, the tumor volume of each mouse was detected and grouped (6 mice per group), with the dosage and administration method shown in Table 9.

[0305] [Table 16] h-IgG, IBI308, 2124 and 2063 were used at concentrations of 1 mg / mL, 1 mg / mL, 0.6 mg / mL and 1 mg / mL, respectively, and were administered once a week for a total of three times (QWx3) on days 8, 15 and 22 after B16F10 cell inoculation, respectively. As shown in Figures 11A-11C, the tumor volume and body weight of the mice were monitored twice a week, and the monitoring was terminated after 28 days.

[0306] Because B16F10 cells easily metastasize and induce mouse death, the relative tumor inhibition rate (TGI%) was calculated on the 22nd day after inoculation, and the calculation formula was TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group). Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 Body weight was measured using an electronic balance. 3 If mortality within a group exceeded half, the mouse was euthanized. If mortality within a group exceeded half, tumor growth curves for the entire group were not presented at this time point.

[0307] The results of tumor inhibition rate are shown in Table 10. On the 22nd day after inoculation, compared with the h-IgG group, the tumor inhibition rates of IBI308, IBI308+2214, and 2063 were 2%, 84%, and 99%, respectively, and the CR rate of 2063 was obviously superior to IBI308 and the combination of IBI308 and non-targeting IL2m-Fc molecule (2124). At the same time, the detection results of mouse body weight (Figure 11C) showed that there was no significant difference in the body weight of the mice during the monitoring period.

[0308] [Table 17]

[0309] To further prove the in vivo efficacy of the inventive αPD-1 / IL2m immunoconjugate 2149, MC38 cells (mouse colon cancer cell line, Shanghai Hegen Bio) were used to inoculate hPD-1 knock-in mice to measure the antitumor efficacy of the inventive immunoconjugate (2149). The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010006762.

[0310] MC38 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS (1x) to a cell concentration of 5x106 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank area of ​​hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.

[0311] Eight days after tumor cell inoculation, the tumor volume of each mouse was detected and the mice were divided into groups (8 mice per group), with the dosage and administration method shown in Table 11.

[0312] [Table 18]

[0313] h-IgG, 2149,10mg / kg, 2149,20mg / kg, and 2149,40mg / kg were used at concentrations of 4mg / mL, 1mg / mL, 2mg / mL, and 4mg / mL, respectively, and were administered once a week for a total of three times (QWx3). They were administered on days 8, 15, and 22 after MC38 cell inoculation, respectively, and the tumor volumes and body weights of the mice were monitored twice a week, as shown in Figures 12A-B. When the tumors reached 2000mm 3 Mice were euthanized when tumor volumes exceeded 2000 mm and were monitored throughout the experiment until the end of the 61-day period. 3 The mice were euthanized after exceeding the age of 10 days, so the relative tumor inhibition rate (TGI%) was calculated on the 36th day after inoculation. The calculation formula was TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group). Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated using the following formula: V = L × W 2 Body weight was measured using an electronic balance.

[0314] As shown in the tumor growth curves and survival curves in Figure 12A and Figure 12B, the antitumor effects of different dose groups of 2149 molecules were dose-dependent, and the mice tumors completely regressed in the 20 mg / kg and 40 mg / kg groups. This advantage was also reflected in the survival curve in Figure 12B, where the mice in these two groups experienced 100% tumor regression, while in the 10 mg / kg group, only two of the eight mice experienced complete tumor regression. The results of tumor inhibition rate were shown in Table 12. On the 36th day after inoculation, the tumor inhibition rates of 2149, 10 mg / kg, 2149, 20 mg / kg, and 2149, 40 mg / kg were 84%, 103%, and 103%, respectively, compared with the h-IgG group. At the same time, the detection results of mouse body weight (Figure 12C) showed that on the 36th day after inoculation, the mouse body weight was not significantly different.

[0315] [Table 19]

[0316] To prove the in vivo efficacy of the immunoconjugate 2149, PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) were used to inoculate hPD-1 knock-in mice to measure the antitumor efficacy of the bifunctional PD-1 antibody and IL-2 mutant molecule fusion protein (2149) of the present invention. The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010007909.

[0317] B16F10 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS (1x) until the cell concentration reached 2.5x10 6 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0318] Six days after tumor cell inoculation, the tumor volume of each mouse was detected and divided into groups (8 mice per group), with the dosage and administration method shown in Table 13.

[0319] [Table 20]

[0320] h-IgG, IBI308,20mg / kg, IBI308,40mg / kg, 2149,20mg / kg and 2149,40mg / kg were used at concentrations of 4mg / mL, 2mg / mL, 4mg / mL, 2mg / mL and 4mg / mL, respectively, and were administered once a week for a total of three times (QWx3). They were administered on the 8th, 15th and 22nd days after B16F10 cell inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 13A-13B, and the monitoring was terminated after 22 days. Because B16F10 cells easily metastasize and induce the death of mice, the relative tumor inhibition rate (TGI%) was calculated on the 15th day after inoculation, and the calculation formula was TGI%=100%×(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume before administration of control group). Measurement of tumor volume: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 Body weight was measured using an electronic balance. 3 When the body weight exceeded 100 mg / kg, the mouse was euthanized.

[0321] As shown in the mouse tumor growth curves in Figures 13A and 13B, in the PD1-resistant model, IBI308 showed little efficacy, whereas the 20 mg / kg and 40 mg / kg groups of 2149 showed some antitumor effect, and the antitumor effect of the high dose was better than that of the low dose. This effect was also reflected in the mouse survival curve, and in the high dose group, the tumors of two mice had not yet completely regressed at the end of the experiment (Figure 13C and Table 14).

[0322] The results of tumor inhibition rate are shown in Table 12. On the 15th day after inoculation, compared with the 40 mg / kg group of h-IgG, the tumor inhibition rates of IBI308, 20 mg / kg, IBI308, 40 mg / kg, 2149, 20 mg / kg, and 2149, 40 mg / kg were 29%, 27%, 82%, and 86%, respectively. At the same time, the detection results of mouse body weight (Figure 13D) showed that on the 22nd day after inoculation, the mouse body weight was not significantly different.

[0323] [Table 21]

[0324] To prove that the in vivo efficacy of αPD-1 / IL2m immune complex 2149 is superior to the control drug PD-1-IL2v (molecule number 2061, sequence is from US20180326010A1, see also sequence listing), PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) were used to inoculate hPD-1 knock-in mice to measure the antitumor efficacy of the bifunctional PD-1 antibody and IL-2 mutant molecule fusion protein of the present invention (2149). The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010008942.

[0325] B16F10 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS (1x) until the cell concentration reached 2.5x10 6 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0326] Eight days after tumor cell inoculation, the tumor volume of each mouse was detected and divided into groups (7 mice per group), with the dosage and administration method shown in Table 15.

[0327] [Table 22]

[0328] h-IgG, IBI308, 40mg / kg, 2061, 10mg / kg, 2061, 20mg / kg, 2061, 40mg / kg, 2149, 10mg / kg, 2149, 20mg / kg, and 2149, 40mg / kg were used at concentrations of 4mg / mL, 2mg / mL, 4mg / mL, 2mg / mL, and 4mg / mL, respectively, and were administered once a week for a total of three times (QWx3) on days 8, 15, and 22 after B16F10 cell inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week, and the monitoring was terminated after 33 days, as shown in Figure 14A. Because B16F10 cells easily metastasize and cause mouse death, the relative tumor inhibition rate (TGI%) was calculated on the 19th day after inoculation, and the calculation formula was TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group). Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a caliper, and the tumor volume was calculated according to the following formula: V = L * W 2 Body weight was measured using an electronic balance. 3 If mortality within a group exceeded half, the mouse was euthanized. If mortality within a group exceeded half, tumor growth curves for the entire group were not presented at this time point.

[0329] The results of tumor inhibition rate are shown in Table 16. On the 19th day after inoculation, the tumor inhibition rates of IBI308, 40 mg / kg, 2061, 10 mg / kg, 2149, 10 mg / kg, 2149, 20 mg / kg, and 2149, 40 mg / kg were 21%, 96%, 79%, 87%, and 97%, respectively, compared with the h-IgG, 40 mg / kg group. For 2061, 20 mg / kg and 40 mg / kg, the body weight was significantly reduced and the mice died after the first injection, so the TGI of this group was not calculated.

[0330] In addition, a statistical analysis was performed on the survival rate of mice (Figure 14B). As shown in the figure, when comparing the maximum dose / tolerance dose of 2061 and 2149 in this experiment, the survival rate of mice was high at a molecular dose of 40 mg / kg for 2149 and 10 mg / kg for 2061. For 2149, the tumors of three of the seven mice completely regressed, while the tumors of only one mouse completely regressed for 2061. At the same time, the results of detecting the weight of mice (Figure 14C) showed that on the 29th day after inoculation, none of the mice in each dose group of 2149 lost weight, but the mice in the 2061 group lost weight, and at the low dose (10 mg / kg), the weight of the mice decreased by an average of more than 5%, and at the medium dose (20 mg / kg) and high dose (40 mg / kg), the mice died, with a total of seven mice in each group, and six died. The details are shown in Table 12. 2149 was relatively safe, with no obvious weight loss at low, medium or high doses, only one mouse died at low and medium doses, but no mice died at high doses, and had a higher complete tumor remission rate than 2061 (Table 16). Therefore, 2149 was more effective, safer and had a higher therapeutic window than 2061.

[0331] [Table 23]

[0332] To verify the in vivo efficacy of αPD-1 / IL2m immune complex 2214, MC38 cells (mouse colon cancer cell line, Shanghai Hegen Bio) were used to inoculate hPD-1 knock-in mice to measure the antitumor efficacy of the bifunctional PD-1 antibody and IL-2 mutant molecule fusion protein (2214) of the present invention. The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010010829.

[0333] MC38 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and MC38 cells were resuspended in PBS (1x) to a cell concentration of 5x10 6 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank area of ​​hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.

[0334] Seven days after tumor cell inoculation, the tumor volume of each mouse was detected and grouped (7 mice per group), with the dosage and administration method shown in Table 17.

[0335] [Table 24]

[0336] h-IgG and 2214 were all used at a concentration of 4 mg / mL and were administered once a week for a total of three times (QWx3). They were administered on days 7, 14, and 21 after MC38 cell inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 15A, and monitoring was terminated after 56 days. The tumor volume of the control group was 2000 mm 3 Since the tumor volume exceeded 100%, the relative tumor inhibition rate (TGI%) was calculated on the 28th day after inoculation, and the calculation formula was TGI% = 100% × (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume before administration of the control group). Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated using the following formula: V = L × W 2 Body weight was measured using an electronic balance.

[0337] The results of the tumor inhibition rate are shown in Table 18. On the 28th day after inoculation, the tumor inhibition rate of 2214 was 104% compared with the h-IgG, 20 mg / kg group. At the same time, the detection results of mouse body weight (FIG. 15C) showed that on the 28th day after inoculation, there was no significant difference in the mouse body weight.

[0338] [Table 25]

[0339] To prove the in vivo efficacy of αPD-1 / IL2m immune complex 2214, PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) were used to inoculate hPD-1 knock-in mice to measure the antitumor efficacy of the bifunctional PD-1 antibody and IL-2 mutant molecule fusion protein (2214) of the present invention. The experiment used SPF grade female hPD-1 knock-in mice (purchased from SHANGHAI MODEL ORGANISMS), with the identification number NO.20170010010829.

[0340] B16F10 cells were subcultured periodically for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS (1x) until the cell concentration reached 2.5x10 6 A cell suspension was prepared at 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0341] Seven days after tumor cell inoculation, the tumor volume of each mouse was detected and divided into groups (7 mice per group), with the dosage and administration method shown in Table 19.

[0342] [Table 26]

[0343] h-IgG, 2214, 20mg / kg and 2214, 40mg / kg were used at concentrations of 4mg / mL, 2mg / mL, and 4mg / mL, respectively, and were administered once a week for a total of three times (QWx3). They were administered on the 7th, 14th, and 21st days after B16F10 cell inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 16A-16B, and the monitoring was terminated after 63 days. Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated according to the following formula: V = L × W 2Body weight was measured using an electronic balance. 3 When the body weight exceeded 100 mg / kg, the mouse was euthanized.

[0344] As shown in Fig. 16A, the mouse survival curves showed that both doses of 2214 could significantly extend the survival time of mice. At the same time, the mouse body weight detection results (Fig. 16C) showed that on the 22nd day after inoculation, the mouse body weights were not significantly different.

[0345] [Table 27-1]

[0346] [Table 27-2]

[0347] [Table 27-3]

[0348] [Table 27-4]

[0349] [Table 27-5]

[0350] [Table 27-6]

[0351] [Table 27-7]

[0352] [Table 27-8]

[0353]

Table 27-9

[0354]

Table 27-10

[0355]

Table 27-11

[0356]

Table 27-12

Claims

1. An immunoconjugate comprising: (i) an anti-PD-1 antibody or antigen-binding fragment thereof; and (ii) an IL-2 mutein; The anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in the amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively, and a light chain comprising a light chain mutant region, wherein the light chain mutant region comprises LCDR1, LCDR2, and LCDR3 set forth in the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; and The IL-2 mutein comprises an amino acid sequence comprising a mutation referenced to an amino acid position in SEQ ID NO:3, wherein the mutation is (a) T3A, N88R, and S130R; and (b) An immunoconjugate comprising a deletion of amino acids at positions 73 to 83 and a substitution with the amino acid sequence of AGDASIH (SEQ ID NO: 39) or AQSKNFH (SEQ ID NO: 41).

2. The immune complex of claim 1, wherein the substituted amino acid sequence of (b) is AGDASIH (SEQ ID NO: 39).

3. The immune complex of claim 1, wherein the substituted amino acid sequence of (b) is AQSKNFH (sequence number 41).

4. The immune complex of claim 1, wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:

23.

5. The immunoconjugate of claim 1, wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:

25.

6. The immunoconjugate of claim 1, wherein the IL-2 mutein is fused to an Fc fragment directly or via a linker.

7. The immune complex of claim 6, wherein the IL-2 mutein is fused to the Fc fragment via a linker comprising the amino acid sequence GGGGSGGGGS (SEQ ID NO: 5).

8. the IL-2 mutein comprises an N-terminus and a C-terminus; the Fc fragment comprises an N-terminus and a C-terminus; and The immune complex of claim 6, wherein the C-terminus of the IL-2 mutein is fused to the N-terminus of the Fc fragment directly or via a linker.

9. The immune complex of claim 6, wherein the Fc fragment to which the IL-2 mutein is fused is linked to the Fc fragment of the PD-1 antibody.

10. The immune complex of claim 6, wherein the Fc fragment to which the IL-2 mutein is fused contains one or more mutations that form knobs.

11. The immune complex of claim 10, wherein the Knob comprises one or more mutations selected from T366W, S354C, and combinations thereof.

12. The immune complex of claim 10, wherein the Fc fragment of the PD-1 antibody comprises one or more hole-forming mutations.

13. The immune complex of claim 12, wherein the Hole comprises one or more mutations selected from Y349C, T366S, L368A, Y407V, and combinations thereof.

14. The immune complex of claim 6, wherein the Fc fragment to which the IL-2 mutein is fused comprises the amino acid sequence shown in SEQ ID NO:

42.

15. The immune complex of claim 6, wherein the Fc fragment to which the IL-2 mutein is fused comprises the amino acid sequence shown in SEQ ID NO:

6.

16. The immune complex of claim 12, wherein the Fc fragment of the anti-PD-1 antibody comprises the amino acid sequence shown in SEQ ID NO:

12.

17. the anti-PD-1 antibody or antigen-binding fragment thereof a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; and A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15 The immune complex of claim 1, comprising:

18. A pharmaceutical composition comprising an immunoconjugate, the immunoconjugate comprising: (i) a first monomer comprising an anti-PD-1 antibody and an Fc fragment; and (ii) a second monomer comprising an IL-2 mutein fused to the Fc fragment directly or via a linker; The anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in the amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively, and a light chain comprising a light chain mutant region, wherein the light chain mutant region comprises LCDR1, LCDR2, and LCDR3 set forth in the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; and The IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:25; where (a) the Fc fragment of the first monomer comprises Knob and the Fc fragment of the second monomer comprises Hole; or (b) A pharmaceutical composition, wherein the Fc fragment of the first monomer comprises a Hole and the Fc fragment of the second monomer comprises a Knob.

19. The pharmaceutical composition of claim 18, wherein the Fc fragment of (a) or (b) containing the Knob comprises the amino acid sequence shown in SEQ ID NO:

6.

20. The pharmaceutical composition of claim 18, wherein the Fc fragment (a) or (b) containing the hole comprises the amino acid sequence shown in SEQ ID NO:

12.

21. the anti-PD-1 antibody or antigen-binding fragment thereof a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; and 19. The pharmaceutical composition of claim 18, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

15.

22. 19. The pharmaceutical composition of claim 18, wherein the IL-2 mutein is fused to the Fc fragment via a linker comprising the amino acid sequence set forth in SEQ ID NO:

5.

23. the IL-2 mutein comprises an N-terminus and a C-terminus; the Fc fragment comprises an N-terminus and a C-terminus; and The pharmaceutical composition of claim 18, wherein the C-terminus of the IL-2 mutein is fused to the N-terminus of the Fc fragment directly or via a linker.

24. A pharmaceutical composition comprising an immunoconjugate, the immunoconjugate comprising: (i) a first monomer, (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 14; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; an anti-PD-1 antibody comprising a first monomer comprising: (ii) a second monomer, (a) a second monomer comprising an IL-2 mutein, a linker, and an Fc fragment, said second monomer comprising the amino acid sequence set forth in SEQ ID NO:26; A pharmaceutical composition comprising:

25. 25. A method of treating cancer in a subject, said method comprising administering to said subject the pharmaceutical composition of claim 24.

26. 26. The method of claim 25, wherein the cancer is a PD-1 resistant cancer.

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