PEGylated IL-2 fusion protein of targeted immune checkpoint and application of PEGylated IL-2 fusion protein

Abstract

The invention relates to the field of immunotherapy, in particular to a PEGylated IL-2 fusion protein of a targeted immune checkpoint and application of the PEGylated IL-2 fusion protein in tumor immunotherapy. The PEGylated IL-2 fusion protein disclosed by the invention can simultaneously act on an immune checkpoint and an IL2 receptor on an activated T cell in a targeting manner through a homeopathic effect, the differentiation of the activated T cell to a depleted T cell (Tex) is inhibited, and the PEGylated IL-2 fusion protein has selectivity on Teff and Treg and does not preferentially activate the Treg, so that the anti-tumor effect is remarkably enhanced, and the PEGylated IL-2 fusion protein has an important clinical value on tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy, and more specifically, to a PEGylated IL-2 fusion protein targeting immune checkpoints and its application in tumor immunotherapy. Background Technology

[0002] Interleukin-2 (IL2) is an essential cytokine for maintaining T cell development and function. IL2 receptors exist in three forms: IL2-Rα monomer, IL2-Rβγ dimer, and IL2-Rαβγ trimer. IL2 has a weaker affinity for the α monomer (10-1). -8 M has a moderate affinity for the βγ dimer, around 10. -9 M has the highest affinity for the αβγ trimer, at 10. -11 The distribution of IL-2 receptors varies across different immune cells: IL-2-Rαβγ trimer is long-term expressed on the surface of regulatory T cells (Tregs), thus exhibiting the highest affinity for IL-2; IL-2-Rβγ dimer is expressed on the surface of resting effector T cells (Teffs) and cytotoxic T cells (CTLs), showing moderate affinity for IL-2. However, after Teff cell activation, IL-2-Rαβγ trimer expression is upregulated, enhancing its affinity for IL-2. In tumor treatment, on the one hand, under the same IL-2 concentration, IL-2-Rαβγ trimer preferentially binds, activating Treg cells and inhibiting Teff cells, thus weakening anti-tumor activity; on the other hand, while high concentrations of IL-2 can activate Teff after activating Tregs, the excessively strong STAT5 signaling from IL-2-Rαβγ trimer leads to T cell exhaustion, similarly weakening anti-tumor activity.

[0003] Therefore, a non-α IL-2 receptor that can block IL-2 from binding to the IL-2-Rα receptor was designed to reduce the binding affinity of IL-2 to the IL-2-Rαβγ trimer. Compared to IL-2, the non-α IL-2 does not preferentially activate Treg cells, but instead simultaneously activates both Treg and Teff cells, resulting in enhanced anti-tumor activity. However, because the downstream signaling of the IL-2-Rβγ dimer is weaker than that of the IL-2-Rαβγ trimer, the activation level of Teff cells is insufficient, preventing them from fully exerting their anti-tumor effect. This demonstrates that IL-2's regulation of T cells is extremely stringent. The key to using IL-2 in tumor immunotherapy lies in how to modify it so that it neither binds to the IL-2-Rα receptor nor fails to transmit sufficient downstream signals, while simultaneously avoiding excessive T cell activation leading to T cell exhaustion.

[0004] T cell exhaustion refers to the progressive decline in T cell function caused by continuous stimulation of the TCR under sustained antigen exposure. This leads to a weakened ability to secrete functional cytokines and an upregulation of various inhibitory receptors such as PD-1, LAG-3, TIGIT, and TIM3. Compared to bystander T cells in the body, these T cells expressing exhaustion markers are rich in tumor-reactive CD8+ T cells capable of recognizing tumor cells. Therefore, T cell exhaustion is unavoidable in tumor immunotherapy. Reducing CD8+ TIL exhaustion during tumor treatment is a new direction for solid tumor immunotherapy at present. Summary of the Invention

[0005] The inventors of this application have constructed a series of IL2 fusion proteins containing non-natural amino acids that target immune checkpoints using gene codon expansion technology, and have chemically PEGylated the positions where non-natural amino acids are inserted to obtain PEGylated IL2 fusion proteins that target immune checkpoints, thereby providing the following aspects.

[0006] IL-2 fusion protein

[0007] In one aspect, the present invention provides a fusion protein comprising (i) a targeting portion that targets an immune checkpoint and (ii) a site-mutated IL-2, wherein the site-mutated IL-2 is mutated to non-natural amino acids at residues selected from the following amino acid positions compared to wild-type IL-2: F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49.

[0008] Targeting the immune checkpoint

[0009] In some embodiments, the targeting portion has an antigen-binding domain that specifically binds to immune checkpoints.

[0010] In some implementations, the targeting portion is selected from antibodies or antibody fragments.

[0011] In some embodiments, the targeting portion may be a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bispecific antibody, a nanobody, or an antibody fragment (such as Fab, Fab', Fab2, Fab'2, scFab, scFv, dsFv, dAb).

[0012] In some embodiments, the targeting portion is selected from nanobodies, scFv, or single-chain Fab (scFab).

[0013] In some embodiments, the targeting portion is selected from nanobodies.

[0014] In some implementations, the immune checkpoints are selected from LAG-3, TIGIT, TIM-3, PD-1, PD-L1, CLTA-4, CD39, VISTA, and A2AR.

[0015] In some embodiments, the targeting portion is selected from nanobodies that target LAG-3, TIGIT, TIM-3, PD-1, or PD-L1.

[0016] In some embodiments, the LAG-3-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:2-4, respectively. In some embodiments, the LAG-3-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:1, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets mouse LAG-3.

[0017] In some embodiments, the LAG-3-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:22-24, respectively. In some embodiments, the LAG-3-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:21, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets human LAG-3.

[0018] In some embodiments, the TIGIT-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:6-8, respectively. In some embodiments, the TIGIT-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:5, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets mouse TIGIT.

[0019] In some embodiments, the TIM-3-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:10-12, respectively. In some embodiments, the TIM-3-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:9, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets mouse TIM-3.

[0020] In some embodiments, the TIM-3-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:26-28, respectively. In some embodiments, the TIM-3-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:25, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets human TIM-3.

[0021] In some embodiments, the PD-1-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:14-16, respectively. In some embodiments, the PD-1-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:13, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets mouse PD-1.

[0022] In some embodiments, the PD-1-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:30-32, respectively. In some embodiments, the PD-1-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:29, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets human PD-1.

[0023] In some embodiments, the PD-L1-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:18-20, respectively. In some embodiments, the PD-L1-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:17, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets mouse PD-L1.

[0024] In some embodiments, the PD-L1-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:34-36, respectively. In some embodiments, the PD-L1-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:33, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with it. In some embodiments, the nanobody targets human PD-L1.

[0025] Site-directed mutation of IL-2

[0026] In some embodiments, the IL-2-related amino acid positions described herein are determined with reference to the sequence shown in SEQ ID NO:37. In this document, when stating that "the site-directed mutated IL-2 is mutated to a non-natural amino acid at a residue selected from the following amino acid positions compared to wild-type IL-2," the amino acid position refers to the position in the sequence shown in SEQ ID NO:37 and its corresponding position. The corresponding position refers to the position in the sequence to be compared that is equivalent to a given amino acid position in SEQ ID NO:37 when the sequence to be compared is optimally aligned with SEQ ID NO:37 (i.e., to obtain the highest percentage of identity). For example, the expression "Y45" refers to the 45th amino acid residue in the sequence shown in SEQ ID NO:37 and its corresponding position.

[0027] In some embodiments, the position where the amino acid is mutated to a non-natural amino acid is selected from F42, Y45, E62, K64, P65, and E68.

[0028] In some embodiments, the wild-type IL-2 has the amino acid sequence shown in SEQ ID NO:37.

[0029] In some embodiments, the site-directed mutated IL-2, compared to the wild-type IL-2 shown in SEQ ID NO:37, has residues at amino acid positions selected from F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, and K49 mutated to non-natural amino acids.

[0030] In some embodiments, the site-directed mutated IL-2, compared to the wild-type IL-2 shown in SEQ ID NO:37, has residues at amino acid positions selected from F42, Y45, E62, K64, P65, and E68 mutated to non-natural amino acids.

[0031] In some embodiments, the non-natural amino acids contained in the site-directed mutated IL-2 have side chains that allow linkage with PEG groups.

[0032] In some embodiments, the non-natural amino acid contains a chemical functional group (e.g., carbonyl, alkynyl, or azide group); the PEG group contains a labeling group that can chemically react with the chemical functional group, thereby allowing the PEG group to be attached to the non-natural amino acid.

[0033] In some embodiments, the non-natural amino acid contains an azide group, and the PEG group contains a labeling group that can undergo a click chemical reaction with the azide group, thereby allowing the PEG group to be attached to the non-natural amino acid.

[0034] In some embodiments, the labeling group capable of undergoing a click chemical reaction with the azide group is a chemical moiety containing a dibenzocyclooctyn group, such as DBCO, DIBO, or BCN.

[0035] In some embodiments, the non-natural amino acid is a lysine derivative or tyrosine derivative containing an azide group, such as Nε-2-azidoethoxycarbonyl-L-lysine (NAEK) or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

[0036] structure

[0037] In some implementations, the targeting portion of the targeted immune checkpoint is directly fused to the N-terminus of the site-directed mutated IL-2.

[0038] In some embodiments, the targeting portion of the immune checkpoint is fused to the N-terminus of the site-directed mutated IL-2 via a peptide linker. In some embodiments, the peptide linker is a flexible peptide linker, such as a peptide linker comprising one or more glycine residues and / or one or more serine residues.

[0039] In some exemplary embodiments, the fusion protein comprises the amino acid sequence described in any one of SEQ ID NO:41-49.

[0040] Preparation of fusion proteins

[0041] The fusion protein of the present invention can be prepared by any method known in the art of inserting non-natural amino acids.

[0042] In some implementations, non-natural amino acids can be inserted at specific points using orthogonal translation techniques for non-natural amino acids, detailed in teachings for which see, for example, PCT / CN2022 / 129975, the entire contents of which are incorporated herein by reference.

[0043] Orthogonal translation of non-natural amino acids is well known to those skilled in the art. This technique utilizes stop codons to insert non-natural amino acids into the amino acid sequence of a protein during translation, effectively expanding the number of amino acid codons; therefore, it is also known as genetic codon expansion. Typically, a non-natural amino acid orthogonal translation system involves tRNA, aminoacyl-tRNA synthetase, and a target nucleic acid sequence with one or more stop codons. The system is introduced into host cells and cultured in a medium containing appropriate nutrients and the one or more non-natural amino acids to be inserted. The host cells are then maintained under conditions that allow for the expression of the target protein. In response to a non-natural codon, one or more non-natural amino acids are incorporated into the polypeptide chain.

[0044] In some implementations, non-natural amino acids can be inserted at specific points using orthogonal translation techniques. These non-natural amino acids may contain chemical functional groups, such as carbonyl, alkynyl, and azide groups. These groups can generally form stable covalent bonds effectively and selectively, and then the PEG group can be modified at specific points by forming covalent bonds through a chemical reaction.

[0045] In one aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of the present invention, wherein the codon corresponding to the position mutated to a non-natural amino acid is TAG.

[0046] In one aspect, the present invention provides a vector comprising isolated nucleic acid molecules as described above; preferably, the vector is an expression vector.

[0047] In one aspect, the present invention provides a host cell comprising isolated nucleic acid molecules or vectors as described above.

[0048] In one aspect, the present invention provides a method for preparing the fusion protein of the present invention, comprising the following steps:

[0049] - Co-transfect host cells with the isolated nucleic acid molecules or vectors as described above, along with a vector encoding amber codon-repressed tRNA and aminoacyl-tRNA synthetase specific to non-natural amino acids.

[0050] - The host cells are cultured in a culture medium containing non-natural amino acids.

[0051] In some embodiments, the non-natural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine (NAEK). In some embodiments, the aminoacyl-tRNA synthetase specific to the non-natural amino acid is a NAEK-specific aminoacyl-tRNA synthetase.

[0052] In some embodiments, the vector encoding the amber codon-repressive tRNA and the aminoacyl-tRNA synthetase specific to non-natural amino acids is pSURAR-YAV (also known as pSUPAR-YAV-tRNA / PylRS), which is obtained from Escherichia coli containing the plasmid pSUPAR-YAV-tRNA / PylRS. This strain is deposited at the China General Microbiological Culture Collection Center (No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 8, 2013, with accession number CGMCC No: 7432, and classified as Escherichia coli.

[0053] In some embodiments, the site-mutated IL-2 contained in the fusion protein, compared to wild-type IL-2, has residues at specified amino acid positions (e.g., F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49) substituted with a structure of Formula I:

[0054]

[0055] The direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence, where the Nth amino acid is the residue at the specified amino acid position, R1 is the amino acid residue from the 1st to the (N-1)th position of the IL-2 amino acid sequence, and R2 is the amino acid residue from the (N+1)th position to the C-terminus of the IL-2 amino acid sequence.

[0056] Conjugate

[0057] In one aspect, the present invention provides a conjugate comprising the fusion protein of the present invention and a PEG group linked to a non-natural amino acid of said fusion protein. In some embodiments, the conjugate of the present invention may be referred to as a "PEGylated IL2 fusion protein targeting immune checkpoints" or simply as a "PEGylated IL2 fusion protein".

[0058] In some embodiments, the average molecular weight of the PEG-modified group is 5 to 60 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa.

[0059] In some embodiments, the average molecular weight of the PEG-modified group is 5–40 kDa, for example 5–30 kDa, 5–25 kDa, 5–20 kDa, 10–40 kDa, 10–30 kDa, 15–30 kDa, 10–25 kDa, or 15–25 kDa, for example 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or 40 kDa.

[0060] In some embodiments, the average molecular weight of the PEG-modified group is 5 kDa, 10 kDa, or 20 kDa.

[0061] In some embodiments, the average molecular weight of the PEG-modified group is 20 kDa.

[0062] In some embodiments, the non-natural amino acids contained in the fusion protein of the present invention contain chemical functional groups, such as carbonyl, alkynyl, and azide groups, which are generally capable of forming stable covalent bonds effectively and selectively; the PEG group contains a labeling group that can chemically react with the chemical functional group to form a covalent bond, thereby linking the PEG group to the non-natural amino acid.

[0063] In some embodiments, the non-natural amino acid contains an azide group, and the PEG group contains a labeling group capable of undergoing a click chemical reaction with the azide group, thereby linking the PEG group to the non-natural amino acid.

[0064] In some embodiments, the non-natural amino acid is a lysine derivative containing an azide group. In some embodiments, the non-natural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine (NAEK).

[0065] In some embodiments, the non-natural amino acid is a tyrosine derivative containing an azide group. In some embodiments, the non-natural amino acid is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

[0066] In some embodiments, the labeling group capable of undergoing a click chemical reaction with the azide group is a chemical moiety containing a dibenzocyclooctyne group, such as dibenzocyclooctyne (DBCO), 4-dibenzocyclooctynol (DIBO), or BCN (bicyclo[6.1.0]nonyne).

[0067] In some embodiments, the labeling group capable of undergoing a click reaction with the azide group is DBCO, and the site-modified IL-2 contained in the fusion protein, compared to wild-type IL-2, has residues at specified amino acid positions (e.g., F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49) replaced by a structure of formula IIa:

[0068]

[0069] Wherein, the direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence, wherein the Nth amino acid is the residue at the specified amino acid position, R1 is the amino acid residue from the 1st to the (N-1)th position of the IL-2 amino acid sequence, R2 is the amino acid residue from the (N+1)th position to the C-terminus of the IL-2 amino acid sequence, and R3 is the PEG group.

[0070] In some embodiments, the labeling group capable of undergoing a click reaction with the azide group is DIBO, and the site-modified IL-2 contained in the fusion protein, compared to wild-type IL-2, has residues at specified amino acid positions (e.g., F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49) replaced by a structure of formula IIb:

[0071]

[0072] Wherein, the direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence, wherein the Nth amino acid is the residue at the specified amino acid position, R1 is the amino acid residue from the 1st to the (N-1)th position of the IL-2 amino acid sequence, R2 is the amino acid residue from the (N+1)th position to the C-terminus of the IL-2 amino acid sequence, and R3 is the PEG group.

[0073] In some embodiments, the labeling group capable of undergoing a click reaction with the azide group is BCN, and the site-modified IL-2 contained in the fusion protein, compared to wild-type IL-2, has residues at specified amino acid positions (e.g., F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49) replaced by a structure of formula IIc:

[0074]

[0075] Wherein, the direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence, wherein the Nth amino acid is the residue at the specified amino acid position, R1 is the amino acid residue from the 1st to the (N-1)th position of the IL-2 amino acid sequence, R2 is the amino acid residue from the (N+1)th position to the C-terminus of the IL-2 amino acid sequence, and R3 is the PEG group.

[0076] In some embodiments, the PEGylated IL2 fusion protein targeting immune checkpoints involved in this application is named as follows: [Immune checkpoint targeted by the targeting portion] - [Position of non-natural amino acid mutated compared to SEQ ID NO:37] - [Average molecular weight of the PEG group to which it is attached].

[0077] In some exemplary embodiments, the conjugate is selected from mLag3-Y45-20K, mTim3-Y45-20K, mTigit3-Y45-20K, mPD1-Y45-20K, mPDL1-Y45-20K, hLag3-Y45-20K, hTim3-Y45-20K, hPD1-Y45-20K, hPDL1-Y45-20K, mLag3-P65-20K, mTim3-P65-20K, mTigit3-P65-20K, mPD1-P65-20K, mPDL1-P65-20K, hLag3-P65-20K, hTim3-P65-20K, hPD1-P65-20K, hPDL 1-P65-20K, mLag3-F42-20K, mTim3-F42-20K, mTigit3-F42-20K, mPD1-F42-20 K, mPDL1-F42-20K, hLag3-F42-20K, hTim3-F42-20K, hPD1-F42-20K, hPDL1-F42 -20K, mLag3-E62-20K, mTim3-E62-20K, mTigit3-E62-20K, mPD1-E62-20K, mPD L1-E62-20K, hLag3-E62-20K, hTim3-E62-20K, hPD1-E62-20K, hPDL1-E62-20K. Taking mLag3-Y45-20K as an example, it contains, from the N-terminus to the C-terminus, a Lag3-targeting nanobody (mLag3, whose CDR and VHH sequences are shown in Table 1) and an IL-2 variant. The IL-2 variant differs from SEQ ID NO:37 in that Y45 is replaced with the non-natural amino acid NAEK, and a PEG group with an average molecular weight of 20 kDa is further linked at this position.

[0078] Preparation of conjugates

[0079] The conjugates of the present invention can be prepared by any method known in the art of linking PEG to a non-natural amino acid, see, for example, PCT / CN2022 / 129975, the entire of which is incorporated herein by reference.

[0080] In some embodiments, the non-natural amino acids contained in the fusion protein of the present invention may contain chemical functional groups, such as carbonyl, alkynyl, azide, etc., which are generally capable of effectively and selectively forming stable covalent bonds, and then site-specifically modifying the PEG group by forming covalent bonds through chemical reactions.

[0081] In some embodiments, the PEG group is modified at specific sites via click chemistry. In some embodiments, the non-natural amino acid and the PEG group each comprise chemical groups capable of undergoing click chemistry.

[0082] In one aspect, a method for preparing the conjugates of the present invention is provided, the method comprising:

[0083] - Provided: (a) the fusion protein of the present invention; (b) a PEG group modified by a labeling group, said labeling group being capable of forming a covalent bond with a non-natural amino acid in the fusion protein;

[0084] - Co-incubate (a) and (b) to couple non-natural amino acids with PEG groups via a chemical reaction.

[0085] In some embodiments, the non-natural amino acid described in (a) contains a chemical functional group (e.g., carbonyl, alkynyl, azide group), and the PEG group described in (b) contains a labeling group that can chemically react with the chemical functional group to form a covalent bond.

[0086] In some embodiments, the non-natural amino acid described in (a) contains an azide group.

[0087] In some embodiments, the non-natural amino acid described in (a) is a lysine derivative or tyrosine derivative containing an azide group, such as Nε-2-azidoethoxycarbonyl-L-lysine (NAEK) or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

[0088] In some implementations, the method includes:

[0089] - Provided: (a) the fusion protein of the present invention; (b) a PEG group modified with a labeling group, said labeling group being capable of undergoing a click chemical reaction with an azide group;

[0090] - Co-incubate (a) and (b) to couple non-natural amino acids to PEG groups via a click reaction.

[0091] In some embodiments, the click chemistry is a copper-free click chemistry. Copper-free click chemistry is a click reaction achieved by introducing cyclooctyne to maintain cell viability, where the strain of the eight-membered ring allows reaction with azides under catalyst-free conditions. One of these reagents consists of a so-called DBCO compound. Azide-modified macromolecules can now be labeled without metal catalysts, thus enabling not only research in living cells but also preventing damage to proteins.

[0092] In some embodiments, the labeling group capable of undergoing a click reaction with the azide group is a chemical moiety containing an alkynyl group. In some embodiments, the labeling group capable of undergoing a click reaction with the azide group is a chemical moiety containing a dibenzocyclooctynyl group. In some embodiments, the labeling group capable of undergoing a click reaction with the azide group is dibenzocyclooctyne (DBCO), 4-dibenzocyclooctynol (DIBO), or BCN (bicyclo[6.1.0]nonyne). In some embodiments, the labeling group capable of undergoing a click reaction with the azide group is DBCO.

[0093] In some embodiments, the DBCO-labeled PEG group has the structure shown in Formula IIIa, where R3 is a PEG group.

[0094]

[0095] In some embodiments, the DIBO-labeled PEG group has the structure shown in Formula IIIb, where R3 is a PEG group.

[0096]

[0097] Therapeutic applications

[0098] In one aspect, the present invention provides pharmaceutical compositions comprising the conjugates of the present invention, and pharmaceutically acceptable carriers and / or excipients.

[0099] In some embodiments, the pharmaceutical composition further comprises additional pharmaceutically active agents.

[0100] In some embodiments, the additional pharmaceutically active agent is an antitumor agent.

[0101] In some embodiments, the additional pharmaceutically active agent is selected from tumor vaccines, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, antibodies that specifically target tumor cells, or immune checkpoint inhibitors.

[0102] In some implementations, the additional pharmaceutically active agent is a tumor vaccine.

[0103] In some embodiments, the conjugate of the present invention and the additional pharmaceutically active agent may be administered together in a single formulation. In some embodiments, the conjugate of the present invention and the additional pharmaceutically active agent may be administered in different formulations, for example, in any order, such as simultaneously, before, or after administration.

[0104] The conjugates or pharmaceutical compositions of the present invention can be formulated into dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or glucose. pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0105] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (wherein which water is soluble) or dispersions and sterile powders for readily preparing sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, polyoxyethylene castor oil ELTM, or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid sufficient for easy injection. It must be stable under manufacturing and storage conditions and must be preserved against contamination by microorganisms such as bacteria and fungi. Carriers can be solvents or dispersion media including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Protection against microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride, will be preferably included in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0106] In another aspect, the present invention provides a method for enhancing immune responses, preventing and / or treating proliferative diseases (e.g., tumors), comprising administering an effective amount of the conjugate or pharmaceutical composition of the present invention to a subject in need. The use of the conjugate or pharmaceutical composition of the present invention in the preparation of medicaments for enhancing immune responses, preventing and / or treating proliferative diseases is also provided.

[0107] In some embodiments, the immune response is a cellular immune response. In some embodiments, the immune response is a T cell-mediated immune response, particularly an effector T cell (Teff)-mediated immune response. In some embodiments, the enhanced immune response further includes reducing or inhibiting Treg cell function.

[0108] In some embodiments, the proliferative disease is a tumor. In some embodiments, the tumor includes solid tumors or hematologic malignancies. In some embodiments, the tumor includes metastatic cancer, recurrent or refractory cancer.

[0109] In some embodiments, the tumor is selected from melanoma, colon cancer, colorectal cancer, rectal cancer, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, prostate cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.

[0110] In some embodiments, the conjugate is administered in combination with another pharmaceutically active agent.

[0111] In some embodiments, the additional pharmaceutically active agent is an antitumor agent.

[0112] In some embodiments, the additional pharmaceutically active agent is selected from tumor vaccines, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, antibodies that specifically target tumor cells, or immune checkpoint inhibitors.

[0113] In some embodiments, the additional pharmaceutically active agent is a tumor vaccine. In some embodiments, the conjugate is administered in combination with the tumor vaccine, for example, simultaneously, separately, or sequentially.

[0114] In some embodiments, the conjugate and additional pharmaceutically active agents are individually formulated into two or more compositions (e.g., a kit containing each component). The individual components administered in combination may be administered simultaneously, separately, or sequentially. In some embodiments, the individual components administered in combination may be given to the subject at a different time than the other components; for example, as part of a treatment regimen, each administration may be given non-simultaneously (e.g., individually or sequentially) at intervals of a given time period. In some embodiments, the individual components administered in combination may also be administered sequentially, but substantially simultaneously, during the same administration period. Furthermore, the individual components administered in combination may be given to the subject via the same or different routes.

[0115] In some embodiments, the conjugate is formulated together with additional pharmaceutically active agents into a single composition, for example, for simultaneous delivery.

[0116] In some embodiments, the conjugates or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including solutions for injection, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The conjugates or pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the active ingredient into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0117] In some embodiments, the conjugates or pharmaceutical compositions of the present invention may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the conjugates or pharmaceutical compositions of the present invention are administered by intravenous injection or bolus.

[0118] In some implementations, the subject is a mammal, such as a human.

[0119] Terminology Definition

[0120] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, cell culture, biochemistry, and cell biology, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0121] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0122] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0123] As used herein, the term "non-natural amino acid" refers to amino acids other than the 20 amino acids naturally present in proteins. Non-limiting examples of non-natural amino acids include: Nε-2-azidoethoxycarbonyl-L-lysine (NAEK), p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-DOPA, fluorinated phenylalanine, isopropyl... Non-natural analogs of the amino acids p-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronylphenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonylserine, phosphonyltyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and tyrosine; non-natural analogs of glutamine amino acids; non-natural analogs of phenylalanine amino acids; non-natural analogs of serine amino acids; and non-natural analogs of threonine amino acids. Analogs; alkyl, aryl, acyl, azide, cyano, halogen, hydrazine, acylhydrazine, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, selenoyl, ester, thioic acid, borate, boronate, phosphate, phosphonoyl, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, ketone or amino-substituted amino acids or combinations thereof; amino acids with a photoactivated crosslinking agent; spin-labeled amino acids; fluorescent amino acids; metal-bound amino acids; metal-containing amino acids; radioactive amino acids; photocage-encapsulated and / or photoisomerizable amino acids; Amino acids containing biotin or biotin analogues; amino acids containing ketone groups; amino acids containing polyethylene glycol or polyether; amino acids with heavy atom substitutions; chemically or photolytically cleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids; carbon-linked sugar-containing amino acids; redox-active amino acids; acids containing α-hydroxy groups; aminothio acids; α,α-disubstituted amino acids; β-amino acids; cyclic amino acids other than proline or histidine; and aromatic amino acids other than phenylalanine, tyrosine, or tryptophan.

[0124] In some implementations, the non-natural amino acid contains a selective reactive group, or a reactive group for site-selective labeling of the target peptide. The chemical reaction can be a bicorthogonal reaction (e.g., a biocompatibility and selectivity reaction), Cu(I)-catalyzed or “copper-free” yne-azidotriazole formation reaction, Staudinger ligation, inverse-electron-demand Diels-Alder (IEDDA) reaction, “photo-click” chemistry, or a metal-mediated process (such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling), etc.

[0125] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0126] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. In some embodiments, the host cell includes *Escherichia coli*.

[0127] As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant and can include a variety of antibody structures, provided they exhibit the desired antigen-binding activity. Typically, an antibody can be an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding sites.

[0128] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as 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), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the IMGT, Kabat, or Chothia numbering system.

[0129] As used herein, the term "nanobody" is also known as "single-domain antibody (sdAb)," and the two terms are used interchangeably. It refers to an antibody fragment composed of a single variable domain (e.g., a variable region of the heavy chain), typically derived from the variable region of a heavy chain antibody (e.g., a camel or shark antibody). Typically, a nanobody consists of four framework regions and three complementarity-determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0130] As used herein, the term "antibody fragment" refers to a polypeptide that contains a fragment of a full-length antibody, retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding fragment." See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0131] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region; and the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0132] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site.

[0133] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used. In some cases, a disulfide bond may also exist between the VH and VL of the scFv.

[0134] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. DThe "binding rate constant" (Kbinding rate constant) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. A smaller equilibrium dissociation constant indicates a tighter antibody-antigen binding and a higher affinity between the antibody and the antigen. The specific binding properties between the two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "binding rate constant" (Kbinding rate constant or Kkon) and the "dissociation rate constant" (Kdis or Koff) can be calculated from the concentration and the actual rates of association and dissociation. The ratio of Kdis / Kkon is equal to the dissociation constant Kdis. D K can be measured using any effective method. D The values ​​of kon and kdis can be measured, for example, using surface plasmon resonance (SPR) in Biacore to measure the dissociation constant, or using bioluminescent interferometry or Kinexa to measure the dissociation constant.

[0135] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0136] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, delayed absorption agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Delayed absorption agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.

[0137] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0138] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0139] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor.

[0140] Beneficial effects of the invention

[0141] The PEGylated IL2 fusion protein targeting immune checkpoints of this invention addresses current challenges faced by IL2 in the field of tumor immunotherapy. For example, it simultaneously targets both immune checkpoints and IL2 receptors on activated T cells through homeopathic effects, inhibiting the differentiation of activated T cells into exhausted T cells (Tex) and enhancing anti-tumor efficacy. Site-specific PEGylation solves the problem of poor selectivity of IL2 for Teff and Treg cells, preventing preferential activation of Tregs after PEGylation. PEGylation also addresses the short half-life of IL2 drugs. This PEGylated IL2 fusion protein targeting immune checkpoints has significant clinical value for tumor treatment.

[0142] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0143] Figure 1 Coomassie blue staining results of PEGylated IL-2 fusion protein. The left lane in the figure shows the electrophoresis results of the IL-2 fusion protein before and after the 20kDa PEG group is attached; the three lanes on the right show the electrophoresis results of Lag3-Y45 after the PEG groups of 5kDa, 10kDa and 20kDa are attached, respectively.

[0144] Figure 2 Results of surface plasmon resonance (SPR) affinity assays of PEGylated IL-2 fusion protein targeting Lag3 for different subunits of the IL-2 receptor and the Lag3 receptor.

[0145] Figures 3A-3D Results of surface plasmon resonance (SPR) affinity assays of PEGylated IL-2 fusion proteins targeting different immune checkpoints for different subunits of the IL-2 receptor and corresponding immune checkpoint receptors.

[0146] Figure 4 In vitro induction of exhausted CD8+ T cells and exhausted precursor CD8+ T cells.

[0147] Figure 5 Effect of PEGylated IL-2 fusion protein on the phosphorylated STAT5 level of depleted CD8+T.

[0148] Figure 6 PEGylated IL-2 fusion protein on depleted CD8 + T cells, depleted precursor CD8 + The effects of T cell proliferation and function.

[0149] Figure 7 Evaluation of the antitumor activity of PEGylated IL-2 fusion protein in a mouse melanoma model.

[0150] Figures 8A-8B Evaluation of the antitumor activity of PEGylated IL-2 fusion protein in a mouse colon cancer model.

[0151] Figure 9 Evaluation of the antitumor activity of PEGylated IL-2 fusion protein combined with tumor antigen peptide in a mouse melanoma model.

[0152] Sequence information

[0153] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0154] Table 1: Sequence Description

[0155]

[0156]

[0157]

[0158]

[0159] Detailed Implementation

[0160] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0161] Example 1: Construction, expression, and purification of PEGylated IL2 fusion protein expression plasmid

[0162] In this embodiment, five nanobodies targeting different human or mouse T-cell receptors were selected for the preparation of IL2 fusion proteins. These are nanobodies targeting LAG-3 (Lymphocyte activation gene-3) (mLag3 / hLag3), TIGIT (T cell immunoreceptor with Ig and ITIM domain) (mTIGIT), TIM-3 (T cell immunoglobulin domain and mucin domain-3) (mTIM-3 / hTIM-3), PD-1 (programmed death 1) (mPD-1 / hPD-1), and PD-L1 (programmed cell death-Ligand 1) (mPD-L1 / hPD-L1). Their CDR and VHH are summarized in Table 1.

[0163] These nanobodies were fused to the N-terminus of an IL2 protein that had been site-mutated to non-natural amino acids, and then PEG was attached to obtain PEGylated IL2 fusion proteins.

[0164] Mutation sites were selected on the IL-2 protein (SEQ ID NO:37) as shown below, where the positions are those in SEQ ID NO:37: F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49. The codons corresponding to these positions were mutated to TAG, thereby introducing non-natural amino acids through non-natural amino acid orthogonal translation technology.

[0165] The naming convention for the PEGylated IL2 fusion protein involved in this application is: [ICInb] - [position of the non-natural amino acid mutated compared to SEQ ID NO:37] - [average molecular weight of the attached PEG group], where ICINb represents a nanobody selected from anti-LAG-3, TIGIT, TIM-3, PD-1, or PD-L1. Taking ICINb-Y45-20K as an example, this fusion protein contains an immune checkpoint nanobody and a site-directed mutated IL-2 from the N-terminus to the C-terminus. The amino acid sequence of the site-directed mutated IL-2 differs from that of SEQ ID NO:37 in that Y45 is replaced by the non-natural amino acid NAEK, and a PEG group with an average molecular weight of 20 kDa is further attached at this position.

[0166] The following example, using ICINb-Y45-20K, illustrates an exemplary method for preparing PEGylated IL2 fusion proteins:

[0167] 1.1 Construction of fusion protein expression plasmid and site-directed PEGylation

[0168] Using molecular cloning technology, the nucleic acid sequence encoding nanobodies was constructed on the N-terminus of Y45 (IL-2 was mutated to TAG at position 45 for insertion into NAEK, and its nucleotide sequence is shown in SEQ ID NO:40) of the laboratory PET-21a(+)-Y45 plasmid, resulting in a series of ICInb-Y45 fusion protein expression sequences.

[0169] 1.2 Expression of site-directed mutant proteins containing non-natural amino acids

[0170] The aforementioned nucleic acid sequence containing codon substitutions was synthesized and cloned into the pET-21a(+) (laboratory-preserved) *E. coli* plasmid expression vector. This vector, along with the pSURAR-YAV plasmid (laboratory-preserved), was co-transfected into *TransB(DE3)* strain (purchased from TransGen, catalog number: CD811-02). The pSURAR-YAV plasmid encodes amber codon-repressed tRNA and NAEK-specific aminoacyl-tRNA synthetase. Co-transfection with a nucleic acid sequence containing the TAG codon allows the introduction of the non-natural amino acid NAEK at a specific site. The transformed strain was inoculated into 2 ml LB medium containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol, and then cultured at 37°C and 220 rpm. The overnight culture was diluted to the optical density in 2×YT medium and cultured at 37°C for 3 hours until the A600nm value reached approximately 1. After inducing tRNA synthetase expression for half an hour by adding 0.1% L-arabinose, 1 mM UAA (NAEK, synthesized in the laboratory) and 0.5 mM isopropyl β-d-thiogalactopyranoside (IPTG) were added, and the temperature was lowered to 20°C. Approximately 18 hours later, cells were harvested by centrifugation and resuspended in His-Bind buffer (20 mM phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole). Protein was extracted twice by passing the cells through a Micofluidizer at 1200 bar and 4°C. The supernatant was then collected by centrifugation at 10,000 g for 40 min and frozen at -80°C until further processing. The sequence of the obtained ICInb-Y45 protein is shown in SEQ ID NO:41-49.

[0171] 1.3 PEGylation of ICINb-Y45-20K protein

[0172] His-tagged ICINb-Y45 in the lysed supernatant was enriched using Ni-NTA His-Bind Resin (R90101, Invitrogen). DBCO-PEG-10K / 20K / 30K was added to the elution buffer (20 mM phosphate, pH 8.0, 500 mM NaCl, 300 mM imidazole) to a final concentration of 500 μM. The reaction was carried out at 4°C with gentle shaking for 12–16 hours. The PEGylated ICINb-Y45-20K protein was then purified by cation exchange chromatography (Resource S, GE Healthcare) and size exclusion chromatography (Superdex 200 increase10 / 300 GL, GE Healthcare) to remove unreacted PEG and other contaminating proteins. The main elution peak was collected using a 10 kDa centrifugal filter unit (Millipore), concentrated, and exchanged into PBS buffer. The purity of the PEGylation product was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions and stained with Coomassie blue.

[0173] Electrophoresis results as follows Figure 1 As shown, the PEGylated ICIB-Y45 protein has a purity exceeding 95%. PEGylated ICIB-F42, ICIB-E62, ICIB-K64, ICIB-P65, ICIB-E68, ICIB-K35, ICIB-T37, ICIB-R38, ICIB-T41, ICIB-K48, and ICIB-K49 proteins were also prepared using the same method, all with a purity exceeding 95%.

[0174] Example 2: Assay of the binding activity of PEGylated IL2 fusion protein to nanobody receptors and IL-2 receptors

[0175] In this embodiment, the affinity of PEGylated IL2 fusion protein for nanobody receptors and IL-2 receptor IL-2Rα, IL-2Rβγ dimers and IL-2Rαβγ trimer complexes was evaluated using surface plasmon resonance (SPR).

[0176] 2.1 Assay of the binding activity of PEGylated IL2 fusion protein to IL-2 receptor

[0177] Human IL-2Rα (Fc tag, ILA-H5251), IL-2Rβγ (Fc tag & Fc tag, ILG-H5254), and IL-2Rβαγ (Fc tag & Fc tag, ILG-H5257) were all purchased from AcroBiosystems. First, the anti-human IgG (Fc) antibody (SSA015, Sino Biological) was diluted to 25 μg / ml with 10 mM sodium acetate solution at pH 5.0. It was then coupled to a CM5 chip activated with 400 mM EDC and 100 mM NHS (cytiva) at a flow rate of 10 μl / min until the response value RU was between 9000 and 14000. Finally, it was blocked with 1 M EDC (cytiva). Subsequently, the three receptors were diluted to 10 μg / ml with running buffer (1×PBS-P) and loaded at a flow rate of 10 μl / min until the capture response value reached approximately 500 RU. Then, 100 μl of a 1 nM sample of the target protein was used for a preliminary experiment to determine the activity of the protein coupled to the chip and to serve as a concentration reference. The regeneration conditions were measured using pH 2.5 Glysine (Cytiva). Finally, based on the preliminary experiment, each protein sample was diluted twofold, resulting in a total of nine concentrations. Through cycles of receptor capture, binding, dissociation, and regeneration, the affinity of each PEGylated IL-2 fusion protein for the three different IL-2 receptors was determined. Data were analyzed using Biacore 8K evaluation software, and a 1:1 stable affinity model was used to fit the data to determine the KD value and other kinetic parameters. Protein concentration was measured using the BCA (Pierce) method. All sample concentrations represent the mass of IL-2 without PEG conjugation.

[0178] 2.2 Assay of the binding activity of PEGylated IL2 fusion protein to nanobody receptor

[0179] The extracellular domains of mouse-derived LAG3 (53069-M02H), TIGIT (50939-M38H), TIM3 (51152-M02H), PD-1 (50124-M08H), and PD-L1 (50010-M03H) proteins were purchased from Sinocare. All experiments were performed at 25°C using 1×PBS-P solution (Cytiva). Target proteins were immobilized on a CM5 chip via amino-coupling until the protein response value (RU) reached above 1000. Subsequently, approximately 100 μl of 1 nM protein samples was used for preliminary experiments to determine the activity of the protein coupled to the chip and to serve as a concentration reference. The regeneration condition was determined to be 5 mM NaOH. Finally, based on the preliminary experiments, each protein sample was diluted twofold, resulting in nine gradients, and then analyzed using the Biacore 8K system (Cytiva). The results were then analyzed.

[0180] Taking the murine Lag3 nanobody fusion protein with PEG modification at 20K as an example, PEG modification at different sites on IL-2 resulted in different binding abilities and shielding effects compared to IL-2Rα. Figure 2 Among these, PEG modifications at sites Y45, F42, E62, K64, P65, and E68 do not bind to α, indicating a bias towards non-α receptors. K35, T37, R38, T41, K48, and K49 exhibit weak binding affinity to α, ranging from 1e⁻⁷ to 1e⁻⁶, but their affinity for dimer IL-2R is significantly higher than that for trimer IL-2R. Alternatively, while their affinity for dimer IL-2R is weaker than that for trimer IL-2R, the difference (e.g., the ratio) is significantly smaller than the difference in affinity between native IL-2 for dimer and trimer IL-2R, demonstrating a significantly improved bias towards dimer IL-2R. Therefore, they also exhibit a bias towards non-α receptors and thus possess the ability to shield IL-2Rα. Furthermore, all these fusion proteins demonstrate good binding activity to Lag3.

[0181] Variants containing amino acids PEGylated at positions 45 (Y45), 65 (P65), 42 (F42), and 62 (E62) on IL-2 were selected for substitution with different nanobodies, and their binding activity was detected. The results are as follows: Figures 3A-3D As shown, the results indicate that different nanobody fusion proteins all achieved the effect of shielding IL-2Rα and effectively binding to the corresponding immune checkpoint receptors.

[0182] Example 3: PEGylated IL2 fusion protein on depleted CD8 + The effect of T cells

[0183] Mouse T cells were cultured in vitro using different ICINb-Y45-20K proteins, and their effects on mouse CD8 were examined. + The effects of T activation, proliferation, and differentiation.

[0184] 3.1 Mouse CD8 depletion + T cells, depleted precursor CD8 + T cell in vitro induction

[0185] To better simulate T cell exhaustion in the tumor microenvironment, the antigenic peptide OVA was used. 257-264 Stimulation of OVA-specific CD8 + T cells were used to construct an exhaustion model. Spleens from OT-1 mice were harvested, ground, and subjected to erythropoiesis to obtain splenic lymphocytes. These lymphocytes were then processed at a concentration of 3 × 10⁻⁶ cells / mL. 6 Cells / ml were resuspended in T cell culture medium (RPMI 1640 + 10% FBS + 100 U / ml penicillin + 100 mg / ml streptomycin + 1 mmol / L sodium pyruvate + 1× NEAA + 2 mmol / L L-glutamine + 50 mM β-mercaptoethanol), and 1 μM OVA was added as a final concentration. 257-264 Cells were cultured for three days with peptide (invivogen), IL-2 (20 ng / ml), and mIL-7 (5 ng / ml). Live cells were then enriched by gradient centrifugation using Ficoll-Paque Plus (GE Healthcare) at a concentration of 1 × 10⁻⁶ cells / ml. 6 CD8 cells / mL were resuspended in T cell culture medium for further enrichment and culture, allowing CD8 cells / mL to be further enriched. + The T ratio increases to over 95%. Activated CD8 + T cells were passaged twice to day 7. They were then induced for two days with a mixture of bivalent anti-CD3 antibody (prepared at a molar ratio of anti-CD3 antibody (17A2, Bio X cell): anti-rat IgG (abcam) = 2:1) and IL-2 (10 ng / mL). Cells were collected for flow cytometry analysis to identify their exhaustion state, and the Tpex and Tex cell populations were separated and cultured separately using an Aria Sorp flow cytometer. Based on the literature, stem T cells (T stem cells) were preliminarily defined as CD8+. + PD-1 - TIM-3 - T pex (precursor exhausted T cell) is defined as CD8. + PD-1 + TIM-3- ; Define T ex (exhausted T cell) as CD8 + PD-1 + TIM-3 + .

[0186] The results are as follows Figure 4 As shown, two CD8 groups characterized by Tpex (PD1+TIM3-) and Tex (PD1+TIM3+) were successfully induced in vitro using antigen peptides and bivalent CD3 antibodies. + T cells were used. After separating the cells into two groups using a flow cytometry system, subsequent experiments were conducted.

[0187] 3.2 Mouse CD8 depletion + Detection of T phosphorylated STAT5 levels

[0188] The depleted CD8 after overnight rest was prepared according to 3.1. + T cells were resuspended in PBS containing 0.5% BSA to a concentration of 2 × 10⁻⁶ cells. 6 Cells were collected at 50 μl / well in 96-well V-bottom plates and incubated at 37°C for 2 h. IL-2 or each ICINb-Y45-20K protein were serially diluted 10-fold with PBS and added to the 96-well plates. Cells were stimulated at 37°C for 20 min, and then immediately fixed by adding 50 μl / well of fIxation buffer (BD) and incubating at room temperature for 20 min. After one wash with PBS, cells were permeabilized by resuspending in pre-chilled permeabilization buffer (BD) at 4°C for 30 min. Cells were then washed twice with FACS buffer and incubated with 1:100 dilutions of anti-mouse CD8-APC and pSTAT5-PE antibodies. Cells were then washed twice with staining buffer and mean fluorescence intensity (MFI) was measured using a CytoFLEX flow cytometer (Beckman-Coulter). Data were plotted as MFI minus background and normalized to the maximum signal (IL-2, 100 μM). Background was defined as pSTAT5 MFI in unstimulated cells. After subtracting the MFI of unstimulated cells and normalizing to the maximum signal intensity, the dose-response curve was fitted to a logistic model using GraphPad Prism data analysis software, and the half-maximum effective concentration (EC50 value) and the corresponding 95% confidence interval were calculated.

[0189] The results are as follows Figure 5As shown, the EC50 of different fusion proteins is correlated with the expression level of immune checkpoints on the surface of T cells. The higher the expression level of immune checkpoints, the more obvious the enhancement effect of the fusion protein on Y45. Overall, the activity is stronger than that of Y45 but weaker than that of wild-type IL-2.

[0190] 3.3 Mouse CD8 depletion + T cells, depleted precursor CD8 + T cell in vitro culture, proliferation, and functional detection

[0191] The Tpex and Tex cells prepared in step 3.1 were divided into 7 equal groups and cultured in medium containing 10 nM IL-2, Y45-20K, or ICINb-Y45-20K, respectively. Continuous stimulation and culture with 0.5 μg / mL of bivalent anti-CD3 antibody were added. After three days, cell counts were performed, and cells were analyzed using OVA. 257-264 Cells in each group were restimulated with peptides for 1 hour, treated with Brefeildin A for 3 hours, and then fixed. Following the manufacturer's instructions, infiltration and staining were performed using the eBioscience Cell Fixation and Infiltration Buffer Kit (Thermo Fisher Scientific). Cells were then incubated with specific antibodies at 4°C for 1 hour. In addition to the specific fluorescent antibodies mentioned in the previous examples, anti-mouse Granzyme B-PE, anti-mouse IFN-γ-FITC, anti-mouse CD8-APC, anti-mouse PD-1-BV421, and anti-mouse TIM-3-BV605 were added for flow cytometry staining to detect the state of T cells and the expression of intracellular transcription factors and cytokines.

[0192] The results are as follows Figure 6 As shown, compared to Y45 and wild-type IL-2, the fusion protein can significantly improve the expansion efficiency of Tex cells and slightly improve the expansion efficiency of Tpex cells. Figure 6 A), and after Tpex or Tex cells proliferate, they maintain better cell function when encountering antigens compared to wild-type IL-2. Figure 6 B).

[0193] Example 4: Experiment on in vivo treatment of mouse tumors with ICInb-Y45-20K protein

[0194] 3.1 Mouse melanoma model

[0195] C57BL / 6 mice (6 to 8 weeks old, purchased from the Laboratory Animal Center of Peking University Health Science Center) underwent subcutaneous implantation of B16-OVA melanoma cells (3 x 10 cells per animal) in the right axilla. 5(cells), this is Day 0. When the tumor reaches 1500 mm... 3 Mice were sacrificed when the tumor reached a certain size. Tumor volume was calculated as follows: V = a² * b / 2, where a is the width and b is the tumor length (both in millimeters). Tumor volume was measured until 7 days after B16-OVA cell implantation, when the tumor measured 60-80 mm. 3 Animals were administered Y45-20K (0.25 mg / kg × 3, every other day), ICInb-Y45-20K (0.5 mg / kg × 3, every other day), IL-2 (0.25 mg / kg daily × 5), or PBS. Tumor size was monitored and tumor growth curves were plotted throughout the treatment. On day 19, spleens and lymph nodes from each group of mice were extracted and split to prepare single-cell suspensions. Tumor tissue was digested and chopped in a buffer solution containing 2 mg / mL type II and IV collagenase (GIBCO BRL) and 0.5 mg / mL DNase (Sigma Aldrich) at 37°C for 60 minutes to form single-cell suspensions. Subsequently, cytotoxic CD8+ cytotoxicity in mouse lymph nodes and tumor tissues was measured by flow cytometry. + The number and differentiation phenotype of T cells.

[0196] The results are as follows Figure 7 As shown, TIM3-Y45 and LAG3-Y45 can effectively inhibit tumor growth in mice compared to IL-2 and Y45. Figure 7 A) The proportion of T cells and CD8 / Treg cells infiltrating the tumor was significantly increased. Figure 7 BC). Further studies have shown that IL2, TIM3-Y45, and LAG3-Y45 can all effectively promote the proliferation of intratumoral Terex cells compared to Y45. However, in terms of the function of intratumoral Terex cells, the IL2 group showed almost no function of intratumoral Terex cells, indicating a state of terminal exhaustion, while the TIM3-Y45 and LAG3-Y45 groups maintained Terex function better. Figure 7 D).

[0197] 3.2 Mouse colon cancer model

[0198] C57BL / 6 mice (6 to 8 weeks old, purchased from the Laboratory Animal Center of Peking University Health Science Center) were subcutaneously implanted with MC38 colon adenocarcinoma cells (Chinese Academy of Sciences Cell Bank (Shanghai, China), 5 x 10 cells per animal) in the right axilla. 5 (cells). When the tumor reaches 1500mm 3 Mice were sacrificed when the tumor was small. Tumor volume was calculated as follows: V = a 2 *b / 2, where a is the width and b is the tumor length (both in millimeters). 7 days post-implantation (Day 0), when the tumor measures 60-80mm... 3During the treatment, animals were administered Y45-20K (0.25 mg / kg × 3, every other day), ICInb + Y45-20K (0.25 mg / kg × 3 each, ICInb intraperitoneally, Y45-20K subcutaneously, every other day), ICInb-Y45-20K (0.5 mg / kg × 3, every other day), or IL-2 (0.25 mg / kg daily × 5). Tumor size was monitored and tumor growth curves and mouse survival curves were plotted throughout the treatment.

[0199] The results are as follows Figure 8A As shown, the TIM3-Y45-20K and LAG3-Y45-20K groups effectively inhibited tumor growth, while the mixed administration group (i.e., ICINb+Y45-20K) did not show an effective therapeutic effect, illustrating the importance of fusion proteins acting cis-on the same T cells.

[0200] Furthermore, in the aforementioned mouse colon cancer model, the in vivo antitumor activity of PEGylated IL2 fusion proteins formed with mLAG3 or mTIM3 nanobodies using PEGylated variants at positions 42 (F42), 62 (E62), and 65 (P65) was also examined. The dosage was 0.5 mg / kg × 3, administered every other day. Results are as follows... Figure 8B As shown, all the PEGylated IL2 fusion proteins detected significantly inhibited tumor growth.

[0201] 3.3 Mouse melanoma model combined with tumor antigen peptide

[0202] C57BL / 6 mice (6 to 8 weeks old, purchased from the Laboratory Animal Center of Peking University School of Medicine) underwent subcutaneous implantation of B16-OVA melanoma cells (donated by Professor Gong Changyang's research group at the State Key Laboratory of Biotherapy, Sichuan University, 3 x 10 cells per animal) in the right axilla. 5 (cells), this is Day 0. When the tumor reaches 1500 mm... 3 Mice were sacrificed when the tumor reached a certain size. Tumor volume was calculated as follows: V = a² * b / 2, where a is the width and b is the tumor length (both in millimeters). Tumor volume was measured until 7 days after B16-OVA cell implantation, when the tumor measured 60-80 mm. 3 During treatment, animals were administered Y45-20K (0.25 mg / kg × 3, every other day), ICINb-Y45-20K (0.5 mg / kg × 3, every other day), or IL-2 (0.25 mg / kg daily × 5). Simultaneously, each group of mice received a tumor vaccine subcutaneously on Day 7, Day 9, and Day 11, with 90 μg of OVA. 257-264+1.25 nM CpG / mouse (OVA sequence: SIINFEKL synthesized by GenScript; CpG adjuvant: ODN1826 (MCE, HY-146245)). Tumor size was monitored and tumor growth curves were plotted throughout the study. On day 21, spleens and lymph nodes from each group of mice were extracted and split to prepare single-cell suspensions. Tumor tissue was digested and chopped in a buffer containing 2 mg / mL type II and IV collagenase (GIBCO BRL) and 0.5 mg / mL DNase (Sigma Aldrich) at 37°C for 60 minutes to form single-cell suspensions. The antigen-specific CD8 antigens infiltrating the mouse tumor tissue were then detected by flow cytometry. + The number and functional phenotype of T cells.

[0203] The results are as follows Figure 9 As shown, the ICINb-Y45-20K group combined with the tumor vaccine had a significantly higher efficacy compared to IL-2 and Y45 alone. Figure 9 A) The number of antigen-specific T cells infiltrating the tumor is significantly increased, and their function is stronger. Figure 9 B).

Claims

1. A fusion protein comprising (i) a targeting portion targeting an immune checkpoint and (ii) a site-mutated IL-2, wherein the site-mutated IL-2 is mutated to non-natural amino acids at residues selected from the following amino acid positions compared to wild-type IL-2: F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49.

2. The fusion protein of claim 1, wherein, The target portion of the targeted immune checkpoint is selected from antibodies or antibody fragments; Preferably, the targeting portion is selected from nanobodies, scFv, or single-chain Fab (scFab).

3. The fusion protein according to claim 1 or 2, wherein, The immune checkpoints are selected from LAG-3, TIGIT, TIM-3, PD-1, PD-L1, CLTA-4, CD39, VISTA, and A2AR.

4. The fusion protein according to any one of claims 1-3, wherein, The targeting portion of the immune checkpoint is selected from nanobodies that target LAG-3, TIGIT, TIM-3, PD-1, or PD-L1; Preferably, wherein: (ia) The LAG-3-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:2-4 respectively; preferably, the LAG-3-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:1 or a sequence having at least 80% identity with it. (ib) The LAG-3-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:22-24, respectively; preferably, the LAG-3-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:21 or a sequence having at least 80% identity with it. (ii) The TIGIT-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:6-8, respectively; preferably, the TIGIT-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:5 or a sequence having at least 80% identity with it. (iii-a) The nanobody targeting TIM-3 comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:10-12, respectively; preferably, the nanobody targeting TIM-3 comprises a VHH sequence as shown in SEQ ID NO:9 or a sequence having at least 80% identity with it. (iii-b) The TIM-3-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:26-28, respectively; preferably, the TIM-3-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:25 or a sequence having at least 80% identity with it. (iv-a) The PD-1-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:14-16, respectively; preferably, the PD-1-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:13 or a sequence having at least 80% identity with it. (iv-b) The PD-1-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:30-32, respectively; preferably, the PD-1-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:29 or a sequence having at least 80% identity with it. (va) The PD-L1-targeting nanobody comprises the CDR1-CDR3 sequences as shown in SEQ ID NOs:18-20, respectively; preferably, the PD-L1-targeting nanobody comprises the VHH sequence as shown in SEQ ID NO:17 or a sequence having at least 80% identity with it; or (vb) The PD-L1-targeting nanobody comprises CDR1-CDR3 sequences as shown in SEQ ID NOs:34-36, respectively; preferably, the PD-L1-targeting nanobody comprises a VHH sequence as shown in SEQ ID NO:33 or a sequence having at least 80% identity with it.

5. The fusion protein according to any one of claims 1-4, wherein, The positions where the amino acids were mutated to non-natural amino acids were selected from F42, Y45, E62, K64, P65, and E68.

6. The fusion protein according to any one of claims 1-5, wherein, The non-natural amino acid has a side chain that allows it to be linked to a PEG group.

7. The fusion protein of claim 6, wherein, The non-natural amino acid contains a chemical functional group (e.g., carbonyl, alkynyl, or azide group); the PEG group contains a labeling group that can chemically react with the chemical functional group, thereby allowing the PEG group to be attached to the non-natural amino acid; Preferably, the non-natural amino acid contains an azide group, and the PEG group contains a labeling group that can undergo a click chemical reaction with the azide group, thereby allowing the PEG group to be attached to the non-natural amino acid; Preferably, the labeling group capable of undergoing a click chemical reaction with the azide group is a chemical moiety containing a dibenzocyclooctyn group, such as DBCO, DIBO, or BCN; Preferably, the non-natural amino acid is a lysine derivative or tyrosine derivative containing an azide group, such as Nε-2-azidoethoxycarbonyl-L-lysine (NAEK) or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

8. The fusion protein according to any one of claims 1-7, wherein, The targeting portion of the targeted immune checkpoint is fused directly or via a peptide linker to the N-terminus of the site-directed mutated IL-2.

9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein according to any one of claims 1-8, wherein the codon corresponding to the position where the amino acid is mutated to a non-natural amino acid is TAG.

10. A vector comprising the isolated nucleic acid molecule of claim 9; preferably, the vector is an expression vector.

11. A host cell comprising the isolated nucleic acid molecule of claim 9 or the vector of claim 10.

12. A method for preparing the fusion protein according to any one of claims 1-8, comprising the following steps: - Co-transfect host cells with the isolated nucleic acid molecule of claim 9 or the vector of claim 10 and a vector encoding amber codon-repressed tRNA and aminoacyl-tRNA synthetase specific to non-natural amino acids; - The host cells are cultured in a culture medium containing non-natural amino acids; Preferably, the non-natural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine (NAEK); preferably, the aminoacyl-tRNA synthetase specific to the non-natural amino acid is a NAEK-specific aminoacyl-tRNA synthetase.

13. A conjugate comprising the fusion protein of any one of claims 1-8 and a PEG group linked to a non-natural amino acid of the fusion protein.

14. The conjugate of claim 13, wherein, The average molecular weight of the PEG group is 5 to 60 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa or 60 kDa. Preferably, the average molecular weight of the PEG-modified group is 5-40 kDa, for example 5-30 kDa, 5-25 kDa, 5-20 kDa, 10-40 kDa, 10-30 kDa, 15-30 kDa, 10-25 kDa, or 15-25 kDa, for example 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or 40 kDa.

15. A method for preparing the conjugate of claim 13 or 14, the method comprising: - Provided: (a) the fusion protein according to any one of claims 1-8; (b) A PEG group modified by a labeling group, wherein the labeling group can form a covalent bond with a non-natural amino acid in the fusion protein; - Co-incubate (a) and (b) to couple non-natural amino acids with PEG groups via a chemical reaction.

16. The method of claim 15, wherein, The non-natural amino acid described in (a) contains a chemical functional group (e.g., carbonyl, alkynyl, azide group), and the PEG group described in (b) contains a labeling group that can chemically react with the chemical functional group to form a covalent bond; Preferably, the non-natural amino acid described in (a) contains an azide group; Preferably, the non-natural amino acid described in (a) is a lysine derivative or tyrosine derivative containing an azide group, such as Nε-2-azidoethoxycarbonyl-L-lysine (NAEK) or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

17. The method of claim 16, comprising: - Provides: (a) the fusion protein according to any one of claims 1-8; (b) a PEG group modified with a labeling group, said labeling group being capable of undergoing a click chemical reaction with an azide group; - Co-incubate (a) and (b) to couple non-natural amino acids to PEG groups via a click reaction; Preferably, the click chemical reaction is a copper-free click chemical reaction; Preferably, the labeling group capable of undergoing a click chemical reaction with the azide group is a chemical moiety containing an alkynyl group, such as a chemical moiety containing a dibenzocyclooctynyl group, such as DBCO, DIBO, or BCN; Preferably, the labeling group capable of undergoing a click chemical reaction with the azide group is DBCO.

18. A pharmaceutical composition comprising the conjugate of claim 13 or 14, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents, such as antitumor agents; Preferably, the additional pharmaceutically active agent is selected from tumor vaccines, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive nuclides, radiosensitizers, antiangiogenic agents, cytokines, specific tumor cell-targeting antibodies, or immune checkpoint inhibitors. Preferably, the additional pharmaceutically active agent is a tumor vaccine.

19. Use of the conjugate of claim 13 or 14 or the pharmaceutical composition of claim 18 in the preparation of a medicament for enhancing immune response, preventing and / or treating proliferative diseases; Preferably, the proliferative disease is a tumor; Preferably, the tumor includes a solid tumor or a hematologic malignancy; Preferably, the tumor includes metastatic cancer, recurrent or refractory cancer; Preferably, the tumor is selected from melanoma, colon cancer, colorectal cancer, rectal cancer, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, prostate cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.

20. The use as described in claim 19, wherein, The conjugate is used in combination with other pharmaceutically active agents; Preferably, the additional pharmaceutically active agent is an antitumor agent; Preferably, the additional pharmaceutically active agent is selected from tumor vaccines, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive nuclides, radiosensitizers, antiangiogenic agents, cytokines, specific tumor cell-targeting antibodies, or immune checkpoint inhibitors. Preferably, the additional pharmaceutically active agent is a tumor vaccine.

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