Construct for protein degradation and use thereof

Abstract

Provided in the present application are a new lysosome-targeting fusion protein based on the fusion of an IGF2 mutant with an antibody, and a method for degrading a protein by using the new protein construct.

Description

Constructs for protein degradation and their applications Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a construct for lysosomal targeted protein degradation and its application. Background Art

[0002] Lysosomes are the degradation hubs of intracellular substances, using acidic hydrolases to degrade proteins, organelles, and even invading microorganisms. Enzymes in mammalian lysosomes are synthesized in the cytoplasm. When lysosomal enzymes or lysosomal-localized proteins traverse the endoplasmic reticulum, they undergo N-mannose (mannose carbohydrate) glycosylation. Ultimately, in the Golgi apparatus, the proteins or enzymes are further modified with mannose-6-phosphate (M6P). M6P-modified proteins are transported to lysosomes via binding to their receptor, IGF2R (also known as M6PR, CI-MPR, or CD222), which then returns to the cell surface to continue its trafficking function. IGF2R is a 300 kDa membrane protein. Its extracellular region consists of 15 domains, each composed of 147 amino acids. Sequence similarity among these domains ranges from 15% to 38%. IGF2R plays a role in protein trafficking, lysosomal biogenesis, and cell growth regulation. Clinically, recombinant enzymes are delivered to lysosomes by targeting IGF2R for the treatment of lysosomal storage diseases.

[0003] Of the 15 extracellular domains of the IGF2R, domains 3, 5, and 9 bind mannose-6-phosphate (M6P) or M6P-modified proteins, while domains 11 and 13 bind insulin-like growth factor 2 (IGF2). When proteins or enzymes carrying M6P bind to the IGF2R, they induce IGF2R-mediated targeted endocytosis, thereby transporting the M6P-modified protein or enzyme to the lysosome. Similarly, when IGF2 binds to the IGF2R, the IGF2R transports IGF2 to the lysosome through endocytosis, thereby mediating its degradation. This maintains the balance of IGF2 outside the cell, prevents excessive IGF2 from binding to the IGF1R, and attenuates the activation of the IGF1R tyrosine kinase domain.

[0004] Based on the principle of IGF2R-mediated protein or enzyme endocytosis and lysosomal delivery, researchers in the prior art have modified target proteins or enzymes with M6P or its analogs, thereby transporting them to lysosomes in the hope of treating lysosomal enzyme deficiencies. Similarly, researchers in the prior art have used IGF2 mutants as a medium to deliver target proteins or enzymes to lysosomes in the hope of treating lysosomal deficiencies. These methods involve fusion expression of the IGF2 mutant with the target protein to produce a fusion protein. Binding of the IGF2 mutant to the IGF2R mediates the targeted transport of the fused protein to the lysosome, where it can then exert its biological function (see US Patent Applications US2003 / 0082176A1, US2004 / 0006008A1, and US2005 / 0244400A1).

[0005] In addition to using IGF2R to deliver exogenous proteins, some researchers have also used IGF2R as a shuttle receptor to conduct research on membrane protein degradation. In 2020, the Bertozzi group at Stanford University in the United States first reported a lysosomal targeted degradation technology (LYTAC) based on glycosylated antibodies. Its mechanism of action is as follows: (1) LYTAC molecules consist of two parts: an antibody and a polysaccharide. By coupling the polysaccharide to the antibody, a LYTAC molecule that can recognize two receptors is prepared, namely, the antibody recognizes the target membrane protein (the target to be degraded) and the polysaccharide recognizes the IGF2R receptor; (2) The LYTAC double-headed molecule simultaneously binds to the lysosomal shuttle receptor IGF2R on the cell membrane and the target membrane protein to form a ternary complex, which then induces endocytosis and transports the ternary complex into the lysosome; (3) Under the action of lysosomal enzymes, the target protein is degraded by the lysosomal enzymes (see US patent application US2022 / 0023434A1), while the IGF2R returns to the cell membrane and continues to perform its transport function. However, this technology has the following disadvantages: it requires the use of chemical reactions to connect the M6P polysaccharide to the antibody. Since the coupling of M6P polysaccharide to antibodies requires the use of lysine on the antibody, and there are many lysines on the antibody, this method cannot guarantee the uniformity of the prepared LYTAC molecules, making it difficult to achieve application and transformation.

[0006] To solve this problem, in 2021, the Bertozzi team used codon expansion technology to introduce non-natural amino acids at specific positions of antibodies, and achieved site-specific labeling of polysaccharides on antibodies through photo-click technology, thereby preparing a two-headed LYTAC molecule that specifically recognizes the ASGPR lysosomal shuttle receptor and the target protein (Ahn, G., et al., Nat Chem Biol, 2021.17(9): p.937-946). The principle of this technology is: during the translation of the antibody, a cyclic alkyne-modified non-natural amino acid is introduced at a specific position of the antibody to solve its homogeneity problem. This technology requires two key steps to achieve the preparation of site-specific glycosylated modified antibodies (LYTAC): (1) genetically encoded non-natural amino acid technology and (2) click chemistry-mediated antibody coupling technology. Compared with the first-generation LYTAC technology (U.S. patent application US2022 / 0023434A1), this technology has improved product uniformity, but there are still many problems: (1) Click chemistry is an efficient reaction with a high reaction rate, but in the absence of a catalyst or under light conditions, the reaction rate of cycloalkyne azide cannot guarantee 100% coupling; (2) The site-specific insertion technology of non-natural amino acids requires the use of stop codons (TAG / TGA / TAA or quadruple codons), which leads to a decrease in expression. After the introduction of non-natural amino acids, the expression level of most proteins decreases by about 10 times, and the non-natural amino acids are not 100% inserted. The fidelity of their insertion has also become an aspect affecting uniformity; (3) The product preparation process is complex and the output is low, which is not conducive to the subsequent industrial promotion.

[0007] By fusion-expressing wild-type IGF2 with a specific antibody, a dual-headed molecule that targets both the IGF2R and the desired target can be created. While LYTACs can be constructed, studies have shown that wild-type IGF2 binds to both the IGF2R and IGF1R simultaneously. Furthermore, upon binding to the IGF1R, IGF2 activates the tyrosine kinase activity of the IGF1R, leading to tumorigenesis. Previous studies have also shown that wild-type IGF2 promotes the proliferation of tumor cells (BT-474 and SK-Hep1). Therefore, constructing a protein degradation system using wild-type IGF2 carries a significant risk of carcinogenesis.

[0008] Therefore, there is an urgent need for technologies for protein degradation that meet the requirements of product uniformity, simple preparation process, high yield, higher affinity and better selectivity, while simplifying the preparation process and avoiding the risk of inducing tumors. Summary of the Invention

[0009] The present invention uses directed evolution to provide a novel lysosomal targeting fusion protein (LYTAF) based on the fusion of an IGF2 mutant and an antibody, as well as a method for degrading proteins using the novel protein construct.

[0010] In a first aspect, the present invention provides an IGF2 mutant having improved affinity for IGF2R.

[0011] Preferably, the IGF2 mutant does not bind to IGF1R, or the affinity of the IGF2 mutant to IGF1R is reduced compared to the wild type.

[0012] In some embodiments, the IGF2 mutant has a deletion of amino acids 1-7 (Δ1-7) compared to wild-type IGF2.

[0013] In some embodiments, any one of amino acids 1 to 7 of the IGF2 mutant can be independently replaced with any one amino acid.

[0014] In some embodiments, the amino acid at position 6 of the IGF2 mutant can be replaced with a positively charged amino acid, preferably R or K. For example, the IGF2 mutant can have the mutation E6R or E6K.

[0015] In some embodiments, the amino acid at position 6 of the IGF2 mutant can be replaced with an amino acid whose side chain forms a hydrogen bond with the side chain of another amino acid, preferably, replaced with Q, S, T, C or Y. For example, the IGF2 mutant can have the mutation E6Q, E6S, E6T, E6C or E6Y.

[0016] In some embodiments, the amino acid at position 7 of the IGF2 mutant is replaced by an aliphatic amino acid with a hydrophobic side chain, preferably, by A, I, L or V.

[0017] In some embodiments, the amino acid at position 14 of the IGF2 mutant can be replaced with an amino acid whose side chain can form a hydrogen bond, preferably, replaced with T, S, C or Y. For example, the IGF2 mutant can have mutations V14T, V14S, V14C or V14Y.

[0018] In some embodiments, the amino acid at position 15 of the IGF2 mutant can be replaced by an aliphatic amino acid, preferably, by G, A, V, L, I, F, W, N or Q. For example, the IGF2 mutant can have the mutation D15G, D15A, D15V, D15L, D15I, D15F, D15W, D15N or D15Q.

[0019] In some embodiments, the amino acid at position 15 of the IGF2 mutant can be substituted with an amino acid whose side chain can form hydrogen bonds with the side chains of other amino acids, preferably, with C, S, T, or Y. For example, the IGF2 mutant can have mutations D15C, D15S, D15T, or D15Y.

[0020] In some embodiments, the amino acid at position 18 of the IGF2 mutant can be replaced by an aliphatic amino acid with a side chain length of 1-4 carbon atoms, preferably, by Y, G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have the mutation Q18Y, Q18G, Q18A, Q18V, Q18L, Q18I, Q18M, Q18N, Q18Q, Q18T, Q18C or Q18S.

[0021] In some embodiments, the amino acid at position 19 of the IGF2 mutant can be replaced with an aliphatic amino acid having a side chain length of 1-4 carbon atoms, preferably, G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have the mutation F19G, F19A, F19V, F19L, F19I, F19M, F19N, F19Q, F19T, F19C or F19S.

[0022] In some embodiments, the amino acid at position 27 of the IGF2 mutant can be replaced by an aliphatic amino acid with a side chain length of 1-4 carbon atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have the mutations Y27G, Y27A, Y27V, Y27L, Y27I, Y27M, Y27N, Y27Q, Y27T, Y27C or Y27S.

[0023] In some embodiments, the amino acid at position 37 of the IGF2 mutant can be replaced by an aliphatic amino acid with a side chain length of 1-4 carbon atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have the mutation R37G, R37A, R37V, R37L, R37I, R37M, R37N, R37Q, R37T, R37C or R37S.

[0024] In some embodiments, the amino acid at position 43 of the IGF2 mutant can be replaced with an aliphatic amino acid having a side chain length of 1-4 carbon atoms, preferably, G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have the mutation V43G, V43A, V43V, V43L, V43I, V43M, V43N, V43Q, V43T, V43C or V43S.

[0025] In some embodiments, the IGF2 mutant may comprise one or more mutations of E6R / E6Q, T7A, V14T, D15A, Q18Y, F19L, Y27L, R37A, and V43M.

[0026] In some embodiments, the IGF2 mutant comprises Δ1-7, mutations Y27L and R37A.

[0027] In some embodiments, the IGF2 mutant comprises mutations R37A and V43M.

[0028] In some embodiments, the IGF2 mutant comprises mutations E6R, R37A, and V43M.

[0029] In some embodiments, the IGF2 mutant comprises mutations E6R, Y27L, and V43M.

[0030] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, Y27L, and R37A.

[0031] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, and V14T.

[0032] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, and D15A.

[0033] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, V14T, and D15A.

[0034] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, and F19L.

[0035] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, V14T, and F19L.

[0036] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, D15A, and F19L.

[0037] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, V14T, D15A, and F19L.

[0038] In some embodiments, the IGF2 mutant comprises Δ1-7, mutations Y27L, R37A, and V43M.

[0039] In some embodiments, the IGF2 mutant comprises mutations E6Q, T7A, Q18Y, F19L, Y27L, and R37A.

[0040] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, and F19L.

[0041] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, V14T, and F19L.

[0042] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, D15A, and F19L.

[0043] In some embodiments, the IGF2 mutant comprises Δ1-7 and / or mutation Y27L and / or mutation R37A, and mutation V43M.

[0044] In some embodiments, the IGF2 mutant comprises mutations E6Q and / or T7A and / or Q18Y and / or F19L and / or Y27L.

[0045] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, and F19L.

[0046] In some embodiments, the IGF2 mutant comprises mutations V43M, E6R, R37A, V14T, and F19L.

[0047] In some embodiments, the mutant has an amino acid sequence as shown in any one of SEQ ID NO:70 and SEQ ID NO:76, SEQ ID NO:80, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity thereto, or consists of the same.

[0048] In some embodiments, the IGF2 precursor mutant has the following mutations or deletions compared to the wild-type IGF2 precursor: the IGF2 precursor mutant comprises mutations E6R, R37A, V43M, and R68A.

[0049] The amino acid numbering of the wild-type IGF2 (1-67) mentioned in the present disclosure corresponds to the amino acid sequence shown in SEQ ID NO: 1.

[0050] The amino acid numbering of the wild-type IGF2 precursor (1-104) mentioned in the present disclosure corresponds to the amino acid sequence shown in SEQ ID NO:117.

[0051] The amino acid numbering of the wild-type IGF2 precursor (1-156) mentioned in the present disclosure corresponds to the amino acid sequence shown in SEQ ID NO:118.

[0052] In a second aspect, the present invention provides a construct comprising the IGF2 mutant of the first aspect and a cleavable portion.

[0053] In some embodiments, the construct comprises the following structure:

[0054] AB,

[0055] Wherein, A represents IGF2 mutant; B represents linker.

[0056] Those skilled in the art will understand that the AB structure only means that the construct is composed of two parts, A and B, and does not limit the amino acid sequence of the construct. AB and BA are equivalent in this article.

[0057] In some embodiments, the construct further comprises a target molecule binding moiety.

[0058] In some embodiments, the construct comprises the following structure:

[0059] ABC,

[0060] Wherein, A represents an IGF2 mutant; B represents a linker; and C represents a target molecule binding portion or Fc.

[0061] Those skilled in the art will understand that the ABC structure only means that the construct is composed of three parts, A, B and C, and A and C are connected by B, and does not limit the amino acid sequence of the construct. ABC and CBA are equivalent in this article.

[0062] In some embodiments, the linker may be a linker conventionally used in the art. In some embodiments, the linker may be (G) n 、(G m S) n, poly(glycine-alanine), poly(alanine-serine), poly(alanine-proline), wherein n and m are each independently selected from an integer of 1 to 20. In some specific embodiments, the linker can be (G)2, (G)3, (G)4, (G)5, (G)8, (GGGGS)3, (GGGGS)4, ASTKGP, TVAAP or GGGGS.

[0063] It should be understood by those skilled in the art that the structures shown in the present disclosure are only for illustration and do not indicate the connection order thereof.

[0064] In some embodiments, the Fc domain is selected from the Fc domain of IgG, IgM, IgE, IgA, or IgD.

[0065] In some embodiments, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 domain.

[0066] Preferably, the Fc domain may be derived from human.

[0067] In some embodiments, the Fc domain is a human IgG1 Fc domain, preferably including an Fc domain mutation such as a substitution at position N297 (such as N297G or N297Q).

[0068] In some embodiments, the construct has, or consists of, a sequence as set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 38, 40, 42, 44, 46, 48, 52, 58, 66, 68, 70, 72, 74, 76, 78, 80, 82, or 84, or an amino acid sequence at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical thereto.

[0069] In some embodiments, the target molecule binding moiety comprises an antibody, a ligand, a receptor, a polypeptide, or a protein.

[0070] In some embodiments, the target molecule binding portion can be a receptor, such as a full-length receptor, a receptor fragment, or a functional variant thereof.

[0071] In some embodiments, the target molecule binding portion can be an antigenic protein or a fragment thereof. In some embodiments, the target molecule binding portion can be an antigenic protein or a fragment thereof that recognizes pathogenic autoantibodies. In some embodiments, the antigenic protein or its fragment can include, for example, but not limited to, a disintegrin and metalloproteinase member 13 with thrombospondin type 1 motif (ADAMTS13), steroidogenic cytochrome P450 enzyme 21-hydroxylase, N-methyl-d-aspartate-(NMDA)-receptor, red blood cells, anti-smooth muscle antibodies (ASMA), actin, platelets, signal recognition particles (SRP), 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), myosin, sperm, amylase α2, type XVII collagen (col17), kallikrein 13, type VII collagen (col7), myeloperoxidase (MPO), type IV collagen, proteinase 3 (PR3), thyroid stimulating hormone receptor (TSHR), thyroid globulin, thyroid peroxidase (TPO), thyroglobulin, thyroid peroxidase (TPO), platelets, myeloperoxidase (MPO), muscle nicotinic acetylcholine receptor, muscle-specific kinase (MuSK), low-density lipoprotein receptor protein 4 (LRP4), myosin, β1-adrenergic receptor, adenine nucleotide translocase, aquaporin-4, myelin oligodendrocyte glycoprotein (MOG), heat shock protein 90 (HSP90), heat shock protein A5 (HSPA5), desmoglein-3, parietal cells, mitochondria, phospholipase A2 receptor (PLA2R), thrombospondin type 1 domain-containing 7A (THSD7A), cyclic citrullinated protein, RNA-binding protein (Ros), La, double-stranded DNA (dsDNA), angiotensin II type 1 receptor (AT1R), endothelin-1A receptor (ETAR), insulin, glutamate decarboxylase, or protein tyrosine phosphatase.

[0072] In some embodiments, the target molecule binding moiety comprises an antibody or an antigen-binding fragment thereof.

[0073] In some embodiments, the antibody comprises a full-length antibody or an antigen-binding fragment thereof.

[0074] In some embodiments, the antibody may include a multispecific antibody or an antigen-binding fragment thereof. In some embodiments, the antibody may be a monospecific antibody or an antigen-binding fragment thereof. In some embodiments, the antibody may be a bispecific antibody or an antigen-binding fragment thereof.

[0075] In some embodiments, the antigen-binding fragment comprises the heavy chain or light chain of an antibody, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, Fd, dAb (single domain antibody), affibody, HCAb (heavy chain antibody), Nb (nanobody), VHH, and any combination thereof.

[0076] In some embodiments, the antibody or antigen-binding fragment thereof can bind to a protein (soluble and membrane-associated proteins, such as an antibody or fragment thereof, a receptor, a growth factor, a cytokine, a chemokine, an enzyme, or a hormone), a lipoprotein, a liposome, a nucleic acid (e.g., an oligonucleotide, DNA, RNA), a toxin, a viral particle, or a cell (e.g., a prokaryotic cell, a eukaryotic cell).

[0077] In some embodiments, the target molecule is a cell surface protein or an extracellular protein.

[0078] In some embodiments, the target molecule may be an immune checkpoint molecule, such as PD-L1, or may be a "tumor molecule" associated with tumor occurrence and / or development and / or metastasis.

[0079] In some embodiments, the target molecule is a pathogenic target molecule, e.g., a protein that is harmful or unwanted to a sample (e.g., a cell) or a subject. In some embodiments, the pathogenic target molecule is a pathogenic autoantibody or a fragment thereof. In some embodiments, the pathogenic target molecule is a cell surface receptor.

[0080] In some embodiments, the target molecule may be an immune checkpoint molecule, such as PD-L1, or may be a "tumor molecule" associated with tumor occurrence and / or development and / or metastasis.

[0081] In some embodiments, the target molecule can be a receptor tyrosine kinase, a growth factor receptor, a cytokine, a mucin, a Siglec receptor or an immune checkpoint regulator. In some embodiments, the target molecule can include, but is not limited to, human epidermal growth factor receptor 2 (HER2), HER3, epidermal growth factor (EGFR), fibroblast growth factor (FGFRs), vascular endothelial growth factor (VEGFA), mesenchymal epithelial transition factor (c-Met), platelet-derived growth factor receptor (PDGFR), FZD, interleukin-1 receptor (IL1R), PD-L1 / PD-1, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), extracellular matrix metalloproteinases (MEMs), cytokines, and cytokines. Protein α-synuclein, CD20, TIM3, LAG3, TIGIT, CEACAM1, CD25, Ig-like transcription factor 2 (ILT-2), ILT-3, ILT-4, ILT-5, leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), platelet endothelial cell adhesion molecule (PECAM-1, CD31), paired immunoglobulin-like receptor (PILR-α / β), SIRL-1 or SIRP-α, or functional fragments thereof.

[0082] In some embodiments, the IGF2 mutant is linked to the N-terminus or C-terminus of the target molecule binding moiety or Fc.

[0083] In some embodiments, the IGF2 mutant is linked to the N-terminus of an antibody, polypeptide, protein, or Fc.

[0084] In some embodiments, the IGF2 mutant is linked to the N-terminus of an antibody, polypeptide, protein or Fc via a linker.

[0085] In some embodiments, the IGF2 mutant is linked to the C-terminus of an antibody, polypeptide, protein, or Fc.

[0086] In some embodiments, the IGF2 mutant is linked to the C-terminus of an antibody, polypeptide, protein or Fc via a linker.

[0087] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody heavy chain.

[0088] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody heavy chain via a linker.

[0089] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody heavy chain.

[0090] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody heavy chain via a linker.

[0091] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody light chain.

[0092] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody light chain via a linker.

[0093] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody light chain.

[0094] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody light chain via a linker.

[0095] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody light chain and to the C-terminus of the heavy chain.

[0096] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody light chain and to the C-terminus of the heavy chain via a linker.

[0097] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody light chain and to the N-terminus of the heavy chain.

[0098] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody light chain and to the N-terminus of the heavy chain via a linker.

[0099] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody light chain and to the C-terminus of the heavy chain.

[0100] In some embodiments, the IGF2 mutant is linked to the N-terminus of the antibody light chain and to the C-terminus of the heavy chain via a linker.

[0101] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody light chain and to the N-terminus of the heavy chain.

[0102] In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody light chain and to the N-terminus of the heavy chain via a linker.

[0103] In some embodiments, the A comprises two or more IGF2 mutants, and the two or more IGF2 mutants are two or more identical or different IGF2 mutants.

[0104] In some embodiments, the two or more repeats of the same IGF2 mutant are linked to the C-terminus or N-terminus of the antibody light chain.

[0105] In some embodiments, the two or more repeated forms of the same IGF2 mutant are connected to the C-terminus or N-terminus of the antibody light chain via a linker.

[0106] In some embodiments, the two or more repeats of the same IGF2 mutant are linked to the C-terminus or N-terminus of the antibody heavy chain.

[0107] In some embodiments, the two or more repeated forms of the same IGF2 mutant are connected to the C-terminus or N-terminus of the antibody heavy chain via a linker.

[0108] In a third aspect, the present invention provides a nucleic acid molecule encoding the IGF2 mutant of the first aspect or the construct of the second aspect.

[0109] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid or nucleic acid group of the third aspect.

[0110] In some embodiments, the expression vector may include a eukaryotic expression vector and a prokaryotic expression vector. In some embodiments, the expression vector is selected from expression vectors for mammalian host cells, such as, but not limited to, BPV-1, pHyg, pRSV, pSV2, pTK2, pIRES, pRc / CMV2, pRc / RSV, pSFV1, pVPakc vector, pCMV vector, pSG5 vector, retroviral vector (e.g., pFB vector), pcDNA-3, adenoviral vector, adeno-associated viral vector, baculoviral vector, yeast vector (e.g., pESC vector), etc.

[0111] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid or nucleic acid group of the third aspect or the expression vector of the fourth aspect.

[0112] In a sixth aspect, the present invention provides a pharmaceutical composition comprising: the IGF2 mutant of the first aspect, the construct of the second aspect, the nucleic acid or nucleic acid group of the third aspect, the expression vector of the fourth aspect, and / or the host cell of the fifth aspect; and a pharmaceutically acceptable carrier.

[0113] In some embodiments, the pharmaceutical composition is used to treat a disease.

[0114] In some embodiments, the disease is a disease associated with the expression or overexpression of the target molecule.

[0115] In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or cervical cancer, salivary gland cancer, kidney cancer or ureteral cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, bile duct cancer, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, The present invention includes but is not limited to: melanoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (such as that associated with brain tumors) and Meigs syndrome, brain tumors and brain cancer, and head or neck cancer and related metastatic cancers.

[0116] In a seventh aspect, the present invention provides a method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the IGF2 mutant of the first aspect, the construct of the second aspect, the nucleic acid or nucleic acid group of the third aspect, the expression vector of the fourth aspect, the host cell of the fifth aspect, and / or the pharmaceutical composition of the sixth aspect.

[0117] In an eighth aspect, the present invention provides the use of the IGF2 mutant of the first aspect, the construct of the second aspect, the nucleic acid or nucleic acid group of the third aspect, the expression vector of the fourth aspect, the host cell of the fifth aspect, and / or the pharmaceutical composition of the sixth aspect in the preparation of a drug for treating a disease.

[0118] In some embodiments, the disease is a disease associated with the expression or overexpression of the target molecule.

[0119] In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or cervical cancer, salivary gland cancer, kidney cancer or ureteral cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, bile duct cancer, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, The present invention includes but is not limited to: melanoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (such as that associated with brain tumors) and Meigs syndrome, brain tumors and brain cancer, and head or neck cancer and related metastatic cancers.

[0120] In a ninth aspect, the present invention provides use of the IGF2 mutant of the first aspect in the delivery of substances such as proteins, nucleic acids, small molecule drugs and polypeptides targeted to lysosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 exemplifies a comparison of LYTAC and LYTAF provided by the present disclosure. LYTAC requires a chemical reaction to link the antibody to the polysaccharide, while LYTAF can be obtained through expression without the need for chemical modification.

[0122] FIG2 exemplarily shows the principle of LYTAF-mediated membrane protein degradation.

[0123] FIG3 shows the results of affinity testing of IGF2 mutants to IGF2R-D11 (the 11th domain of IGF2R).

[0124] Figure 4 shows the results of the detection of different IGF2 mutants and Pertuzumab fusion proteins inhibiting the growth of breast cancer cells SK-BR-3. The vertical axis represents the coverage (or density) of cells in the culture dish, and the horizontal axis represents the incubation time.

[0125] FIG5 shows the results of the detection of the affinity of wild-type and mutant IGF2 to IGF2R.

[0126] FIG6 shows the results of the detection of the affinity of IGF2 mutants to IGF1R.

[0127] FIG7 shows the detection results of HER2-targeted LYTAF-M5.6-mediated degradation of HER2 membrane protein in multiple breast cancer cell lines.

[0128] Figure 8 shows LYTAF-M5.6-mediated internalization of fluorescently labeled antibody IgG-647 into lysosomes. A) Schematic diagram of LYTAF-M5.6-mediated internalization of fluorescent antibody IgG-647. B) Confocal laser scanning microscopy was used to assess the efficiency of LYTAF-M5.6 in delivering extracellular IgG-647 into lysosomes. Note: IgG-647 is a red fluorescent-labeled secondary antibody, Lysotracker is a lysosomal localization indicator, and Hoechst dye specifically stains cell nuclei.

[0129] Figure 9 shows that LYTAF-M5.6 inhibits the growth of SK-BR-3 breast cancer cells. D0: Photo taken immediately after placement in the Incucyte; D2: Photo taken 48 hours after placement in the Incucyte; D4: Photo taken 96 hours after placement in the Incucyte; D6: Photo taken 144 hours after placement in the Incucyte; D8: Photo taken 192 hours after placement in the Incucyte. The vertical axis represents the LYTAF-M5.6 concentration or the control.

[0130] Figure 10 shows that LYTAF-M5.6 inhibits the proliferation of the breast cancer cell line BT-474. Figure 10A. Cell proliferation assay using the Incucyte assay six days after LYTAF (0-100 nM) administration. Vertical plots show three replicates. Figure 10B. Cell viability assay using the Celltiters assay six days after LYTAF (0-100 nM) administration.

[0131] Figure 11 shows the effects of LYTAF-M5.6 on breast cancer cell JIMT-1 tumors and mouse body weight. Figure 11A shows that LYTAF-M5.6 inhibits the growth of breast cancer cell JIMT-1 tumors, and Figure 11B shows that LYTAF-M5.6 does not affect the body weight of mice.

[0132] FIG12 shows that LYTAF mediates degradation of HER2 and EGFR in animals.

[0133] FIG13 shows that LYTAF mediates EGFR degradation in liver cancer cell SNU-449.

[0134] FIG14 shows that LYTAF mediates degradation of EGFR and c-Met in the non-small cell lung cancer cell line NCI-H1975.

[0135] FIG15 shows that LYTAF mediates degradation of HER2 membrane protein in breast cancer cells SUM159PT.

[0136] FIG16 shows that LYTAF mediates degradation of HER2 membrane protein in breast cancer cells T47D.

[0137] FIG. 17 shows that LYTAF mediates degradation of HER2 membrane protein in breast ductal carcinoma cells HCC 1954.

[0138] FIG18 shows that LYTAF mediates degradation of HER2 membrane protein in breast cancer cells MDA-MB-435.

[0139] Figure 19 shows that LYTAF mediates the degradation of c-Met membrane protein in MHCC-97H hepatoma cells. Figure 19A. c-Met control antibody does not mediate c-Met degradation. Figure 19B. c-Met control antibody linked to IGF2-M5.6 to obtain a fusion protein LYTAF-mediated c-Met degradation.

[0140] Figure 20 shows the results of testing that wild-type IGF2 promotes the proliferation of breast cancer and liver cancer cells. Figure 20a shows the cell growth of BT-474 cells treated with different concentrations of wild-type IGF2 protein over 72 hours. Figure 20b compares the cell proliferation of BT-474 cells treated with PBS at 0 hours and 72 hours, and with groups treated with different concentrations of IGF2, using the coverage of the PBS-treated group as a reference. Figure 20c shows photographs of the cell growth density of BT-474 cells treated with PBS and hFc-IGF2 at 0 hours and 72 hours. Figure 20d shows the cell growth of SK-Hep1 cells treated with different concentrations of wild-type IGF2 protein over 72 hours. Figure 20e shows the cell proliferation of SK-Hep1 cells treated with PBS at 0 hours and 72 hours, and with groups treated with different concentrations of hFc-IGF2, using the coverage of the PBS-treated group as a reference. Figure 20f shows the cell growth density of SK-Hep1 cells treated with PBS and hFc-IGF2 at 0 hours and 72 hours.

[0141] Figure 21 shows the results of the test that the IGF2 mutant M5.6 had no effect on breast cancer cells and liver cancer cells. Figure 21a shows the effect of different concentrations of IGF2-M5.6-hFc protein (also known as a fusion protein of IGF2 mutant M5.6 and IgG1 antibody hFc) on the proliferation of breast cancer cells BT-474 and liver cancer cells SK-Hep1. Figure 21b shows the effect of different concentrations of IGF2 mutant M5.6 on the proliferation of breast cancer cells SK-BR-3. DETAILED DESCRIPTION

[0142] While glycosylation-modified antibodies (LYTACs) (Figure 1, left) can achieve protein degradation, they also have significant drawbacks. These primarily require chemical reactions to glycosylate the antibody, making it difficult to obtain uniform molecules. Furthermore, the process is relatively complex, hindering subsequent drug development and industrial applications.

[0143] The present invention obtains a ligand with high affinity to IGF2R by transforming and screening IGF2 mutants, thereby increasing its endocytosis efficiency, and obtains an IGF2 mutant that selectively recognizes IGF2R by means of protein evolution.

[0144] Wild-type IGF2 binds not only to IGF2R but also to IGF1R. When IGF2 binds to IGF1R, it induces IGF1R tyrosine kinase activity, activating tumor signaling pathways and inducing tumor proliferation. The present disclosure modifies IGF2 to obtain an IGF2 mutant with low affinity for IGF1R, thus avoiding the risk of inducing tumors. Therefore, the present disclosure utilizes genetic engineering methods to design and screen an IGF2 mutant with high affinity for IGF2R and low affinity for IGF1R, which helps avoid inducing tumors.

[0145] Compared with previously reported IGF2 mutants used for protein-targeted lysosomal delivery and the treatment of lysosomal enzyme deficiency, the IGF2 mutant provided by the present disclosure not only greatly improves its affinity with IGF2R, but also reduces its affinity with IGF1R, and has higher selectivity. It is a new type of IGF2 mutation and has better advantages in applications such as drug delivery and protein degradation.

[0146] The IGF2 mutants obtained by the present disclosure screening can be expressed by fusion with antibodies that specifically recognize target proteins (such as HER2) through a linker (Figure 1, right). This new fusion protein double-headed molecule is named Lysosomal Targeting Fusion Protein (LYTAF) by us. LYTAF uses the IGF2 mutant at one end of the linker to recognize the lysosomal shuttle receptor IGF2R, and uses the antibody at the other end of the linker to recognize the target protein to be degraded (such as HER2), forming a ternary complex, and then inducing the ternary complex to enter the lysosome, thereby mediating the degradation of the target protein in the lysosome (Figure 2). Compared with the previously reported LYTAC, LYTAF can be obtained through mammalian cell expression, without the need for further chemical reaction steps, the process is simple, and the problem of product uniformity is solved, making it easier to apply and transform.

[0147] Our research shows that LYTAF can mediate the targeted degradation of multiple target proteins, laying the foundation for the subsequent development of drugs based on target protein degradation technology or drug delivery.

[0148] In a specific embodiment, the IGF2 mutant disclosed herein can be linked to antibodies targeting target proteins (e.g., EGFR, HER2, HER3, c-Met, HGF, EGF, FGF19, EGFA, etc.) or their receptors or ligands through different linkers to mediate the efficient degradation of the target protein or other proteins that interact with the target protein.

[0149] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0150] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0151] As used herein, the expression "about" is understood by one of ordinary skill in the art and varies within certain limits depending on the context in which it is used. If the use of the term is not understood by one of ordinary skill in the art based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0152] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. These terms also include polypeptides with co-translational modifications (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like.

[0153] The term "antibody" generally refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen, including but not limited to polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. An antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. The basic four-chain antibody unit can be a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain can be linked to an H chain by a covalent disulfide bond, and the two H chains can be linked to each other by one or more disulfide bonds. Each H chain generally includes an N-terminal heavy chain variable region and a subsequent heavy chain constant region. The heavy chain variable region can include a variable domain (VH), and the heavy chain constant region generally includes three to four constant domains (CH). Each L chain also includes a variable region containing a variable domain (VL) and a constant region containing a constant domain (CL). VL corresponds to VH, and CL can correspond to the first constant domain (CH1) of the heavy chain. Antibodies typically include six CDRs; three heavy chain CDRs in VH, and three light chain CDRs in VL.

[0154] An "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (target protein). Examples of binding fragments encompassed by the term "antigen-binding portion / fragment" of an antibody include: (i) a Fab fragment - a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment - a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and (v) a dAb fragment consisting of the VH domain. An isolated complementarity-determining region (CDR) or a combination of two or more isolated CDRs linked by a synthetic linker may comprise the antigen-binding domain of an antibody, if capable of binding to an antigen.

[0155] The term "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region (e.g., CHI or CL) of the antibody excluding the first constant region immunoglobulin domain. In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains in each of the two heavy chains of the antibody; the IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position C226 or P230 of the heavy chain (or the amino acid between these two amino acids) to the carboxyl terminus, wherein numbering is performed according to the EU index in Kabat. The CH2 domain of the human IgG Fc region extends from approximately amino acid 231 to approximately amino acid 340, while the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, i.e., it extends from approximately amino acid 341 of IgG to approximately amino acid 447 (including the C-terminal lysine). As used herein, the Fc region can be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region in an isolate or in the context of a protein polypeptide comprising Fc (such as a "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin)).

[0156] The terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to include a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides.

[0157] Suitable host cells for the present disclosure can contain expression vectors (constructs) such as plasmids, etc., introduced, for example, via transformation, transfection, infection or injection. The vector has a coding sequence or a portion thereof encoding the protein expressed and produced during culture. Such expression vectors contain the necessary elements for transcribing and translating the inserted coding sequence. Expression vectors can be constructed using methods well known and practiced by those skilled in the art, which contain sequences encoding the produced proteins and polypeptides, and appropriate transcription and translation control elements. These methods include in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination. Such technology is described in, for example, Sambrook, J. et al., Molecular Cloning Laboratory Manual (4th Edition) (Cold Spring Harbor Laboratory Press).

[0158] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). After importing the host cell, other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included, which have equivalent functions.

[0159] "Specific binding" means that the antibody binds to a specific antigen with greater affinity than to other antigens. D ) is approximately 1×10 -8 M or less, an antibody is considered to "specifically bind", for example, about 1 × 10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 M or smaller, usually K D At least higher than its K for binding to nonspecific antigens (such as BSA, casein) D 100 times smaller. K D Can be measured using standard procedures.

[0160] A "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0161] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0162] Unless otherwise specified, an "individual" or "subject" is a human. In some cases, where specified, an "individual" or "subject" is or includes a non-human mammal (e.g., a "mammalian subject" or "non-human mammalian subject"). Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0163] The term "treat" refers to obtaining a desired pharmacological and / or physiological effect. If the disease or its symptoms can be completely or partially prevented, the effect is preventive, and if the disease and / or adverse effects caused by the disease can be partially or completely cured, the effect is therapeutic. As used herein, "treat" encompasses any treatment of a disease in a mammal (e.g., a human) and includes (a) preventing the disease from occurring in a subject who may be ill but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., stopping its development; and (c) relieving the disease, i.e., causing the disease to regress.

[0164] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to mammals, including but not limited to mice (e.g., rats, mice), civets (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0165] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, solid tumors, hematological tumors, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastomas, sarcomas, e.g., squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, gliomas, cervical cancer, ovarian cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, peritoneal cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, leukemias and other lymphoproliferative disorders, and various types of head and neck cancer.

[0166] The three-letter or one-letter abbreviations of amino acids used herein are shown in Table 1 below.

[0167] Table 1

[0168] Before further describing the present disclosure, it is to be understood that the present disclosure is not limited to the particular embodiments described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting.

[0169] As used herein, the term "cell surface protein" refers to a protein located on the cell surface, for example, a transmembrane protein having an extracellular domain, or a protein that is otherwise localized to the cell surface, such as a protein that is associated with a membrane protein.

[0170] In some embodiments, the linker can be any suitable linker that can be easily selected by a person skilled in the art. Exemplary linkers include but are not limited to the sequences shown in Table 2:

[0171] Table 2

[0172] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Similarly, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0173] Percentage sequence identity can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain percent sequence identity. The comparison of sequences and the determination of percent sequence identity between the two sequences can be accomplished using software that is easy to use online and download. Suitable software programs are available from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining percent sequence identity is bl2seq, which is a part of the BLAST suite of programs available from the BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, a part of the EMBOSS suite of Needle, Stretcher, Water or Matcher, bioinformatics programs, and also available at www.ebi.ac.uk / Tools / psa.

[0174] In some embodiments, the pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of the active ingredient as described above, which is typically formulated into the desired composition after sufficient purification together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include excipients, diluents, antioxidants, preservatives, colorants, flavorings, diluents, emulsifiers, suspending agents, solvents, fillers, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, antimicrobial agents, and surfactants.

[0175] The compositions can be in liquid or powder or lyophilized form and can include one or more lyoprotectants, excipients, surfactants, or fillers. The compositions of the present invention can be suitable for parenteral administration, for example, by injection or infusion into an animal by any of the following routes: intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral, and inhalation routes.

[0176] Example

[0177] Example 1

[0178] (1) Design and screening of IGF2 mutants that specifically recognize IGF2R

[0179] (1.1) Based on the amino acid sequence of wild-type human IGF2 (IGF2-WT) (SEQ ID NO: 1), the first round of IGF2 mutations was designed and attached to the C-terminus of the human IgG1 antibody hFc. The details are shown in Table 3.

[0180] Table 3. Amino acid and nucleic acid sequence numbers of IGF2 mutations and fusion proteins designed in the first round.

[0181] (1.2) The wild-type IGF2 or its mutant is fused to the C-terminus of the hFc of the IgG1 antibody through the linker (G)5. Its amino acid sequence is shown in seq2.1 to seq8.1, and its encoding DNA sequence is shown in sequences seq2.2 to seq8.2.

[0182] (1.3) Wild-type IGF2 or its mutant is fused to the C-terminus of the heavy chain of the anti-HER2 antibody Pertuzumab through a linker (G)5, and co-expressed with the light chain (PtzL) of the Pertuzumab antibody to obtain a HER2-targeted LYTAF molecule. The amino acid sequence of the Pertuzumab heavy chain after linkage to IGF2 or its mutant is shown in seq9.1 to seq15.1, and its encoding DNA sequence is shown in sequences seq9.2 to seq15.2. The amino acid and DNA sequences of the light chain (PtzL) of the Pertuzmab antibody are shown in seq16.1 and seq16.2. The amino acid and DNA sequences of the heavy chain (PtzH) of the Pertuzmab antibody are shown in seq17.1 and seq17.2.

[0183] (1.4) Synthesize seq2.2 to seq8.2 genes and ligate them into the EcoRV and XbaI restriction sites of the pCDNA3.4 plasmid to obtain seq2.2-pCDNA3.4 to seq8.2-pCDNA3.4.

[0184] (1.5) Sequences 2.2-pCDNA3.4 to 8.2-pCDNA3.4 were transfected into HEK293-F cells, and the following fusion proteins were expressed and purified: hFc-IGF2-WT, hFc-IGF2-M1, hFc-IGF2-M2, hFc-IGF2-M3, hFc-IGF2-M4, hFc-IGF2-M5, and hFc-IGF2-M6. The transfection and purification methods are as follows:

[0185] (1.5.1) Expi-293F cells were cultured in suspension in OPM-293 CD05 Medium (OPM). The cell culture conditions were 37°C, 8% CO2, and a shaker at 100 rpm. When the cells grew to 2.5 × 10 6 When the density reached 1 / mL, transfection was started. The transfection reagent used was PEI MAX-Transfection Grade Linear Polyethylenimine Hydrochloride (MW40,000) solution produced by Polysciences, hereinafter referred to as PEI.

[0186] (1.5.2) PEI and plasmid were mixed in a 1:1 ratio, and the final concentration of the plasmid was 1 μg / mL. That is, 1 mg of PEI solution and 1 mg of plasmid were added to 1 L of culture medium, and the ratio of the two was 1:1. The plasmid and PEI mixture was slowly added dropwise to the culture medium and continued to be cultured in a shaker at 37°C, 8% CO2, and a speed of 100 rpm. 24 hours after transfection, protein expression enhancer (1 mL of 1 M sodium butyrate was added to each 1 L of culture medium) and 200 mL of fresh culture medium were added. After 5-7 days of culture, the culture supernatant was collected and purified after centrifugation.

[0187] (1.5.3) hFc-IGF2-WT, hFc-IGF2-M1, hFc-IGF2-M2, hFc-IGF2-M3, hFc-IGF2-M4, hFc-IGF2-M5, and hFc-IGF2-M6 were purified using Protein A filler (Protein At Beads 4FF, Tiandi Renhe, SA023025). Elution was performed using 100 mM glycine (pH 3.0). Before elution, the corresponding volume of Tris-HCl (pH 8.5) neutralizing solution was added to the collection tube (eluent:neutralizing solution = 10:1). After desalting by dialysis, the target protein was obtained.

[0188] (1.6) Synthesize seq9.2 to seq17.2 genes and connect them to the EcoRV and XbaI restriction sites of pCDNA3.4 plasmid to obtain seq9.2-pCDNA3.4 to seq17.2-pCDNA3.4. Seq9.2-pCDNA3.4, seq10.2-pCDNA3.4, seq11.2-pCDNA3.4, seq12.2-pCDNA3.4, seq13.2-pCDNA3.4, seq14.2-pCDNA3.4, seq15.2-pCDNA3.4, seq17.2-pCDNA3.4 plasmid and seq16.2-pCDNA3.4 plasmid were co-transfected into HEK293-F cell line respectively. The following fusion proteins were obtained by purification: PtzHL-IGF2-M7, PtzHL-IGF2-M8, PtzHL-IGF2-M9, PtzHL-IGF2-M10, PtzHL-IGF2-M11, PtzHL-IGF2-M12, PtzHL-IGF2-M13 (the C-terminus of the Pertuzumab antibody was connected to the wild-type sequence or mutant sequence of IGF2 via a linker, see Table 3) and PtzHL (Pertuzumab antibody control).

[0189] (1.6.1) Expi-293F cells were cultured in suspension in OPM-293 CD05 Medium (OPM) at 37°C, 8% CO2, and a shaker at 100 rpm. 6 When the density of cells reached 1 / mL, transfection was started. The transfection reagent used was PEI solution from Polysciences.

[0190] (1.6.2) The concentrations of PEI and plasmid are mixed in a 1:1 ratio, with a final concentration of 1 μg / mL. This means that 1 mg of PEI solution and 1 mg of plasmid (0.5 mg of antibody heavy and light chains, respectively) need to be added to 1 L of culture medium. Slowly add the plasmid and PEI mixture dropwise to the culture medium and continue culturing in a shaker at 37°C, 8% CO2, and 100 rpm. 24 hours after transfection, add the protein expression enhancer and 200 mL of fresh culture medium. After 5-7 days of culture, collect the supernatant for purification.

[0191] (1.6.3) Purify PtzHL-IGF2-M7, PtzHL-IGF2-M8, PtzHL-IGF2-M9, PtzHL-IGF2-M10, PtzHL-IGF2-M11, PtzHL-IGF2-M12, PtzHL-IGF2-M13, and PtzHL using Protein A media. Elution was performed using 100 mM glycine (pH 3.0). Before elution, add the appropriate volume of Tris-HCl (pH 8.5) neutralizing solution to the collection tube (eluent:neutralizer = 10:1). After desalting by dialysis and purification using molecular sieves or ion exchange columns, the above proteins were obtained.

[0192] (1.7) Screening of IGF2 mutants with high affinity for IGF2R using ELISA

[0193] (1.7.1) Expression and purification of the 11th domain of IGF2R

[0194] The gene encoding the 11th domain of IGF2R (D11-IGF2R) was synthesized and inserted into the pCDNA3.4 plasmid under the EcoRV and XbaI double restriction sites to generate the expression plasmid D11-IGF2R-pCDNA3.4. D11-IGF2R-pCDNA3.4 was transfected into HEK-293F mammalian cells for expression. The IGF2R-D11 protein was purified using a nickel column and molecular sieves for future use. The amino acid sequence of IGF2R-D11 is shown in seq 18.1 (SEQ ID NO: 36), and its DNA sequence is shown in seq 18.2 (SEQ ID NO: 37).

[0195] (1.7.2) The mutants with high affinity for IGF2R-D11 protein were screened using ELISA as follows.

[0196] (1.7.2.1) Dilute the IGF2R-D11 protein to 1 μg / μL using coating buffer (0.05 M carbonate buffer, pH 9.6). Add 150 μL of coating buffer to each well of a 96-well ELISA plate and incubate overnight at 4°C.

[0197] (1.7.2.2) Wash four times with PBST (0.05% Tween 20 + PBS).

[0198] (1.7.2.3) Add 200 μL of blocking solution (0.2% Tween 20 + 2% BSA in PBS) to each well and incubate at 37°C for 1.5 h for blocking.

[0199] (1.7.2.4) Wash four times with PBST (0.05% Tween 20 + PBS).

[0200] (1.7.2.5) Add 100 μL of serially diluted hFc-IGF2 mutant samples at different concentrations to each well and incubate at 37°C for 1.5 h.

[0201] (1.7.2.6) Wash four times with PBST (0.05% Tween 20 + PBS).

[0202] (1.7.2.7) Dilute the secondary antibody (anti-hFc-HRP) in 2% BSA at a ratio of 1:5000, add 100 μL per well, and incubate at 37°C for 1.5 h.

[0203] (1.7.2.8) Wash 5 times with PBST (0.05% Tween 20 + PBS).

[0204] (1.7.2.9) Add 100 μL / well of colorimetric reagent (TMB, Solebro) and incubate at room temperature in the dark for 1-10 min.

[0205] (1.7.2.10) Add 50 μL of stop solution (Solar Bio) to each well to terminate the reaction.

[0206] (1.7.2.111) Detect the absorbance at 450 nm using a microplate reader.

[0207] (1.7.3) The results of affinity screening of IGF2 mutants are shown in Figure 3.

[0208] As can be seen from the results in Figure 3, the IGF2 mutation corresponding to Seq7.1 (E6R / R37A / V43M) has the best affinity with IGF2R, which is better than wild-type IGF2 (Seq2.1) and the reported IGF2 mutation for lysosomal protein delivery (Seq3.1). Among them, the E6R mutation significantly enhances the affinity of IGF2 to IGF2R.

[0209] (1.8) Through tumor cell proliferation inhibition experiments, we screened and verified that pertuzumab, which has an IGF2 mutant containing the amino acid sequence shown in Seq7.1 linked to its C-terminus, has better anti-tumor activity.

[0210] As shown in Table 3, in Seq9.1 to Seq15.2, we linked different IGF2 mutants to the C-terminus of the heavy chain of Pertuzumab, and obtained a molecule fused with the antibody and IGF2 mutant by expressing it with the light chain PtzL of Pertuzumab antibody. The molecule contains the complete H chain and L chain of Pertuzumab, and the IGF2 mutant linked to the H chain of Pertuzumab.

[0211] We co-incubated different concentrations of Pertuzumab (PtzHL) or Pertuzumab fused with different IGF2 mutations (PtzHL-IGF2-M7 to PtzHL-IGF2-M13) with the breast cancer cell line SK-BR-3. Using an Incucyte instrument, we monitored cell growth in real time. We screened for IGF2 mutants that could enhance the anti-tumor activity of Pertuzumab based on the cell growth patterns. The specific steps are as follows:

[0212] (1.8.1) SK-BR-3 cells were seeded at 2000 cells per well in a 96-well cell culture plate. After 12 hours, PBS, pertuzumab, or proteins expressing different pertuzumab fusions with IGF2 mutants (PtzHL-IGF2-M7 to PtzHL-IGF2-M13) were added to the cell culture medium. The cells were then placed in an Incucyte instrument for real-time cell growth monitoring.

[0213] (1.8.2) Set the Incucyte instrument parameters, take photos every 6 hours, monitor continuously for 96 hours, collect statistics, and perform analysis.

[0214] (1.8.3) Experimental results

[0215] As shown in Figure 4, PtzHL-M12 demonstrated superior anti-tumor activity compared to the control antibody Pertuzumab (PtzHL). Since M12 and M7 share the same IGF2 mutation, this suggests that the IGF2 mutation (E6R / R37A / V43M) warrants further development. Furthermore, we found that wild-type IGF2 promoted tumor growth compared to the PBS-treated group, confirming our previous hypothesis that wild-type IGF2 promotes tumor growth and that the application prospects of unmodified IGF2 are very limited.

[0216] (1.9) Summary:

[0217] (1.9.1) We initially screened the affinity of IGF2 mutants for the receptor IGF2R using ELISA. We found that a fusion protein containing the IGF2-M5 mutant linked to the C-terminus of hFc showed significantly improved affinity compared to wild-type IGF2 and exhibited the best affinity of all mutants.

[0218] (1.9.2) Through cell proliferation inhibition experiments, we preliminarily screened fusion proteins containing different IGF2 mutations linked to the C-terminus of the pertuzumab heavy chain. The results showed that the fusion protein (PtzH-IGF2-M12) containing the C-terminus of the pertuzumab heavy chain linked to IGF2-M5 had the best anti-breast cancer cell proliferation effect among all tested proteins.

[0219] (2.0) Based on IGF2-M5 (E6R / R37A / V43M), new IGF2 mutations were screened to obtain IGF2 mutations with higher affinity for IGF2R and lower affinity for IGF1R.

[0220] (2.0.1) Using IGF2-M5 (E6R / R37A / V43M) as a template, we further designed secondary mutations as shown in Table 4. IGF2-M5.1 is a mutation based on the IGF2 precursor (see Seq 1.3, SEQ ID NO: 3 for the new amino acid sequence of the IGF2 precursor). IGF2-M5.2 is a mutation based on the IGF2 precursor (see Seq 1.2, SEQ ID NO: 2 for the new amino acid sequence of the IGF2 precursor).

[0221] Table 4. Design of new IGF2 mutants based on the IGF2-M5 mutation.

[0222] The screening was performed based on the IGF2 mutants shown in Table 4, whose sequences and numbers are as above.

[0223] (2.0.2) The codon-optimized DNA sequences corresponding to seqM5.1-2 to seqM5.10-2 were ligated into the pCDNA3.4 plasmid at the EcoRV and XbaI restriction sites to generate seqM5.1-pCDNA3.4 to seqM5.10-pCDNA3.4. The IGF2 mutants were linked to the hFc fragment using a GGGGS linker.

[0224] (2.0.3) SeqM5.1-pCDNA3.4 to seqM5.10-pCDNA3.4 were transfected into HEK293-F cell lines, and the following fusion proteins were obtained after expression and purification: hFc-IGF2-M5.1, hFc-IGF2-M5.2, hFc-IGF2-M5.3, hFc-IGF2-M5.4, hFc-IGF2-M5.5, hFc-IGF2-M5.6, hFc-IGF2-M5.7, hFc-IGF2-M5.8, hFc-IGF2-M5.9, and hFc-IGF2-M5.10. The transfection and purification methods are as follows:

[0225] (2.0.3.1) Expi-293F cells were cultured in suspension in OPM-293 CD05 Medium (OPM) at 37°C, 8% CO2, and a shaker at 100 rpm. 6 When the density reaches 400 μg / mL, transfection was started.

[0226] (2.0.3.2) Mix PEI and plasmid in a 1:1 ratio, with a final concentration of 1 μg / mL. This means that 1 mg of PEI solution and 1 mg of plasmid should be added to 1 L of culture medium, resulting in a 1:1 ratio. Slowly add the plasmid and PEI mixture dropwise to the culture medium and continue incubating in a shaker at 37°C, 8% CO2, and 100 rpm. 24 hours after transfection, add the protein expression enhancer and culture for 5-7 days. Collect the supernatant for purification.

[0227] (2.0.3.3) hFc-IGF2-M5.1, hFc-IGF2-M5.2, hFc-IGF2-M5.3, hFc-IGF2-M5.4, hFc-IGF2-M5.5, hFc-IGF2-M5.6, hFc-IGF2-M5.7, hFc-IGF2-M5.8, hFc-IGF2-M5.9, and hFc-IGF2-M5.10 were purified using Protein A filler (Protein At Beads 4FF, Tiandi Renhe, SA023025). Elution was performed using 100 mM glycine (pH 3.0) as eluent. Before elution, the corresponding volume of Tris-HCl (pH 8.5) as neutralizer was added to the collection tube (eluent:neutralizer = 10:1). The proteins were obtained after dialysis, desalting, and molecular sieve purification.

[0228] (2.0.4) ELISA was used to screen IGF2 mutants (hFc-IGF2-M5.1 to hFc-IGF2-M5.10) with high affinity to IGF2R.

[0229] From the results in FIG5 , it can be seen that each mutant has an improved affinity for IGF2R, among which mutant M5.6 has the highest affinity for IGF2R (about 0.42 nM).

[0230] (2.0.5) Screening of IGF2 mutants with low affinity for IGF1R using ELISA

[0231] The affinity of the IGF2 mutants for IGF1R was tested by ELISA. The amino acid sequence of the IGF1R protein is shown in Seq32-1 (SEQ ID NO:64), and its DNA sequence is shown in Seq32-2 (SEQ ID NO:65). The DNA sequence encoding IGF1R was ligated into pCDNA3.4, expressed in HEK293F mammalian cells, and purified via nickel column.

[0232] The results are shown in Figure 6 . M2 represents a protein expressed by fusion with hFc and an IGF2 mutant reported in prior patents (US 2003 / 0082176 A1, US 2004 / 0006008 A1, and US 2005 / 0244400 A1). As can be seen from the results in Figure 6 , M2 exhibits similar affinity for IGF1R as the wild-type. All mutants disclosed herein significantly reduce the affinity of IGF2 for IGF1R, with the IGF2-M5.6 mutant exhibiting the lowest affinity, showing no affinity at all. With the exception of IGF2-M2, which exhibits similar affinity to wild-type IGF2, all other mutants selected for this experiment exhibit significantly reduced affinity for IGF1R.

[0233] (2) Designing a fusion protein of IGF2-M5.6 and Pertuzumab, LYTAF, and testing its ability to mediate the degradation of the membrane protein HER2

[0234] (2.1) Expression and purification of LYTAF and antibodies

[0235] The IGF2 mutant IGF2-M5.6 was linked to the C-terminus of the Pertuzumab antibody heavy chain via a linker (GGGGS) 3 (amino acid sequence, see seq19-1 (SEQ ID NO: 38), DNA sequence, see seq19-2 (SEQ ID NO: 39)) to prepare a fusion protein PtzH-IGF2-M5.6 linked to the C-terminus of the Pertuzumab heavy chain. By genetic engineering, the gene encoding the fusion protein was ligated to the EcoRV and XbaI restriction sites of the plasmid pCDNA3.4 to obtain PtzH-IGF2-M5.6-pCDNA3.4. Simultaneously, the gene encoding the Pertuzumab antibody light chain (amino acid sequence, see seq16-1, DNA sequence, see seq16-2) was ligated to the EcoRV and XbaI restriction sites of the plasmid pCDNA3.4 to obtain PtzL-pCDNA3.4. The PtzL-pCDNA3.4 plasmid and the PtzH-IGF2-M5.6-pCDNA3.4 plasmid were co-transfected into mammalian cells HEK293-F for expression, and the HER2-targeting LYTAF-M5.6 protein was purified.

[0236] (2.2) Purified LYTAF-M5.6 protein or pertuzumab was co-incubated with HER2-positive breast cancer cell lines BT474, T47D, Sum159P, and JIMT-1 for 36 hours. The cells were washed three times with PBS, lysed, and harvested. Total HER2 content in the cells was assayed by Western blot. As shown in Figure 7, the control antibody pertuzumab did not mediate HER2 degradation at varying concentrations, whereas LYTAF-M5.6 efficiently mediated HER2 degradation at low concentrations.

[0237] (3) LYTAF efficiently mediates HER2 protein internalization

[0238] FIG8A exemplarily shows a schematic diagram of LYTAF-mediated antibody internalization into lysosomes.

[0239] To verify this process, this example co-incubated SK-BR-3 breast cancer cells with IgG-647 secondary antibody and LYTAF-M5.6. The antibody on one end of LYTAF-M5.6 recognized the secondary antibody IgG-647, while the other end (IGF2-M5.6) recognized the IGF2R on the cell membrane, forming a ternary complex. This ternary complex then endocytosed, carrying IgG-647 into lysosomes.

[0240] Briefly, mammalian SK-BR-3 cells were seeded into glass-bottomed cell culture dishes. After 24 hours, IgG-647 (rabbit anti-human) and 10 nM LYTAF or the control antibody Pertuzumab were added to a final concentration of 20 nM and incubated in a 37°C cell culture incubator for 1 hour. The cells were washed five times with HBSS solution, then 50 nM Lysotracker was added and incubated in a 37°C cell culture incubator for 10 minutes. After washing three more times with HBSS solution, Hoechst was added and incubated at room temperature for 5 minutes. The localization of IgG-647, lysosomes, and nuclei was examined using confocal microscopy. The results are shown in Figure 8B.

[0241] As shown in Figure 8B, we can see that IgG-647 co-localizes with the lysosomal dye lysotracker, which fully demonstrates that LYTAF can mediate the endocytosis of IgG-647 and its entry into the lysosome.

[0242] (4) LYTAF-M5.6 significantly inhibits the proliferation of tumor cells

[0243] (4.1) SK-BR-3 cells were seeded at 2000 cells / well in a 96-well plate. 12 hours later, different concentrations of LYTAF-M5.6 were added, and the cells were placed in an Incucyte instrument to monitor cell growth in real time. As shown in Figure 9, LYTAF-M5.6 significantly inhibited cell growth, and cell proliferation decreased with increasing LYTAF-M5.6 concentration. At a LYTAF-M5.6 concentration of 200 nM, SK-BR-3 cells stopped growing at all.

[0244] (4.2) Unlike LYTAF, which significantly inhibited SK-BR-3 cell proliferation, Pertuzumab and hFC-IGF2 did not significantly inhibit cell proliferation. This fully demonstrates that LYTAF significantly inhibited tumor growth compared to the control antibody.

[0245] (5) To verify the above results, we further studied the effect of LYTAF-M5.6 in inhibiting cell proliferation in the BT-474 cell line.

[0246] (5.1) As shown in Figure 10, we used two experiments to test the inhibitory activity of LYTAF in breast cancer cells BT-474. The specific methods are as follows: BT-474 cells were seeded at 2000 cells / well in a 96-well transparent plate. After 24 hours, different concentrations of LYTAF were added and the cells were placed in an Incucyte instrument to monitor cell growth in real time and take photos. The results are shown in Figure 10A.

[0247] (5.2) At the same time, we used celltiters reagent to detect the activity of BT-474 cells treated with different concentrations of LYTAF. The specific method is: BT-474 cells were inoculated into a 96-well non-transparent plate at a rate of 2000 / well, and different concentrations of LYTAF were added after 24 hours. Three replicates were made for each concentration, and the cells were placed in a CO2 incubator for culture for 5 days. Celltiters reagent (Promega) was added to each well, and then the fluorescence intensity was detected. The fluorescence value of the PBS group was taken as 100% to normalize the data, and the proportion of live cells in the groups treated with different concentrations of LYTAF was calculated. The results are shown in Figure 10B. The specific method is as follows:

[0248] (5.2.1) BT474 cells were evenly plated in a 384-well plate, with 800 cells per well.

[0249] (5.2.2) After 6 hours, pipette a total of 15 μL of serially diluted 4-fold LYTAF-M5.6 and cell culture medium into a 384-well plate containing 45 μL of culture medium sample (with 4% FBS).

[0250] (5.2.3) After incubation at 37°C in a CO2 cell culture incubator for 5 days, remove the cell culture plate and equilibrate it at room temperature for 30 minutes.

[0251] (5.2.4) Add half the volume of cell culture medium 2.0 reagent (e.g., for a 384-well plate, add 30 μL of 2.0 reagent).

[0252] (5.2.5) Mix on an orbital shaker for 2 minutes to induce cell lysis.

[0253] (5.2.6) After incubation at room temperature for 10-15 minutes, measure and record the luminescence signal on a microplate reader.

[0254] (5.3) Experimental results

[0255] This experiment used two methods to detect the inhibition of LYTAF-M5.6 on the growth of breast cancer cells BT474. As shown in Figure 10A, the growth rate of cells was monitored using the Incucyte instrument. When LYTAF-M5.6 reached 25nM, the growth of cells was significantly inhibited. As shown in Figure 10B, the growth rate of cells was significantly inhibited using the Incucyte instrument. 2.0 reagent was used to detect cell activity and it was found that LYTAF-M5.6 significantly inhibited the proliferation of tumor cells.

[0256] (6) LYTAF inhibits the proliferation of xenograft tumors in nude mice

[0257] (6.1) Experimental animals

[0258] Fifty specific pathogen free (SPF) healthy female Balb / c nude mice, weighing 15-25 g and aged 4 weeks, were purchased from Jicui Yaokang.

[0259] (6.2) Animal model establishment

[0260] A breast cancer model was established in Balb / c nude mice using JIMT-1 cells as a xenograft to evaluate the efficacy and safety of LYTACs. JIMT-1 cells were digested and centrifuged at 1000 rpm for 5 minutes, washed twice with PBS, and resuspended in PBS containing 50% Matrigel. Each mouse was inoculated with 5e6 cells / 100 μl. Mouse body weights were recorded every 3-4 days after inoculation. Mice had free access to food during the experiment. All mouse husbandry, care, and sacrifice adhered to animal welfare principles and ethical standards.

[0261] (6.3) Animal grouping

[0262] 40 Balb / c nude mice were fed with normal feed and tap water, and kept at room temperature of 25℃±1℃, relative humidity of 60%-70%, and kept under level 11 conditions for three days to adapt to the new environment. 3 Balb / c nude mice were randomly divided into four groups, including a model group (PBS group), a control antibody group (Pertuzumab group and Transtuzumab group), and a LYTAF group, with 5-6 mice in each group. The details are shown in Table 5.

[0263] Table 5

[0264] Note: JIMT-1 cells are a transtuzumab-resistant cell line, so we added a transtuzumab group while testing pertuzumab and LYTAF-M5.6.

[0265] (6.4) Administration

[0266] Tumor grows to 70-100 mm 2 Dosing was then initiated. For the first three weeks, dosing was performed twice weekly, with an interval of 3-4 days between doses. Dosing was continued once weekly for the next two weeks, for a total of five weeks. The active agent and LYTAC were prepared at 1 mg / mL according to the protocol, with a dosing volume of 200 μL per dose.

[0267] (6.5) Animal Sacrifice and Specimen Collection

[0268] (6.5.1) Animal sacrifice and blood collection

[0269] On the second day after dosing, blood was collected using the ocular bleeding method. The specimen was placed in a polyethylene tube containing anticoagulant and centrifuged at 6500 rpm for 15 minutes. The supernatant was collected and placed in an EP tube and stored in a -80°C refrigerator until testing. After collection, the mice were sacrificed by cervical dislocation.

[0270] (6.6) Tumor tissue isolation

[0271] The tumor tissue was separated, washed with PBS and the surface moisture of the tissue was removed. A portion was fixed with 4% paraformaldehyde to prepare paraffin sections, and the other portion was quickly frozen in liquid nitrogen for later use.

[0272] (6.7) Visceral separation

[0273] The liver and kidney tissues were separated, washed with PBS and the surface moisture of the tissues was removed, and then fixed with 4% paraformaldehyde to prepare paraffin sections for later use.

[0274] (6.8) Experimental results

[0275] The experimental results are shown in Figure 11. As can be seen from Figure 11, LYTAF significantly inhibited the growth of tumor-bearing mice compared with control antibodies Pertuzumab (Ptz) or Trantuzuma (TRZ) (Figure 11A), but did not affect the body weight of mice (Figure 11B).

[0276] (7) LYTAF mediates HER2 degradation in the JIMT1 xenograft Balb / c nude mouse breast cancer model

[0277] (7.1) Construction of a JIMT1 xenograft Balb / c nude mouse breast cancer model

[0278] JIMT-1 cells were digested and centrifuged at 1000 rpm for 5 minutes. The cells were washed twice with PBS and resuspended in PBS containing 50% Matrigel. Each mouse was inoculated with 5e6 cells / 100 μl. Mouse weights were recorded every 3-4 days after inoculation.

[0279] During the experiment, mice were fed freely. The feeding and killing of mice were in accordance with animal welfare principles and ethical standards.

[0280] (7.2) Administration

[0281] When the tumor grows to 70-100 mm 2 Then the drug was administered at a single dose of 30 mg / kg. After 48 hours, the mice were killed by cervical dislocation, and the tumor tissues were obtained and frozen in a -80°C refrigerator.

[0282] (7.3) Extraction method of total tissue protein:

[0283] Take the tissue out of the -80°C freezer;

[0284] Add an appropriate volume of RIPA lysis buffer (PMSF is added before use) and disrupt the cells using a tissue cell homogenizer (80 Hz, 120 s);

[0285] Centrifuge at 12,000 rpm for 30 min at 4°C and collect the supernatant.

[0286] Repeat the centrifugation once and take the supernatant.

[0287] Add 4× loading buffer (containing DTT), incubate at 100°C in a metal bath for 10 min, centrifuge instantaneously, and store at -20°C for later use.

[0288] (7.4) Tissue protein quantification

[0289] Protein quantification was performed using the BCA protein quantification kit (Pierce TM Follow the instructions of BCA Protein Assay Kits (Cat. No.: A55864).

[0290] Draw a BCA standard curve: the concentration of the standard BSA is 2 mg / mL. Dilute the 2 mg / mL standard to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL using deionized water by serial dilution. Use deionized water as the blank control group.

[0291] Sample dilution: Dilute the total protein sample of the tissue 5 times (10uL sample dissolved in 40uL water) as the working solution of the sample to be tested;

[0292] Prepare BCA working solution: Mix solution A and solution B in the BCA kit at a volume ratio of 50:1 to prepare an appropriate volume of BCA working solution, mix thoroughly, and use immediately;

[0293] Add 200 μl of BCA working solution to each well, then add 10 μl of the sample or standard to be tested, shake it slightly to make the liquid uniform, and place it in a 37°C incubator for 30 minutes;

[0294] The absorbance at 570 nm was measured by microplate reader;

[0295] A standard curve was drawn with A570 as the ordinate and BSA concentration as the abscissa;

[0296] Calculate the sample concentration according to the standard curve.

[0297] (7.5) Western blot detection of HER2 degradation

[0298] Protein separation by SDS-PAGE: 80 μg of the extracted soluble protein was loaded per lane and subjected to constant voltage electrophoresis on a 10% SDS-PAGE gel at 80 V for 15 min, then adjusted to 150 V for about 1 h.

[0299] Transfer: Cut a PVDF membrane of appropriate width and length based on the size of the desired gel block. Soak the PVDF membrane in methanol to activate the surface active groups. After electrophoresis, remove the gel and locate the target protein according to the protein marker. Cut the gel. Place the gel in the order of sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge. Be careful to avoid bubbles between the gel and PVDF membrane. If bubbles occur, flush them with transfer buffer using a 1mL pipette tip or press them out with a test tube. Finally, place the gel into the vertical transfer tank with the negative side (black side) facing the transfer tank. Place the transfer tank in a 4°C refrigerator, fill it with transfer buffer, insert the electrodes, set the current to a constant cross-current of 260mA, and transfer for 100 minutes.

[0300] Blocking: After electrotransfer, remove the PVDF membrane immediately and place it in 5% BSA blocking solution with the surface in contact with the gel facing up to block nonspecific binding sites. Incubate on a shaker at room temperature for 1 hour.

[0301] Antibody hybridization: Anti-HER2 primary antibody was added at a 1:2000 ratio in blocking buffer and incubated overnight at 4°C; then washed eight times with TBST, 5 minutes each time. Secondary antibody incubation: The corresponding HPR-conjugated goat anti-rabbit antibody (1:10000) was added and incubated at room temperature for 1 hour; then washed eight times with TBST, 5 minutes each time.

[0302] Development: Automated development on a gel imager: Turn on the gel imager and set the development program. Based on the antibody titer and target protein expression level, slowly add the pre-mixed colorimetric solution dropwise to the PVDF membrane and allow it to react for 1-2 minutes. Then, transfer the PVDF membrane to the gel imager and begin automated development. Once the development program is complete, save the appropriate image.

[0303] (7.6) Experimental results

[0304] The experimental results are shown in Figure 12. The results in Figure 12 show that LYTAF can also effectively degrade the target HER2 in animals, while the control antibody Pertuzumab (Ptz) does not mediate HER2 degradation, which fully demonstrates that our developed LYTAF can also degrade the target protein in vivo.

[0305] Example 2

[0306] LYTAF mediates the degradation of membrane protein EGFR in hepatocellular carcinoma cells

[0307] (1) The IGF2-M5.6 mutation was linked to the C-terminus of the heavy chain of the EGFR antibody Panitumumab via a linker (GGGGS)3 to obtain the targeted PanH-M5.6. The coding gene of PanH-M5.6 was synthesized by codon optimization and gene synthesis, and ligated to the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The correctness of the sequence was verified by sequencing to obtain PanH-M5.6-pCDNA3.4. The amino acid sequence of PanH-M5.6 is detailed in seq24-1 (SEQ ID NO:48), and its DNA sequence is detailed in seq24-2 (SEQ ID NO:49).

[0308] (2) The gene encoding the panitumumab light chain was synthesized and ligated into the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The sequence was then sequenced to verify the correctness of the sequence, thereby obtaining PanL-pCDNA3.4. The amino acid sequence corresponding to the panitumumab light chain is shown in seq25-1 (SEQ ID NO: 50), and the DNA sequence is shown in seq25-2 (SEQ ID NO: 51).

[0309] (3) PanH-M5.6-pCDNA3.4 and PanL-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain Pan_LYTAF.

[0310] (4) The gene encoding the heavy chain of Panitumumab was synthesized and ligated into the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites, and the sequence was sequenced to verify the correctness of the sequence, thereby obtaining PanH-pCDNA3.4.

[0311] (5) PanL-pCDNA3.4 and PanH-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain Pan_LYTAF and the control antibody PanHL.

[0312] (6) Pan_LYTAF or PanHL at different concentrations were co-incubated with liver cancer cell lines for 36 h, and the EGFR content in the cells was detected by western blot.

[0313] (7) Experimental results: As shown in Figure 13, Pan_LYTAF can significantly mediate EGFR degradation, but the control antibody cannot mediate EGFR degradation, which fully demonstrates that LYTAF mediates membrane protein degradation, which is also applicable to liver cancer cell lines.

[0314] Example 3

[0315] LYTAF mediates simultaneous degradation of EGFR and c-Met in non-small cell lung cancer cell lines

[0316] (1) The IGF2-M5.6 mutation was linked to the C-terminus of the heavy chain of the EGFR antibody Certuximab via a linker (GGGGS)3 to obtain the targeted CetH-M5.6. The CetH-M5.6 coding gene was synthesized by codon optimization and gene synthesis, and ligated to the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The correctness of the sequence was verified by sequencing to obtain CetH-M5.6-pCDNA3.4. The amino acid sequence of CetH-M5.6 is shown in seq26-1 (SEQ ID NO: 52), and its DNA sequence is shown in seq26-2 (SEQ ID NO: 53).

[0317] (2) The gene encoding the certuximab light chain was synthesized and ligated into the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The sequence was then sequenced to verify the correctness of the sequence, thereby obtaining CetL-pCDNA3.4. The amino acid sequence corresponding to the certuximab light chain is shown in seq27-1 (SEQ ID NO: 54), and its DNA sequence is shown in seq27-2 (SEQ ID NO: 55).

[0318] (3) CetH-M5.6-pCDNA3.4 and CetL-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain Cet_LYTAF.

[0319] (4) The gene encoding the certuximab heavy chain was synthesized and ligated into the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The sequence was then sequenced to verify the correctness of the sequence, thereby obtaining CetH-pCDNA3.4. The amino acid sequence of the certuximab heavy chain is shown in seq28-1 (SEQ ID NO: 56), and the corresponding DNA sequence is shown in seq28-2 (SEQ ID NO: 57).

[0320] (5) CetL-pCDNA3.4 and CetH-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain Cet_LYTAF and the control antibody CetHL.

[0321] (6) Different concentrations of Cet_LYTAF or CetHL were co-incubated with non-small cell lung cancer cell lines for 36 h, and the EGFR content in the cells was detected by western blot.

[0322] Experimental results: As shown in Figure 14, Cet_LYTAF can significantly mediate EGFR degradation, but the control antibody cannot mediate EGFR degradation, which fully demonstrates that LYTAF mediates membrane protein degradation, which is also applicable in non-small cell lung cancer cell lines.

[0323] Example 4

[0324] IGF2-M5.6 was linked to the N-terminus of the pertuzumab heavy chain, and the ability of LYTAF to degrade HER2 was tested in the breast cancer cell line Sum159PT.

[0325] (1) The IGF2-M5.6 mutant was linked to the N-terminus of the heavy chain of the HER2 antibody Pertuzumab via a linker (ASTKGP) to obtain IGF2-M5.6-PtzH. The coding gene for IGF2-M5.6-PtzH was synthesized by codon optimization and gene synthesis, and ligated into the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The correctness of the sequence was verified by sequencing to obtain IGF2-M5.6-PtzH-pCDNA3.4. The amino acid sequence of IGF2-M5.6-PtzH is shown in sequence seq20-1 (SEQ ID NO: 40), and its DNA sequence is shown in sequence seq20-2 (SEQ ID NO: 41).

[0326] (2) IGF2-M5.6-PtzH-pCDNA3.4 and PtzL-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain the fusion expression protein IGF2-M5.6H_LYTAF of the antibody and IGF2-M5.6.

[0327] (3) Different concentrations of IGF2-M5.6H_LYTAF were co-incubated with the breast cancer cell line Sum159PT for 36 hours, and the HER2 content in the cells was detected by Western Blot.

[0328] (4) Experimental results: As shown in FIG15 , IGF2-M5.6H_LYTAF can significantly mediate HER2 degradation, but the control antibody cannot mediate HER2 degradation.

[0329] Example 5

[0330] IGF2-M5.6 was linked to the N-terminus of the pertuzumab light chain to test the ability of LYTAF to degrade HER2 in the breast cancer cell line T47D.

[0331] (1) The IGF2-M5.6 mutation was linked to the N-terminus of the light chain of the HER2 antibody Pertuzumab via a linker (TVAAP) to obtain IGF2-M5.6-PtzL. The coding gene for IGF2-M5.6-PtzL was synthesized by codon optimization and gene synthesis, and ligated to the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The correctness of the sequence was verified by sequencing to obtain IGF2-M5.6-PtzL-pCDNA3.4. The amino acid sequence of IGF2-M5.6-PtzL is shown in sequence seq21-1 (SEQ ID NO: 42), and its DNA sequence is shown in sequence seq21-2 (SEQ ID NO: 43).

[0332] (2) IGF2-M5.6-PtzL-pCDNA3.4 and PtzH-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain IGF2-M5.6L_LYTAF, which is a fusion expression of IGF2-M5.6 and Pertuzumab.

[0333] (3) Different concentrations of IGF2-M5.6L_LYTAF or Pertuzumab were co-incubated with the breast cancer cell line T47D for 36 h, and the HER2 content in the cells was detected by Western Blot.

[0334] (4) Experimental results: As shown in Figure 16, IGF2-M5.6L_LYTAF can significantly mediate HER2 degradation.

[0335] Example 6

[0336] IGF2-M5.6 was linked to the C-terminus of the pertuzumab light chain, and the ability of LYTAF to degrade HER2 was tested in the human breast ductal carcinoma cell line HCC1954.

[0337] (1) The IGF2-M5.6 mutation was linked to the C-terminus of the light chain of the HER2 antibody Pertuzumab via a linker (GGGGS) 3 to obtain PtzL-IGF2-M5.6. The coding gene of PtzL-IGF2-M5.6 was synthesized by codon optimization and gene synthesis, and ligated to the mammalian expression vector pCDNA3.4 at the Xba1 and EcoRV restriction sites. The correctness of the sequence was verified by sequencing to obtain PtzL-IGF2-M5.6-pCDNA3.4. The amino acid sequence of PtzL-IGF2-M5.6 is shown in sequence seq22-1 (SEQ ID NO: 44), and its DNA sequence is shown in sequence seq22-2 (SEQ ID NO: 45).

[0338] (2) PtzL-IGF2-M5.6-pCDNA3.4 and PtzH-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain LYTAF-IGF2-M5.6L expressing the fusion of IGF2-M5.6 and Pertuzumab.

[0339] (3) Different concentrations of LYTAF-IGF2-M5.6L or Pertuzumab were co-incubated with the breast ductal cancer cell line HCC1954 for 36 h, and the HER2 content in the cells was detected by Western Blot.

[0340] (4) Experimental results: As shown in FIG17 , LYTAF-IGF2-M5.6L can significantly mediate HER2 degradation.

[0341] Example 7

[0342] IGF2-M5.8 was linked to the C-terminus of the heavy chain of Pertuzumab, and the ability of LYTAF to degrade HER2 was tested in the breast cancer cell line MDMB435.

[0343] (1) The IGF2-M5.8 mutation was linked to the C-terminus of the light chain of the HER2 antibody Pertuzumab via a linker (GGGGS) 3 to obtain PtzH-IGF2-M5.8. The coding gene of PtzH-IGF2-M5.8 was synthesized by codon optimization and gene synthesis, and ligated to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4. The correctness of the sequence was verified by sequencing to obtain PtzH-IGF2-M5.8-pCDNA3.4. The amino acid sequence of PtzH-IGF2-M5.8 is shown in sequence seq23-1 (SEQ ID NO: 46), and its DNA sequence is shown in sequence seq23-2 (SEQ ID NO: 47).

[0344] (2) PtzH-IGF2-M5.8-pCDNA3.4 and PtzL-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain LYTAF-IGF2-M5.8H, which is a fusion expression of IGF2-M5.8 and Pertuzumab.

[0345] (3) Different concentrations of LYTAF-IGF2-M5.8H were co-incubated with the breast cancer cell line MDMB435 for 36 h, and the HER2 content in the cells was detected by Western Blot.

[0346] (4) Experimental results: As shown in FIG18 , LYTAF-IGF2-M5.8H can significantly mediate HER2 degradation.

[0347] Example 8

[0348] IGF2-M5.6 was linked to the C-terminus of the heavy chain of the c-Met antibody, and the ability of LYTAF to degrade c-Met was detected after incubation in the liver cancer cell line MHCC-97H for 48 hours.

[0349] (1) The IGF2-M5.6 mutation was linked to the C-terminus of the c-Met antibody heavy chain via a linker (GGGGS) 3 to obtain Anti-cMet-IGF2-M5.6. The coding gene for Anti-cMet-IGF2-M5.6 was synthesized by codon optimization and gene synthesis, and ligated to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4. The correctness of the sequence was verified by sequencing to obtain Anti-cMet-IGF2-M5.6-pCDNA3.4. The amino acid sequence of Anti-cMet-IGF2-M5.6 is shown in sequence seq29-1 (SEQ ID NO: 58), and its DNA sequence is shown in sequence seq29-2 (SEQ ID NO: 59).

[0350] (2) The c-Met antibody light chain coding gene Anti-cMetL was ligated to the Xba1 and EcoRV restriction sites of pCDNA3.4, and the sequence was sequenced to verify the correctness of the sequence to obtain the Anti-cMetL-pCDNA3.4 plasmid. The amino acid sequence of Anti-cMetL is shown in seq30-1 (SEQ ID NO: 60), and the corresponding DNA sequence is shown in seq30-2 (SEQ ID NO: 61) (the coding sequence of the c-Met antibody light chain is from patent US2019360029).

[0351] (3) Anti-cMet-IGF2-M5.6-pCDNA3.4 and Anti-cMetL-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain LYTAF expressing the fusion of IGF2-M5.6 and Anti-cMet.

[0352] (4) The gene encoding the cMet antibody heavy chain, Anti-cMetH, was ligated into the Xba1 and EcoRV restriction sites of pCDNA3.4, and sequenced to verify the correctness of the sequence, thereby obtaining the Anti-cMetH-pCDNA3.4 plasmid. The amino acid sequence of Anti-cMetH is shown in seq31-1 (SEQ ID NO: 62), and the corresponding DNA sequence is shown in seq31-2 (SEQ ID NO: 63).

[0353] (5) Anti-cMetH-pCDNA3.4 plasmid and Anti-cMetL-pCDNA3.4 plasmid were co-transfected into HEK293-F cells for expression, and the Anti-cMet antibody was obtained after purification (shown as control Ab in FIG19 ).

[0354] (6) Anti-cMet antibodies or corresponding LYTA at different concentrations were co-incubated with the liver cancer cell line MHCC-97H for 48 h, the cells were collected, and the c-Met content in the cells was detected by Western Blot.

[0355] (7) Experimental results: As shown in Figure 19, the Anti-cMet antibody cannot degrade c-Met, while its corresponding LYTAF can significantly degrade c-Met.

[0356] Example 9

[0357] The wild-type IGF2 sequence was linked to the N-terminus of the hFc of an IgG1 antibody and its effect on cell proliferation was detected after co-incubation with the breast cancer cell line BT-474 or the liver cancer cell line SK-Hep1. The specific steps include:

[0358] (1) IGF2-WT (SEQ ID NO: 1) was linked to the N-terminus of the hFc (SEQ ID NO: 116) of the IgG1 antibody via a linker (GGGGS). The gene encoding the fusion protein was synthesized through codon optimization and gene synthesis, and ligated into the mammalian expression vector pCDNA3.4 under the Xba1 and EcoRV restriction sites. The sequence was then sequenced to verify correctness. This yielded IGF2-WT-hFc-pCDNA3.4.

[0359] (2) IGF2-WT-pCDNA3.4 was transfected into HEK293-F cells for expression to obtain hFc-IGF2 protein.

[0360] (3) Following the steps described in Example 1, different concentrations of IGF2-WT-hFc protein (shown as hFc-IGF2 in FIG. 20 ) were co-incubated with the liver cancer cell line SK-Hep1 or the breast cancer cell line BT-474, and the cell growth was monitored in real time using Incucyte.

[0361] (4) Experimental results: As shown in FIG20 , compared with the control PBS, IGF2-WT-hFc significantly promoted the proliferation of breast cancer cells BT-474 and liver cancer cells SK-Hep1.

[0362] Example 10

[0363] The IGF2-M5.6 sequence was linked to the N-terminus of the hFc of an IgG1 antibody and its effect on cell proliferation was examined after co-incubation with breast cancer cell lines BT-474, SK-BR-3 or liver cancer cell line SK-Hep1.

[0364] (1) IGF2-M5.6 (SEQ ID NO: 76) was linked to the N-terminus of the hFc (SEQ ID NO: 116) of the IgG1 antibody via a linker (GGGGS) 3. The gene encoding the fusion protein was synthesized through codon optimization and gene synthesis, and ligated into the mammalian expression vector pCDNA3.4 under the Xba1 and EcoRV restriction sites. The sequence was then sequenced to verify the correctness of the sequence. This yielded IGF2-M5.6-hFc-pCDNA3.4.

[0365] (2) IGF2-M5.6-pCDNA3.4 was transfected into HEK293-F cells for expression to obtain IGF2-M5.6-hFc protein.

[0366] (3) According to the steps described in Example 1, different concentrations of IGF2-M5.6-hFc protein were co-incubated with the liver cancer cell line SK-Hep1 or the breast cancer cell line BT-474, respectively, and the effect of different concentrations of IGF2-M5.6-hFc on cell growth was detected using Celltiters-glo reagent according to the instructions.

[0367] (4) Following the steps described in Example 1, the breast cancer cell line SK-BR-3 was co-incubated with different concentrations of IGF2-M5.6-hFc protein, and the effect of different concentrations of IGF2-M5.6-hFc on cell growth was detected in real time using an Incucyte instrument.

[0368] (5) Experimental results: As shown in FIG21 , compared with the control PBS, IGF2-M5.6-hFc had no effect on the proliferation of breast cancer cells or liver cancer cells even at high concentrations.

Claims

1. An insulin-like growth factor 2 (IGF2) mutant, characterized in that: The IGF2 mutant has an increased affinity for the insulin-like growth factor 2 receptor (IGF2R).

2. The IGF2 mutant according to claim 1, characterized in that The IGF2 mutant does not bind to IGF1R, or has reduced affinity to IGF1R.

3. The IGF2 mutant according to claim 1, characterized in that The IGF2 mutant has one or more of the following: Compared with wild-type IGF2, The amino acid at position 6 is replaced by a positively charged amino acid or an amino acid whose side chain forms a hydrogen bond with the side chain of another amino acid, preferably by R, K, Q, S, T, C or Y; The amino acid at position 37 is replaced by an aliphatic amino acid with a side chain length of 1-4 carbon atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S; The amino acid at position 43 is replaced by an aliphatic amino acid having a side chain length of 1-4 C atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S.

4. The IGF2 mutant according to claim 1, characterized in that The IGF2 mutant has one or more of the following: Compared with wild-type IGF2, amino acids 1-7 are missing; The amino acid at position 6 is replaced by a positively charged amino acid or an amino acid whose side chain forms a hydrogen bond with the side chain of another amino acid, preferably by R, K, Q, S, T, C or Y; The amino acid at position 7 is replaced with an aliphatic amino acid with a hydrophobic side chain, preferably, with A, I, L or V; The amino acid at position 14 is replaced with an amino acid whose side chain can form a hydrogen bond, preferably, replaced with T, S, C or Y; The amino acid at position 15 is replaced by an aliphatic amino acid or an amino acid whose side chain can form a hydrogen bond with the side chain of other amino acids, preferably by G, A, V, L, I, F, W, N, Q, C, S, T or Y; The amino acid at position 18 is replaced with an aliphatic amino acid having a side chain length of 1-4 carbon atoms, preferably, with Y, G, A, V, L, I, M, N, Q, T, C or S; The amino acid at position 19 is replaced by an aliphatic amino acid with a side chain length of 1-4 carbon atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S; The amino acid at position 27 is replaced by an aliphatic amino acid with a side chain length of 1-4 C atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S; The amino acid at position 37 is replaced by an aliphatic amino acid with a side chain length of 1-4 carbon atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S; The amino acid at position 43 is replaced by an aliphatic amino acid having a side chain length of 1-4 C atoms, preferably, by G, A, V, L, I, M, N, Q, T, C or S.

5. The IGF2 mutant according to claim 1, characterized in that The IGF2 mutant comprises one or more mutations selected from E6R / E6Q, T7A, V14T, D15A, Q18Y, F19L, Y27L, R37A, V43M and R68A, Preferably, compared with wild-type IGF2, the IGF2 mutant has one or more of the following mutations or deletions: (1) The IGF2 mutant comprises a deletion of amino acids 1-7 and mutations Y27L and R37A; (2) the IGF2 mutant comprises mutations R37A and V43M; (3) the IGF2 mutant comprises mutations E6R, R37A and V43M; (4) the IGF2 mutant comprises mutations E6R, R37A, V43M and R68A; (5) the IGF2 mutant comprises mutations E6R, Y27L and V43M; (6) the IGF2 mutant comprises mutations V43M, E6R, Y27L and R37A; (7) The IGF2 mutant comprises mutations V43M, E6R, R37A and V14T; (8) The IGF2 mutant comprises mutations V43M, E6R, R37A and D15A; (9) The IGF2 mutant comprises mutations V43M, E6R, R37A, V14T and D15A; (10) The IGF2 mutant comprises mutations V43M, E6R, R37A and F19L; (11) The IGF2 mutant comprises mutations V43M, E6R, R37A, V14T and F19L; (12) The IGF2 mutant comprises mutations V43M, E6R, R37A, D15A and F19L; (13) The IGF2 mutant comprises mutations V43M, E6R, R37A, V14T, D15A and F19L; (14) The IGF2 mutant comprises a deletion of amino acids 1-7, mutations Y27L, R37A and / or V43M; (15) The IGF2 mutant comprises mutations E6Q, T7A, Q18Y, F19L, Y27L and R37A; (16) The IGF2 mutant comprises a deletion of amino acids 1-7 and / or a Y27L mutation and / or a R37A mutation, as well as a V43M mutation; (17) The IGF2 mutant comprises mutations E6Q and / or T7A and / or Q18Y and / or F19L and / or Y27L.

6. The IGF2 mutant according to any one of claims 3 to 5, characterized in that The wild-type IGF2 has an amino acid sequence as shown in SEQ ID NO: 1, 117 or 118.

7. A construct, characterized in that The construct comprises the IGF2 mutant of any one of claims 1 to 6 and a linker, The construct includes the following structures: AB, Wherein, A represents IGF2 mutant; B represents linker.

8. The construct according to claim 5, characterized in that The construct further comprises a target molecule binding moiety or an Fc domain of an antibody. Preferably, the construct comprises the following structure: ABC, Wherein, A represents an IGF2 mutant; B does not exist or represents a linker; C represents a target molecule binding portion or an Fc domain of an antibody, Preferably, the linker is (G) n , (G m S) n , poly(glycine-alanine), poly(alanine-serine), poly(alanine-proline), wherein n and m are each independently selected from an integer of 1 to 20; Preferably, the linker has GG, GGG or a sequence as shown in any one of SEQ ID NOs: 86 to 115; Preferably, the linker is (G)2, (G)3, (G)4, (G)5, (G)8, (GGGGS)3, (GGGGS)4, ASTKGP, TVAAP or GGGGS; Preferably, the construct has a sequence as shown in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82 or 84.

9. The construct according to claim 7, characterized in that The Fc domain includes the Fc domain of IgG, IgM, IgE, IgA or IgD, preferably, the Fc domain is IgG1, IgG2, IgG3 or IgG4 domain, or The target molecule binding portion is an antibody or an antigen binding fragment thereof, preferably, the target molecule binding portion comprises a multispecific antibody or an antigen binding fragment thereof, more preferably, the antigen binding fragment comprises an antibody heavy chain or light chain, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, Fd, dAb, affibody, HCAb, nanobody, VHH and any combination thereof; or The target molecule binding portion is an antigen protein or a fragment thereof, which recognizes pathogenic autoantibodies or fragments thereof.

10. The construct according to claim 7, characterized in that The target molecule includes a protein, a pathogenic target molecule or a non-protein, preferably includes a soluble protein, a membrane-associated protein, a lipoprotein, a liposome, a nucleic acid, a toxin, a viral particle or a cell. Preferably, the target molecule is a cell surface protein or an extracellular protein; Preferably, the target molecule is a tumor molecule associated with tumor occurrence and / or development and / or metastasis; Preferably, the target molecule is a receptor tyrosine kinase, a growth factor receptor, a cytokine, a mucin, a Siglec receptor or an immune checkpoint regulator; Preferably, the target molecule is human epidermal growth factor receptor 2 (HER2), HER3, epidermal growth factor (EGFR), fibroblast growth factor (FGFRs), vascular endothelial growth factor (VEGFA), mesenchymal epithelial transition factor (c-Met), platelet-derived growth factor receptor (PDGFR), FZD, interleukin-1 receptor (IL1R), PD-L1 / PD-1, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), extramembrane protein α-synuclein, CD20, TIM3, LAG3, TIGIT, CEACAM1, CD25, Ig-like transcription factor 2 (ILT-2), ILT-3, ILT-4, ILT-5, leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), platelet endothelial cell adhesion molecule (PECAM-1, CD31), paired immunoglobulin-like receptor (PILR-α / β), SIRL-1 or SIRP-α, or a functional fragment thereof.

11. The construct according to claim 7, characterized in that The IGF2 mutant is directly or via a linker connected to the N-terminus or C-terminus of the target molecule binding portion or the Fc domain; or The IGF2 mutant is directly or via a linker connected to the N-terminus of the antibody or Fc; or The IGF2 mutant is directly or via a linker connected to the C-terminus of the antibody or Fc; or The IGF2 mutant is directly or via a linker connected to the N-terminus of the antibody heavy chain; or The IGF2 mutant is directly or via a linker connected to the C-terminus of the antibody heavy chain; or The IGF2 mutant is directly or via a linker connected to the N-terminus of the antibody light chain; or The IGF2 mutant is directly or via a linker connected to the C-terminus of the antibody light chain; or The IGF2 mutant is directly or via a linker connected to the C-terminus of the antibody light chain, and connected to the C-terminus of the heavy chain; The IGF2 mutant is directly or via a linker connected to the N-terminus of the antibody light chain, and is connected to the N-terminus of the heavy chain; The IGF2 mutant is directly or via a linker connected to the N-terminus of the antibody light chain, and connected to the C-terminus of the heavy chain; The IGF2 mutant is directly or via a linker connected to the C-terminus of the antibody light chain, and connected to the N-terminus of the heavy chain; In some embodiments, the IGF2 mutant is linked to the C-terminus of the antibody light chain and to the N-terminus of the heavy chain.

12. The construct according to claim 7, characterized in that The A comprises one or more IGF2 mutants.

13. The construct according to claim 12, characterized in that The two or more IGF2 mutants include two or more identical or different IGF2 mutants, Preferably, the two or more IGF2 mutants are linked to the C-terminus or N-terminus of the target molecule binding portion or Fc domain.

14. A nucleic acid molecule encoding the IGF2 mutant of any one of claims 1 to 6 or the construct of any one of claims 7 to 13. An expression vector comprising the nucleic acid molecule of claim 14 .

16. A host cell comprising the nucleic acid molecule of claim 14 or the expression vector of claim 15.

17. A pharmaceutical composition comprising: the IGF2 mutant according to any one of claims 1 to 6, the construct according to any one of claims 7 to 13, the nucleic acid molecule according to claim 14, the expression vector according to claim 15, and / or the host cell according to claim 16; and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, characterized in that The pharmaceutical composition is used to treat a disease; Preferably, the disease is a disease associated with the expression or overexpression of the target molecule. Preferably, the disease is cancer, More preferably, the cancer is selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or cervical cancer, salivary gland cancer, kidney cancer or ureteral cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penis cancer, melanoma, bile duct cancer, central nervous system tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, neuroblastoma, Theca tumors, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas and Ewing's sarcoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphomas, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorders, as well as abnormal vascular proliferation associated with keloids, edema and Meigs' syndrome, brain tumors and brain cancers, and head or neck cancers and related metastatic cancers.

19. A method for treating a disease, characterized in that: The method comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 17 or 18 to a subject in need thereof.

20. The method according to claim 19, characterized in that The disease is a disease associated with the expression or overexpression of the target molecule, Preferably, the disease is cancer, More preferably, the cancer is selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or cervical cancer, salivary gland cancer, kidney cancer or ureteral cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penis cancer, melanoma, bile duct cancer, central nervous system tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, neuroblastoma, Theca tumors, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas and Ewing's sarcoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphomas, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorders, as well as abnormal vascular proliferation associated with keloids, edema and Meigs' syndrome, brain tumors and brain cancers, and head or neck cancers and related metastatic cancers.

21. Use of the IGF2 mutant according to any one of claims 1 to 6 or the construct according to any one of claims 7 to 13 in the delivery of proteins, nucleic acids, small molecule drugs and polypeptides targeted to lysosomes.

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