Constructs for protein degradation and uses thereof

Abstract

The invention provides a novel lysosomal targeting fusion protein based on fusion of an IGF2 mutant and an antibody, and a method for degrading a protein by using the novel protein construct.

Description

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 center of intracellular material degradation, degrading proteins, organelles, and even invading microorganisms through acid hydrolases. Enzymes in mammalian lysosomes are synthesized in the cytoplasm. When lysosomal enzymes or lysosomal localized proteins pass through the endoplasmic reticulum, they undergo N-mannose (mannose carbohydrate) glycosylation modification, and finally in the Golgi apparatus, the protein or enzyme is further modified by mannose-6-phosphate (M6P). M6P-modified proteins are transported to lysosomes by binding to their receptor IGF2R (also called M6PR, CI-MPR or CD222), while IGF2R returns to the cell surface and continues to perform its transport function. IGF2R is a membrane protein with a molecular weight of 300kDa. Its extracellular region contains 15 domains, each of which is composed of 147 amino acids, and the sequence similarity of each domain is between 15-38%. IGF2R plays functions such as protein transport, lysosomal biogenesis, and cell growth regulation. Clinically, IGF2R is targeted to deliver infused recombinant enzymes to lysosomes for the treatment of lysosomal storage diseases.

[0003] Among the 15 extracellular domains of IGF2R, domains 3, 5, and 9 bind to mannose-6-phosphate (M6P) or M6P-modified proteins, while domains 11 and 13 bind to insulin-like growth factor 2 (IGF2). When proteins or enzymes carrying M6P bind to IGF2R, IGF2R-mediated targeted lysosomal endocytosis is induced, thereby transporting M6P-modified proteins or enzymes to lysosomes. Similarly, when IGF2 binds to IGF2R, IGF2R transports IGF2 to lysosomes through endocytosis, thereby mediating the degradation of IGF2, thereby maintaining the balance of IGF2 outside the cell, preventing excessive IGF2 from binding to IGF1R, and then weakening the activation of the IGF1R tyrosine kinase domain.

[0004] Based on the principle of IGF2R-mediated protein or enzyme endocytosis and lysosomal delivery, in the prior art, people modify the target protein or enzyme by M6P or its analogs, so as to transport the target protein or enzyme to the lysosome, in order to treat lysosomal enzyme deficiency. Similarly, in the prior art, there are also researchers who use IGF2 mutants as a medium to deliver the target protein or enzyme to the lysosome, in order to treat lysosomal deficiency; the method is to express the IGF2 mutant fusion with the target protein to prepare a fusion protein of the IGF2 mutant and the target protein. After the IGF2 mutant binds to IGF2R, it mediates the targeted transport of the protein fused with the IGF2 mutant to the lysosome, thereby exerting a biological function (see US patent applications US2003 / 0082176A1, US2004 / 0006008A1, 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 team at Stanford University in the United States first reported a lysosomal targeted degradation technology (LYTAC) based on glycosylated antibodies. Its working principle is as follows: (1) LYTAC molecules are composed of two parts: antibodies and polysaccharides. By coupling polysaccharides to antibodies, LYTAC molecules that can recognize two receptors are prepared, that is, antibodies recognize target membrane proteins (targets to be degraded) and polysaccharides recognize IGF2R receptors; (2) LYTAC double-headed molecules simultaneously bind to the lysosomal shuttle receptor IGF2R and target membrane proteins on the cell membrane to form a ternary complex, induce endocytosis, and then transport the ternary complex into the lysosome; (3) Under the action of lysosomal enzymes, the target protein is degraded by lysosomal enzymes (see US patent application US2022 / 0023434A1), while IGF2R returns to the cell membrane and continues to perform its transport function. However, this technology has the following disadvantages: it is necessary to use chemical reaction means to connect M6P polysaccharides to antibodies. Since the coupling of M6P polysaccharide to antibody requires the help 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, Bertozzi's research group 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 cycloalkyne-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 glycosylation-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. In addition, non-natural amino acids are not 100% inserted, and the fidelity of their insertion has also become an aspect affecting uniformity; (3) The product preparation process is complex and the yield is low, which is not conducive to the subsequent industrial promotion.

[0007] By fusion-expressing wild-type IGF2 with specific antibodies, a dual-head molecule that can target IGF2R and the target can be prepared. Although LYTAC can be constructed, studies have shown that wild-type IGF2 will bind to both IGF2R and IGF1R. And after IGF2 binds to IGF1R, it will activate the tyrosine kinase activity of IGF1R, leading to the occurrence of tumors. We also found in previous studies that wild-type IGF2 will promote the proliferation of tumor cells (BT-474 and SK-Hep1). Therefore, there is a greater risk of carcinogenesis when constructing a protein degradation system using wild-type IGF2.

[0008] Therefore, there is an urgent need for a protein degradation technology that can 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, and a method for degrading proteins using the novel protein construct.

[0010] In a first aspect, the present invention provides an IGF2 mutant having an 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 the amino acids 1 to 7 of the IGF2 mutant can be independently replaced by any one amino acid.

[0014] In some embodiments, the amino acid at position 6 of the IGF2 mutant can be replaced by a positively charged amino acid, preferably by 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 by an amino acid whose side chain forms a hydrogen bond with the side chain of other amino acids, preferably, replaced by Q, S, T, C or Y. For example, the IGF2 mutant can have mutations 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 by an amino acid whose side chain can form a hydrogen bond, preferably, replaced by 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, replaced by G, A, V, L, I, F, W, N or Q. For example, the IGF2 mutant can have mutations 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 replaced by an amino acid whose side chain can form a hydrogen bond with the side chain of other amino acids, preferably, replaced by 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 C atoms, preferably, replaced by Y, G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have mutations 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 by an aliphatic amino acid with a side chain length of 1-4 C atoms, preferably, replaced by G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have mutations 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 C atoms, preferably, replaced by G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have 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 C atoms, preferably, replaced by G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have mutations 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 by an aliphatic amino acid with a side chain length of 1-4 C atoms, preferably, replaced by G, A, V, L, I, M, N, Q, T, C or S. For example, the IGF2 mutant can have mutations 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 IGF2 wild type (1-67) mentioned in the present disclosure has its amino acid numbering corresponding to the amino acid sequence shown in SEQ ID NO:1.

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

[0051] The wild-type IGF2 precursor (1-156) mentioned in the present disclosure has its amino acid numbering corresponding 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 integers 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] Those skilled in the art should understand 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 a sequence as shown 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 having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity thereto, or consists thereof.

[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 moiety can be an antigenic protein or a fragment thereof. In some embodiments, the target molecule binding moiety can be an antigenic protein or a fragment thereof that recognizes pathogenic autoantibodies. In some embodiments, the antigenic protein or a fragment thereof can include, for example, but not limited to, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 13 (ADAMTS13), steroidogenic cytochrome P450 enzyme 21-hydroxylase, N-methyl-D-aspartate-(NMDA)-receptor, red blood cells, anti-smooth muscle antibody (ASMA), actin, platelets, signal recognition particle (SRP), 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), myosin, sperm, amylase alpha 2, type XVII collagen (col17), kallikrein 13, type VII collagen (col7), myeloperoxidase (MPO), type IV collagen, proteinase 3 (PR3), thyroid stimulating hormone receptor (TSHR), thyroglobulin, thyroid peroxidase (TPO), thyroglobulin, thyroid peroxidase (TPO), platelets, myeloperoxidase (MPO), muscle nicotinic acetylcholine receptor, muscle-specific kinase (MuSK), low density lipoprotein receptor-related protein 4 (LRP4), myosin, beta1-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), citrullinated cyclic protein, RNA-binding protein (Ro), La, double-stranded DNA (dsDNA), angiotensin II type 1 receptor (AT1R), endothelin-1A receptor (ETAR), insulin, glutamic acid 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 includes a full-length antibody or an antigen-binding fragment thereof.

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

[0075] In some embodiments, the antigen-binding fragment includes a heavy 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 (MEM), 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 their functional fragments.

[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 repeated forms 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 repeated forms 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 an expression vector for a mammalian host cell, 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, penis cancer, melanoma, bile duct cancer, central nervous system (CNS) tumors, spinal axis tumors, brain stem gliomas, multiforme glioblastomas, astrocytic tumors, The invention relates 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 vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors) and Meigs syndrome, brain tumors and brain cancers, and head or neck cancers and related metastatic cancers.

[0116] In the seventh aspect, the present invention provides a method for treating a disease, the method 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 medicament 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, penis cancer, melanoma, bile duct cancer, central nervous system (CNS) tumors, spinal axis tumors, brain stem gliomas, multiforme glioblastomas, astrocytic tumors, The invention relates 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 vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors) and Meigs syndrome, brain tumors and brain cancers, and head or neck cancers 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 LYTAC and LYTAF provided by the present disclosure are compared in an exemplary manner. Among them, LYTAC needs to undergo a chemical reaction to connect the antibody to the polysaccharide, while LYTAF can be obtained by expression without chemical modification.

[0122] Figure 2 The schematic diagram of LYTAF-mediated membrane protein degradation is shown as an example.

[0123] Figure 3 The results of the affinity test of IGF2 mutants to IGF2R-D11 (the 11th domain of IGF2R) are shown.

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

[0125] Figure 5The results of the tests on the affinity of wild-type and mutant IGF2 to IGF2R are shown.

[0126] Figure 6 The results of the tests on the affinity of IGF2 mutants to IGF1R are shown.

[0127] Figure 7 The results of the detection of HER2-targeted LYTAF-M5.6-mediated degradation of HER2 membrane protein in multiple breast cancer cell lines are shown.

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

[0129] Fig. 9 It shows that LYTAF-M5.6 inhibits the growth of breast cancer cells SK-BR-3. D0: Photo taken just after being placed in Incucyte; D2: Photo taken after the cell culture plate was placed in Incucyte for 48 hours; D4: Photo taken after the cell culture plate was placed in Incucyte for 96 hours; D6: Photo taken after the cell culture plate was placed in Incucyte for 144 hours; D8: Photo taken after the cell culture plate was placed in Incucyte for 192 hours. The vertical axis is the concentration of LYTAF-M5.6 or the control.

[0130] Fig.10 It was shown that LYTAF-M5.6 inhibited the proliferation of breast cancer cell line BT-474. Fig.10 A. Results of cell proliferation assay by Incucyte on day 6 after administration of LYTAF (0-100 nM). The vertical plot was repeated three times. Fig.10 B. Six days after administration of LYTAF (0-100 nM), the cell activity was detected using Celltiters reagent.

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

[0132] Fig.12 LYTAF was shown to mediate degradation of HER2 and EGFR in animals.

[0133] Fig.13 It was shown that LYTAF mediated the degradation of EGFR in hepatoma cell SNU-449.

[0134] Fig.14 LYTAF was shown to mediate degradation of EGFR and c-Met in the non-small cell lung cancer cell line NCI-H1975.

[0135] Fig.15 LYTAF is shown to mediate degradation of HER2 membrane protein in breast cancer cells SUM159PT.

[0136] Fig.16 LYTAF was shown to mediate degradation of HER2 membrane protein in breast cancer T47D cells.

[0137] Fig.17 LYTAF was shown to mediate degradation of HER2 membrane protein in breast ductal carcinoma cells HCC 1954.

[0138] Fig.18 LYTAF was shown to mediate degradation of HER2 membrane protein in breast cancer cells MDA-MB-435.

[0139] Fig.19 It was shown that LYTAF mediates the degradation of c-Met membrane protein in hepatoma cell MHCC-97H. Fig.19 The Ac-Met control antibody did not mediate the degradation of c-Met. Fig.19 After the Bc-Met control antibody was connected with IGF2-M5.6, the fusion expressed protein LYTAF mediated the degradation of c-Met.

[0140] Fig. 20 The results show that wild-type IGF2 promotes the proliferation of breast cancer cells and liver cancer cells. Fig. 20 a shows the cell growth of BT-474 cells treated with different concentrations of IGF2 wild-type protein within 72 hours. Fig. 20 b Taking the coverage rate of the PBS-treated group as a reference, the proliferation of BT-474 cells in the PBS-treated group at 0h and 72h and the groups treated with different concentrations of IGF2 were compared. Fig. 20 c Photographs showing the growth density of BT-474 cells in the PBS-treated group and the hFc-IGF2-treated group at 0 h and 72 h. Fig. 20 d shows the cell growth of SK-Hep1 cells treated with different concentrations of IGF2 wild-type protein within 72 hours. Fig. 20e Using the coverage rate of the PBS-treated group as a reference, the proliferation of SK-Hep1 cells in the PBS-treated group at 0h and 72h and in the hFc-IGF2-treated groups at different concentrations were compared. Fig. 20 f Photographs showing the growth density of SK-Hep1 cells in the PBS-treated group and the hFc-IGF2-treated group at 0 h and 72 h.

[0141] Fig.21 The results show that the IGF2 mutant M5.6 has no effect on breast cancer cells and liver cancer cells. Fig.21 a shows the effect of different concentrations of IGF2-M5.6-hFc protein (also known as the 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. Fig.21 b shows the effect of different concentrations of IGF2 mutant M5.6 on the proliferation of breast cancer cells SK-BR-3. DETAILED DESCRIPTION

[0142] Glycosylation-modified antibody LYTAC ( Figure 1 , left) can achieve protein degradation, but its disadvantages are also obvious. It mainly manifests in the need to modify the antibody by glycosylation through chemical reaction, making it difficult to obtain uniform molecules, and the process is relatively complicated, which is not conducive to subsequent drug development and industrial application.

[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 not only binds to IGF2R, but also binds to IGF1R. After binding to IGF1R, IGF2 will induce the tyrosine kinase activity of IGF1R, activate the tumor signaling pathway, and induce tumor proliferation. The present disclosure obtains an IGF2 mutant with low affinity to IGF1R by modifying IGF2, thereby avoiding the risk of inducing tumor occurrence. Therefore, the present disclosure uses genetic engineering methods to design and screen an IGF2 mutant with high affinity to IGF2R and low affinity to IGF1R, which is beneficial to avoid inducing tumor occurrence.

[0145] Compared with the previously reported IGF2 mutants used for protein targeted lysosomal delivery and thus 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, has higher selectivity, and is a new type of IGF2 mutation with better advantages in applications such as drug delivery and protein degradation.

[0146] The IGF2 mutants screened in the present disclosure can be fused and expressed with an antibody that specifically recognizes a target protein (such as HER2) through a linker ( Figure 1 , right). This new fusion protein double-headed molecule is named Lysosomal Targeting Fusion Protein (LYTAF). LYTAF uses the IGF2 mutant at one end of the linker to recognize the lysosomal shuttling 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 previously reported LYTAC, LYTAF can be obtained through mammalian cell expression without further chemical reaction steps. The process is simple and solves the problem of product uniformity, making it easier to apply and transform.

[0147] Our research shows that LYTAF can mediate the targeted degradation of a variety of 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 in the present invention can be connected 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] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of 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] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term 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, etc.

[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. The antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. The basic 4-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 connected to the H chain by a covalent disulfide bond, and the two H chains can be connected 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] The "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) Fab fragments - monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments - bivalent fragments comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, and (v) dAb fragments 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 an antigen-binding domain of an antibody if it is 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 of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises CH2 and CH3 constant domains in each of the two heavy chains of the antibody; 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 immunoglobulin domains Cγ2 and Cγ3 and a 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 the C226 or P230 position of the heavy chain (or the amino acid between these two amino acids) to the carboxyl terminus, wherein the numbering is performed according to the EU index in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, that is, it extends from about amino acid 341 of IgG to about amino acid 447 (including the C-terminal lysine). As used herein, the Fc region can be a native sequence Fc, including any allotype variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to the 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] Host cells suitable for the present disclosure may contain expression vectors (constructs), such as plasmids, etc., introduced, for example, via transformation, transfection, infection or injection. The vector has a coding sequence or part thereof encoding a 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 techniques are described in, for example, Sambrook, J. et al., Molecular Cloning Laboratory Guide (Fourth 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 being introduced into the host cell, other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell, thereby replicating 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"). Generally, expression vectors useful in recombinant DNA technology are usually 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 are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

[0159] "Specific binding" means that the antibody binds to a specific antigen with greater affinity than to other antigens. Generally, when the equilibrium dissociation constant (K D ) is about 1×10 -8 M or less, an antibody is considered to "specifically bind", for example, about 1 × 10 - 9 M or smaller, about 1×10 -10 M or smaller, about 1×10 -11 M or less, 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 effective form permitting the biological activity of the active ingredient contained therein, 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, which 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 "treatment" 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 the adverse effects caused by the disease can be partially or completely cured, the effect is therapeutic. As used herein, "treatment" covers 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., preventing its development; (c) relieving the disease, i.e., causing the disease to regress.

[0164] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and 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] Amino Acids Three letters Single letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamate Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0169] Before further describing the present disclosure, it should be understood that the present disclosure is not limited to the particular embodiments described, as it may also vary. It should also be understood that the terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting.

[0170] 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 otherwise localized to the cell surface, such as a protein associated with a membrane protein.

[0171] 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:

[0172] Table 2

[0173]

[0174]

[0175] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching 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. Likewise, 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.

[0176] The sequence identity percentage 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 the sequence identity percentage. The comparison of the sequence and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, a part of the EMBOSS suite of Needle, Stretcher, Water or Matcher, bioinformatics programs, and can also be obtained at www.ebi.ac.uk / Tools / psa.

[0177] In some embodiments, the pharmaceutical composition may include a therapeutically or preventively effective amount of an active ingredient as described above, which is usually formulated into a desired composition together with a pharmaceutically acceptable carrier after sufficient purification. Pharmaceutically acceptable carriers include excipients, diluents, antioxidants, preservatives, colorants, flavoring agents, diluents, emulsifiers, suspending agents, solvents, fillers, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, antimicrobial agents, and surfactants.

[0178] The composition can be in liquid or powder or lyophilized form, and can include one or more lyoprotectants, excipients, surfactants, or fillers. The composition of the present invention can be suitable for parenteral administration, such as injection or infusion into an animal by any of the following routes: intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intralesional, rectal, percutaneous, oral and inhalation routes.

[0179] Example

[0180] Example 1

[0181] (1) Design and screening of IGF2 mutants that specifically recognize IGF2R

[0182] (1.1) Based on the amino acid sequence of human wild-type IGF2 (IGF2-WT) (SEQ ID NO: 1), the first round of mutations of IGF2 were designed, and these mutations were connected to the C-terminus of the human IgG1 antibody hFc. The specific information is shown in Table 3.

[0183] Table 3. Amino acid and nucleic acid sequence numbers of IGF2 mutations and fusion proteins designed in the first round.

[0184]

[0185] (1.2) Wild-type IGF2 or its mutant is connected via a linker (G) 5 The amino acid sequence of the hFc fused to the C-terminus of the IgG1 antibody is shown in seq2.1 to seq8.1, and the encoding DNA sequence is shown in seq2.2 to seq8.2.

[0186] (1.3) Wild-type IGF2 or its mutant is connected via a linker (G) 5 The LYTAF molecule targeting HER2 is obtained by fusion expression to the C-terminus of the heavy chain of the anti-HER2 antibody Pertuzumab antibody and co-expression with the light chain (PtzL) of the Pertuzmab antibody. The amino acid sequence of the Pertuzumab heavy chain after connection with 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.

[0187] (1.4) Synthesize seq2.2 to seq8.2 genes and ligate them to the EcoRV and XbaI restriction sites of pCDNA3.4 plasmid to obtain seq2.2-pCDNA3.4 to seq8.2-pCDNA3.4.

[0188] (1.5) Seq2.2-pCDNA3.4 to seq8.2-pCDNA3.4 were transfected into HEK293-F cell lines respectively, and the following fusion proteins were obtained after expression and purification: 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:

[0189] (1.5.1) Expi-293F cells were cultured in suspension in OPM-293CD05 Medium (OPM). The cell culture conditions were 37°C, 8% CO 2 The shaking speed was 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.

[0190] (1.5.2) PEI and plasmid were mixed in a ratio of 1:1. The final concentration of the plasmid was 1 μg / mL. That is, 1 mg PEI solution and 1 mg plasmid were added to 1 L of culture medium. 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 placed at 37°C and 8% CO. 2 , continue to culture in a shaker at 100 rpm. 24 hours after transfection, add protein expression enhancer (1 mL of 1 M sodium butyrate per 1 L of culture medium) and 200 mL of fresh culture medium. After 5-7 days of culture, centrifuge and collect the culture supernatant for purification.

[0191] (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). 100 mM glycine (pH = 3.0) was used as eluent for elution. Before elution, a corresponding volume of Tris-HCl pH = 8.5 neutralizing solution was added to the collection tube (eluent: neutralizing solution = 10:1). After dialysis and desalting, the target protein was obtained.

[0192] (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 through a linker, see Table 3) and PtzHL (Pertuzumab antibody control).

[0193] (1.6.1) Expi-293F cells were cultured in suspension in OPM-293CD05 Medium (OPM) at 37°C and 8% CO 2 , the shaking speed was 100 rpm. When the cells grew to 2.5×10 6 When the density of the cells reached 1 / mL, transfection was started. The transfection reagent used was PEI solution from Polysciences.

[0194] (1.6.2) The concentrations of PEI and plasmid are mixed in a 1:1 ratio, and the final concentration is 1 μg / mL, that is, 1 mg PEI solution and 1 mg plasmid (the amount of antibody heavy chain and light chain is 0.5 mg respectively) need to be added to 1L of culture medium. The plasmid and PEI mixture is slowly added dropwise to the culture medium and continued to be placed at 37°C and 8% CO 2 Continue to culture in a shaker at 100 rpm. 24 hours after transfection, add protein expression enhancer and 200 mL of fresh culture medium. After 5-7 days of culture, collect the supernatant for purification.

[0195] (1.6.3) PtzHL-IGF2-M7, PtzHL-IGF2-M8, PtzHL-IGF2-M9, PtzHL-IGF2-M10, PtzHL-IGF2-M11, PtzHL-IGF2-M12, PtzHL-IGF2-M13 and PtzHL were purified using Protein A filler. 100 mM glycine (pH = 3.0) was used as eluent for elution. Before elution, a corresponding volume of Tris-HCl pH = 8.5 neutralizing solution was added to the collection tube (eluent: neutralizing solution = 10:1). After dialysis desalting and purification by molecular sieve or ion exchange column, the above proteins were obtained.

[0196] (1.7) Screening of IGF2 mutants with high affinity to IGF2R using ELISA

[0197] (1.7.1) Expression and purification of the 11th domain of IGF2R

[0198] The coding gene of the 11th domain of IGF2R (D11-IGF2R) was synthesized and constructed into the EcoRV and XbaI double restriction sites of the pCDNA3.4 plasmid to obtain the expression plasmid D11-IGF2R-pCDNA3.4. After transfection of D11-IGF2R-pCDNA3.4 into mammalian cells HEK-293F for expression, the IGF2R-D11 protein was obtained after purification by nickel column and molecular sieve for standby use. The amino acid sequence of IGF2R-D11 is shown in seq18.1 (SEQ ID NO: 36), and its DNA sequence is shown in seq18.2 (SEQ ID NO: 37).

[0199] (1.7.2) The mutants with high affinity to IGF2R-D11 protein were screened using the ELISA method as follows.

[0200] (1.7.2.1) Use coating solution (0.05M carbonate buffer, pH 9.6) to dilute IGF2R-D11 protein to 1μg / μL, add to 96-well ELISA plate, add 150μL of coating solution to each well, and incubate at 4℃ overnight.

[0201] (1.7.2.2) Wash 4 times with PBST (0.05% Tween 20 + PBS).

[0202] (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.

[0203] (1.7.2.4) Wash 4 times with PBST (0.05% Tween 20 + PBS).

[0204] (1.7.2.5) Add 100 μL of different concentrations of gradient diluted hFc-IGF2 mutant samples to each well and incubate at 37°C for 1.5 h.

[0205] (1.7.2.6) Wash 4 times with PBST (0.05% Tween 20 + PBS).

[0206] (1.7.2.7) Dilute the secondary antibody (anti-hFc-HRP) with 2% BSA at a ratio of 1:5000, add 100 μL to each well, and incubate at 37°C for 1.5 h.

[0207] (1.7.2.8) Wash 5 times with PBST (0.05% Tween 20 + PBS).

[0208] (1.7.2.9) Add 100 μL / well of colorimetric reagent (TMB, Solebol) and incubate at room temperature in the dark for 1-10 min.

[0209] (1.7.2.10) Add 50 μL of stop solution (Solarbio) to each well to terminate the reaction;

[0210] (1.7.2.111) Detect the absorbance at 450 nm using an ELISA reader.

[0211] (1.7.3) The results of affinity screening of IGF2 mutants are as follows Figure 3 shown.

[0212] from Figure 3 The results show that 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 and IGF2R.

[0213] (1.8) Through the tumor cell proliferation inhibition experiment, it was screened and verified that Pertuzumab with an IGF2 mutant including the amino acid sequence shown in Seq7.1 linked to the C-terminus had better anti-tumor activity.

[0214] As shown in Table 3, in Seq9.1 to Seq15.2, we connected different IGF2 mutants to the C-terminus of the heavy chain of Pertuzumab, and obtained a molecule fused with the antibody and the IGF2 mutant by expressing it with the light chain PtzL of the Pertuzumab antibody. The molecule contains the complete H chain and L chain of Pertuzumab, and the IGF2 mutant connected to the H chain of Pertuzumab.

[0215] We co-incubated different concentrations of Pertuzumab (PtzHL) or Pertuzumab fused with different IGF2 mutations (PtzHL-IGF2-M7 to PtzHL-IGF2-M13) with breast cancer cell line SK-BR-3, and monitored the cell growth in real time using the Incucyte instrument. We screened IGF2 mutants that could enhance the anti-tumor activity of Pertuzumab based on the cell growth. The specific steps are as follows:

[0216] (1.8.1) SK-BR-3 cells were seeded into 96-well cell culture plates at a number of 2000 cells per well. After 12 hours, PBS or Pertuzumab or different Pertuzumab and IGF2 mutant fusion proteins (PtzHL-IGF2-M7 to PtzHL-IGF2-M13) were added to the cell culture medium, and the cells were placed in the Incucyte instrument to monitor the cell growth in real time.

[0217] (1.8.2) Set the Incucyte instrument parameters, take photos every 6 hours, monitor continuously for 96 hours, collect statistics, and perform analysis.

[0218] (1.8.3) Experimental results

[0219] like Figure 4 As shown, PtzHL-M12 has a better anti-tumor growth effect than the control antibody Pertuzumab (PtzHL). Since M12 and M7 have the same IGF2 mutation, it shows that the IGF2 mutation (E6R / R37A / V43M) has a basis for further development. At the same time, we found that compared with the PBS-treated group, wild-type IGF2 promoted tumor proliferation, verifying our previous assumption that wild-type IGF2 has the effect of promoting tumor growth, and the application prospects of unmodified IGF2 are very limited.

[0220] (1.9) Summary:

[0221] (1.9.1) We preliminarily screened the affinity of IGF2 mutations with the receptor IGF2R by ELISA. We found that the fusion protein with IGF2-M5 mutation linked to the C-terminus of hFc had significantly improved affinity compared to wild-type IGF2, and among all mutants, it had the best affinity.

[0222] (1.9.2) Through cell proliferation inhibition experiments, we preliminarily screened the fusion proteins with different IGF2 mutations linked to the C-terminus of the Pertuzumab heavy chain. The results showed that the fusion protein with IGF2-M5 linked to the C-terminus of the Pertuzumab heavy chain (PtzH-IGF2-M12) had the best anti-breast cancer cell proliferation effect in all tests.

[0223] (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.

[0224] (2.0.1) Further using IGF2-M5 (E6R / R37A / V43M) as a template, we further designed second-site mutations of mutants as shown in Table 4 below. Among them, IGF2-M5.1 was mutated based on the precursor of IGF2 (the new amino acid sequence of the IGF2 precursor is shown in Seq1.3, SEQ ID NO: 3). Among them, IGF2-M5.2 was the mutant made based on the precursor of IGF2 (the new amino acid sequence of the IGF2 precursor is shown in Seq1.2, SEQ ID NO: 2).

[0225] Table 4. Design new IGF2 mutants based on IGF2-M5 mutations.

[0226]

[0227]

[0228] Screening work was carried out based on the IGF2 mutants shown in Table 4, and their sequences and numbers are as above.

[0229] (2.0.2) After codon optimization of the DNA sequences corresponding to seqM5.1-2 to seqM5.10-2, they were ligated to the EcoRV and XbaI restriction sites of the pCDNA3.4 plasmid to obtain seqM5.1-pCDNA3.4 to seqM5.10-pCDNA3.4. The IGF2 mutants and the hFc fragment were linked by a GGGGS linker.

[0230] (2.0.3) SeqM5.1-pCDNA3.4 to seqM5.10-pCDNA3.4 were transfected into HEK293-F cell lines respectively, 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, hFc-IGF2-M5.10. The transfection and purification methods are as follows:

[0231] (2.0.3.1) Expi-293F cells were cultured in suspension in OPM-293CD05 Medium (OPM) at 37°C and 8% CO 2 , the shaking speed was 100 rpm. When the cells grew to 2.5×10 6 When the density reaches 1.5447 / mL, transfection was started.

[0232] (2.0.3.2) The concentrations of PEI and plasmid are mixed in a 1:1 ratio, and the final concentration is 1 μg / mL. That is, 1 mg PEI solution and 1 mg plasmid need to be added to 1 L of culture medium, and the ratio of the two is 1:1. Slowly add the plasmid and PEI mixture dropwise to the culture medium and continue to place at 37°C and 8% CO 2 , and continue culturing in a shaker at 100 rpm. 24 hours after transfection, add protein expression enhancer and culture for 5-7 days, then collect the supernatant for purification.

[0233] (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). 100 mM glycine (pH = 3.0) was used as eluent for elution. Before elution, a corresponding volume of Tris-HCl pH = 8.5 neutralizing solution was added to the collection tube (eluent: neutralizing solution = 10:1). After dialysis desalting and molecular sieve purification, the protein was obtained.

[0234] (2.0.4) The ELISA method was used to screen IGF2 mutants (hFc-IGF2-M5.1 to hFc-IGF2-M5.10) with high affinity to IGF2R.

[0235] from Figure 5 From the results, 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).

[0236] (2.0.5) Screening of IGF2 mutants with low affinity for IGF1R using ELISA

[0237] The affinity of IGF2 mutants to IGF1R was tested by ELISA. The amino acid sequence of 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 to pCDNA3.4, expressed in mammalian cells HEK293F, and purified by nickel column.

[0238] The results are shown in Figure 6 Wherein M2 is a protein expressed by fusion of IGF2 mutant and hFc reported in previous patents (US2003 / 0082176 A1, US2004 / 0006008A1, US2005 / 0244400A1). Figure 6 The results show that the affinity of M2 to IGF1R is similar to that of the wild type, and all mutants provided in the present disclosure significantly reduce the affinity of IGF2 to IGF1R, among which the IGF2-M5.6 mutation has the lowest affinity to IGF1R and has no affinity to IGF2R. Except for IGF2-M2, whose affinity is similar to that of wild-type IGF2, the other mutants selected in this experiment significantly reduce their affinity to IGF1R.

[0239] (2) Designing a fusion protein of IGF2-M5.6 and pertuzumab, LYTAF, and testing its ability to mediate the degradation of membrane protein HER2

[0240] (2.1) Expression and purification of LYTAF and antibodies

[0241] The IGF2 mutant IGF2-M5.6 was linked to a linker (GGGGS) 3The fusion protein PtzH-IGF2-M5.6 connected to the C-terminus of the heavy chain of the Pertuzumab antibody (amino acid sequence see seq19-1 (SEQ ID NO: 38), DNA sequence see seq19-2 (SEQ ID NO: 39)) was prepared. The coding gene of the fusion protein was connected to the EcoRV and XbaI restriction sites of the plasmid pCDNA3.4 by genetic engineering means to obtain PtzH-IGF2-M5.6-pCDNA3.4. At the same time, the coding gene of the light chain of the Pertuzumab antibody (amino acid sequence see seq16-1, DNA sequence see seq16-2) was connected 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.

[0242] (2.2) The purified LYTAF-M5.6 protein or Pertuzumab antibody was co-incubated with HER2-positive breast cancer cell lines BT474, T47D, Sum159P, and JIMT-1 for 36 h, respectively. The cells were washed three times with PBS, lysed and collected, and the total HER2 content in the cells was detected by Western blot. The results are shown in Table 1. Figure 7 As shown, different concentrations of the control antibody Pertuzumab do not mediate the degradation of HER2, while the LYTAF-M5.6 molecule can efficiently mediate the degradation of HER2 at low concentrations.

[0243] (3) LYTAF efficiently mediates HER2 protein internalization

[0244] like Figure 8 A is a schematic diagram showing LYTAF-mediated antibody internalization into lysosomes.

[0245] To verify this process, this example used IgG-647 secondary antibody and LYTAF-M5.6 to co-incubate breast cancer cells SK-BR-3 cells. The antibody at one end of LYTAF-M5.6 recognized the secondary antibody IgG-647, and the other end (IGF2-M5.6) recognized the IGF2R on the cell membrane, thereby forming a ternary complex. The ternary complex carries IgG-647 into the lysosome through endocytosis.

[0246] Briefly, mammalian cells SK-BR-3 were inoculated into a cell culture dish (glass bottom dish). After 24 hours, IgG-647 (rabbit anti-human) with a final concentration of 20nM and LYTAF or control antibody Pertuzumab were added, and incubated in a cell culture incubator at 37°C for 1 hour. The cells were washed 5 times with HBSS solution, and then 50nM Lysotracker was added and incubated in a cell culture incubator at 37°C for 10 minutes. After washing 3 times with HBSS solution, Hoechest was added and incubated at room temperature for 5 minutes, and the localization of IgG-647, lysosomes and cell nuclei was detected using a confocal microscope. The results are shown in Figure 8 In B.

[0247] like Figure 8 B shows that we can see that IgG-647 co-localizes with the lysosomal dye lysotracker, which fully demonstrates that LYTAF can mediate the internalization of IgG-647 and enter the lysosome.

[0248] (4) LYTAF-M5.6 significantly inhibits tumor cell proliferation

[0249] (4.1) SK-BR-3 cells were seeded into 96-well plates at a rate of 2000 cells / well. LYTAF-M5.6 of different concentrations was added 12 hours later and placed in an Incucyte instrument to monitor cell growth in real time. Fig. 9 As shown, LYTAF-M5.6 significantly inhibited cell growth, and cell proliferation decreased with increasing LYTAF-M5.6 concentrations. When the concentration of LYTAF-M5.6 reached 200 nM, SK-BR-3 cells did not grow at all.

[0250] (4.2) Unlike LYTAF, which significantly inhibited the proliferation of SK-BR-3 cells, Pertuzumab and hFC-IGF2 could not significantly inhibit cell proliferation, which fully demonstrated that LYTAF significantly inhibited tumor growth compared with the control antibody.

[0251] (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.

[0252] (5.1) Fig.10 As shown in Figure 2, we used two experiments to detect the inhibitory activity of LYTAF on breast cancer cells BT-474. The specific method is as follows: BT-474 cells were inoculated into a 96-well transparent plate at a concentration of 2000 cells / well. After 24 hours, different concentrations of LYTAF were added and placed in the Incucyte instrument to monitor the growth of cells in real time, and the results were obtained by taking photos. Fig.10 As shown in A.

[0253] (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 concentration of 2000 / well, and different concentrations of LYTAF were added after 24 hours. Each concentration was repeated 3 times and placed in a CO 2 The cells were cultured in an incubator 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 Fig.10 B. The specific method is as follows:

[0254] (5.2.1) BT474 cells were evenly plated in a 384-well plate, with 800 cells per well.

[0255] (5.2.2) After 6 hours, a total of 15 μL of serially diluted 4-fold concentration LYTAF-M5.6 and cell culture medium was pipetted into a 384-well plate containing 45 μL of culture medium sample (with a FBS content of 4%).

[0256] (5.2.3) In CO 2 After incubation at 37°C in a cell culture incubator for 5 days, the cell culture plate was taken out and equilibrated at room temperature for 30 minutes.

[0257] (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).

[0258] (5.2.5) Mix on an orbital shaker for 2 min to induce cell lysis.

[0259] (5.2.6) After incubation at room temperature for 10-15 min, measure and record the luminescent signal on a microplate reader.

[0260] (5.3) Experimental results

[0261] This experiment used two methods to detect the inhibitory effect of LYTAF-M5.6 on the growth of breast cancer cells BT474. Fig.10 As shown in A, the growth rate of cells was monitored using the Incucyte instrument. When LYTAF-M5.6 reached 25 nM, the growth of cells was significantly inhibited. Fig.10 As shown in B, using 2.0 reagent was used to detect the activity of cells, and it was found that LYTAF-M5.6 significantly inhibited the proliferation of tumor cells.

[0262] (6) LYTAF inhibits the proliferation of xenograft tumors in nude mice

[0263] (6.1) Experimental animals

[0264] Fifty specific pathogen free (SPF) healthy female Balb / c nude mice, weighing 15-25 g and aged 4 weeks, were purchased from Jicui Yaokang.

[0265] (6.2) Animal model establishment

[0266] A breast cancer model was constructed using JIMT-1 cells in xenografted Balb / c nude mice to evaluate the efficacy and safety of LYTAC against breast cancer. JIMT-1 cells were digested and centrifuged at 1000rpm for 5min, washed twice with PBS, and resuspended in PBS containing 50% matrix gel. Each mouse was inoculated with 5e6 cells / 100ul / mouse. The weight of the mice was recorded every 3-4 days after inoculation. During the experiment, the mice were free to eat. The feeding and killing of mice were in accordance with animal welfare principles and ethical standards.

[0267] (6.3) Animal grouping

[0268] 40 Balb / c nude mice were fed with ordinary feed and tap water, and the room temperature was controlled at 25℃±1℃, relative humidity was 60%-70%, and the feeding conditions were level 11. They were allowed to adapt to the new environment for three days. 3 Balb / c nude mice were randomly divided into 4 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.

[0269] Table 5

[0270]

[0271]

[0272] Note: JIMT-1 cells are a Transtuzumab-resistant cell line, so we added a Transtuzumab group while testing Pertuzumab and LYTAF-M5.6.

[0273] (6.4) Administration

[0274] Tumor grows to 70-100 mm 2 Then the drug was started. In the first 3 weeks, the drug was administered twice a week, with an interval of 3-4 days. In the next two weeks, the drug was administered once a week, for a total of 5 weeks. According to the protocol, the positive drug and LYTAC were prepared to 1 mg / mL, and the volume of each administration was 200 μL.

[0275] (6.5) Animal killing and specimen collection

[0276] (6.5.1) Animal sacrifice and blood collection

[0277] On the second day after administration, blood was collected using the eyeball blood sampling method. The specimen was placed in a polyethylene test tube containing an anticoagulant, centrifuged at 6500rpm for 15 minutes, and the supernatant was placed in an EP tube after centrifugation and stored in a -80℃ refrigerator for testing. After collection, the mice were killed by cervical dislocation.

[0278] (6.6) Tumor tissue isolation

[0279] The tumor tissue was separated, washed with PBS and the moisture on the surface of the tissue was removed. A portion of the tissue was fixed with 4% paraformaldehyde to prepare paraffin sections, and the other portion was quickly frozen in liquid nitrogen for later use.

[0280] (6.7) Viscera separation

[0281] The liver and kidney tissues were separated, washed with PBS and the surface moisture of the tissues was removed, then fixed with 4% paraformaldehyde and prepared into paraffin sections for later use.

[0282] (6.8) Experimental results

[0283] The experimental results are shown in Fig.11 In. From Fig.11 It can be seen that LYTAF significantly inhibited the growth of tumor-bearing mice compared with the control antibodies Pertuzumab (Ptz) or Trantuzuma (TRZ) ( Fig.11 A), but did not affect the body weight of mice ( Fig.11 B).

[0284] (7) LYTAF mediates HER2 degradation in the JIMT1 xenograft Balb / c nude mouse breast cancer model

[0285] (7.1) Construction of JIMT1 xenograft Balb / c nude mouse breast cancer model

[0286] JIMT-1 cells were digested and centrifuged at 1000 rpm for 5 min, washed twice with PBS, and resuspended in PBS containing 50% Matrigel. Each mouse was inoculated with 5e6 cells / 100ul / mouse. The weight of the mice was recorded every 3-4 days after inoculation.

[0287] During the experiment, mice were fed freely. The feeding and killing of mice were in accordance with animal welfare principles and ethical standards.

[0288] (7.2) Administration

[0289] 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.

[0290] (7.3) Extraction method of total tissue protein:

[0291] Take the tissue out of the -80°C freezer;

[0292] Add an appropriate volume of RIPA lysis buffer (PMSF is added before use) and disrupt with a tissue cell homogenizer (80HZ, 120S);

[0293] Centrifuge at 12,000 rpm for 30 min at 4°C and collect the supernatant.

[0294] Repeat the centrifugation once and take the supernatant.

[0295] Add 4× loading buffer (containing DTT), place in a 100°C metal bath for 10 min, centrifuge instantly, and store at -20°C for later use.

[0296] (7.4) Tissue protein quantification

[0297] Protein quantification was performed using the BCA protein quantification kit (Pierce TM BCA Protein Assay Kits (Cat. No.: A55864) instructions.

[0298] Draw a BCA standard curve: The concentration of the standard BSA is 2 mg / mL. Use deionized water to 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 respectively according to the doubling method. Use deionized water as the blank control group.

[0299] 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;

[0300] 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 it immediately after preparation;

[0301] 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 reaction for 30 minutes;

[0302] The absorbance value at 570 nm was measured by an enzyme-labeled instrument;

[0303] A standard curve was drawn with A570 as the ordinate and BSA concentration as the abscissa;

[0304] Calculate the sample concentration according to the standard curve.

[0305] (7.5) Western blot detection of HER2 degradation

[0306] Protein separation by SDS-PAGE: Load the extracted soluble protein at 80 μg total protein / lane and perform 10% SDS-PAGE gel electrophoresis at constant voltage of 80V for 15min, then adjust to 150V and perform electrophoresis for about 1h.

[0307] Transfer: Cut the PVDF membrane of corresponding width and length according to the size of the target gel block, soak the PVDF membrane in methanol solution to activate the surface active groups; after the electrophoresis, remove the gel, find the position of the target protein according to the protein marker, and cut the gel. Install it in the order of sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge, and pay attention to avoid bubbles between the gel and the PVDF membrane. If bubbles are generated, use a 1mL gun tip to absorb the electrotransfer buffer to flush out the bubbles or use a test tube to press out the bubbles; finally, put the gel into the vertical transfer tank facing the negative electrode side (black side of the transfer tank); place the transfer tank in a 4℃ refrigerator, fill it with transfer buffer, plug in the electrode, set the current to a constant cross current of 260mA, and electrotransfer for 100min.

[0308] 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 non-specific binding sites. Incubate on a shaker at room temperature for 1 hour.

[0309] Antibody hybridization: Add anti-HER2 primary antibody at a ratio of 1:2000 in blocking solution, incubate overnight at 4°C; wash 8 times with TBST, 5 minutes each time. Secondary antibody incubation: Add corresponding HPR-labeled goat anti-rabbit (1:10000) and incubate at room temperature for 1 hour; wash 8 times with TBST, 5 minutes each time.

[0310] Development: Automatic development of gel imager: Turn on the gel imager and set the development program. According to the titer of the antibody and the expression level of the target protein, slowly add the color development solution prepared in proportion to the PVDF and react for 1-2 minutes. Then move the PVDF membrane to the gel imager and start automatic development. After the development program is completed, save the appropriate picture.

[0311] (7.6) Experimental results

[0312] The experimental results are shown in Fig.12 In. From Fig.12 The results showed that LYTAF can also efficiently degrade the target HER2 in animals, while the control antibody Pertuzumab (Ptz) does not mediate the degradation of HER2, which fully demonstrates that the LYTAF we developed can also degrade the target protein in vivo.

[0313] Example 2

[0314] LYTAF mediates the degradation of membrane protein EGFR in hepatocellular carcinoma cells

[0315] (1) Through the linker (GGGGS) 3 The IGF2-M5.6 mutation was connected to the heavy chain C-terminus of the EGFR antibody Panitumumab to obtain the targeted PanH-M5.6. The coding gene of PanH-M5.6 was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and 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).

[0316] (2) The gene encoding the light chain of Panitumumab was synthesized and ligated to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and the sequence was sequenced to verify the correctness of the sequence to obtain PanL-pCDNA3.4. The amino acid sequence corresponding to the light chain of Panitumumab is shown in seq25-1 (SEQ ID NO: 50), and its DNA sequence is shown in seq25-2 (SEQ ID NO: 51).

[0317] (3) PanH-M5.6-pCDNA3.4 and PanL-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain Pan_LYTAF.

[0318] (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, thereby obtaining PanH-pCDNA3.4.

[0319] (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.

[0320] (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.

[0321] (7) Experimental results: Fig.13 As shown, 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.

[0322] Example 3

[0323] LYTAF mediates degradation of both EGFR and c-Met in non-small cell lung cancer cell lines

[0324] (1) Through the linker (GGGGS) 3 The IGF2-M5.6 mutation was connected to the heavy chain C-terminus of the EGFR antibody Certuximab to obtain the targeted CetH-M5.6. The coding gene of CetH-M5.6 was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and 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).

[0325] (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, and the sequence was 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).

[0326] (3) CetH-M5.6-pCDNA3.4 and CetL-pCDNA3.4 were co-transfected into HEK293-F cells for expression to obtain Cet_LYTAF.

[0327] (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, and the sequence was sequenced to verify the correctness of the sequence, thereby obtaining CetH-pCDNA3.4. The amino acid sequence of the Certuximab heavy chain is seq28-1 (SEQ ID NO: 56), and the corresponding DNA sequence is shown in seq28-2 (SEQ ID NO: 57).

[0328] (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.

[0329] (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.

[0330] Experimental results: Fig.14 As shown, 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.

[0331] Example 4

[0332] 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.

[0333] (1) The IGF2-M5.6 mutant was connected to the N-terminus of the heavy chain of the HER2 antibody Pertuzumab through a linker (ASTKGP) to obtain IGF2-M5.6-PtzH. The coding gene of IGF2-M5.6-PtzH was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and 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 seq20-2 (SEQ ID NO: 41).

[0334] (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.

[0335] (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.

[0336] (4) Experimental results: Fig.15 As shown, IGF2-M5.6H_LYTAF can significantly mediate HER2 degradation, but the control antibody cannot mediate HER2 degradation.

[0337] Example 5

[0338] 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

[0339] (1) The IGF2-M5.6 mutation was connected to the N-terminus of the light chain of the HER2 antibody Pertuzumab through a linker (TVAAP) to obtain IGF2-M5.6-PtzL. The coding gene of IGF2-M5.6-PtzL was synthesized by codon optimization and gene synthesis, and ligated to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and 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 seq21-2 (SEQ ID NO: 43).

[0340] (2) IGF2-M5.6-PtzL-pCDNA3.4 and PtzH-pCDNA3.4 were co-transfected into HEK293-F cells for expression, thereby obtaining IGF2-M5.6L_LYTAF, a fusion expression of IGF2-M5.6 and Pertuzumab.

[0341] (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.

[0342] (4) Experimental results: Fig.16 As shown, IGF2-M5.6L_LYTAF can significantly mediate HER2 degradation.

[0343] Example 6

[0344] 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.

[0345] (1) Through the linker (GGGGS) 3 , the IGF2-M5.6 mutation was connected to the C-terminus of the light chain of the HER2 antibody Pertuzumab to obtain PtzL-IGF2-M5.6. The coding gene of PtzL-IGF2-M5.6 was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and 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 seq22-2 (SEQ ID NO: 45).

[0346] (2) PtzL-IGF2-M5.6-pCDNA3.4 and PtzH-pCDNA3.4 were co-transfected into HEK293-F cells for expression, thereby obtaining LYTAF-IGF2-M5.6L expressing the fusion of IGF2-M5.6 and Pertuzumab.

[0347] (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.

[0348] (4) Experimental results: Fig.17 As shown, LYTAF-IGF2-M5.6L can significantly mediate HER2 degradation.

[0349] Example 7

[0350] 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

[0351] (1) Through the linker (GGGGS) 3, the IGF2-M5.8 mutation was connected to the C-terminus of the light chain of the HER2 antibody Pertuzumab to obtain PtzH-IGF2-M5.8. The coding gene of PtzH-IGF2-M5.8 was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and 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 seq23-2 (SEQ ID NO: 47).

[0352] (2) PtzH-IGF2-M5.8-pCDNA3.4 and PtzL-pCDNA3.4 were co-transfected into HEK293-F cells for expression, and LYTAF-IGF2-M5.8H expressing the fusion of IGF2-M5.8 and Pertuzumab was obtained.

[0353] (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.

[0354] (4) Experimental results: Fig.18 As shown, LYTAF-IGF2-M5.8H can significantly mediate HER2 degradation.

[0355] Example 8

[0356] 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 48 h of incubation in the liver cancer cell line MHCC-97H.

[0357] (1) Through the linker (GGGGS) 3 , the IGF2-M5.6 mutation was connected to the C-terminus of the heavy chain of the c-Met antibody to obtain Anti-cMet-IGF2-M5.6. The coding gene of Anti-cMet-IGF2-M5.6 was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of the mammalian expression vector pCDNA3.4, and 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 seq29-2 (SEQ ID NO: 59).

[0358] (2) The c-Met antibody light chain coding gene Anti-cMetL was connected 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).

[0359] (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.

[0360] (4) The cMet antibody heavy chain coding gene Anti-cMetH was connected 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-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).

[0361] (5) Anti-cMetH-pCDNA3.4 plasmid and Anti-cMetL-pCDNA3.4 were co-transfected into HEK293-F cells for expression, and the Anti-cMet antibody was obtained after purification (in Fig.19 (shown as control Ab)

[0362] (6) Different concentrations of Anti-cMet antibodies or corresponding LYTA 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.

[0363] (7) Experimental results: Fig.19 As shown, Anti-cMet antibodies cannot degrade c-Met, while its corresponding LYTAF can significantly degrade c-Met.

[0364] Example 9

[0365] The wild-type sequence of IGF2 was connected to the N-terminus of the hFc of the 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:

[0366] (1) Through the linker (GGGGS) 3 , IGF2-WT (SEQ ID NO: 1) was connected to the N-terminus of hFc (SEQ ID NO: 116) of IgG1 antibody. The coding gene of the fusion protein was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of mammalian expression vector pCDNA3.4, and the correctness of the sequence was verified by sequencing. IGF2-WT-hFc-pCDNA3.4 was obtained.

[0367] (2) IGF2-WT-pCDNA3.4 was transfected into HEK293-F cells for expression to obtain hFc-IGF2 protein.

[0368] (3) According to the steps described in Example 1, different concentrations of IGF2-WT-hFc protein (in Fig. 20 hFc-IGF2 (hFc-IGF2) was co-incubated with liver cancer cell line SK-Hep1 or breast cancer cell line BT-474, and the cell growth was monitored in real time using Incucyte.

[0369] (4) Experimental results: Fig. 20 As shown, IGF2-WT-hFc significantly promoted the proliferation of breast cancer cells BT-474 and liver cancer cells SK-Hep1 compared with the control PBS.

[0370] Example 10

[0371] The IGF2-M5.6 sequence was linked to the N-terminus of the hFc of the IgG1 antibody and its effect on cell proliferation was detected after co-incubation with breast cancer cell lines BT-474, SK-BR-3 or liver cancer cell line SK-Hep1.

[0372] (1) Through the linker (GGGGS) 3 , IGF2-M5.6 (SEQ ID NO: 76) was connected to the N-terminus of hFc (SEQ ID NO: 116) of IgG1 antibody. The coding gene of the fusion protein was synthesized by codon optimization and gene synthesis, and connected to the Xba1 and EcoRV restriction sites of mammalian expression vector pCDNA3.4, and the correctness of the sequence was verified by sequencing. IGF2-M5.6-hFc-pCDNA3.4 was obtained.

[0373] (2) IGF2-M5.6-pCDNA3.4 was transfected into HEK293-F cells for expression to obtain IGF2-M5.6-hFc protein.

[0374] (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, and the Celltiters-glo reagent was used to detect the effect of different concentrations of IGF2-M5.6-hFc on cell growth according to the instructions.

[0375] (4) According to the steps described in Example 1, different concentrations of IGF2-M5.6-hFc protein were co-incubated with the breast cancer cell line SK-BR-3, and the effect of different concentrations of IGF2-M5.6-hFc on cell growth was detected in real time using the Incucyte instrument.

[0376] (5) Experimental results: Fig.21 As shown, IGF2-M5.6-hFc had no effect on the proliferation of breast cancer cells or liver cancer cells even at high concentrations compared to the control PBS.

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 is 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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