Complexes targeting target cells and uses thereof

Abstract

The present disclosure discloses a complex targeting a target cell and use thereof, the complex comprising (1) a first molecule: a protein comprising an Fc fragment and (2) a second molecule: an enzyme and a fragment capable of being connected to the first molecule in a non-covalent manner to form a stable structure, and the complex retains the activity of each part, and is capable of playing a role of targeting the protein and the enzyme by administering the targeting complex to a subject.

Description

Complexes targeting target cells and uses thereof

[0001] Priority claim

[0002] The present disclosure claims priority to International Application No. PCT / CN2024 / 112583, filed on August 16, 2024, and Chinese Patent Application No. CN2024119553147, filed on December 27, 2024, the entire contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biological medicine, in particular to complexes targeting target cells and uses thereof. BACKGROUND

[0004] The rise of immunotherapy has brought a paradigm shift for intractable tumors. Immune cells integrate signals from activating and inhibitory receptors to determine their response to challenging targets. Tumors often impede immune cell recognition by overexpressing ligands for inhibitory receptors. This discovery has led to new therapeutic strategies aimed at blocking inhibitory immune cell signaling, such as the clinically approved T cell checkpoint inhibitors targeting PD-1 and CTLA-4. Ongoing preclinical studies focus on therapies that target multiple immune pathways in combination. For example, antibodies against PD-1 / PD-L1 in combination with antibodies targeting other T cell checkpoint inhibitors exhibit improved anti-tumor activity in syngeneic tumor models. Complementary to these interventions are therapies targeting innate immune cells, particularly natural killer (NK) cells, macrophages, and dendritic cells.

[0005] Cancer immunotherapy using immune checkpoint inhibitors, including antibodies that block the PD-1 / PD-L1 pathway, has improved outcomes for many cancer patients. However, despite the progress made so far, many patients do not respond to currently available immune checkpoint inhibitors.

[0006] Abnormal increase or abnormal expression of cell surface molecules or extracellular matrix molecules such as sialic acid, hyaluronic acid, heparin, collagen, and chondroitin often exist on or around the surface of tumor cells. For example, high sialylation is often observed in tumor tissues compared to corresponding normal tissues. The total sialic acid level in serum or glycolipid-bound sialic acid is significantly increased in patients with various cancers such as ovarian cancer, leukemia, colorectal cancer, and breast cancer. High sialylation leads to accelerated cancer progression and poor prognosis. The increase of sialic acid in tumor cells is mainly caused by abnormal metabolic amount and abnormal expression of sialyltransferase / sialidase. It is reported that in highly metastatic breast tumors, sialic acid metabolism is up-regulated, and knocking out the key node CMAS gene of sialic acid metabolism can inhibit sialic acid activation and reduce the formation of lung metastatic tumors in vivo. On the other hand, abnormal expression of sialyltransferase and sialidase accelerates and maintains the high sialylation state of glycoconjugates. Sialylation of tumor cells further promotes their immune escape, enhances the proliferation and metastasis of tumors, helps tumor angiogenesis, and helps resist apoptosis and cancer treatment.

[0007] Abnormal increase or abnormal expression of cell surface molecules or extracellular matrix molecules not only occurs in tumor cells, but also widely exists in various diseases and corresponding cell types. For example, overexpression of hyaluronic acid leads to joint swelling and pain, HIV virus depends on the sialic acid metabolic pathway of host cells for envelope formation, and over-secretion of collagen and hyaluronic acid in hepatitis leads to fibrotic diseases. Although these diseases can be treated by antibodies, the abnormal increase or abnormal expression of these cell surface molecules or extracellular matrix molecules greatly reduces the efficacy of antibody therapy.

[0008] Therefore, only targeting drugs such as antibodies are not enough to achieve therapeutic effect, and it may also be necessary to simultaneously remove the "disguise" on the surface of target cells and in the matrix. There is an urgent need for a new treatment strategy to enable immunotherapy to break through the limitations of the barrier formed by cell surface molecules or extracellular matrix molecules, so as to truly restore and amplify the immune response. SUMMARY

[0009] The purpose of the present disclosure is to provide a complex targeting target cells and applications thereof.

[0010] The first aspect of the present disclosure provides a targeting complex comprising a first molecule and a second molecule, the first molecule comprising an Fc fragment portion and a targeting binding fragment portion, and the second molecule comprising a first portion of an enzyme and a second portion capable of non-covalently binding to the first molecule. In an alternative embodiment, the first molecule is a targeting protein comprising an Fc fragment portion and a targeting binding fragment portion, and the second molecule is a fusion protein.

[0011] In some embodiments, the first molecule comprises an antibody or a fusion protein comprising an Fc fragment. In an alternative embodiment, the antibody is selected from an anti-Her2 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0012] In some embodiments, the non-covalent binding is an inter-protein interaction. In an alternative embodiment, the inter-protein interaction is selected from an antigen-antibody binding mode, a Protein A / Protein G-antibody Fc binding mode, or an antibody Fc receptor-antibody Fc binding mode.

[0013] In some embodiments, the second molecule retains an enzymatic activity, and / or the first molecule retains a targeting activity. In an alternative embodiment, upon binding to a target molecule, the second molecule can act on a substrate molecule of the enzyme surrounding the target molecule.

[0014] In some embodiments, the enzyme is an extracellular molecule-modifying enzyme.

[0015] In an alternative embodiment, the enzyme is selected from a hyaluronidase, a sialidase, an N-glycosidase, an O-glycosidase, a collagenase, a heparinase, a chondroitinase, or a chondroitinase.

[0016] In an alternative embodiment, the sialidase is selected from a Salmonella typhimurium sialidase, a Vibrio cholerae sialidase, a human neuraminidase 1, a human neuraminidase 2, a human neuraminidase 3, or a human neuraminidase 4.

[0017] In an alternative embodiment, the N-glycosidase is selected from Endo H, PNGase F, PNGase A, Endo S, Endo S2, Endo D, Endo F2, or Endo F3.

[0018] In an alternative embodiment, the O-glycosidase is selected from Endo-O-Glycosidase or Exo-O-Glycosidase.

[0019] In an alternative embodiment, the hyaluronidase is selected from HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, or PH20 / SPAM1.

[0020] In an alternative embodiment, the chondroitinase is selected from chondroitinase C, chondroitinase B, hyaluronidase ACIII, chondroitinase ACII, chondroitinase AC, or chondroitinase ABC.

[0021] In an alternative embodiment, the heparinase is selected from heparinase I, heparinase II, or heparinase III.

[0022] In an alternative embodiment, the collagenase is selected from collagenase 1, collagenase 2, collagenase 3 or serine protease Cathepsin L.

[0023] In some embodiments, the second portion of the second molecule is capable of non-covalently binding to the constant region of the first molecule. In an alternative embodiment, the second portion of the second molecule is capable of non-covalently binding to the Fc fragment portion of the first molecule. In an alternative embodiment, the affinity of the second portion of the second molecule to the Fc fragment portion of the first molecule is KDof 1 x 10 -7 M. In an alternative embodiment, the affinity of the second portion of the second molecule to the Fc fragment portion of the first molecule is KDof 1 x 10 -12 M. In an alternative embodiment, the affinity of the second portion of the second molecule to the Fc fragment portion of the first molecule is KDof 1 x 10 -8 M. In an alternative embodiment, the affinity of the second portion of the second molecule to the Fc fragment portion of the first molecule is KDof 1 x 10 -11 M. In an alternative embodiment, the second portion of the second molecule is selected from an antibody or an antigen-binding fragment thereof (e.g. VHH, scFv, Fab or Fab2) capable of binding to an antibody constant region fragment, or selected from Protein A or an Fc-binding fragment thereof, an affibody or an Fc-binding fragment thereof, Protein G or an Fc-binding fragment thereof, or an FcR or an Fc-binding fragment thereof, or is an Fc-binding peptide.

[0024] In some embodiments, the second portion of the second molecule is located at the N-terminus or the C-terminus of the first portion. Preferably, the second portion of the second molecule is located at the N-terminus of the first portion.

[0025] In an alternative embodiment, the enzyme of the second molecule is connected to the second portion by a peptide bond or a linker. In an alternative embodiment, the linker is a peptide linker.

[0026] In some embodiments, the second portion of the second molecule is a wild-type Z33 or a polypeptide having amino acid mutations relative to the wild-type Z33.

[0027] In some embodiments, the site of the amino acid mutations includes at least one of the 1st, 4th and 29th positions, and the wild-type Z33 has the amino acid sequence shown in SEQ ID NO: 1.

[0028] In some embodiments, the polypeptide having an amino acid mutation relative to wild type Z33 has an amino acid substitution relative to wild type Z33. In an alternative embodiment, the substituted amino acid at position 1 is selected from F or L. In an alternative embodiment, the substituted amino acid at position 4 is selected from Q, F, E, R or L. In an alternative embodiment, the substituted amino acid at position 29 is selected from S, Q, R or K.

[0029] In some embodiments, the polypeptide having an amino acid mutation relative to wild type Z33 has an amino acid sequence that is further at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95% or at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0030] In some embodiments, the polypeptide having an amino acid mutation relative to wild type Z33 comprises an amino acid sequence of: 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 ,

[0031] wherein,

[0032] X1is selected from F or L;

[0033] X4is selected from Q, F, E, R or L;

[0034] X 29 is selected from S, Q, R or K;

[0035] further,

[0036] X2is selected from N, E or S;

[0037] X3is selected from M, K or E;

[0038] X5is selected from Q or C;

[0039] X6 is selected from Q, W, L, K or A;

[0040] X7 is selected from R, L, K, N, A, D or C;

[0041] X8 is selected from R or A;

[0042] X9 is selected from F, M or A;

[0043] X 10 is selected from Y, A or S;

[0044] X 11 is selected from E, A, D or S;

[0045] X 12 is selected from A, I, T or E;

[0046] X 13 is selected from L, A, G or K;

[0047] X 14 is selected from H, A, V, F, W or G;

[0048] X 15 is selected from D, V, C, N or L;

[0049] X 16 is selected from P, L or A;

[0050] X 17 is selected from N, L, T or G;

[0051] X 18 is selected from L, M or V;

[0052] X 19 is selected from N, G or T;

[0053] X 20 is selected from E, K, A or Q;

[0054] X 21 is selected from E or Q;

[0055] X 22 is Q;

[0056] X 23 is R;

[0057] X 24 is selected from N, W, A or K;

[0058] X 25 is selected from A or G;

[0059] X 26 is selected from K, R or F;

[0060] X 27 is I;

[0061] X 28 is selected from K, C, Q or R;

[0062] X 30 is selected from I, F or L;

[0063] X 31 is selected from R, M or K;

[0064] X 32 is selected from D, N, K or E; and

[0065] X 33 is selected from D, N or E.

[0066] In some embodiments, X7is R; X9is F; X 10 is Y; X 13 is L; X 23 is R; and / or X 30 is I.

[0067] In some embodiments, X 11 is E or D; X 12 is A or T; X 14 is H or W; X 16 is P, A or L; X 17 is N, L or T; X 18 is N or M; and / or X 23 is R.

[0068] In some embodiments, X1is L; X4is L; and / or X 29 is Q, K or R.

[0069] In some embodiments, the hyaluronidase amino acid sequence is set forth in any one of SEQ ID NO. 6, 7, or 65-71, the sialidase amino acid sequence is set forth in any one of SEQ ID NO. 4, 5, or 55-60, the collagenase amino acid sequence is set forth in SEQ ID NO. 8, the heparinase amino acid sequence is set forth in SEQ ID NO. 9, the chondroitin sulfatease amino acid sequence is set forth in SEQ ID NO. 10, and the chondroitinase amino acid sequence is set forth in SEQ ID NO. 11.

[0070] In some embodiments, the second portion of the second molecule is an antibody or antigen-binding fragment thereof comprising heavy chain variable region CDR1-CDR3, the amino acid sequences of which, according to the Kabat numbering scheme, comprise SEQ ID NOs. 31-33, respectively.

[0071] In an alternative embodiment, the second portion of the molecule comprises a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO. 12-18 and SEQ ID NO. 34-39, or an Fc binding fragment thereof.

[0072] In an alternative embodiment, the second portion of the second molecule has an amino acid sequence selected from a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO. 34-39, or an Fc binding fragment thereof.

[0073] In an alternative embodiment, the second portion of the second molecule has an amino acid sequence as set forth in SEQ ID NO. 34, 36, 38, or 39.

[0074] In some embodiments, the fusion protein as the second molecule is selected from a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO. 19-30 or SEQ ID NO. 61-64.

[0075] A second aspect of the present disclosure provides a molecule comprising a first portion of an enzyme and a second portion capable of non-covalently binding to an antibody constant region, in an alternative embodiment, the molecule is a fusion protein.

[0076] In some embodiments, the non-covalent binding is an inter-protein interaction, in an alternative embodiment, the inter-protein interaction is selected from an antigen-antibody binding mode, a Protein A / Protein G-antibody Fc binding mode, or an antibody Fc receptor-antibody Fc binding mode.

[0077] In some embodiments, the second portion has an affinity KD of 1 x 10 -07 M to 1 x 10 -12 M to 1 x 10 -8 M to 1 x 10 -11 M.

[0078] In some embodiments, the molecule retains enzyme activity. In an alternative embodiment, the molecule, upon binding to a first molecule, modifies a substrate molecule of the enzyme surrounding a target of the first molecule, the first molecule comprising an Fc fragment portion and a targeting binding portion. In an alternative embodiment, the first molecule comprises an antibody or a fusion protein comprising an Fc fragment.

[0079] In some embodiments, the enzyme is an extracellular molecule modifying enzyme.

[0080] In an alternative embodiment, the enzyme is selected from hyaluronidase, sialidase, N-glycosidase, O-glycosidase, collagenase, heparinase, chondroitinase, or chondroitinase.

[0081] In an alternative embodiment, the sialidase is selected from Salmonella typhimurium sialidase, Vibrio cholerae sialidase, human neuraminidase 1, human neuraminidase 2, human neuraminidase 3, or human neuraminidase 4.

[0082] In an alternative embodiment, the N-glycosidase is selected from Endo H, PNGase F, PNGase A, Endo S, Endo S2, Endo D, Endo F2, or Endo F3.

[0083] In an alternative embodiment, the O-glycosidase is selected from Endo-O-Glycosidase or Exo-O-Glycosidase.

[0084] In an alternative embodiment, the hyaluronidase is selected from HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, or PH20 / SPAM1.

[0085] In an alternative embodiment, the chondroitinase is selected from Chondroitinase C, Chondroitinase B, Hyaluronidase ACIII, Chondroitinase ACII, Chondroitinase AC, or Chondroitinase ABC.

[0086] In an alternative embodiment, the heparinase is selected from Heparinase I, Heparinase II, or Heparinase III.

[0087] In an alternative embodiment, the collagenase is selected from Collagenase 1, Collagenase 2, Collagenase 3, or Serine protease Cathepsin L.

[0088] In some embodiments, the second part of the molecule is capable of non-covalently binding to the constant region of the first molecule.

[0089] In an alternative embodiment, the second part of the molecule is capable of non-covalently binding to the Fc region of the first molecule. In an alternative embodiment, the second part of the molecule is selected from an antibody or an antigen-binding fragment thereof (e.g. VHH, scFv, Fab, or Fab2) capable of binding to an antibody constant region fragment, or from Protein A or an Fc-binding fragment thereof, Protein G or an Fc-binding fragment thereof, or FcR or an Fc-binding fragment thereof, or is an Fc-binding peptide.

[0090] In some embodiments, the second portion of the molecule is the second portion of the second molecule in the targeting complex as previously described.

[0091] In some embodiments, the second portion of the molecule is located at the N-terminus or the C-terminus of the first portion. Preferably, the second portion of the molecule is located at the N-terminus of the first portion.

[0092] In some embodiments, the second portion of the molecule is a polypeptide that is wild-type Z33 or has amino acid mutations relative to wild-type Z33. In some embodiments, the site of the amino acid mutation comprises at least one of position 1, position 4, and / or position 29, wherein the wild-type Z33 has an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the polypeptide that has amino acid mutations relative to wild-type Z33 has amino acid substitutions relative to wild-type Z33. In an alternative embodiment, the substituted amino acid at position 1 is selected from F or L. In an alternative embodiment, the substituted amino acid at position 4 is selected from Q, F, E, R or L. In an alternative embodiment, the substituted amino acid at position 29 is selected from S, Q, R or K.

[0093] In some embodiments, the polypeptide that has amino acid mutations relative to wild-type Z33 has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0094] In some embodiments, the polypeptide that has amino acid mutations relative to wild-type Z33 comprises an amino acid sequence of: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 ,

[0095] wherein,

[0096] X1is selected from F or L;

[0097] X4is selected from Q, F, E, R or L;

[0098] X 29 selected from S, Q, R or K;

[0099] further,

[0100] X2is selected from N, E or S;

[0101] X3is selected from M, K or E;

[0102] X5is selected from Q or C;

[0103] X6is selected from Q, W, L, K or A;

[0104] X7is selected from R, L, K, N, A, D or C;

[0105] X8is selected from R or A;

[0106] X9is selected from F, M or A;

[0107] X 10 selected from Y, A or S;

[0108] X 11 selected from E, A, D or S;

[0109] X 12 selected from A, I, T or E;

[0110] X 13 selected from L, A, G or K;

[0111] X 14 selected from H, A, V, F, W or G;

[0112] X 15 selected from D, V, C, N or L;

[0113] X 16 selected from P, L or A;

[0114] X 17 selected from N, L, T or G;

[0115] X 18 selected from L, M or V;

[0116] X 19 selected from N, G or T;

[0117] X 20 selected from E, K, A or Q;

[0118] X 21 is selected from E or Q;

[0119] X 22 is Q;

[0120] X 23 is R;

[0121] X 24 is selected from N, W, A or K;

[0122] X 25 is selected from A or G;

[0123] X 26 is selected from K, R or F;

[0124] X 27 is I;

[0125] X 28 is selected from K, C, Q or R;

[0126] X 30 is selected from I, F or L;

[0127] X 31 is selected from R, M or K;

[0128] X 32 is selected from D, N, K or E; and X 33 is selected from D, N or E.

[0129] In some embodiments,

[0130] X7is R;

[0131] X9is F;

[0132] X 10 is Y;

[0133] X 13 is L;

[0134] X 23 is R; and / or X 30 is I.

[0135] In some embodiments,

[0136] X 11 is E or D;

[0137] X 12 is A or T;

[0138] X 14 is H or W;

[0139] X 16 is P, A or L;

[0140] X 17 is N, L or T;

[0141] X 18 is N or M; and / or X 23 is R.

[0142] In some embodiments,

[0143] X1is L;

[0144] X4is L; and / or

[0145] X 29 is Q, K or R.

[0146] In some embodiments, the hyaluronidase amino acid sequence is set forth in any one of SEQ ID NO. 6, 7, or 65-71, the sialidase amino acid sequence is set forth in any one of SEQ ID NO. 4, 5, or 55-60, the collagenase amino acid sequence is set forth in SEQ ID NO. 8, the heparinase amino acid sequence is set forth in SEQ ID NO. 9, the chondroitin sulfatease amino acid sequence is set forth in SEQ ID NO. 10, and the chondroitinase amino acid sequence is set forth in SEQ ID NO. 11.

[0147] In some embodiments, the second portion of the molecule is an antibody or antigen binding fragment thereof comprising heavy chain variable region CDR1-CDR3 having an amino acid sequence comprising, respectively, SEQ ID NO. 31-33 according to the Kabat numbering convention.

[0148] In an alternative embodiment, the second portion of the molecule comprises a polypeptide selected from the group consisting of SEQ ID NO. 12-18 and 34-39, or an Fc binding fragment thereof.

[0149] In an alternative embodiment, the second portion of the second molecule has an amino acid sequence selected from the group consisting of a polypeptide set forth in any one of SEQ ID NO. 34-39, or an Fc binding fragment thereof.

[0150] In an alternative embodiment, the second portion of the second molecule has an amino acid sequence of SEQ ID NO. 34, 36, 38, or 39.

[0151] In some embodiments, the molecule is selected from the group consisting of a polypeptide having an amino acid sequence set forth in any one of SEQ ID NO. 19-30 or SEQ ID NO. 61-64.

[0152] The third aspect of the present disclosure provides a polypeptide, wherein the polypeptide is the second part of the second molecule in the targeting complex according to any one of the preceding embodiments, and the second part of the second molecule is the polypeptide having an amino acid mutation relative to wild type Z33.

[0153] In some embodiments, the polypeptide is a polypeptide having an amino acid mutation relative to wild type Z33. In some embodiments, the site of the amino acid mutation comprises at least one of position 1, position 4 and / or position 29, wherein the wild type Z33 has the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the polypeptide having an amino acid mutation relative to wild type Z33 has an amino acid substitution relative to wild type Z33. In an alternative embodiment, the substituted amino acid at position 1 is selected from F or L. In an alternative embodiment, the substituted amino acid at position 4 is selected from Q, F, E, R or L; and in an alternative embodiment, the substituted amino acid at position 29 is selected from S, Q, R or K.

[0154] In some embodiments, the polypeptide having an amino acid mutation relative to wild type Z33 has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95% or at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0155] In some embodiments, the polypeptide having an amino acid mutation relative to wild type Z33 comprises an amino acid sequence of: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 ,

[0156] wherein,

[0157] X1is selected from F or L;

[0158] X4 is selected from Q, F, E, R or L;

[0159] X 29 selected from S, Q, R or K;

[0160] Further,

[0161] X2 is selected from N, E or S;

[0162] X3 is selected from M, K or E;

[0163] X5 is selected from Q or C;

[0164] X6 is selected from Q, W, L, K or A;

[0165] X7 is selected from R, L, K, N, A, D or C; X8 is selected from R or A;

[0166] X9 is selected from F, M or A;

[0167] X 10 selected from Y, A or S;

[0168] X 11 selected from E, A, D or S;

[0169] X 12 selected from A, I, T or E;

[0170] X 13 selected from L, A, G or K;

[0171] X 14 selected from H, A, V, F, W or G;

[0172] X 15 selected from D, V, C, N or L;

[0173] X 16 selected from P, L or A;

[0174] X 17 selected from N, L, T or G;

[0175] X 18 selected from L, M or V;

[0176] X 19 selected from N, G or T;

[0177] X 20 selected from E, K, A or Q;

[0178] X 21 selected from E or Q;

[0179] X 22 is Q;

[0180] X 23 is R;

[0181] X 24 is selected from N, W, A, or K;

[0182] X 25 is selected from A or G;

[0183] X 26 is selected from K, R, or F;

[0184] X 27 is I;

[0185] X 28 is selected from K, C, Q, or R;

[0186] X 30 is selected from I, F, or L;

[0187] X 31 is selected from R, M, or K;

[0188] X 32 is selected from D, N, K, or E; and X 33 is selected from D, N, or E.

[0189] In some embodiments,

[0190] X7is R;

[0191] X9is F;

[0192] X 10 is Y;

[0193] X 13 is L;

[0194] X 23 is R; and / or

[0195] X 30 is I.

[0196] In some embodiments,

[0197] X 11 is E or D;

[0198] X 12 is A or T;

[0199] X 14 is H or W;

[0200] X 16 is P, A, or L;

[0201] X 17 is N, L, or T;

[0202] X18 is N or M; and / or

[0203] X 23 is R.

[0204] In some embodiments,

[0205] X1is L;

[0206] X4is L; and / or

[0207] X 29 is Q, K or R.

[0208] In an alternative embodiment, the amino acid sequence of the polypeptide having an amino acid mutation relative to wild type Z33 comprises an amino acid sequence selected from any one of SEQ ID NO. 34-39. In an alternative embodiment, the amino acid sequence of the polypeptide having an amino acid mutation relative to wild type Z33 is selected from any one of SEQ ID NO. 34-39. In an alternative embodiment, the amino acid sequence of the polypeptide having an amino acid mutation relative to wild type Z33 is SEQ ID NO. 34, 36, 38 or 39.

[0209] A fourth aspect of the disclosure provides a fusion protein comprising the polypeptide having an amino acid mutation relative to wild type Z33. In an alternative embodiment, the fusion protein comprises the polypeptide having an amino acid mutation relative to wild type Z33 and an ASGPR binding peptide. In an alternative embodiment, the amino acid sequence of the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NO. 42-54. In an alternative embodiment, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO. 42-54.

[0210] A fifth aspect of the disclosure provides a composition comprising: the targeting complex according to any one of the preceding embodiments, the molecule according to any one of the preceding embodiments or the polypeptide according to any one of the preceding embodiments; and a pharmaceutically acceptable carrier.

[0211] A sixth aspect of the disclosure provides a nucleic acid encoding the targeting complex according to any one of the preceding embodiments, the molecule according to any one of the preceding embodiments or the polypeptide according to any one of the preceding embodiments.

[0212] A seventh aspect of the disclosure provides an expression vector comprising the nucleic acid according to any one of the preceding embodiments. In an alternative embodiment, the expression vector comprises the nucleic acid according to any one of the preceding embodiments and a promoter operably linked to the nucleic acid.

[0213] The eighth aspect of the present disclosure provides a host cell comprising the nucleic acid as previously described or the expression vector as previously described.

[0214] In some embodiments, the host cell is a mammalian host cell, a prokaryotic host cell, or a yeast host cell.

[0215] The ninth aspect of the present disclosure provides a method comprising administering the targeting complex as any one of the preceding embodiments, the molecule as any one of the preceding embodiments, or the composition as any one of the preceding embodiments to an individual in need thereof.

[0216] The tenth aspect of the present disclosure provides a method of treating cancer, the method comprising administering the targeting complex as any one of the preceding embodiments, or the molecule as any one of the preceding embodiments, or the composition as any one of the preceding embodiments to an individual having cancer, in an alternative embodiment, the cancer is breast cancer.

[0217] The eleventh aspect of the present disclosure provides use of the targeting complex as any one of the preceding embodiments, or the molecule as any one of the preceding embodiments, or the composition as any one of the preceding embodiments in the manufacture of a medicament for treating cancer in a subject. In one embodiment, the use comprises administering the targeting complex as any one of the preceding embodiments, or the molecule as any one of the preceding embodiments, or the composition as any one of the preceding embodiments to a subject having cancer, in an alternative embodiment, the cancer is breast cancer.

[0218] In some embodiments, the tumor inhibitory activity of the targeting complex is significantly better than that of the first molecule in the targeting complex administered alone. In an alternative embodiment, the targeting complex is selected from the group consisting of "Keytruda and NEU2-M-anFcVH", "Atezolizumab and Z33-mutl-PH20", "Antibody-1 and NEU2-M-anFcVH", or "Antibody-1 and PH20-anFcVH".

[0219] The twelfth aspect of the present disclosure provides use of the polypeptide as previously described, the use is selected from the group consisting of:

[0220] (a) detecting an Fc-containing molecule; or

[0221] (b) purifying an Fc-containing molecule;

[0222] In an alternative embodiment, the Fc-containing molecule is an IgG.

[0223] The advantages of some embodiments provided by the present disclosure are: providing a general technical platform, which can target extracellular molecule modifying enzymes to the surrounding of target molecules by means of target molecules such as antibodies, effectively remove enzyme substrate molecules around target cells or target molecules, and synergistically improve the therapeutic effect of target molecules and enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0224] Figure 1 shows the results of sialidase NEU2-M-anFcVH fusion protein enzyme activity detection.

[0225] Figure 2 shows the NEU2-M-anFcVH and Keytruda form a complex to detect the activity of the enzyme on the surface of Jurkat-T cells (PD-1 positive cells).

[0226] Figure 3 shows the NEU2-M-anFcVH and antibody-1 form a complex to detect the activity of the enzyme on the surface of SK-BR-3 cells (HER2 positive cells).

[0227] Figure 4 shows the results of sialidase enzyme activity detection in the fusion protein formed by hNEU2-M and Z33.

[0228] Figure 5 shows the results of in vitro imaging of the complex in tumor-bearing mice.

[0229] Figure 6 shows the results of agarose gel electrophoresis after the fusion protein enzyme cuts the substrate sodium hyaluronate molecule.

[0230] Figure 7 shows the results of the Keytruda+Neu2-M-anFcVH efficacy of each group in the CT26-hPDL1 model study.

[0231] Figure 8 shows the results of the Atezolizumab+PH20-anFcVH efficacy of each group in the PBMC NCI-H292 model study.

[0232] DETAILED DESCRIPTION

[0233] I. DEFINITIONS

[0234] Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present disclosure, the following terms are defined with the following meanings.

[0235] The term "fusion protein" generally refers to a protein resulting from the fusion of two or more proteins or polypeptides. The genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to one another to form a fusion gene or a fused nucleic acid molecule, which can encode the fusion protein. Translation of the fusion gene results in a single polypeptide having the properties of at least one, and optionally each, of the two or more proteins or polypeptides prior to fusion. Fusion proteins described in the present disclosure generally comprise at least two domains (A and B), and optionally a third component, e.g., a linker between the two domains. Generation of recombinant fusion proteins is known in the art and generally involves removing the stop codon from the cDNA sequence encoding the first protein or polypeptide, and then attaching the cDNA sequence of the second protein in-frame by ligation or overlap extension PCR. The cDNA sequence is then expressed by a cell as a single protein. The protein can be engineered to include the entire sequence of both original proteins or polypeptides, or only a portion of either.

[0236] A "targeting protein" refers to an antibody or fusion protein having a targeting binding fragment portion that is capable of binding to a target molecule. In certain aspects, the targeting protein specifically binds to a cell surface molecule. As used herein, a targeting protein that "specifically binds to a cell surface molecule" or has "specificity for a cell surface molecule" refers to a targeting protein that binds to a cell surface molecule with greater affinity than to other cell surface molecules. According to certain embodiments, the targeting protein exhibits a binding affinity KD for the cell surface molecule of less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, or less than or equal to about 10 -7 M, or less than or equal to about 10 -8 M, or less than or equal to about 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M or less. Such affinities can be readily determined using routine techniques, e.g., by equilibrium dialysis, surface plasmon resonance (SPR) techniques (e.g., a BIAcore 2000 instrument, using the general procedures outlined by the manufacturer), radioimmunoassay, or by another method.

[0237] According to certain embodiments, the targeting protein is an antibody. The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgGl, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of tetramers, which in turn are composed of two dimers of heavy and light chain polypeptides); including but not limited to single chain Fv, scFv, VHH, Fab, F(ab'), F(ab')2, di-scFv, and affibody; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments); and fusion proteins.

[0238] In certain aspects, when the targeting protein is an antibody, the antibody can be a therapeutic antibody, e.g., an antibody that is efficacious in the treatment of cancer (e.g., through antibody-dependent cellular cytotoxicity and / or other mechanisms), immune-related disorders, endothelial cell-related disorders, etc., even in the absence of an extracellular molecular modification enzyme. For example, the antibody can be a therapeutic antibody that specifically binds to a tumor-associated cell surface molecule or a tumor-specific cell surface molecule.

[0239] In certain aspects, when the targeting protein is an Fc-containing fusion protein, the portion of the targeting protein that targets binding to the target molecule is capable of specifically binding to the target molecule.

[0240] "Protein-protein interaction" refers to a non-covalent, mutual affinity or binding relationship between proteins, and the manner of protein-protein interaction includes, but is not limited to, a manner of binding selected from the group consisting of antigen-antibody, Protein A / Protein G-antibody Fc, and antibody Fc receptor-antibody Fc, and the above-mentioned manner of binding includes a manner of binding between an antigen and an antibody, and a manner of binding between an antigen fragment (such as an antigen epitope) and an antibody and an antigen-binding fragment thereof; the manner of binding between Protein A / Protein G-antibody Fc includes a manner of binding between Protein A / Protein G-antibody Fc, and a manner of binding between an Fc-binding fragment of Protein A / Protein G and antibody Fc; the manner of binding between antibody Fc receptor-antibody Fc includes a manner of binding between antibody Fc receptor-antibody Fc, and a manner of binding between an Fc-binding fragment of antibody Fc receptor and antibody Fc.

[0241] "Enzyme" refers to a class of proteins with catalytic ability.

[0242] An "extracellular molecule-modifying enzyme" is an enzyme that, upon binding of the targeting protein to the corresponding cell surface molecule of the target cell, effects one or more structural modifications or changes of a substrate molecule of the enzyme at the surface of the target cell or in the extracellular matrix of the target cell. In certain aspects, the structural change is of the cell surface molecule to which the targeting protein binds. In other aspects, the structural change is of a molecule on the surface of the target cell other than the cell surface molecule to which the targeting protein binds or of an extracellular matrix molecule.

[0243] In certain aspects, the extracellular molecule-modifying enzyme is a wild-type enzyme (i.e., a naturally-occurring enzyme). In other aspects, the enzyme is not a wild-type enzyme. For example, the extracellular molecule-modifying enzyme can be a non-natural derivative of a wild-type enzyme. Such a derivative at least partially retains the enzymatic activity of the corresponding wild-type enzyme. Enzyme derivatives that can be used include enzyme derivatives having fewer amino acids or more amino acids than the corresponding wild-type enzyme. Alternatively or additionally, the enzyme derivative can include one or more amino acid substitutions or amino acid modifications relative to the corresponding wild-type enzyme.

[0244] An example of a structural change effected by an extracellular molecule-modifying enzyme in a molecule on the surface of a target cell is cleavage of the molecule. In certain aspects, the molecule cleaved by the extracellular molecule-modifying enzyme is a polymer. Cell surface polymers that can be cleaved (e.g., degraded) by an extracellular molecule-modifying enzyme include, but are not limited to, polypeptides, polysaccharides, glycoproteins, and the like. For example, the extracellular molecule-modifying enzyme can be a protease that cleaves polypeptides (or a subset thereof of interest) on the surface of a target cell. In certain aspects, the polymer cleaved by the extracellular molecule-modifying enzyme is a polysaccharide (or "glycan"), i.e., a molecule containing glycosidically linked monosaccharides. In such embodiments, the extracellular molecule-modifying enzyme can be, for example, a glycoside hydrolase (e.g., a hyaluronidase).

[0245] According to certain embodiments, the extracellular molecule-modifying enzyme cleaves (e.g., hydrolyzes) a terminal residue of a molecule (e.g., a polymer) on the surface of a target cell. In certain aspects, the terminal residue is present in a molecule selected from the group consisting of an oligosaccharide, a polysaccharide, a glycoprotein, a glycolipid, and a ganglioside. In some embodiments, the terminal residue is a terminal sialic acid residue. When the terminal residue is a terminal sialic acid residue, the extracellular molecule-modifying enzyme can be a sialidase (or a derivative thereof as described above) that cleaves the glycosidic bond of sialic acid (neuraminic acid) to release the terminal sialic acid residue from oligosaccharides, polysaccharides, glycoproteins, glycolipids, and other substrates.

[0246] According to certain embodiments, the extracellular molecule-modifying enzyme cleaves (e.g., hydrolyzes) a molecule (e.g., a polymer) that is extracellular. In certain aspects, the extracellular molecule is a molecule that is present in the extracellular matrix (ECM) that is extracellular to the target cell, such as hyaluronan, collagen, heparin, chondroitin sulfate, chondroitin, etc.

[0247] Hyaluronic acid (HA) is found primarily in mammalian connective tissue, skin, cartilage, and synovial fluid. Hyaluronic acid is also a major component of the vitreous humor of the eye. In connective tissue, the hydration associated with hyaluronic acid creates space between tissues, creating a favorable environment for cell movement and proliferation. Hyaluronic acid plays a key role in biological phenomena associated with cell movement, including rapid development, regeneration, repair, embryogenesis, embryonic development, wound healing, angiogenesis, and tumorogenesis (Toole 1991 Cell Biol. Extracell. Matrix, Hay (ed.), Plenum Press, New York, 1384-1386; Bertrand et al. 1992 Int. J. Cancer 52:1-6; Knudson et al. 1993 FASEB J. 7:1233-1241). In addition, hyaluronic acid levels have been associated with tumor invasion (Ozello et al. 1960 Cancer Res. 20:600-604; Takeuchi et al. 1976, Cancer Res. 36:2133-2139; Kimata et al. 1983 Cancer Res. 43:1347-1354).

[0248] "Hyaluronidase" is a class of glycosidases that degrade hyaluronic acid and some glycosaminoglycans, found throughout the animal kingdom, including wild-type hyaluronidases and mutant hyaluronidases.

[0249] There are three major classes of hyaluronidases:

[0250] 1. Mammalian-type hyaluronidases (EC 3.2.1.35), which are endo-beta-N- acetylhexosaminidases that produce tetrasaccharides and hexoses as major end products. They have both hydrolytic and transglycosidase activities and degrade hyaluronic acid and chondroitin sulfate (CS), particularly C4-S and C6-S.

[0251] 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronic acid and, to varying degrees, CS and DS. They are endo-beta-N-acetylhexosaminidases that act by beta-elimination, producing mainly disaccharide end products.

[0252] 3. Hyaluronidases (EC 3.2.1.36) from leeches, other parasites and crustaceans, which are endo-beta-glucuronidases that produce tetrasaccharides and hexaose end products by hydrolysis of β1-3 linkages.

[0253] Mammalian hyaluronidases can be further divided into two groups: neutral active and acid active enzymes. There are six hyaluronidase-like genes in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20 / SPAM1. HYALP1 is a pseudogene, HYAL3 has not shown enzymatic activity on any known substrate. HYAL4 is a chondroitinase, lacking activity on hyaluronan. HYAL1 is the prototype acid active enzyme, PH20 is the prototype neutral active enzyme. Acid active hyaluronidases, such as HYAL1 and HYAL2 lack catalytic activity at neutral pH. For example, HYAL1 has no catalytic activity above pH 4.5 in vitro (Frost et al., Anal Biochemistry, 1997). HYAL2 is an acid active enzyme with very low specific activity in vitro.

[0254] Hyaluronidase-like enzymes can also be characterized as those that are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor. However, there is variation from species to species: for example, bovine PH20 is very loosely attached to the plasma membrane and is not anchored by a phospholipase sensitive anchor (Lalancette et al., Biol Reprod. 2001 Aug;65(2):628-36). This unique feature of bovine hyaluronidase has allowed the use of soluble bovine testicular hyaluronidase as an extract for clinical applications. PH20s of other species are lipid-anchored enzymes that are not soluble without the use of detergents or lipases. For example, human PH20 is anchored to the plasma membrane by a GPI anchor. Attempts have been made to make human PH20 DNA constructs that do not introduce a lipid anchor, but the result is either catalytically inactive enzymes, or enzymes that are not soluble (Arming et al., Eur J Biochem. 1997 Aug 1;247(3):810-4). Naturally occurring rhesus sperm hyaluronidase was found to be both soluble and membrane-bound. The 64 kDa membrane-bound form has enzymatic activity at pH 7.0, while the 54 kDa form has activity only at pH 4.0 (Cherr et al., Dev Biol. 1996 Apr 10;175(1):142-53). Thus, the soluble form of PH20 lacks enzymatic activity at neutral conditions. Examples of hyaluronidases include, but are not limited to, hyaluronidases as disclosed in WO2020022791A1, WO2013102144A2.

[0255] Chondroitinases are enzymes found throughout the animal kingdom. These enzymes degrade glycosaminoglycans through an endoglycosidase reaction. Specific examples of known chondroitinases include chondroitinase ABC (from Proteus vulgaris; Japanese Patent Laid-Open No. 6-153947, T. Yamagata, H. Saito, O. Habuchi and S. Suzuki, J. Biol. Chem., 243, 1523 (1968), S. Suzuki, H. Saito, T. Yamagata, K. Anno, N. Seno, Y. Kawai and T. Furuhashi, J. Biol. Chem., 243, 1543 (1968)); chondroitinase AC (from Flavobacterium heparinum; T. Yamagata, H. Saito, O. Habuchi and S. Suzuki, J. Biol. Chem., 243, 1523 (1968)); chondroitinase ACII (from Arthrobacter aurescens; K. Hiyama and S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama and S. Okada, J. Biochem. (Tokyo), 80, 1201 (1976)); hyaluronidase ACIII (from Flavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1989)); chondroitinase B (from Flavobacterium heparinum; Y. M. Michelacci and C. P. Dietrich, Biochem. Biophys. Res. Commun., 56, 973 (1974), Y. M. Michelacci and C. P. Dietrich, Biochem. J., 151, 121 (1975), Kenichi Maeyama, Akira Tawada, Akiko Ueno and Keiichi Yoshida, Seikagaku, 57, 1189 (1985)); chondroitinase C (from Flavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Kelichi Yoshida, Kiyoshi Morikawa and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1939)); and the like.

[0256] “Chondroitinase (chondroitin sulfate lyase, chondroitinase or chondroitin sulfateyase, sometimes also referred to as “ChSase” below)”, is a class of lyase enzymes that can degrade glycosaminoglycans such as chondroitin sulfate, chondroitin, hyaluronic acid, etc. into unsaturated disaccharides (ΔDi and oligosaccharides).

[0257] “Heparinase”, is a class of polysaccharide lyase enzymes that act on heparin or heparan sulfate, and is found in a variety of microorganisms, among which the heparinase from Flavobacterium heparinum is the most common. There are only three heparinases from Flavobacterium heparinum, namely heparinase I (EC 4.2.2.7), heparinase II (No EC code) and heparinase III (EC 4.2.2.8) (Robert J. Linhardt et al., Purification and characterization of heparin lyases from Flavobacterium heparinum, JBC 1992 Vol. 267:24347-24355).

[0258] Heparinase III mainly acts on heparan sulfate, and has a molecular weight of 73 kDa. Studies have shown that compared with the other two heparinases, heparinase III can act on heparin-like substances in the extracellular matrix to produce small heparin molecules with activity, which can inhibit the proliferation of capillary epithelial cells, thereby inhibiting the growth of tumor cells and reducing the metastasis and spread of cancer cells (patent US6869789B2, CN114703168A).

[0259] “Collagenase” is an endopeptidase that can specifically recognize the Pro-X-Gly-Pro sequence (which frequently occurs in collagen and is rarely found in other proteins) and cut the peptide bond between the neutral amino acid (X) and glycine (Gly) in the sequence. Many proteases can hydrolyze single-chain and denatured collagen polypeptides, but collagenase is the only protease that can degrade natural collagen fibers with a triple-helical superstructure, which is widely present in connective tissue. Common collagenases such as collagenase 1 (MMP1), collagenase 2 (MMP8) and / or collagenase 3 (MMP13), serine protease Cathepsin L, etc.

[0260] “Sialidase”, sialidase (EC 3.2.1.18, neuraminidase) catalyzes the hydrolysis of terminal sialic acid residues of glycoconjugates, sialidases and polypeptides having sialidase activity thereof in patent applications WO2018006034A1, WO2019136167A1, WO2022150521A, WO2022150516A1, WO2022150507A1, WO2022006492A2, WO2021003469A2, WO2021003468A are incorporated herein.

[0261] “Targeting complex”, refers to a protein complex formed by the fusion of a targeting protein containing an Fc fragment of an antibody and an extracellular molecule modifying enzyme and a targeting protein binding fragment, through non-covalent linkage. The formation of this complex does not affect or lose the binding of the targeting protein to its target molecule, nor does it affect or lose the degradation activity of the extracellular molecule modifying enzyme to the extracellular molecule.

[0262] “Target cell” refers to a specific cell population that is actively recognized and targeted for action in drug, gene or cell therapy. They are specifically bound by the corresponding targeting protein, ligand or modifying enzyme through surface markers, metabolic characteristics or functional status, so as to achieve precise intervention. According to the type of target cell, the corresponding targeting protein and extracellular molecule modifying enzyme can be selected. According to certain embodiments, the target cell is selected from the group consisting of cancer cells, immune cells, endothelial cells and epithelial cells. Target cells of interest include, but are not limited to, cells associated with a particular disease or condition. For example, the target cell can be a normally functioning cell (e.g., a normally functioning immune cell, etc.), and the extracellular molecule modifying enzyme modulates the function of the cell in a manner that is therapeutically beneficial to the individual in need, e.g., enhances the function of the cell.

[0263] In other aspects, the target cell is not a normal cell. Non-normal target cells of interest include, but are not limited to, cancer cells. “Cancer cell” refers to a cell that exhibits a tumor cell phenotype, which can be characterized by, for example, one or more of abnormal cell growth, abnormal cell proliferation, loss of density-dependent growth inhibition, nonadherent dependent growth potential, ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any appropriate indicator of cellular transformation. “Cancer cell” is used interchangeably with “tumor cell”, “malignant cell” or “cancerous cell” in the present disclosure, and includes cancer cells of solid tumors, semi-solid tumors, primary tumors, metastatic tumors, etc. In certain aspects, the cancer cell is a carcinoma cell. According to certain embodiments, the cancer cell is selected from the group consisting of breast cancer cells, ovarian cancer cells, gastric cancer cells, colon cancer cells, etc.

[0264] In certain aspects, when the target cell is a cancer cell, the molecule targeted by the protein binding on the surface of the cancer cell is a tumor-associated cell surface molecule or a tumor-specific cell surface molecule. A "tumor-associated cell surface molecule" refers to a cell surface molecule that is expressed on malignant cells with limited expression on cells of normal tissues, a cell surface molecule that is expressed at a higher density on malignant cells compared to normal cells, such as but not limited to PD-L1 or HER2, or a molecule that is specifically expressed during embryonic development, silenced in adulthood but reactivated in tumors.

[0265] In certain aspects, when the target cell is an immune cell, the molecule targeted by the protein binding on the surface of the immune cell is a tumor-immune recognition associated cell surface molecule or a tumor-immune escape associated surface molecule, such as but not limited to PD-1.

[0266] The "second moiety non-covalently bound to the constant region of the protein molecule" refers to a protein, an antibody, an Fc binding fragment of an antibody, an Fc binding fragment of a polypeptide, which can bind to the constant region of an antibody (including the CH region (including the Fc region, the CH1 region, the hinge region) and / or the CL region of an antibody), an Fc fragment or a protein containing an Fc fragment of an antibody by non-covalent binding, including but not limited to Protein A, Protein G, Fc receptor (FcR), an antibody that can specifically bind to the constant region fragment of an antibody, other Fc binding peptides, and Fc binding fragments of the above proteins or polypeptides. The Fc binding proteins or fragments thereof disclosed in the article published by Weonu Choe et al. in 2016 (Weonu Choe et al., Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials (Basel). 2016 Dec; 9(12): 994.) are all incorporated into the present disclosure.

[0267] "Protein A", also known as staphylococcal protein A or protein A, refers to a cell wall-anchored surface protein derived from Staphylococcus aureus, which enables the bacteria to escape innate and adaptive immune responses. Protein A binds to the Fc portion of immunoglobulin. The Protein A and Fc binding fragments thereof disclosed in the prior art, such as CN106422418A, EP2655404A1 and other patent documents, are all incorporated into the present disclosure. An exemplary Fc binding fragment of Protein A is Z33, the amino acid sequence of which is shown as SEQ ID NO. 18, which is a fragment of Protein A binding to Fc and can bind to Fc when present alone. The Fc binding peptides shown as SEQ ID NO. 16-17 are other polypeptides that can bind to Fc.

[0268] "Protein G", also known as Streptococcal Protein G (SPG), is a protein isolated from the cell wall of Streptococcus bacteria and culture supernatant, which can bind to the Fc of antibodies. Protein G and its Fc-binding fragments disclosed in the prior art, such as US5108894A, EP1054061A1 and the like, are incorporated into the present disclosure.

[0269] "FcR" is a class of cell surface proteins that can specifically bind to the Fc fragment of antibodies. FcR that binds to IgG antibodies includes FcyR, FcRn, etc.

[0270] The term "about" when used in association with a numerical value means a numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0271] The term "and / or" should be understood to mean either one of the items or a combination of any two or more of the items.

[0272] As used in the present disclosure, the term "comprising" or "including" means including the recited elements, integers or steps, but not excluding any other elements, integers or steps. In the present disclosure, when the term "comprising" or "including" is used, unless otherwise specified, the case of consisting of the recited elements, integers or steps is also encompassed. For example, when referring to an antibody variable region "comprising" a certain specific sequence, an antibody variable region consisting of the certain specific sequence is also intended to be covered.

[0273] In the present disclosure, the term "antibody" refers to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. The term encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), fully human antibodies or chimeric antibodies or humanized antibodies, full-length antibodies and antibody fragments, antibody conjugates, as long as they exhibit the desired antigen-binding activity.

[0274] The term "antigen binding fragment" of an antibody, which can be used interchangeably with "antibody fragment" and "antigen binding portion" in the present disclosure, refers to a molecule that is not an intact antibody, which comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. As will be understood by those in the art, antigen binding portions of antibodies typically comprise amino acid residues that form the "complementarity determining regions" or "CDRs." Antigen binding fragments can be made by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab, scFv, F(ab'), F(ab')2, Fab'-SH, Fv, single chain Fv, diabody, triabody, tetrabody, minibody, single domain antibody VHH. For more detailed description of antibody fragments, see: Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993); Shao Rong et al. (ed.), Antibody Drug Research and Application, People's Medical Publishing House (2013); Hollinger et al., PNAS USA 90:6444-6448 (1993); Hudson et al., Nat. Med. 9:129-134 (2003).

[0275] The term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al. Kuby Immunology, 6th Ed., W.H. Freeman and Co. p. 91 (2007)). A single VHor VLdomain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a VHor VLdomain from an antibody that binds the antigen to screen a library of complementary VLor VHdomains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0276] The variable region generally exhibits the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from each pair of chains are aligned by the framework regions, which contribute to the specificity of antigen recognition. The two light and three heavy chain variable regions generally comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N- to C-terminus. The part of an antibody other than the variable region of an antibody is the constant region of an antibody, in a fusion protein with a targeting binding capacity of the Fc fragment, the part of the constant region of an antibody is the part of the Fc fragment comprised by the fusion protein.

[0277] A "pharmaceutical composition" refers to a preparation or combination of preparations which contain one, two or more active ingredients, which permits the active ingredients contained therein to exist in an effective form for biological activity and does not contain additional ingredients which are unacceptable for the subject to whom the preparation is administered. When the "pharmaceutical composition" exists in the form of a combination of separate preparations containing different, two or more active ingredients, they can be administered simultaneously, sequentially, separately or at intervals, with the aim of exerting the biological activity of the plurality of active ingredients together for the treatment of a disease.

[0278] In a particular aspect, the bifunctional molecule according to the present disclosure further comprises a peptide linker connecting the antigen binding domain and the IL-7m to the Fc chain. The peptide linker generally has sufficient length and flexibility to ensure sufficient spatial freedom for the IL-7m and the antigen binding domain connected by the linker in between to exert their functions.

[0279] A "linker", "peptide linker" or "linker" refers to a sequence of at least one amino acid. Such a peptide linker can serve to prevent steric hindrance. The length of the peptide linker is generally 3-44 amino acid residues. Preferably, the peptide linker has 3-30 amino acid residues. In some aspects, the peptide linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues.

[0280] The peptide linker sequence can be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the peptide linker is preferably non-immunogenic in a subject to which the bifunctional molecule is practically administered. One group of useful peptide linker sequences are peptide linkers derived from heavy chain antibody hinge regions as described in WO 96 / 34103 and WO 94 / 04678, another group of useful peptide linker sequences are derived from "peptide linkers" as described in CN110520444A. Other examples are polyalanine linker sequences.

[0281] Further preferred examples of linker sequences are flexible linkers (e.g., (Gly4Ser) n , (Gly) n ), rigid linkers (e.g., (EAAAK)n, (XP)n) and cleavable linkers. For example, Gly / Ser linkers of varying lengths, including (Gly4Ser) n (n = 1-9), (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser and (Gly3Ser2)3, particularly (Gly4Ser)3. Preferably, the peptide linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)5, (Gly4Ser)3 and (Gly3Ser2)3. Even more preferably, the peptide linker is (Gly4Ser)5.

[0282] In one aspect, the peptide linker comprised in the fusion protein is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser and (Gly3Ser2)3, preferably (Gly4Ser)3. Preferably, the peptide linker is selected from the group consisting of (Gly4Ser)5, (Gly4Ser)4, (Gly4Ser)3 and (Gly3Ser2)3.

[0283] An "effective amount" is generally an amount that is sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or their underlying cause, prevent symptoms and / or their underlying cause from occurring, or ameliorate or improve damage that results from or is associated with the disease state (e.g., a lung disease). In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount that is sufficient to treat a disease state or symptoms, particularly states or symptoms associated with the disease state, or otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable symptoms associated with the disease in any way. A "prophylactically effective amount" is an amount that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of the disease state, or reducing the likelihood of the onset (or reoccurrence) of the disease state or associated symptoms. A full therapeutic or prophylactic effect can not be achieved with a single dose, and may

[0284] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny can not be completely identical to the parental cell both in genetic and phenotypic characteristics, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the present disclosure.

[0285] "Transfection" refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by a variety of means known in the art, including electroporation, microinjection, liposome fusion, etc.

[0286] "Nucleic acid molecule encodes," "coding DNA sequence," and "coding DNA" refer to the order of deoxyribonucleotides along the chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0287] The term "tumor" as used in the present disclosure refers to a disease characterized by the pathological proliferation of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is generally uncontrolled and progressive, and does not induce or suppress normal cell proliferation. Tumors include "cancer," which refers to all malignant tumors.

[0288] "Treatment" refers to clinical intervention in an attempt to alter the disease course of the individual or cell involved, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any pathological consequences of the disease either directly or indirectly, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating disease state, remission or improved prognosis, etc.

[0289] "Combination" relates to a treatment regimen in which at least two or more different therapies are provided to achieve a specified therapeutic effect, which can be physical, such as radiation therapy, or chemical, such as administration of a drug to a subject, which also includes co-drugs. "Co-drugs" refer to a combination of two or more pharmaceutical preparations each having an active ingredient, which are intended to be used in conjunction with each other in the administration to a subject. The active ingredients can be mixed together to form a single administration unit, or can be separate administration units, used separately; in administration, the different pharmaceutical preparations can be administered substantially simultaneously, concurrently or sequentially.

[0290] The term "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms which are suitable for use with humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio as well as the edibility of the final product.

[0291] The "anti-PD1 antibody" used in the present disclosure refers to an antibody or antigen-binding fragment thereof targeting the immune checkpoint protein PD1 or PD-1, capable of blocking or affecting the binding of PD1 to its ligand (such as PD-L1). The selection range of anti-PD1 antibody includes but is not limited to Nivolumab (Keytruda, Pembrolizumab). "PD1 (Programmed Death 1)", also known as CD279, belongs to the immunoglobulin superfamily.

[0292] The term "amino acid mutation" encompasses amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at the final construct, as long as the final construct possesses the desired properties. Amino acid sequence deletions and insertions include amino and / or carboxyl terminal deletions and amino acid insertions. In certain embodiments of the present disclosure, the specific amino acid mutation is an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., the replacement of one amino acid by another amino acid of different structure and / or chemical properties. Amino acid substitutions include replacement by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Genetic or chemical methods well known in the art can be used to generate amino acid mutations. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. Methods other than genetic engineering to alter the side chain groups of amino acids, such as chemical modifications, can also be useful. Various names can be used in the present disclosure to refer to the same amino acid mutation.

[0293] The term "affinity" or "binding affinity" refers to the strength of specific binding between two biological molecules (such as ligand and receptor, antibody and antigen, enzyme and substrate, etc.) through non-covalent interactions (such as hydrogen bonds, van der Waals forces, hydrophobic interactions, electrostatic attractions, etc.), which is usually quantitatively characterized by the equilibrium dissociation constant (KD). The smaller the KD value (such as picomolar to nanomolar level), the tighter the binding between molecules and the higher the affinity. For example, in antibody engineering, the affinity of Fc region to Protein A / Z33 mutant directly affects the efficiency of antibody purification; while in the development of therapeutic antibodies, high affinity can enhance the target binding capacity and prolong the residence time of the drug in the target tissue, thereby improving the therapeutic effect. Affinity can be accurately determined by techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and biolayer interferometry (BLI), among which the preferred determination method is biolayer interferometry (BLI).

[0294] The term "polynucleotide" is used interchangeably herein with the term "nucleic acid" and refers to a polymeric form of deoxyribonucleotides or ribonucleotides that are either linked together to form single- or double- stranded structures. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties to the reference nucleotide and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0295] The term "sequence identity" means degrees (percentages) to which the amino acid / nucleic acid of two sequences are the same at equivalent positions, when the two sequences are optimally aligned, gaps are introduced as necessary to achieve the maximum percent sequence identity, and no conservative substitutions are counted as part of the sequence identity. One of skill in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the PILEUP and BLAST algorithms can be used to calculate identity or align sequences such as identifying equivalent or corresponding sequences (typically on their default settings). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words buried in the sequence of the query via a similarity search using a word comparison algorithm. These initial needle neighbourhood hits act as seeds for initiating searches to find HSPs containing them. While the seeds are extended in both directions over the comparison window, the number of matches of each extension is counted using the criteria set. Extension stop when: the number of matches is lower than the value of the length of the comparison window divided by 2; the cumulative alignment score falls off by the value of the product of the ambient gap penalty and the number of mismatched base pairs; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. Methods of alignment of sequences for comparison are well known in the art, such as GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.

[0296] The BLAST algorithm statistically analyzes the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which an alignment between two polynucleotide or amino acid sequences could have occurred by chance. For example, a sequence is considered similar to another if the smallest sum probability in comparison of the two sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG software package provides the BESTFIT program (e.g., using its default settings) which can be used to calculate identity.

[0297] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any way, i.e. to obtain preferred embodiments of the present disclosure. The reagents and materials used in the present disclosure are commercially available. DETAILED DESCRIPTION

[0298] The technical solutions of the present disclosure are further illustrated below by means of specific embodiments. Those skilled in the art should understand that the embodiments described are only used to help understand the present disclosure, and should not be regarded as specific limitations on the present disclosure.

[0299] The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to conventional methods and conditions, or according to the instructions of the commercial products.

[0300] Example 1: Construction, expression and preparation of fusion proteins

[0301] DNA fragments encoding fusion proteins (the amino acid sequences of different proteins are shown in Table 1) were synthesized entirely, and were cloned into the constructed expression vectors, respectively. The expression vectors were composed of the following elements:

[0302] (1) glutamine synthetase gene, as a selection marker;

[0303] (2) replication origin: ori, which allows the plasmid to replicate in E. coli;

[0304] (3) β-lactamase gene, which confers ampicillin resistance in E. coli,

[0305] (4) enhancer and promoter from human cytomegalovirus,

[0306] (5) human 1 -immunoglobulin polyadenylation ("poly A") signal sequence, and

[0307] As described above, the target gene containing the fusion protein was prepared by gene synthesis, and was cloned into the pDrG(Amp) plasmid using Hind III and Pac I.

[0308] The constructed fusion protein vectors were transformed into E. coli DH5α, and positive clones were inoculated into 500 ml LB medium for amplification. The DNA was extracted and purified using the Ultrapure Plasmid DNA Purification Kit from Qiagen, according to the manufacturer's instructions. The plasmid DNA containing the fusion protein coding sequence was transfected into CHO-K1 (Chinese hamster ovary cells, purchased from ATCC) using the X-porator H1 electroporator from Yidabiotech, according to the manufacturer's instructions.

[0309] The cell culture medium was replaced with a screening medium containing a screening drug 24-48 hours after transfection, and the screening medium was replaced every 3-4 days until the cell viability was greater than 95%, after which the volume was expanded for expression culture, and the medium was replaced with an expression medium for fed-batch culture. After 3-4 days of culture, the feed medium and glucose were added every other day, and the cell density was greater than 1 x 10 7 After the cell viability was less than 80%, the cells were centrifuged at 9500 rpm for 20 minutes to collect the culture supernatant, and the fusion protein sample was purified by nickel affinity chromatography. The purified sample was filtered through a 0.22 μm membrane, the protein concentration was detected by nanodrop, and the sample was stored at 2-8°C.

[0310] Table 1. Amino acid sequences of enzymes, antibodies, and Fc-binding polypeptides

[0311] Example 2: Detection of the activity of human sialidase NEU2-M-anFcVH (SEQ ID NO. 29) fusion protein

[0312] The purified NEU2-M-anFcVH was diluted to 100 μg / ml with PBS, and then diluted 3-fold to obtain 5 concentration points, with PBS as a blank control. The enzyme substrate 4-MU-NANA was diluted to 2 mM, mixed with NEU2-M-anFcVH at a volume ratio of 1:1, and reacted at 37°C. After reaction, 0.1 M Na2CO3 at pH 10.5 was added to terminate the reaction. The λ ex 365nm, λem 450nm, the higher the enzyme activity. The sample fluorescence value minus the blank control fluorescence value is fitted by a four-parameter curve, and the EC 50 .

[0313] The results are shown in Figure 1, NEU2-M-anFcVH enzyme activity at 100 μg / ml is close to the upper limit of detection, EC 50 about 8.0 μg / ml, with high enzyme activity.

[0314] Example 3: Sialidase NEU2-M-anFcVH fusion protein (SEQ ID NO. 29) and IgG1, IgG4 affinity

[0315] Fortebio was used to detect the affinity of NEU2-M-anFcVH with IgG1 and IgG4, respectively. NEU2-M-anFcVH has a his tag, so 20 μg / ml of NEU2-M-anFcVH protein was loaded with HIS1K probe, and the loading height was set to 0.5 nm. Antibody-1 (anti-Her2 antibody, the amino acid sequences of the light and heavy chains are shown in SEQ ID NO. 1-3) and Keytruda (anti-PD-1 antibody) are antibodies of IgG1 and IgG4 types, respectively, and are diluted 2-fold from 1500 nM to 5 concentration points, and the qualified data are selected for affinity calculation.

[0316] Table 2. NEU2-M-anFcVH and IgG1, IgG4 affinity

[0317] The results are shown in Table 2, NEU2-M-anFcVH binds to antibody-1 and Keytruda, and the affinity is both in the order of 10 -8 M, sufficient to bind to the target antibody to form a complex.

[0318] Example 4: In vitro sialidase activity detection of complex target cell surface

[0319] Take the SKBR3 and Jurkat T cells in the logarithmic growth phase, count the total culture medium, and centrifuge at 300g for 5 minutes, resuspend with the corresponding serum-free medium to adjust the cell density to 2×10 6 / ml, 100 μL of cells were added into a flat-bottom 96-well plate. 100 μL of different concentrations of target protein (antibody) complex (NEU2-M-anFcVH and Keytruda (purchased from Merck) as target complex for HER2 and PD-1 respectively, non-targeted IgG1 antibody as non-targeted complex as negative control, bacterial-derived sialidase α2-3,6,8,9 Neuraminidase (purchased from RhinoBio) as positive control P) were incubated with cells in a 37°C incubator for 1 hour, 100 μL of each well was taken out and added into a new 96-well plate after mixing the head of each well, 300g centrifugation for 5 minutes, and washed with PBS for 2 times. 100 μL of PNA-AF647 reagent or blocking solution was added to resuspend the cells, and the cells were incubated in the dark at 4°C for 30 minutes, 300g centrifugation for 5 minutes, and washed with blocking solution for 2 times. The cells were resuspended in 200 μL of blocking solution and then detected by machine.

[0320] The effect of exposing galactose after sialic acid cleavage by Neu2 enzyme was shown by PNA staining, and the effect of sialic acid cleavage on target cells after binding of target Neu2 was detected, and SK-BR-3 (HER2 + + +) and Jurkat T (PD-1 effect) cells were selected (see FIG. 2 and FIG. 3). It is proved that the same lower concentration of sialidase, target protein (antibody) complex has very clear degradation ability for sialic acid on the surface of target cells, while the non-targeted antibody complex cannot effectively degrade the sialic acid on the surface of target cells.

[0321] Example 5: Detection of enzyme activity in sialidase-Z33 fusion protein

[0322] According to the quantitative results, the supernatant of the fusion protein (hNEU2-M-Z33 or Z33-hNEU2-M) was diluted with PBS, and PBS was used as a blank control. The enzyme substrate 4-MU-NANA was diluted to 2 mM and mixed with hNEU2-M-Z33 or Z33-hNEU2-M at a volume ratio of 1:1, and then the reaction was allowed to stand at 37°C for 2 hours. Then, 0.1M Na2CO3 with pH 10.5 was added to terminate the reaction. The fluorescence values of λ ex 365nm, λ em 450nm were detected by using an enzyme marker, and the higher the fluorescence value, the higher the enzyme activity.

[0323] The results are shown in FIG. 4, and the supernatant of the two proteins has sufficient enzyme activity.

[0324] Example 6: Fortebio confirms the affinity of fusion protein to Fc

[0325] Fortebio was used to detect the affinity of fusion protein to Fc. Two supernatants were loaded with HIS1K probe respectively, and the response value was set to 0.5-0.8 nm after loading, then different concentrations of antibodies were combined, the antibody (antibody-1) was diluted by 2 times from 183.5 nM, and there were 5 concentration points, and the qualified data was selected for affinity calculation.

[0326] Table 3. Affinity of fusion protein to Fc

[0327] Note: * literature data

[0328] The results are shown in Table 3, and Z33 can still bind to the antibody after fusion with hNEU2-M. Compared with the Z33 molecule alone, the affinity of the fusion protein to the antibody is higher, reaching the pM level.

[0329] Example 7: Affinity detection

[0330] The method of biofilm interference binding (BLI) was used to detect the affinity of Z33 mutant and antibody-1. The experiment was carried out on Fortebio Octet Red384 instrument according to the standard operation manual. The samples were stored in PBS containing 0.1% BSA and 0.05% Tween (PBST BSA). Compared with the wild type sequence, the relevant sequence details are shown in Table 4. Among them, the sequences with improved affinity are SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 39, i.e. Z33 Q4L or Z33 S29Q single mutation, Z33 Q4L-S29Q and Z33 Q4L-S29K double mutation. The Z33 Q6W-S29K and Z33 F1L-Q6W double mutants cannot be detected for binding.

[0331] Table 4. Affinity detection of Z33 mutant and antibody-1

[0332] The fusion protein of Z33 mutant fused with LIGAND A also showed excellent affinity. Compared with the affinity of wild type Z33 and antibody-1 in Table 5, the affinity of the fusion protein of Z33 Q4L mutant and Z33 S29Q mutant to antibody-1 was significantly improved. The affinity of Z33 F1L mutant was lower than that of wild type. In addition, the mutation of Z33 S29R / K and Q4L can significantly improve the effect of affinity decrease caused by mutation at other sites in Z33.

[0333] Table 5. Affinity detection of Z33 mutant fusion protein and IgG

[0334] These results demonstrate that the fusion proteins constructed with Z33 and its mutants also have the potential to be used for purifying, detecting Fc-containing molecules, suitable for large-scale production of such compounds, or as Fc engineering tools for drug design and development.

[0335] Fortebio confirmed the affinity of the fusion proteins to Fc

[0336] Fortebio was used to detect the affinity of the fusion proteins to atezolizumab. Four proteins were loaded with HIS1K probe respectively, and the load response value was set to 0.5 nm. Different concentrations of antibodies were combined, with atezolizumab starting from 100 nM, 2-fold dilution, a total of 6 dilution points, and appropriate data were selected for affinity calculation.

[0337] Table 6. Affinity of fusion proteins to Fc

[0338] Example 8: Immunogenicity prediction

[0339] The MHC I immunogenicity of each polypeptide sequence was predicted using the publicly available bioinformatics tool NetMHCpan-4.1, and the MHC II immunogenicity was predicted using NetMHCIIpan-4.0 (https: / / doi.org / 10.1093 / nar / gkaa379). The MHC I and II immunogenicity scores of SEQ ID NO: 36, 37, 38, 39 relative to SEQ ID NO: 18 all decreased.

[0340] Example 9: Detection of the targeting complex of PH20-anFcVH and antibody-1 for HER2+ tumor cells in tumor-bearing mice

[0341] The purified PH20-anFcVH fusion protein and the anti-HER2 antibody antibody-1 were mixed to form a targeting complex.

[0342] Protein labeling fluorescence: 1 mg of PH20-anFcVH fusion protein (2.45 mg / ml) sample was transferred to a 1.5 mL Ep tube, and a Cy5-nhs solution with a concentration of 5 mg / mL was added according to the dye labeling molar ratio of 10 / 1 (dye / labeled sample). At the same time, 10 μl of triethylamine was added, and the sample was labeled overnight on a 3D shaker at room temperature.

[0343] Fluorescent protein purification:

[0344] a. Place a spin column in a 2 mL Ep tube, mix the purification resin, make sure the suspension is even, add 250 μΐ of the suspension to the spin column, and remove the plug at the bottom of the spin column. Centrifuge at 1000 g for 2 minutes, remove the storage solution, add 250 μΐ of PBS, and centrifuge again at 1000 g for 2 minutes. Discard the used collection tube and place the column in a new collection tube.

[0345] b. Add 250 μΐ of the labeled solution to the spin column, and mix the sample with the resin by brief vortexing. Centrifuge at 1000 g for 2 minutes, collect the purified sample, and combine samples from the same sample. Obtain PH20-anFc VH fusion protein-Cy5 molecules.

[0346] Human breast cancer HCC1569 cell line was subcutaneously transplanted into NOD / SCID female mice, and the tumor volume was about 200 mm 3 Group tail vein injection of 100 μΐ tumor-bearing mice (G1 : PBS, G2: PH20-anFc VH fusion protein-Cy5, G3: Isotype-IgG+PH20-anFc VH-Cy5, G4: antibody-1 +PH20-anFc VH-Cy5), the same dose of PH20-anFc VH-Cy5 in each group. 54.5 hours after administration, the tumor, heart, spleen, and kidney were collected and placed in a black paper shell for fluorescence imaging detection of the Cy5 channel (649 / 666).

[0347] The results are shown in Figure 5: the G4 group (antibody-1 +PH20-anFc VH-Cy5 fluorescence) is superior to the G2 and G3 groups in targeting hHER2-HCC1569 tumor cells. The targeting complex targets and binds to HER2 molecules on the surface of tumor cells, and binds less to other organs that are not positive for HER2 molecules, reflecting the enrichment of the complex on the cell surface where the targeting molecule is located.

[0348] Example 10: Detection of the affinity of different hyaluronidases fused with VHH to Fc

[0349] Fortebio was used to detect the affinity of the fusion protein to Fc. The expression supernatant of four hyaluronidase-VHH fusion protein molecules (Table 7, Nos. 1-4) was loaded with HIS1K probe, and the loading response value was set to 0.5-0.8 nm. Different concentrations of antibodies were combined, starting from 1000 nM, 2-fold dilution for 4 concentration points, and affinity calculation was performed.

[0350] Table 7. Affinity results of four hyaluronidase-VHH fusion protein molecules to Fc

[0351] The results are shown in Table 7, and all fusion proteins have strong binding to antibodies.

[0352] Example 11: Detection of enzyme activity in hyaluronidase-VHH fusion proteins

[0353] Take 4 kinds of expression supernatant respectively with enzyme diluent dilution 50 times, mixed with 0.5 mg / ml high molecular weight hyaluronic acid sodium solution 1:1, 37℃ incubation 1 hour. Preparation of 0.5% TAE agarose gel, take the mixed liquid after reaction for agarose gel electrophoresis, using 50V voltage electrophoresis 2-3 hours. Preparation of 50% ethanol 0.005% Stains-All staining solution, electrophoresis after overnight staining in the dark. Using purified water light decolorization 1 hour, scanning and taking pictures.

[0354] The results are shown in Figure 6, the supernatant of the four proteins is diluted 100 times, all have enzyme activity, PH20 fusion protein (Figure 6 lanes 3, 7, corresponding to Table 7 molecules 2, 4) can completely degrade the substrate hyaluronic acid sodium, consistent with the nature of PH20 alone; HYAL1 fusion protein (Figure 6 lanes 1, 5, corresponding to Table 7 molecules 1, 3) is consistent with the nature of HYAL1 alone, can degrade large molecular weight hyaluronic acid sodium into small molecules.

[0355] Example 12: Effect detection of targeted complex Keytruda+NEU2-M-anFcVH in mouse model

[0356] To detect the efficacy of the targeted complex in BALB / c-hPD1 / hPDL1 model, the administration method and study groups as shown in Table 8 were set up, 6 mice in each group, 2 times a week for a total of 3 weeks. The anti-tumor effect of the targeted complex was studied.

[0357] Table 8. Administration method of each group for studying the efficacy of Keytruda+Neu2-M-anFcVH in CT26-hPDL1 model

[0358] The results (Figure 7) show that Keytruda+NEU2-M-anFcVH shows good anti-tumor activity, significantly better than Keytruda.

[0359] Example 13: Effect detection of targeted complex Atezolizumab+Z33-mut1-PH20 in PBMC NCI-H292 model

[0360] To detect the efficacy of the targeted complex in PBMC NCI-H292 model, the administration method and study groups as shown in Table 9 were set up. 6 NOG mice in each group, 2 times a week for 2 consecutive weeks, and tumor growth was detected.

[0361] Table 9. Dosing schedule of each group for the study of Atezolizumab + Z33-mutl-PH20 efficacy in PBMC NCI-H292 model

[0362] The results (Figure 8) show that Atezolizumab + Z33-mutl-PH20 shows good tumor inhibition activity, which is significantly better than Atezolizumab.

[0363] Example 14: Detection of the effect of the targeting complex antibody-1 + NEU2-M-anFcVH in the EMT6-HER2 model

[0364] To detect the therapeutic effect of the targeting complex in the EMT6-hHER2 model, the dosing schedule and study groups shown in Table 10 were set, 6 mice per group, 2 times per week for a total of 3 weeks. The tumor inhibition effect of the targeting complex was studied.

[0365] Table 10. Dosing schedule of each group for the study of antibody-1 + NEU2-M-anFcVH efficacy in EMT6-hHER2 model

[0366] Example 15: Detection of the effect of the targeting complex antibody-1 + PH20-anFcVH in the EMT6-hHER2 model

[0367] To detect the therapeutic effect of the targeting complex in the EMT6-hHER2 model, the dosing schedule and study groups shown in Table 11 were set, 6 mice per group, 2 times per week for a total of 3 weeks.

[0368] Table 11. Dosing schedule of each group for the study of antibody-1 + PH20-anFcVH efficacy in EMT6-hHER2 model

[0369] Although the specific embodiments of the present disclosure are described above, those skilled in the art should understand that these are only illustrative, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the protection scope of the present disclosure is defined by the appended claims.

Claims

1. A targeting complex comprising a first molecule and a second molecule, the first molecule comprising an Fc fragment portion and a targeting binding fragment portion, the second molecule comprising a first portion of an enzyme and a second portion capable of non-covalently binding to the first molecule; optionally, the first molecule is a targeting protein comprising an Fc fragment portion and a targeting binding fragment portion, and the second molecule is a fusion protein.

2. The targeting complex of claim 1, wherein, the first molecule comprises an antibody or a fusion protein comprising an Fc fragment; optionally, the antibody is selected from an anti-Her2 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

3. The targeting complex of claim 1 or 2, wherein, the non-covalent binding is an inter-protein interaction, optionally, the inter-protein interaction is selected from an antigen-antibody binding mode, a Protein A / Protein G-antibody Fc binding mode, or an antibody Fc receptor-antibody Fc binding mode.

4. The targeting complex of any one of claims 1-3, wherein, the second molecule retains the enzymatic activity, and / or the first molecule retains the targeting activity; optionally, upon the targeting complex binding to a target molecule, the second molecule can act on a substrate molecule of the enzyme surrounding the target molecule.

5. The targeting complex of any one of claims 1-4, wherein, the enzyme is an extracellular molecule modifying enzyme; optionally, the enzyme is selected from a hyaluronidase, a sialidase, an N-glycosidase, an O-glycosidase, a collagenase, a heparinase, a chondroitinase, or a chondrosinase; optionally, the sialidase is selected from a Salmonella typhimurium sialidase, a Vibrio cholerae sialidase, a human neuraminidase 1, a human neuraminidase 2, a human neuraminidase 3, or a human neuraminidase 4; optionally, the N-glycosidase is selected from Endo H, PNGase F, PNGase A, Endo S, Endo S2, Endo D, Endo F2, or Endo F3; optionally, the O-glycosidase is selected from Endo-O-Glycosidase or Exo-O-Glycosidase; optionally, the hyaluronidase is selected from HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, or PH20 / SPAM1; optionally, the chondroitinase is selected from Chondroitinase C, Chondroitinase B, Hyaluronidase ACIII, Chondroitinase ACII, Chondroitinase AC, or Chondroitinase ABC; optionally, the heparinase is selected from Heparinase I, Heparinase II, or Heparinase III; and / or optionally, the collagenase is selected from Collagenase 1, Collagenase 2, Collagenase 3, or Serine protease Cathepsin L.

6. The targeting complex of any one of claims 1-5, wherein, the second portion of the second molecule is capable of non-covalently binding to the constant region of the first molecule; optionally, the second portion of the second molecule is capable of non-covalently binding to the Fc fragment portion of the first molecule; Optionally, the second portion of the second molecule has an affinity KDof 1 x 10 -07 M ~ 1 x 10 -12 M; Optionally, the second moiety of the second molecule is selected from an antibody or an antigen-binding fragment thereof (e.g., VHH, affibody, scFv, Fab, or F(ab')2) that can bind to an antibody constant region fragment, or is selected from Protein A or an Fc-binding fragment thereof, an affibody or an Fc-binding fragment thereof, Protein G or an Fc-binding fragment thereof, or an FcR or an Fc-binding fragment thereof, or is an Fc-binding peptide; Optionally, the enzyme of the second molecule is connected to the second moiety by a peptide bond or a linker, optionally, the linker is a peptide linker.

7. The targeting complex of any one of claims 1-6, wherein, The second moiety of the second molecule is a polypeptide that is wild-type Z33 or has an amino acid mutation relative to wild-type Z33.

8. The targeting complex of claim 7, wherein, The position of the amino acid mutation includes at least one of position 1, position 4, and position 29, wherein the wild-type Z33 has an amino acid sequence shown in SEQ ID NO:

1.

9. The targeting complex of claim 7 or 8, wherein, The polypeptide that has an amino acid mutation relative to wild-type Z33 has an amino acid substitution relative to wild-type Z33. Optionally, the substituted amino acid at position 1 is selected from F or L. Optionally, the substituted amino acid at position 4 is selected from Q, F, E, R, or L. Optionally, the substituted amino acid at position 29 is selected from S, Q, R, or K.

10. The targeting complex of any one of claims 7-9, wherein, The polypeptide that has an amino acid mutation relative to wild-type Z33 has an amino acid sequence that is further at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 97% identical to the amino acid sequence shown in SEQ ID NO:

1.

11. The targeting complex of any one of claims 7-10, wherein, The polypeptide having an amino acid mutation relative to wild type Z33 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 , wherein, X1 is selected from F or L; X4 is selected from Q, F, E, R, or L; X 29 selected from S, Q, R or K; further, X2 is selected from N, E, or S; X3 is selected from M, K, or E; X5 is selected from Q or C; X6 is selected from Q, W, L, K, or A; X7 is selected from R, L, K, N, A, D, or C; X8 is selected from R or A; X9 is selected from F, M, or A; X 10 selected from Y, A or S; X 11 selected from E, A, D or S; X 12 selected from A, I, T or E; X 13 selected from L, A, G or K; X 14 is selected from H, A, V, F, W or G; X 15 selected from D, V, C, N or L; X 16 selected from P, L or A; X 17 is selected from N, L, T or G; X 18 selected from L, M or V; X 19 selected from N, G or T; X 20 selected from E, K, A or Q; X 21 selected from E or Q; X 22 Q is H, F, Cl, Br, I, CN, CF X 23 R2is H, F, Cl, Br, I, CF3 X 24 is selected from N, W, A or K; X 25 selected from A or G; X 26 selected from K, R or F; X 27 I; X 28 selected from K, C, Q or R; X 30 selected from I, F or L; X 31 selected from R, M or K; X 32 selected from D, N, K or E; and X 33 is selected from D, N or E.

12. The targeting complex of claim 11, wherein, X7 is R; X9 is F; X 10 Y; X 13 L; X 23 R is H, F, Cl, Br, I, CF3, CN, N02, ox X 30 I.

13. The targeting complex of claim 11 or 12, wherein, X 11 E or D; X 12 is A or T; X 14 is H or W; X 16 P, A or L; X 17 is N, L or T; X 18 is N or M; and / or X 23 R is H, F, Cl, Br, I, CF3, 14. The targeting complex of any one of claims 11-13, wherein, X1 is L; X4 is L; and / or X 29 Q, K or R.

15. The targeting complex of claim 5, wherein, the hyaluronidase amino acid sequence is set forth in any one of SEQ ID NO. 6, 7, or 65-71, the sialidase amino acid sequence is set forth in any one of SEQ ID NO. 4, 5, or 55-60, the collagenase amino acid sequence is set forth in SEQ ID NO. 8, the heparinase amino acid sequence is set forth in SEQ ID NO. 9, the chondroitin sulfatease amino acid sequence is set forth in SEQ ID NO. 10, and the chondroitinase amino acid sequence is set forth in SEQ ID NO.

11.

16. The targeting complex of any one of claims 1-15, wherein, the second part of the second molecule is an antibody or an antigen-binding fragment thereof comprising CDR1-CDR3, the amino acid sequences of which are according to the Kabat coding rule, respectively, SEQ ID NO. 31-33; Optionally, the second part of the second molecule comprises a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO. 12-18 and SEQ ID NO. 34-39, or an Fc-binding fragment thereof; Optionally, the second part of the second molecule comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NO. 34-39, or an Fc-binding fragment thereof; Optionally, the second part of the second molecule has an amino acid sequence of SEQ ID NO. 34, 36, 38 or 39.

17. The targeting complex of any one of claims 1-16, wherein, The fusion protein as the second molecule is selected from a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO. 19-30 or SEQ ID NO. 61-64.

18. A molecule comprising a first part of an enzyme and a second part capable of non-covalently binding to an antibody constant region, optionally, the molecule is a fusion protein.

19. The molecule of claim 18, wherein, The non-covalent binding is an inter-protein interaction, optionally, the inter-protein interaction is selected from an antigen-antibody binding mode, a Protein A / Protein G-antibody Fc binding mode, or an antibody Fc receptor-antibody Fc binding mode; Optionally, the second portion has a KD of 1 x 10 -07 M~1 x 10 -12 M.

20. The molecule of claim 18 or 19, wherein, The molecule retains the enzymatic activity, optionally, the molecule, after binding to a first molecule, modifies a substrate molecule of the enzyme surrounding a target of the first molecule, the first molecule comprising an Fc fragment part and a targeting binding fragment part, optionally, the first molecule comprises an antibody or a fusion protein comprising an Fc fragment.

21. The molecule of any one of claims 18-20, wherein, The enzyme is an extracellular molecule modifying enzyme; Optionally, the enzyme is selected from a hyaluronidase, a sialidase, an N-glycosidase, an O-glycosidase, a collagenase, a heparinase, a chondroitinase, or a chrondosinase; Optionally, the sialidase is selected from a Salmonella typhimurium sialidase, a Vibrio cholerae sialidase, a human neuraminidase 1, a human neuraminidase 2, a human neuraminidase 3, or a human neuraminidase 4; Optionally, the N-glycosidase is selected from Endo H, PNGase F, PNGase A, Endo S, Endo S2, Endo D, Endo F2, or Endo F3; Optionally, the O-glycosidase is selected from Endo-O-Glycosidase or Exo-O-Glycosidase; Optionally, the hyaluronidase is selected from HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, or PH20 / SPAM1; Optionally, the chondroitinase is selected from chondroitinase C, chondroitinase B, hyaluronidase ACIII, chondroitinase ACII, chondroitinase AC, or chondroitinase ABC; Optionally, the heparinase is selected from heparinase I, heparinase II, or heparinase III; and / or Optionally, the collagenase is selected from collagenase 1, collagenase 2, collagenase 3, or serine protease Cathepsin L.

22. The molecule of any one of claims 18-21, wherein, the second part of the molecule is capable of non-covalently binding to the constant region of the first molecule; Optionally, the second part of the molecule is capable of non-covalently binding to the Fc region of the first molecule; Optionally, the second part of the molecule is selected from an antibody or an antigen-binding fragment thereof (e.g. VHH, scFv, Fab or F(ab')2) capable of binding to an antibody constant region fragment, or selected from Protein A or an Fc-binding fragment thereof, Protein G or an Fc-binding fragment thereof, or FcR or an Fc-binding fragment thereof, or is an Fc-binding peptide.

23. The molecule of any one of claims 18-22, wherein, the second part of the molecule is the second part of the second molecule in the targeting complex of any one of claims 7-14.

24. The molecule of claim 21, wherein, the hyaluronidase amino acid sequence is as set forth in any one of SEQ ID NO. 6, 7, or 65-71; the sialidase amino acid sequence is as set forth in any one of SEQ ID NO. 4, 5, or 55-60; the collagenase amino acid sequence is as set forth in SEQ ID NO. 8; the heparinase amino acid sequence is as set forth in SEQ ID NO. 9; the chondroitin sulfatease amino acid sequence is as set forth in SEQ ID NO. 10; the chondroitinase amino acid sequence is as set forth in SEQ ID NO.

11.

25. The molecule of any one of claims 18-24, wherein, the second part of the molecule is an antibody or an antigen-binding fragment thereof comprising heavy chain variable region CDR1-CDR3, the amino acid sequences of which, according to the Kabat coding rules, comprise SEQ ID NO. 31-33, respectively; Optionally, the amino acid sequence of the second part of the molecule comprises a polypeptide or an Fc-binding fragment thereof selected from any one of SEQ ID NO. 12-18 and SEQ ID NO. 34-39; Optionally, the amino acid sequence of the second part of the second molecule is selected from a polypeptide or an Fc-binding fragment thereof as set forth in any one of SEQ ID NO. 34-39; Optionally, the amino acid sequence of the second part of the second molecule is SEQ ID NO. 34, 36, 38, or 39.

26. The molecule of any one of claims 18-25, which is selected from a polypeptide as set forth in any one of SEQ ID NO. 19-30 or SEQ ID NO. 61-64.

27. A polypeptide, wherein, the polypeptide is the second part of the second molecule in the targeting complex of any one of claims 7-14, the second part of the second molecule being the polypeptide having an amino acid mutation relative to wild-type Z33; Optionally, the amino acid sequence of the polypeptide having an amino acid mutation relative to wild-type Z33 comprises an amino acid sequence selected from any one of SEQ ID NO. 34-39; Optionally, the amino acid sequence of the polypeptide having an amino acid mutation relative to wild-type Z33 is selected from any one of SEQ ID NO. 34-39; Optionally, the amino acid sequence of the polypeptide having an amino acid mutation relative to wild type Z33 is SEQ ID NO. 34, 36, 38, or 39.

28. A composition comprising: the targeting complex of any one of claims 1-17, the molecule of any one of claims 18-26, or the polypeptide of claim 27; and a pharmaceutically acceptable carrier.

29. A nucleic acid encoding the targeting complex of any one of claims 1-17, the molecule of any one of claims 18-26, or the polypeptide of claim 27.

30. An expression vector comprising the nucleic acid of claim 29.

31. A host cell comprising the nucleic acid of claim 29 or the expression vector of claim 30.

32. The host cell of claim 31, wherein, the host cell is a mammalian host cell, a prokaryotic host cell, or a yeast host cell.

33. A method comprising administering to an individual in need thereof the targeting complex of any one of claims 1-17 or the molecule of any one of claims 18-26 or the composition of claim 28.

34. A method of treating cancer, the method comprising administering to an individual having cancer the targeting complex of any one of claims 1-17 or the molecule of any one of claims 18-26 or the composition of claim 28, optionally, the cancer is breast cancer.

35. Use of the polypeptide of claim 27, the use selected from: (a) detecting an Fc-containing molecule; or (b) for purifying an Fc-containing molecule; optionally, the Fc-containing molecule is an IgG.

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