CN117510642A

CN117510642A - Double-targeting chimeric body coded by whole genes and application thereof

Info

Publication number: CN117510642A
Application number: CN202311225647.XA
Authority: CN
Inventors: 周合江; 苏凌燕
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2023-09-21
Filing date: 2023-09-21
Publication date: 2024-02-06

Abstract

The present disclosure provides a double-targeting chimera and applications thereof, the chimera comprises a target molecule binding domain and an internalization effector protein binding domain, wherein the target molecule binding domain can specifically bind to a target molecule, and the internalization effector protein binding domain can specifically bind to an internalization effector protein, the chimera is a double-targeting chimera which is completely edited, is easy to synthesize, has no side reaction, overcomes the technical problems of strong immunogenicity, uncertain dosage ratio and connection site when an oligosaccharide is coupled with an antibody, more and complicated chemical synthesis steps of the oligosaccharide and the antibody, and the like, has obvious effect of mediating protein specific degradation, and can significantly inhibit tumor growth of mice.

Description

Double-targeting chimeric body coded by whole genes and application thereof

Technical Field

The invention relates to the technical field of targeted protein degradation, in particular to a double-targeted chimeric and application thereof.

Background

Classical approaches to drug development are generally the discovery of high affinity small molecules and antibodies that recognize molecules that modulate the activity of target proteins. Traditional targets are mostly proteins with defined active sites, and the active sites are suitable for binding small molecules or antibody inhibitors and inhibit the target protein function mainly by occupying a driven pharmacological mode of action. This drug development strategy has resulted in 80-85% of human proteins that are not patentable due to the lack of available binding pockets or chemicals that bind to active pockets, which have been termed "non-patentable proteins" (undruggable protein). In addition, since both small molecules and antibody drugs need to occupy the active site of the target protein continuously to block the function, a sufficiently high concentration is required to be achieved for the action, and the action mechanism inevitably brings about problems of drug resistance or toxic side effects.

Targeted protein degradation selectively and effectively induces consumption or reduction of pathogenic proteins by hijacking endogenous protein degradation mechanisms, thereby providing a new therapeutic option. Since only binding agents are required to recruit proteins of interest for degradation, rather than high affinity inhibitors that bind to the active pocket, targeted protein degradation is likely to target non-patentable proteomes that limit current drug discovery efforts. Targeting protein degradation chimeras are generally composed of a ligand that binds to the target protein, a ligand that mediates degradation, and a linker that connects the two ligands. Natural protein degradation is mediated by two main mechanisms in the cell: ubiquitin-proteasome system and endocytic-lysosomal pathway. Most of the targeted protein degradation technologies currently extend around both systems, with ubiquitin degradation systems targeting primarily intracellular proteins and lysosomal degradation pathways targeting primarily degrading membrane proteins or extracellular proteins.

The lysosome targeting chimeric technology (LYTAC) can effectively realize the degradation of extracellular proteins and membrane proteins by utilizing an endocytic-lysosome approach, but the technology still has the problems of stronger immunogenicity of antibodies or oligosaccharides in molecules, uncertain dosage ratio and connection sites when the oligosaccharides are coupled with the antibodies, more and complicated chemical synthesis steps of the oligosaccharides and the antibodies and the like at present, and still needs to be solved in subsequent researches. Recently reported cytokine receptor targeting chimeras (cytokine receptor-targeting chimera, kineTACs), which are fully genetically encoded bispecific antibodies, contain the cytokine CXCL12 and allow for decoy recovery of the receptor CXCR7, targeting a variety of target proteins to lysosomes for degradation. However, since CXCL12 itself is a functional cytokine, it is not known whether the use of this chimera activates the CXCL12 signaling pathway, causing other side reactions. Therefore, there is a need for a lysosomal targeting chimera that is easy to synthesize and free of side reactions.

Disclosure of Invention

In order to solve at least one of the problems described above, the present disclosure provides a dual targeting chimera and its use.

In a first aspect of the invention, there is provided a dual targeting chimera comprising a target molecule binding domain and an internalization effector protein binding domain, wherein the target molecule binding domain is capable of specifically binding to a target molecule and the internalization effector protein binding domain is capable of specifically binding to an internalization effector protein.

In some embodiments, the target molecule is expressed or secreted extracellular on the cell surface.

In some embodiments, the target molecule comprises a protein or polypeptide, a sugar, a glycoprotein, a lipid, a lipoprotein, a lipopolysaccharide, a nucleic acid, or any combination thereof.

In some embodiments, the target molecule is a protein or polypeptide.

In some embodiments, the target molecule is selected from one or more of EGFR, PDL-1, HER2, CPCR, VEGFR, CTLA4, IL-5Rα, IL-4R, IL-6R, PRLR, nav1.7, GCGR, PD-L1, and HLA-B27.

In some specific embodiments, the target molecule comprises PD-L1.

In some embodiments, the target molecule binding domain comprises an antibody or antigen binding fragment thereof.

In some specific embodiments, the antibody is a humanized antibody.

In some embodiments, the antibody or antigen binding fragment thereof comprises a monoclonal antibody.

In some embodiments, the antibody or antigen binding fragment thereof is selected from one or more of a single chain antibody, a chimeric monoclonal antibody, an anti-idiotype antibody, an scFv, (scFv) 2, fab 'and F (ab') 2, F (ab 1) 2, fv, dAb and Fd fragments, a bifunctional antibody.

In some embodiments, the target molecule binding domain is an anti-PD-L1 antibody or antigen-binding fragment thereof.

In some embodiments, the target molecule binding domain is a single chain variable region antibody against PD-L1.

In some embodiments, the target molecule binding domain comprises (1) an amino acid sequence as set forth in SEQ ID NO. 3, (2) a sequence having greater than 85% sequence identity to SEQ ID NO. 3, or (3) an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO. 7 or a complement thereof, or consists of any of the foregoing (1) - (3).

In some embodiments, the target molecule binding domain is a single chain antibody against PD-L1.

In some embodiments, the anti-PD-L1 single chain antibody has an amino acid sequence as set forth in SEQ ID NO. 3.

In some embodiments, the PD-L1 is expressed from the following cell lines: human colon cancer cell line HCT-116, mouse colon cancer cell line CT26 or mouse melanoma cell line B16F10.

In some embodiments, the internalization effector protein is a cell surface expressed protein that is internalized directly into the lysosome of the cell.

In some embodiments, the internalizing effector proteins include CD63, MHC-I, kremen-1, kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL-related protein 1 receptor, MAL, V-ATPase, ASGR, amyloid precursor protein-like 2 (APLP 2), apelin receptor (APLNR), MAL (myelin and lymphocyte protein, also known as VIP 17), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor (e.g., SCARA1-5, SCARB1-3, CD 36), and the like.

In some embodiments, the internalization effector protein is a transferrin receptor.

In some embodiments, the internalization effector protein binding domain comprises a molecule capable of specifically binding to an internalization effector protein, the molecule comprising a ligand for an internalization effector protein, an anti-internalization effector protein antibody, or an antigen binding fragment thereof.

In some embodiments, the anti-internalizing effector protein antibody is a humanized antibody.

In some embodiments, the anti-internalizing effector protein antibody or antigen-binding fragment thereof comprises a monoclonal antibody.

In some embodiments, the anti-internalizing effector protein antibody or antigen binding fragment thereof is selected from one or more of a single chain antibody, chimeric monoclonal antibody, anti-idiotype antibody, scFv, (scFv) 2, fab 'and F (ab') 2, F (ab 1) 2, fv, dAb, fd fragment, bifunctional antibody.

In some embodiments, the internalizing effector protein binding domain comprises a transferrin receptor binding polypeptide or an anti-transferrin receptor antibody.

In some embodiments, the internalizing effector protein binding domain is a transferrin receptor binding polypeptide.

In some embodiments, the internalizing effector protein binding domain comprises (1) an amino acid sequence as set forth in SEQ ID No. 1, (2) a sequence having greater than 85% sequence identity to SEQ ID No. 1, or (3) an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID No. 5 or a complement thereof, or consists of any of the foregoing (1) - (3).

In some embodiments, the internalizing effector protein binding domain comprises a transferrin receptor binding polypeptide (TFRBP) or an anti-transferrin receptor antibody.

In some embodiments, the internalizing effector protein binding domain comprises a transferrin receptor binding polypeptide.

In some embodiments, the internalization effector protein binding domain of the dual targeting chimera internalizes into the lysosome by interaction with an internalizing cell surface expressed receptor molecule.

In some embodiments, the target molecule binding domain and internalization effector protein binding domain between direct connection or indirect connection, the indirect connection including through a linker connection.

In some embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NOs 9 to 12 or (GmS) n, wherein m=4, n=1 to 10.

In some embodiments, the n=8.

In some embodiments, the linker comprises (1) an amino acid sequence as set forth in SEQ ID NO. 2, (2) a sequence having 85% or more sequence identity to SEQ ID NO. 2, or (3) an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO. 6 or a complement thereof, or consists of any of the foregoing (1) - (3).

In some embodiments, the double targeting chimera comprises (1) an amino acid sequence as set forth in SEQ ID NO. 4, (2) a sequence having 85% or more sequence identity to SEQ ID NO. 4, or (3) an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO. 8 or a complement thereof, or consists of any of the foregoing (1) - (3).

In some embodiments, the dual targeting chimera is linked to the exocrine envelope protein through a cleavable linker containing a cleavage site.

In some embodiments, the dual targeting chimera forms a complex of a "clyA protein-cleavable linker-dual targeting chimera".

In some embodiments, the cleavable linker sequence containing the cleavage site is linked to the internalization effector protein binding domain of the chimera.

In some embodiments, the cleavable linker sequence containing the cleavage site is linked to the N-terminus of the chimera.

In some embodiments, the cleavage site comprises a matrix metalloproteinase 2 (MMP 2) cleavage site.

In some embodiments, the amino acid sequence of the cleavable linker containing the cleavage site is selected from one or more of the sequences set forth in SEQ ID NOS 13-31.

In some embodiments, the cleavable linker containing the cleavage site has the amino acid sequence set forth in SEQ ID NO. 15.

In some embodiments, the exocrine envelope protein is selected from one or more of clyA protein, ompa protein, ompc protein, or ompf protein.

In some embodiments, the exocrine envelope protein comprises a clyA protein.

In some embodiments, the chimera comprises (1) an amino acid sequence as set forth in SEQ ID NO. 36, (2) a sequence that has 85% or more sequence identity to SEQ ID NO. 36, or (3) an amino acid sequence encoded by a nucleotide sequence as set forth in SEQ ID NO. 37 or a complement thereof, or any of the foregoing (1) - (3).

In a second aspect of the invention, there is provided a nucleic acid molecule encoding a dual targeting chimera according to the first aspect.

In some embodiments, the nucleic acid molecule comprises a coding region that encodes a single chain antibody amino acid sequence that is anti-PD-L1 having a sequence set forth in SEQ ID NO. 1.

In some embodiments, the nucleic acid molecule comprises or consists of the sequence: the nucleotide sequence shown in SEQ ID NO. 5, or a sequence with more than 85 percent of sequence identity with the nucleotide sequence, or a complementary sequence thereof.

In some embodiments, the nucleic acid molecule comprises a coding region that encodes an amino acid sequence that is the sequence set forth in SEQ ID NO. 3, or a sequence that has greater than 85% sequence identity thereto, and/or,

in some embodiments, the coding region comprises or consists of the sequence: the nucleotide sequence of the sequence shown in SEQ ID No. 7 or a sequence with more than 85 percent of sequence identity with the nucleotide sequence or a complementary sequence of the nucleotide sequence.

In some embodiments, the nucleic acid molecule comprises a coding region that encodes an amino acid sequence that is the sequence set forth in SEQ ID NO. 4, or a sequence that has greater than 85% sequence identity thereto, or a complement thereof, and/or,

in some embodiments, the coding region comprises or consists of the sequence: the nucleotide sequence of the sequence shown in SEQ ID No. 8 or a sequence with more than 85 percent of sequence identity with the nucleotide sequence or a complementary sequence of the nucleotide sequence.

In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence of an exocrine envelope protein.

In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence comprising a cleavable linker to a cleavage site.

In some embodiments, the nucleotide sequence of the exocrine envelope protein is linked to the nucleotide sequence of the aforementioned double targeting chimera by a cleavable linker nucleotide sequence.

In some embodiments, the nucleic acid molecule encodes a complex of a "clyA protein-cleavable linker-double-targeting chimera".

In some embodiments, the cleavable linker nucleotide sequence containing the cleavage site is linked to the nucleotide sequence of the internalizing effector protein binding domain of the chimera.

In some embodiments, a cleavable linker nucleotide sequence comprising a cleavage site is linked to the 5' end of the chimera.

In some embodiments, the exocrine envelope protein nucleotide sequence is selected from one or more of the nucleotide sequences of clyA, ompa, ompc or ompf proteins.

In some embodiments, the exocrine envelope protein nucleotide sequence comprises a clyA protein nucleotide sequence.

In some embodiments, the coding region comprises or consists of the sequence: (1) a nucleotide sequence shown as SEQ ID NO. 37, (2) a sequence having more than 85% sequence identity with SEQ ID NO. 37, or (3) a sequence complementary to the nucleotide sequence shown as SEQ ID NO. 37, or consisting of any one of the foregoing (1) to (3).

In a third aspect of the invention, there is provided a drug-loaded Outer Membrane Vesicle (OMV) linked to the complex of the aforementioned "clyA protein-cleavable linker-double targeting chimera".

In some embodiments, the outer membrane protein is selected from one or more of clyA protein, ompa protein, ompc protein, or ompf protein.

In some embodiments, the outer membrane protein comprises a ClyA protein.

In some embodiments, the ClyA is linked to the N-terminus of the dual targeting chimera via a cleavable linker, wherein the cleavable linker comprises the amino acid sequence of the sequences set forth in SEQ ID NOS 13-31.

TABLE 1 amino acid sequence listing of cleavable linkers

Name of the name	Sequence(s)	Seq ID NO
			Cuttable joint 1	GPLGVR	13
Cuttable joint 2	PLGLAR	14
			Cuttable joint 3	PLGLAG	15
Cuttable joint 4	IPVSLRSG	16
			Cuttable joint 5	GPLGMLSQ	17
Cuttable joint 6	PLGLAGGGS	18
			Cuttable joint 7	PLGLAGGGGS	19
Cuttable joint 8	IPVSLRSGPLGLAG	20
			Cuttable joint 9	RPKPVEVWRK	21
Cuttable joint 10	VPLSLYS	22
			Cutting joint11	VLVPMAMMAS	23
Cuttable joint 12	LSGRSDNH	24
			Cuttable joint 13	TGRGPSWV	25
Cuttable joint 14	LSGRSDNH	26
			Cuttable joint 15	LSGK	27

Table 1 (subsequent)

Name of the name	Sequence(s)	Seq ID NO
			Cuttable joint 16	AGPR	28
Cuttable joint 17	AANL	29
			Cuttable joint 18	PTNL	30
Cuttable joint 19	TSGRSANP	31

In a fourth aspect of the invention, there is provided a method of preparing said drug-loaded outer membrane vesicles, the method comprising: introducing into said host cell said nucleic acid molecule encoding a complex of an "outer membrane vesicle membrane protein-cleavable linker-dual targeting chimera".

In some embodiments, the introducing is achieved by transfection or transformation.

In some embodiments, the host cell is selected from the group consisting of prokaryotic cells. Wherein the outer membrane vesicle membrane protein is ClyA protein.

In some embodiments, the method further comprises the step of culturing the host cell under suitable conditions and isolating and purifying the drug-loaded outer membrane vesicles by membrane filtration.

In a fifth aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In a sixth aspect of the invention there is provided a cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the fifth aspect of the invention.

In a seventh aspect of the invention, there is provided a composition comprising a double-targeting chimera according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the fifth aspect of the invention and/or a cell according to the sixth aspect of the invention, and a pharmaceutically acceptable carrier.

In an eighth aspect of the invention, there is provided a kit comprising a double-targeting chimera according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the fifth aspect of the invention and/or a cell according to the sixth aspect of the invention.

In a ninth aspect of the invention, there is provided a method for preparing a dual targeting chimera obtained by means of whole gene editing.

In some embodiments, the method uses the vector of the third aspect to load the nucleic acid molecule of the second aspect into the cell of the sixth aspect, expressed under conditions and purified to obtain the double-targeting chimera.

In a tenth aspect of the invention there is provided the use of a dual targeting chimera of the first aspect, a nucleic acid molecule according to the second aspect of the invention, a vector according to the fifth aspect of the invention, a cell according to the sixth aspect of the invention and/or a composition according to the seventh aspect of the invention in the manufacture of a medicament for use in the treatment of a disease in a subject in need thereof.

In some embodiments, the chimera is engineered into an exosome.

In some embodiments, the disease is a disease associated with expression or overexpression of the membrane protein.

In some embodiments, the disease is cancer.

In some embodiments of the present invention, in some embodiments, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or cervical cancer, salivary gland cancer, renal or ureter cancer, prostate cancer, vaginal cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, melanoma, cholangiocarcinoma, central Nervous System (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schlemma, ependymoma, medulloblastoma, squamous cell carcinoma, pituitary adenoma and ewing sarcoma, superficial diffuse melanoma, freckle-like melanoma, acromegaly melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic leukemia, leukemia (l), cancer associated with cancer of the brain cells (e.g., brain and lymphomas), cancer associated with cancer of the brain, malignant cells (e.g., lymphomas) and lymphomas (lymphomas), cancer associated with cancer of the brain and brain, brain and malignant tumor (lymphomas, and cancer associated with cancer of the brain, such as dysfunctional cell-related disorders and malignant tumor (brain-associated with cancer).

In an eleventh aspect of the invention there is provided the use of a dual targeting chimera of the first aspect for the specific degradation of a target molecule. The degradation may be in vitro or in vivo.

In some embodiments, the target molecule comprises PD-L1.

The chimera creatively adopts transferrin receptor binding polypeptide as a receptor for mediating endocytosis, does not activate a downstream signal path of TFR, and has small side effect; the combined antibody Single chain variable region fragment scFv (Single-Chain Fragment Variable) has the advantages of small molecular weight (only 1/6 of the whole antibody), strong penetrability, short in vivo half-life, low immunogenicity, capacity of being expressed in a prokaryotic cell system, easiness in genetic engineering improvement and the like, and meanwhile, the connector is used for connection, has certain flexibility to allow proteins at two sides to complete independent functions besides playing a role of connection, and ensures that the simultaneous identification of TFR and PDL1 and a chimeric body is facilitated by a certain space distance.

Thus, the chimera is a fully genetically edited, easily synthesized, side-reaction-free, double-targeting chimera. The preparation method overcomes the technical problems of over-strong immunogenicity, uncertain dosage ratio and connection site when the oligosaccharide is coupled with the antibody, more and complicated steps of chemical synthesis of the oligosaccharide and the antibody, and the like, has obvious effect of mediating protein specific degradation, has obvious growth inhibition effect on various tumors, remarkably prolongs the survival period of tumor-bearing mice, and even completely eliminates about 60 percent of tumors of mice. Has remarkable technical progress.

Drawings

FIG. 1 shows a schematic diagram of lysosomal targeting chimera structures (A and B) and working principle (C). Specific structures and mechanisms of action of the chimeras of the present disclosure (structures including transferrin receptor binding polypeptide TFRBP-linker-anti-PDL 1 antibody, represented by TfR-LYTAC) are shown. Wherein figure 1A shows the chimeric components of the present disclosure. FIG. 1B shows that the chimera binds both the target protein PDL1 and the lysosomal targeting receptor TFR. FIG. 1C shows the targeting of the chimeric to target protein to lysosomal degradation.

FIG. 2 shows the results of detection of the chimeras of the present disclosure against other control molecules. Among them, FIG. 2A shows the detection electrophoresis patterns of purified PDL1 antibody (left) and chimeric antibody (right), the left is the result of Coomassie brilliant blue staining, and the right is the result of Western blotting detection using his-tag antibody. FIG. 2B shows MST assay for TFR-LYTAC binding to TFR and PDL 1. FIG. 2C shows confocal microscopy to detect TfR-LYTAC binding to PDL1 at the cellular level.

FIG. 3 shows detection of the degradation function of the chimeras of the present disclosure. Wherein FIG. 3A shows that confocal microscopy detects that TfR-LYTAC binds to PDL1 and mediates endocytosis of PDL1 into lysosomes, and FIG. 3B shows that SDS-PAGE immunoblotting detects that TfR-LYTAC mediates degradation of cellular PDL 1.

FIG. 4 shows immunoblotting and flow detection of PDL1 protein from a variety of cell lines, and the relationship of this degradation to time, concentration, lysosomal function and TFR. FIG. 4A shows a flow chart of PD-L1 antibody against phosphate buffer under different concentrations of TfR-LYTAC, FIG. 4B shows a flow chart of PD-L1 antibody against phosphate buffer control under different concentrations of TfR-LYTAC, FIG. 4C shows a flow chart of PD-L1 antibody against phosphate buffer under different reaction time conditions, FIG. 4D shows a flow chart of PD-L1 antibody against phosphate buffer control under different reaction time conditions, FIG. 4E shows a flow chart of PdL1 antibody against phosphate buffer control under different reaction time conditions, FIG. 4E shows a flow chart of TfR-LYTAC, baf (lysosome inhibitor) +TfR-LYTAC, MG132 (proteasome inhibitor) +TfR-LYTAC, respectively, a flow chart of PD-L1 antibody against phosphate buffer control under different reaction time conditions, FIG. 4F shows a flow chart of the respective antibodies under the conditions of TfR-LYNC-TfN, tfSiR+TfNC-L1, tfSiR+TfNC-lyNC, and a flow chart of the respective antibodies under respective conditions of TfR-TfN+ TfNC-TfN.

Figure 5 shows a schematic and test pattern of engineering of the chimera TfR-LYTAC of the present disclosure into bacterial exosome OMVs. Wherein FIG. 5A is a schematic diagram of the structure of the engineering of LYTAC into bacterial exosomes (the material of TfR-LYTAC engineered into bacterial exosomes OMVs is indicated by OMV-LYTAC), and FIG. 5B is the genome of the engineered TfR-LYTAC loaded on pET28a plasmid, the protein coding region comprising a tag comprising clyA, a linker comprising a cleavage site for matrix metalloproteinase 2, LYTAC, 6 x-his. FIG. 5C is a polyacrylamide gel electrophoresis pattern of a western blot detection of the expression product of the genome shown in FIG. 5B.

FIG. 6 shows the relative intensity of the chimeras TfR-LYTAC, exosomes and OMV-LYTAC of the present disclosure over time.

Figure 7 shows the inhibition of melanoma growth by different molecules engineered into bacterial exosomes. Wherein figure 7A shows a graph of tumor volume over days. Fig. 7B shows a plot of tumor volume change with increasing days.

FIG. 8 shows the inhibition of CT26 colon cancer cell growth rate in mice by engineering exosomes, tfR-LYTAC and OMV-LYTAC into bacterial exosomes. The abscissa is the number of days and the ordinate is the tumor volume.

FIG. 9 shows the prolongation effect of exosomes, tfR-LYTAC and OMV-LYTAC engineering into bacterial exosomes on the survival of tumor-bearing mice, with days on the abscissa and survival on the ordinate.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

Definition of the definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise.

The term "dual targeting chimera" as used in this disclosure refers to a chimera comprising two target molecule binding domains that are capable of binding to different target molecules, respectively, wherein one target molecule includes an internalizing effector protein and the other target molecule may be any protein, polypeptide, or other macromolecule that requires a reduction, or elimination of its activity or extracellular concentration. Including proteins or polypeptides, sugars, glycoproteins, lipids, lipoproteins, lipopolysaccharides, or other non-protein polymers or molecules. Examples of cell surface expressed target molecules include cell surface expressed receptors, membrane bound ligands, ion channels, and any other monomeric or multimeric polypeptide component having an extracellular portion that is linked or associated with a cell membrane.

The term "LYTACs" (lysoname-targeting chimeras) or "lysosomal targeting chimera" refers to a dual targeting chimera in which one of the target molecule binding domains is a targeting lysosome, e.g., by a polypeptide capable of binding to a transferrin receptor. In a specific embodiment, such a dual targeting chimera is also known as "TfR-LYTAC".

The term "degradation" as used in the present disclosure may involve the breakdown or disintegration of a protein into smaller amino acid or peptide units. Degradation of a protein may disrupt or ablate its function.

The term "degradation" as used herein includes degradation via both ubiquitin-proteasome system and endocytic-lysosomal system.

The term "target molecule binding domain" as used in the present disclosure means any peptide, polypeptide, nucleic acid molecule, scaffold molecule, peptide display molecule or polypeptide-containing construct capable of specifically binding to a particular target molecule of interest. As used herein, the term "specifically binds" means that a target molecule binding domain forms a complex with a particular target molecule characterized by a dissociation constant (KD) of 500pM or less, and does not bind other unrelated target molecules under ordinary test conditions. An "unrelated target molecule" is a protein, peptide or polypeptide that has less than 95% amino acid sequence identity to each other.

The term "internalization" as used in this disclosure is understood to mean the process by which membrane proteins pass through the cytoplasmic membrane into the cytoplasm.

The term "internalization effector protein" as used in the present disclosure is a protein capable of internalizing into a cell. In some embodiments, the internalization effector protein is a protein that undergoes transcytosis. In some embodiments, the internalizing effector protein is a cell surface expressed protein or a soluble extracellular protein. Internalization effector proteins include proteins that internalize directly into a cell, as well as proteins that internalize indirectly into a cell.

Internalization effector proteins that are directly internalized into a cell include membrane-bound molecules (e.g., transmembrane proteins, GPI-anchored proteins, etc.) having at least one extracellular domain that undergo cellular internalization and are preferably processed by intracellular degradation and/or recycling pathways. Specific non-limiting examples of internalizing effector proteins that are directly internalized into cells include, for example, CD63, MHC-I (e.g., HLA-B27), kremen-1, kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein-2 (APLP 2), apelin receptor (APLNR), MAL (myelin and lymphocyte protein, also known as VIP 17), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor (e.g., SCARA1-5, SCARB1-3, CD 36), and the like.

Internalizing effector proteins that are indirectly internalized into a cell include proteins and polypeptides that do not internalize themselves, but become internalized into the cell upon binding or otherwise associating with a second protein or polypeptide that is directly internalized into the cell. Proteins that are indirectly internalized into a cell include, for example, soluble ligands that bind to internalizing cell surface expressing receptor molecules. A non-limiting example of a soluble ligand that is internalized (indirectly) into a cell by interaction of the soluble ligand with an internalizing cell surface expressing receptor molecule is transferrin.

The term "linker" as used in the present disclosure refers to a (peptide) linker of natural and/or synthetic origin, consisting of linear amino acids. The domains in the dual targeting chimeras of the present disclosure can be linked by linkers, wherein each linker is fused and/or otherwise linked (e.g., via a peptide bond) to at least two domains. In some embodiments, the amino acid sequences of all of the linkers present in the dual targeting chimeras of the present disclosure are identical. In other embodiments, the amino acid sequences of at least two linkers present in the dual targeting chimeras of the present disclosure are different. The linker should have a length suitable for linking two or more monomer domains in this way, the linker being able to ensure that the different domains to which it is linked are correctly folded and properly presented, thereby functioning as a biological activity thereof. In various embodiments, the linker has a flexible conformation. Closing deviceSuitable flexible linkers include, for example, those having glycine, glutamine, and/or serine residues. In some embodiments, the linker may be selected from (GnS) m, (G) n, (EAAAK) n, or (XP) n, wherein n and m are each independently selected from integers from 0 to 5. For example, n is selected from 0, 1, 2, 3, 4 or 5, and m is selected from 1, 2, 3, 4 or 5. In some embodiments, the linker may also include, for example, KESGSVSSEQLAQFRSLD (SEQ ID NO: 9), EGKSSGSGSESKST (SEQ ID NO: 10), (Gly) ₈ (SEQ ID NO: 11), GSAGSAAGSGEF (SEQ ID NO: 12), etc.

The term "antibody" as used in the present disclosure encompasses immunoglobulins (whether naturally occurring or partially or fully synthetically produced) and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. The term antibody is intended to include whole antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and also includes single chain antibodies, humanized antibodies, murine antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments (e.g., scFv, (scFv) 2, fab 'and F (ab') 2, F (ab 1) 2, fv, dAb, and Fd fragments), bifunctional antibodies, and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.

The term "antibody fragment" or "antigen-binding fragment" as used in this disclosure refers to a portion of an intact antibody, and typically comprises the epitope variable region of the intact antibody. Some examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments. "Fab fragment (antigen-binding fragment)" is also known as an antigen-binding fragment, which is a region in an antibody structure that can bind to an antigen. The Fab fragment consists of a complete light chain and a part of heavy chain structure, the light chain and the heavy chain are connected through a disulfide bond, the volume is small, and the molecular weight is 47-48kDa. Fab fragments can be obtained by proteolytic cleavage of full length antibodies. For example, human Immunoglobulin G (lgG) can be degraded into two Fab fragments and one Fc fragment by papain; lgG can be degraded into a F (ab ') 2 fragment and a pFc' fragment under the action of pepsin. The F (ab ') 2 fragment may be further reduced to form two Fab' fragments. Fab fragments can also be prepared by prokaryotic systems (e.g., E.coli systems) and mammalian cell system expression. The escherichia coli expression system has the characteristics of low production cost, high production speed and the like, but inclusion bodies are easy to form, the later purification and renaturation are troublesome, and the activity of the protein obtained by renaturation is very low or even no. The Fab fragment can form disulfide bond smoothly in mammal cell, and has high activity.

The term "single chain antibody" is used interchangeably with the term single chain variable fragment (scFv) in this disclosure and refers to a target-specific binding domain, which is typically a polypeptide that promotes specific binding to a target. A target-specific binding domain is considered specific for a given target if it binds with highest affinity to the given target, while binding with other targets, even targets having related amino acid sequences, is only with lower affinity, e.g. at least 10-fold, preferably at least 100-fold lower. The scFv is not actually a fragment of an antibody, but rather comprises a heavy chain variable domain linked to a light chain variable domain by a linker (Huston et al (1988) Proc. Natl. Acad. Sci. USA85, 5879-5883). The linker is typically glycine-rich to increase flexibility and serine or threonine-rich to increase solubility, and may link the N-terminus of VH to the C-terminus of VL and vice versa. The protein retains the original immunoglobulin specificity despite removal of the constant region and introduction of the linker.

The term "transferrin receptor" or "TfR" as used in the present disclosure refers to transferrin receptor protein 1, a representative transcytosis receptor. The polypeptide sequence of the human transferrin receptor 1 is shown as SEQ ID NO. 3. Transferrin receptor protein 1 from other species is also known (e.g., chimpanzees, accession number xp_003310238.1; macaque, np_001244232.1; dog, np_001003111.1; cow, np_001193506.1; mouse, np_035768.1; rat, np_073203.1; and chicken, np_ 990587.1). The term "transferrin receptor" also encompasses exemplary reference sequences encoded by genes located at the chromosome 1 locus of the transferrin receptor protein, such as allelic variants of human sequences. Full length transferrin receptor proteins include a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: protease-like domains, helical domains and apical domains.

In the present disclosure, "sequence identity" refers to a "percentage of sequence identity" or "percentage of identity" between two polynucleotides, i.e., the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. The matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since the gaps are not nucleotides, the gaps present in the target sequence are not accounted for. Also, since the target sequence nucleotides are counted, and the nucleotides from the reference sequence are not counted, gaps present in the reference sequence are not counted. At least 85% sequence identity includes a contiguous segment having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity over the entire length of the sequence.

The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical formulation that is non-toxic to a subject other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The term "PD-L1" refers to programmed death ligand 1, also known as CD274 and B7H1. The amino acid sequence of full length PD-L1 is provided in GenBank under accession number NP-054862.1. The term "PD-L1" means human PD-L1 unless specified to be from a non-human species.

The term "whole gene editing" as used in the present disclosure means that all of the proteins or polypeptides can be obtained by transcription translation editing using gene information on a DNA vector.

The term "OMV-LYTAC" as used in this disclosure is a molecule having a structure as shown in fig. 5A, clyA protein being one of the most abundant proteins on the OMV surface, clyA being linked to the transferrin receptor-binding polypeptide of TfR-LYTAC via a linker with a cleavable site. TfR-LYTAC is loaded on exosomes by clyA, and cleavage sites are cleaved by matrix metalloproteinase 2 (MMP 2) overexpressed in the tumor environment, exposing binding sites for transferrin receptor binding polypeptides, binding to transferrin, mediating internalization reactions. The OMV-LYTAC is a protein molecule obtained by loading a protein coding region containing clyA, a linker containing a cleavage site of matrix metalloproteinase 2, LYTAC and a 6x-his tag into an expression vector of pET28a plasmid for transcription and translation, as shown in FIG. 5B.

The term "transfection" as used in this disclosure refers to a process of artificially introducing nucleic acid (DNA or RNA) into a cell using a variety of chemical, biological or physical methods. The introduction of exogenous nucleic acids into host cells by transfection can alter the characteristics of the cells, thereby enabling the study of cellular gene function and protein expression. After transfection, the introduced nucleic acid may be transiently present in the cell for only a period of time without replication (transient transfection), or may be stably integrated into the host genome and replicated as the host genome replicates (stable transfection). Methods of transfection include physical-mediated transfection (e.g., electroporation, microinjection, and gene gun), chemical-mediated transfection (e.g., DEAE dextran, calcium phosphate co-precipitation, liposome transfection), and virus-mediated transfection (e.g., recombinant lentivirus, retrovirus, adenovirus, or adeno-associated virus (AAV), or herpes virus-mediated transfection).

Transfection generally involves the use of a vector. As used herein, a "vector" is intended to encompass a variety of substances or constructs for the carrying, or delivery of a nucleic acid of interest, including but not limited to recombinant vectors (e.g., plasmids and viral vectors), exosomes, vesicles, nanoparticles. As used herein, a "recombinant vector" is a non-natural association of nucleic acids from non-homologous sources (typically from different organisms). Recombinant vectors may comprise any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, may be partially synthesized or obtained from natural sources, and may comprise natural, non-natural, or altered nucleotides. Recombinant expression vectors may contain naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

The term "mCherry" as used in the present disclosure refers to red fluorescent proteins from mushroom coral (mushroom coral), which are widely used as tracers for labeling of molecules, localization of cellular components, and the like. mCherry is low in cytotoxicity due to its color and photostability of the monomer molecules. Is more excellent than other fluorescent protein labels, and the maximum excitation light and the emission light are 587nm and 610nm respectively.

The term "micro thermophoresis (MST) detection" as used in this disclosure accurately quantifies interactions between molecules by detecting the laws of motion (changes in molecular mass, charge number, and hydration layer) of the molecules in microscopic temperature gradient fields. When MST experiments are performed, the sample is heated by infrared laser to generate a microscopic temperature gradient field, and the directional movement of the molecules is monitored and quantified by autofluorescence of covalently bound fluorescent dye or tryptophan. The application range includes binding behavior of small molecules, interactions between proteins and interactions between protein complexes.

Examples and figures are provided below to aid in the understanding of the invention. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the invention in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.

Examples

Example 1 binding ability of TfR-LYTAC chimera to target protein PDL1 and receptor TFR.

1. Preparation of recombinant proteins

Chimeric gene TfR-LYTAC (nucleotide sequence shown in SEQ ID NO:4, the chimeric geneThe body structure pattern is shown in FIG. 1) and the 6x-his tagged protein coding region genes were cloned into pET-28a (+) vector and transformed into E.coli Rosetta strain expression. Coli was inoculated into 500ml of LB medium and shaken at 37℃with 220 rpm. When OD is ₆₀₀ When 0.6-0.8 was reached, 1M isopropyl b-D-1-thiogalactonucleoside (IPTG; diluted in LB medium at a ratio of 1:2000) was added to induce expression of the indicator protein. The bacteria were then incubated overnight with shaking (180 rpm) at 18 ℃.

The recombinant proteins were purified using Ni-Sepharose6 fast flow beads (GE healthcare) and further concentrated and desalted using an amicon ultra-1510K centrifugal filter (Merck Millipore). Coomassie blue staining was performed on SDS-PAGE to determine the purity of the chimera, immunoblotting detection (His antibody) was performed to determine the purified protein as the designed chimera, and the detection results are shown in fig. 2A.

2. Detection of

2.1 micro thermophoresis detection

Binding capacity of chimeras (TfRBP, scFv or TfR-LYTAC) to PDL1 was measured using a microphoresis meter (MST monoliths nt.115): proteins were incubated with different concentrations of metabolites in a 20 μl system and loaded into nt.115 standard coated capillaries (nanotempering technique). MST was measured at 25 ℃ and 100% excitation power. All experiments were repeated 2 times per measurement. Data analysis was performed using nanosampler software. The detection result is shown in FIG. 2B.

2.2 confocal microscopy observations

TfR-LYTAC was observed to bind to s-overexpressed PDL1-mCherry and mediate transport of PDL1-mCherry to lysosomes by confocal microscopy systems: the hela cells were placed in glass bottom dishes and transfected with the expression vector described above for 24h. After incubation with FITC-labeled peptides for 2 hours at 4 ℃, the cells were washed twice to remove unbound peptides and fixed with 4% Paraformaldehyde (PFA). Incubation time was 3h for unlabeled peptide. Fluorescent image capture uses an inverted confocal microscope system (LSM 880; zeiss) with a 100X oil objective camera. All images were acquired at room temperature and processed with Zen Blue2 software (Zeiss). The detection result is shown in FIG. 2C.

Example 2 immunoblotting and flow assay degradation function assay for TfR-LYTAC.

The degradation function of the chimeras was detected using confocal scanning microscopy, immunoblotting and flow detection.

1. Confocal scanning microscope detection

Detection of the amount of PDL 1-targeted lysosomes following treatment of cells with TFR binding polypeptides (TFRBP), PDL1 antibodies (scFv), and chimeras described in the present disclosure (TFR-LYTAC) using confocal scanning microscopy. The results are shown in FIG. 3A.

2. Immunoblotting detection

The detection steps of the immunoblotting method are as follows: the samples were dissolved in ice-cold assay buffer (20 mm Tris-HCl, pH7.5,100mM sodium chloride, 0.1% SDS,0.5% sodium deoxycholate, and 1 mm PMSF) containing cOmplet ^TM Protease inhibitor cocktail (rogowski), loading of protein cleavage products into SDS-PAGE wells, transfer of membrane, and immunoaffinity reaction with antibodies bearing the indicator. Western blot was quantified using densitometry and data were processed using Image J software. The results are shown in FIG. 3B.

3. Flow detection

To examine whether TfR-LYTACs are degraded by lysosomal or proteasome systems, we pre-treated HCT116 cells with bafilomycin A1 (a lysosomal acidification inhibitor) or MG132 (a proteasome inhibitor). After 1h of inhibitor treatment, the cells were incubated with TfR-LYTACs for 24h. Using streaming detection: the cell microspheres were washed with cold PBS and centrifuged at 300g for 5 min. Cells were incubated with PBS primary diluted with 3% BSA for 30 min at 4 ℃. Cells were washed once with cold PBS. After washing 3 times with cold PBS of 3% bsa, it was resuspended with cold PBS. Flow cytometry was performed on a BD cytometer or a flow cytometer (beckman coulter). Analysis was performed using FlowJo software (v10.8.0). The detection results are shown in FIG. 4.

Flow cytometry showed that TfR-LYTACs mediated PD-L1 degradation occurred in a dose and time dependent manner (fig. 4A-4D). The bafilomycin pretreatment inhibited the degradation of PD-L1, while MG132 was not active, indicating that TfR-LYTACs were degraded via the lysosomal degradation system (fig. 4E and 4F).

TfR knockout experiments

TfR-siRNA transfected HCT116 cells by Lipo2000 and knockdown efficiency was detected by flow after 72 hours.

The nucleotide sequences of the siRNAs used are shown in Table 2.

TABLE 2 oligonucleotide sequences of siRNA

The experimental results are shown in FIG. 4. Experimental results showed that TfR knockout completely abrogated TfR-LYTACs mediated PD-L1 degradation in HCT116 cells (fig. 4G-4I).

In summary, the chimeric treatment can degrade the protein amount of PDL1 of various cell lines (human colon carcinoma cell line HCT-116, mouse colon carcinoma cell line CT26 and mouse melanoma cell line B16F 10), and the degradation is time, concentration, lysosomal function and TFR dependent.

Example 3 preparation and identification of TfR-LYTAC engineering into bacterial exosomes

1. Preparation

The protein coding region gene (FIG. 5B) containing clyA, linker containing matrix metalloproteinase 2 cleavage site, tfR-LYTAC, 6x-his tag was cloned into pET-28a (+) vector and transformed into low toxicity E.coli strain for expression. The transformed E.coli was inoculated into 500ml of LB medium and shaken at 37℃at 220 rpm. When OD is ₆₀₀ When reaching 0.6-0.8, 1M isopropyl-D-1-thiogalactoside (IPTG; diluted 1:2000 in LB medium) was added to induce expression of indicator protein. The bacteria were then incubated overnight with shaking (180 rpm) at 18 ℃. The sequences are shown in Table 3.

The supernatant was collected by centrifugation at 8000g for 15min, then filtered through a 0.45 μm polyvinylidene fluoride filter (microwell, R8SA47939, USA), and concentrated to 50mL using a 50kDa ultrafiltration membrane (microwell, R3EA06699, USA). OMVs were collected by further filtration through a 0.22 μm polyethersulfone membrane filter (microwells, R9HA36284, usa) using ultracentrifugation at 150000g speed for 3 hours at 4 ℃. Immunoblotting detection (His antibody) was performed by SDS-PAGE to confirm that the designed chimera was fusion expressed on the purified exosomes, and the detection results are shown in FIG. 5C.

TABLE 3 expression products and corresponding amino acid and nucleotide sequences

Expression product name	Amino acid sequence	Nucleotide sequence
			TfR binding polypeptides	SEQ ID NO:1	SEQ ID NO:5
scFv antibody to PDL1	SEQ ID NO:3	SEQ ID NO:7
			TfR-LYTAC chimeras	SEQ ID NO:4	SEQ ID NO:8
clyA-cleavable linker-chimeras	SEQ ID NO:36	SEQ ID NO:37

2. Authentication

The morphology of omv was characterized by transmission electron microscopy (TT 7700 electron microscopy; hitachi, japan). The resuspended omv (8 μl) was loaded onto a grid of one form of coating and incubated for 1 min. After removal of excess OMVs solution, the OMVs-loaded grid was stained with 2% uranyl acetate solution. After removing the excess uranyl acetate solution, images were taken at 80kV using HT7800 (hitachi, japan). OMV size was analyzed by Dynamic Light Scattering (DLS) using nano-floodlight (brookfield, usa).

Example 4 in vivo biological distribution of TfR-LYTAC or OMV-LYTAC

To assess in vivo biodistribution, tfR-LYTAC peptides or vesicles were labeled with NHS-cy5.5 and incubated with dye overnight at 4 ℃. The free Cy5.5 in the vesicle suspension was removed by ultracentrifugation at 150,000 g for 3h at 4 ℃. The peptide suspension was freed from free Cy5.5 using an Amicon Ultra-1510K centrifuge filter (Merck microwell Co.). Cy5.5-labeled peptide (2.5 mg/kg body weight) or vesicles (2.5 mg OMVs protein/kg body weight) were injected into B16F10 tumor-bearing mice by tail vein. Mice were anesthetized at 6, 12, 24, 36h before and after injection, and monitored for cy5.5 signal using the IVIS spectroscopic system (IVIS; united states). Major organs and tumors were harvested 24 or 36 hours after injection for in vitro imaging. Blood was collected at various time points (1 min, 0.5, 1, 2, 4, 8, 12, 24 h) after injection, and the effect of administration on circulation half-life was measured. The detection results are shown in FIG. 6.

Experimental results show that TfR-LYTAC engineered into bacterial outer vesicles significantly enhances drug half-life.

Example 5 in vivo anti-tumor efficacy.

For the B16F10 tumor model, balb/c mice aged 6-8 weeks were subcutaneously injected 4X 10 on the right ⁶ Murine melanoma B16F10 cells. 6 days after tumor cell inoculation, mice were randomly divided into several experimental groups, and the treatment method was as follows: saline control, tfR-LYTAC (1 mg/kg body weight), OMVs (2.5 mg vesicle protein/kg body weight), OMV-TfRBP (2.5 mg vesicle protein/kg body weight), OMV-scFv (2.5 mg vesicle protein/kg body weight), OMV-LYTAC (2.5 mg vesicle protein/kg body weight). For CT26 tumor model, balb/c mice of 6-8 weeks of age were subcutaneously injected 5X 10 on the right ⁶ Mouse colon adenocarcinoma CT26 cells. Tumor cellsAfter 7 days of inoculation, the mice were randomly divided into 4 experimental groups, and the treatment method was: saline control, tfR-LYTAC (1 mg/kg body weight), OMVs (2.5 mg vesicle protein/kg body weight), and OMV-LYTAC (2.5 mg vesicle protein/kg body weight). 150 μl was treated every 3 days by caudal vein. In the treatment process, the size of the tumor is measured by a vernier caliper, and the calculation formula of the tumor volume (V) is as follows: v= (1/2) axb ² Wherein a and b are the major and minor axes of the tumor, respectively. Body weight was additionally measured every 2 days. At the end of treatment, mice were euthanized and tumor xenografts were excised, weighed and sectioned for histological analysis. The detection results are shown in FIGS. 7 to 9. Animal experiments were approved by the ethical committee of the university of agriculture in Yunnan. All mice were purchased from chinese life river laboratory animal technologies limited.

At tissue staining, the major organs (liver, heart, spleen, lung, kidney) and tumors of the mice were fixed with 4% pfa. Then paraffin embedded in3 batches for 1h. The sections were sectioned at 8 μm. The sections were then de-affined in xylene and successively incubated in graded ethanol (100, 90, 95, 80, 70 and 50%, 5min each) for rehydration. For H & E staining, sections were stained with hematoxylin for 18 minutes, washed with distilled water for 3 seconds, alcohol for 2 seconds, and then rinsed with distilled water for 14 minutes. After 70s staining with 0.5% eosin, the sections were washed with 100% ethanol for 2min3 times and xylene for 2min3 times. At immunostaining, the sections were incubated in blocking buffer (5% bsa in PBS) for 30 min, then incubated with primary antibody overnight in blocking buffer at 4 ℃ and extensively washed again. For immunofluorescence, sections were incubated with AlexaFluor 488-conjugated secondary antibody in blocking buffer for 1 hour, while sections were incubated with horseradish peroxidase-conjugated secondary antibody and Immunohistochemistry (IHC) was performed with DAB reaction. The fluorescent image was captured using an inverted confocal microscope system (DMI 3000B; leka) coupled to a camera with a 40X objective lens.

The results show that OMV-LYTAC has obvious growth inhibition effect on various tumors, and remarkably prolongs the survival time of tumor-bearing mice, even about 60% of the mice have complete tumor disappearance.

The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims

1. A dual targeting chimera comprising a target molecule binding domain and an internalizing effector protein binding domain, wherein the target molecule binding domain is capable of specifically binding to a target molecule and the internalizing effector protein binding domain is capable of specifically binding to an internalizing effector protein.

2. The chimera according to claim 1, characterized in that the target molecule is expressed on the cell surface or is secreted extracellularly.

3. The chimera according to claim 1, characterized in that the target molecule comprises a protein or polypeptide, a sugar, a glycoprotein, a lipid, a lipoprotein, a lipopolysaccharide, a nucleic acid, or any combination thereof, preferably a protein or polypeptide, more preferably one or more from the group consisting of EGFR, PDL-1, HER2, CPCR, VEGFR, CTLA4, IL-5rα, IL-4R, IL-6R, PRLR, nav1.7, GCGR, PD-L1 and HLA-B27, further preferably PD-L1.

4. The chimera according to claim 1, characterized in that the target molecule binding domain comprises an antibody or antigen binding fragment thereof,

preferably, the antibody or antigen binding fragment thereof is selected from the group consisting of humanized antibodies,

preferably, the antibody or antigen binding fragment thereof is selected from monoclonal antibodies,

preferably, the antibody or antigen binding fragment thereof is selected from one or more of a single chain antibody, a chimeric monoclonal antibody, an anti-idiotype antibody, an scFv, (scFv) 2, a Fab 'and a F (ab') 2, a F (ab 1) 2, an Fv, a dAb and a Fd fragment, a bifunctional antibody,

preferably, the target molecule binding domain is an anti-PD-L1 antibody or antigen-binding fragment thereof; more preferably, it is a single chain variable region antibody against PD-L1, and still more preferably, the target molecule binding domain comprises (1) an amino acid sequence as shown in SEQ ID NO. 3, (2) a sequence having 85% or more sequence identity to SEQ ID NO. 3, or (3) an amino acid sequence encoded by a nucleotide sequence as shown in SEQ ID NO. 7 or a complementary sequence thereof, or any one of the foregoing (1) to (3).

5. The chimera according to claim 1, wherein said internalizing effector proteins are selected from the group consisting of CD63, MHC-I, kremen-1, kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL-related protein 1 receptor, MAL, V-ATPase, ASGR, amyloid precursor protein-like protein-2, apelin receptor, myelin and lymphocyte proteins, IGF2R, vacuolar H ⁺ Atpase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, or scavenger receptor,

preferably, the internalization effector protein is a transferrin receptor.

6. The chimera according to claim 1, characterized in that the internalizing effector protein binding domain comprises a molecule capable of specifically binding to internalizing effector protein, the molecule comprising a ligand for internalizing effector protein, an anti-internalizing effector protein antibody or antigen binding fragment thereof,

preferably, the anti-internalizing effector protein antibody or antigen-binding fragment thereof is selected from humanized antibodies,

preferably, the anti-internalizing effector protein antibody or antigen-binding fragment thereof is selected from monoclonal antibodies,

preferably, the anti-internalizing effector protein antibody or antigen binding fragment thereof is selected from the group consisting of single chain antibodies, chimeric monoclonal antibodies, anti-idiotype antibodies, scFv, (scFv) 2, fab 'and F (ab') 2, F (ab 1) 2, fv, dAb, fd fragments), bifunctional antibodies,

preferably, the internalizing effector protein binding domain comprises a transferrin receptor binding polypeptide or an anti-transferrin receptor antibody, more preferably a transferrin receptor binding polypeptide, further preferably the internalizing effector protein binding domain comprises (1) an amino acid sequence as shown in SEQ ID No. 1, (2) a sequence having more than 85% sequence identity to SEQ ID No. 1, or (3) an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 5 or the complement thereof, or any one of the foregoing (1) - (3).

7. The chimera of claim 1, wherein the internalization effector protein binding domain of the dual targeting chimera internalizes into a cell lysosome through interaction with the internalization effector protein.

8. The chimera according to claim 1, characterized in that the target molecule binding domain and internalizing effector protein binding domain are linked directly or indirectly, said indirect linkage comprising linking by a linker, preferably said linker comprising an amino acid sequence of the sequences shown as SEQ ID NOs 9-12 or (GmS) n, wherein m = 4, n = 1-10, more preferably n = 8.

9. The chimera according to claim 1, wherein the linker comprises (1) an amino acid sequence as set forth in SEQ ID No. 2, (2) a sequence having 85% or more sequence identity to SEQ ID No. 2, or (3) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID No. 6 or its complement, or any one of the foregoing (1) - (3).

10. The chimera according to claim 1, characterized in that the double targeting chimera comprises (1) an amino acid sequence as shown in SEQ ID No. 4, (2) a sequence having more than 85% sequence identity to SEQ ID No. 4, or (3) an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 8 or its complement, or consists of any of the foregoing (1) - (3).

11. The chimera according to claim 1, characterized in that the double targeting chimera is linked to an exocrine envelope protein by a cleavable linker containing a cleavage site;

preferably, the cleavage site comprises a matrix metalloproteinase 2 (MMP 2) cleavage site;

preferably, the amino acid sequence of the cleavable linker containing the cleavage site is selected from one or more of the sequences shown in SEQ ID NO. 13-31;

preferably, the exocrine envelope protein is selected from one or more of clyA protein, ompa protein, ompc protein or ompf protein, more preferably from clyA protein;

preferably, the chimera comprises (1) an amino acid sequence as set forth in SEQ ID NO. 36, (2) a sequence having 85% or more sequence identity to SEQ ID NO. 36, or (3) an amino acid sequence encoded by a nucleotide sequence as set forth in SEQ ID NO. 37 or its complement, or any of the foregoing (1) - (3).

12. A nucleic acid molecule comprising a coding region encoding the dual targeting chimera of any one of claims 1-11.

13. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a coding region encoding an amino acid sequence as set forth in SEQ ID NO. 1 and/or,

The coding region comprises or consists of the sequence: the nucleotide sequence shown in SEQ ID NO. 5, or a sequence with more than 85 percent of sequence identity with the nucleotide sequence, or a complementary sequence thereof.

14. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a coding region which encodes an amino acid sequence as set forth in SEQ ID NO. 3, or a sequence having more than 85% sequence identity thereto, and/or,

the coding region comprises or consists of the sequence: the nucleotide sequence shown in SEQ ID NO. 7, or a sequence with more than 85 percent of sequence identity with the nucleotide sequence, or a complementary sequence thereof.

15. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a coding region which encodes an amino acid sequence of the sequence shown in SEQ ID NO. 4, or a sequence having more than 85% sequence identity thereto, or a complement thereof, and/or,

the coding region comprises or consists of the sequence: the nucleotide sequence of the sequence shown in SEQ ID No. 8 or a sequence with more than 85 percent of sequence identity with the nucleotide sequence or a complementary sequence of the nucleotide sequence.

16. The nucleic acid molecule of claim 12, further comprising a nucleotide sequence of an exocrine envelope protein;

Preferably, the nucleic acid molecule further comprises a nucleotide sequence comprising a cleavable linker to a cleavage site;

preferably, the exocrine protein nucleotide sequence is selected from one or more of clyA protein, ompa protein, ompc protein or ompf protein nucleotide sequence, more preferably from clyA protein nucleotide sequence;

preferably, the coding region comprises or consists of the sequence: (1) a nucleotide sequence shown as SEQ ID NO. 37, (2) a sequence having more than 85% sequence identity with SEQ ID NO. 37, or (3) a sequence complementary to the nucleotide sequence shown as SEQ ID NO. 37, or consisting of any one of the foregoing (1) to (3).

17. A drug-loaded Outer Membrane Vesicle (OMV) which binds to a dual targeting chimera according to any one of claims 1 to 11.

18. A method of preparing a drug-loaded outer membrane vesicle according to claim 17, the method comprising: introducing the nucleic acid molecule of any one of claims 12 to 16 into a host cell,

preferably, the introduction is achieved by transfection,

preferably, the host cell is selected from the group consisting of prokaryotic cells,

preferably, the method further comprises the step of culturing the host cell under suitable conditions and isolating and purifying the drug-loaded outer membrane vesicles by membrane filtration.

19. A vector comprising the nucleic acid molecule of claims 12-16.

20. A cell comprising the nucleic acid molecule of claims 12-16 or the vector of claim 19.

21. A composition comprising the dual targeting chimera of any one of claims 1-11, the nucleic acid molecule of claims 12-16, the drug-loaded outer membrane vesicle of claim 17, the vector of claim 19 and/or the cell of claim 20, and a pharmaceutically acceptable carrier.

22. Use of a dual targeting chimera according to any one of claims 1-11, a nucleic acid molecule according to claims 12-16, a drug-loaded outer membrane vesicle according to claim 17, a vector according to claim 19, a cell according to claim 20 and/or a composition according to claim 21 for the preparation of a medicament for the treatment of a disease in a subject in need thereof.

23. The use according to claim 22, wherein the disease is a disease associated with the expression or overexpression of the target molecule,

preferably, the disease is a cancer,

more preferably, the cancer is selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or cervical cancer, salivary gland cancer, renal or ureteral cancer, prostate cancer, vaginal cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, melanoma, cholangiocarcinoma, central nervous system tumor, spinal axis tumor, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and ewing's sarcoma, superficial diffuse melanoma, malignant lentigo melanoma, acromelanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myelogenous leukemia and post-transplantation diseases and metastatic and cancer associated with cervical cancer, and malignant tumor-cell proliferation, brain tumor associated with cervical cancer, and malignant tumor.

24. A non-therapeutic method for the specific degradation of a target molecule in vitro, said method comprising the use of a dual targeting chimera according to any one of claims 1-11, a nucleic acid molecule according to claims 12-16, a drug-loaded outer membrane vesicle according to claim 17, a vector according to claim 19, a cell according to claim 20 and/or a composition according to claim 21.

25. The method of claim 24, wherein the target molecule comprises EGFR, PDL-1, HER2, CPCR, VEGFR, CTLA4, IL-5rα, IL-4R, IL-6R, PRLR, nav1.7, GCGR, PD-L1 and HLA-B27, preferably PD-L1.