Fusion protein composition, specific antibody and preparation method and application thereof

Abstract

The invention relates to a fusion protein composition and a monospecific / bispecific / multispecific immune drug constructed by the fusion protein composition. The fusion protein composition comprises a fusion protein A of SpyCatch003-albumin and a fusion protein B of at least one kind of protein-SpyTag003; the fusion protein A is formed by fusing a SpyCatch003 protein and albumin, and the SpyCatch003 protein and the albumin are fused together; the fusion protein B is formed by fusing protein and SpyTag003, and the protein is selected from at least one single-chain antibody. The immune drug is obtained by mixing a nano assembly and at least one fusion protein B and then connecting the mixture through a covalent bond, and the nano assembly is formed by hydrophobic interaction of a fusion protein A and hydrophobic degradable polyester or a derivative thereof. The invention also provides a preparation method of the immune drug and application of the immune drug in tumor immunotherapy or autoimmune disease treatment, inflammation treatment and infection treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a fusion protein composition, a specific antibody, and a preparation method and application thereof. Background Art

[0002] BsAb, bispecific antibodies, is the abbreviation of bispecific antibodies or bifunctional antibodies. It is an artificial antibody that can specifically bind to two antigens or two different epitopes on the same antigen at the same time. Bispecific antibodies have broad application prospects in the fields of tumor immunotherapy and autoimmune diseases. At present, at least more than 30 bispecific antibody drugs are in the clinical research stage. Moreover, the development of bispecific antibodies to multi-antibody technology beyond bispecific antibodies is also under continuous research and development.

[0003] With the development of technology, more and more technologies are used to construct bispecific and multispecific antibody drugs, among which non-natural nanoparticles are also commonly used in the construction of bispecific and multispecific antibody drugs.

[0004] Albumin-polymer composite nanoassemblies can be obtained by combining serum albumin with hydrophobic polymer materials through ultrasonic emulsification and high-pressure homogenization. Therefore, linking single-chain antibody fragments (ScFv) of different antibodies to the surface of nanoparticles by surface fixation is a very promising way to quickly construct dual-multispecific antibody drugs. For example, we have previously constructed fusion proteins and nanoassemblies based on single-chain antibody fragments (CN114516921A), fusion proteins and nanoassemblies based on serum albumin (CN114478800A), and protein nanoparticles for multispecific antibody delivery (CN112336873A).

[0005] In order to make the above-mentioned nanoparticles a universal platform for single / bi / multi-specific drugs, it is necessary to modify the components of the nanoparticles. In the early stage, bi-multi-specific antibodies were directly constructed by constructing ScFv-SA fusion proteins and hydrophobic polymer materials. The technology is simple, but the preparation of different antibodies requires the production of different fusion proteins and then the preparation, which is relatively cumbersome overall; the nanoaptamer constructed by FcγR-SA can effectively adsorb antibodies, thereby quickly constructing single / bi / multi-specific antibodies, but its binding mode is non-covalent binding, and there is a risk of falling. Therefore, it is very necessary to obtain a nanoaptamer with good stability and good biological activity.

[0006] The SpyTag / SpyCatcher system was developed by the Howarth laboratory based on the internal isopeptide bond of the CnaB2 domain of Streptococcus pyogenes fibronectin FbaB. In this domain, an internal isopeptide bond spontaneously forms between the ε-amino group of lysine K3 and the side chain carboxyl group of aspartic acid D117, and the resulting isopeptide bond confers high stability to the CnaB2 domain. The binding domain can be divided into two parts, the larger part named SpyCatcher, which contains 138 amino acid residues; the smaller part named SpyTag, which consists of 13 amino acid residues; these two parts can quickly combine to form a high-affinity covalent bond without exogenous assistance, thereby forming a complex. On this basis, the Howarth laboratory further optimized it into SpyCatcher003 and SpyTag003, and the rate of isopeptide bond formation of the two after optimization increased by more than 100 times. Summary of the invention

[0007] Based on this, one of the objects of the present invention is to provide a fusion protein composition and its application in constructing a bi-multispecific drug, as well as the constructed bi-multispecific drug and its preparation method.

[0008] The first aspect of the present invention provides a fusion protein composition, which includes a fusion protein A of SpyCatcher003-albumin, and a fusion protein B of at least one protein-SpyTag003;

[0009] The fusion protein A is formed by the fusion of SpyCatcher003 protein and albumin;

[0010] The fusion protein B is formed by fusion of a protein and SpyTag003, and the protein is selected from at least one single-chain antibody.

[0011] In some embodiments, the albumin is any one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, and chicken ovalbumin.

[0012] In some embodiments, the single-chain antibody targets at least any one of the following antigens: single-chain antibodies targeting HER2, CD137L, CD137, 4-1BBL, 4-1BB, OX40L, OX40, ICOSL, ICOS, CD86, CD80, CD28, LFA3, CD2, CTLA4, PDL1, PD1, CD70, CD27, GAL9, TIM3, CD111, CD96, CD112, CD226, CD115, CD113, TIGIT, CD39, CD73, CD47, SIRPα, TNFα, IL1β, IL6, TGFβ, IL-10, IL-12 antibodies derived from humans or other species. That is, the single-chain antibody is selected from antibody fragments of at least one of the above antigens. Preferably, the single-chain antibody targets one, two or three antigens, or the fusion protein B is a fusion protein comprising a single-chain antibody, or comprises two fusion proteins, the two fusion proteins are respectively fused with two different single-chain antibodies, or comprises three fusion proteins, the three fusion proteins are respectively fused with three different single-chain antibodies.

[0013] In some of the embodiments, when multiple fusion proteins B comprising multiple different single-chain antibodies are prepared, the amount of each fusion protein B used is most preferably the same.

[0014] In some of the embodiments, in the fusion protein A, the albumin can be linked to the N-terminus or C-terminus of SpyCatcher003 via a flexible connecting peptide.

[0015] In some of the embodiments, in the fusion protein B, the protein can be linked to the N-terminus or C-terminus of SpyTag003 via a flexible linker peptide.

[0016] In some of the embodiments, the residues of the connecting peptide are (GGGGS)n, where n is an integer of 1-4.

[0017] The second aspect of the present invention provides the coding genes of the above-mentioned fusion protein composition, including the coding gene of fusion protein A and the coding gene of fusion protein B.

[0018] In some of the embodiments, the gene encoding fusion protein A is shown in any one of SEQ ID No: 1 and SEQ ID No: 2.

[0019] In some of the embodiments, the gene encoding fusion protein B is shown in any one of SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, and SEQ ID No: 10.

[0020] The third aspect of the present invention provides a gene encoding the above-mentioned recombinant fusion protein composition, or a plasmid vector containing a gene encoding any one of the above-mentioned recombinant fusion protein compositions.

[0021] The fourth aspect of the present invention provides an engineered bacterium, a protoplast, or an in vitro cloning system containing any gene or vector encoding the above-mentioned recombinant fusion protein composition.

[0022] The fifth aspect of the present invention provides a mono / bi / multi-specific immune drug, which is a nanoassembly formed by covalent bonds after fusion protein A in a fusion protein composition interacts with a hydrophobic degradable polyester or its derivative and is then mixed with fusion protein B in the fusion protein composition.

[0023] In some embodiments, the degradable polyester is an aliphatic polyester, or an aliphatic polyester modified with polyethylene glycol.

[0024] In some embodiments, the aliphatic polyester includes, but is not limited to, at least one of polylactide, polyglycolide, poly(lactide-co-glycolide), and polycaprolactone.

[0025] In some embodiments, the block molecular weight of the polylactide is 1 kDa to 1,100 kDa; or the block molecular weight of the poly(lactide-co-glycolide) is 1 kDa to 1,100 kDa, wherein the ratio of lactide to glycolide is in the range of 95 / 5 to 50 / 50.

[0026] In some of the embodiments, the molar ratio of the nanoassembly to the fusion protein B is (1-10):1, preferably the molar ratio of the nanoassembly to the fusion protein B is (1-6):1, and more preferably (2-3):1.

[0027] In some of the embodiments, the ratio of the amount of the fusion protein A to the amount of the fusion protein B is (3-1):1, and most preferably, the ratio of the amount of the fusion protein A to the amount of the fusion protein B is 1:1.

[0028] The sixth aspect of the present invention provides a method for preparing a mono / bi / multi-specific immunological drug, which comprises the following steps:

[0029] (1) mixing the fusion protein A with water or physiological saline at a concentration of 0.5 to 20 mg / mL to obtain an aqueous phase; mixing the hydrophobic degradable polyester and its derivatives with an organic solvent to obtain an oil phase at a concentration of 0.5 to 10 mg / mL;

[0030] (2) preparing an oil-in-water emulsion from the aqueous solution and the oil solution of step (1);

[0031] (3) separating and purifying the emulsion to obtain a nanoassembly;

[0032] (4) Mixing the nanoassembly with at least one of the fusion proteins B at room temperature to obtain the drug.

[0033] In some embodiments, the organic solvent is at least one of chloroform, dichloromethane, and ethyl acetate.

[0034] In some embodiments, the concentration of the recombinant fusion protein A in the aqueous phase is 1 to 10 mg / ml.

[0035] In some embodiments, the concentration of the hydrophobic degradable polyester and its derivatives in the oil phase is

[0036] The concentration is 1~10mg / ml.

[0037] In some embodiments, the weight ratio of the hydrophobic degradable polyester and its derivatives to the fusion protein A is 1:0.1-10, preferably 1:1-10, preferably 1:2-6.

[0038] In some embodiments, the volume ratio of the water phase to the oil phase is 1-10:1, preferably 2-6:1.

[0039] In some embodiments, the molar ratio of the nanoassembly to the total fusion protein B is (1-10):1, preferably (1-6):1, and more preferably (2-3):1.

[0040] The seventh aspect of the present invention provides the use of any of the above-mentioned immune drugs in the preparation of products for tumor immunotherapy, autoimmune disease treatment, inflammation treatment, and infection treatment.

[0041] The present invention creatively constructs a nanoassembly formed by assembling a fusion protein containing SpyCatcher003 and albumin with a hydrophobic degradable polyester through affinity, and then assembling nanomedicines with ScFv containing SpyTag003 to form nanoassemblies with different uses. It is found that SpyCatcher003 on the surface of the assembly can be conveniently and quickly covalently combined with the fusion protein containing SpyTag003 to construct a stable single / bi / multi-specific antibody drug. In addition to maintaining the corresponding biological activity and stable activity, the drug can also simultaneously combine with multiple specific antibodies. The multiple specific antibodies are more firmly combined in the nanoassembly and have stronger stability. Therefore, the single / bi / multi-specific immune drugs can be well applied to the treatment of diseases such as tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Vector construction map.

[0043] Figure 2 : SDS-PAGE characterization of SpyCatcher003-HSA fusion protein.

[0044] Figure 3 :Western Blot characterization of SpyCatcher003-HSA fusion protein.

[0045] Figure 4 : HPLC characterization of the purity of SpyCatcher003-HSA fusion protein after purification.

[0046] Figure 5 : SDS-PAGE characterization of αHER2 ScFv-SpyTag003 fusion protein.

[0047] Figure 6 :Western Blot characterization of αHER2 ScFv-SpyTag003 fusion protein.

[0048] Figure 7 : HPLC characterization of the purity of purified αHER2 ScFv-SpyTag003 fusion protein.

[0049] Figure 8 : ELISA characterization of the affinity of αHER2 ScFv-SpyTag003 fusion protein to HER2 antigen.

[0050] Fig. 9 : SDS-PAGE characterization of αHER2 ScFv-SpyTag003 and SpyCatcher003-HSA binding.

[0051] Fig.10 : Western Blot characterization of αHER2 ScFv-SpyTag003 and SpyCatcher003-HSA binding. Fig.11 :Using Zetasizer to detect NPs prepared from recombinant proteins SCHSA Particle size.

[0052] Fig.12 :Scanning electron microscopy characterization of NPs SCHSA .

[0053] Fig.13 :NP SCHSA Particle stability assessment.

[0054] Fig.14:Zetasizer device detects NPs prepared from recombinant proteins SCHSA Changes after binding of αHER2 ScFv-SpyTag003.

[0055] Fig.15 :Nanoflow cytometry characterization of NPs SCHSA Binding of αHER2 ScFv-SpyTag003.

[0056] Fig.16 : Flow cytometric characterization of NPs covalently bound to SpyTag003 SCHSA Can enhance the ability to bind to target cells.

[0057] Fig.17 : Confocal characterization of NPs covalently bound to SpyTag003 SCHSA Can enhance the ability to bind to target cells.

[0058] Fig.18 : Confocal characterization of αHER2 / CD3 ScFv-SpyTag003 equipped nanoparticles to enhance the binding of immune cells to tumor cells.

[0059] Fig.19 : HPLC characterization of NPs SCHSA Able to bind three different ScFv-SpyTag003 simultaneously.

[0060] Fig. 20 : HPLC characterization of NPs SCHSA The ratio of αCD22-binding ScFv-SpyTag003 fusion protein.

[0061] Fig.21 :NP SCHSA The ratio of αCD22-binding ScFv-SpyTag003 fusion protein. Fig.21 : Schematic diagram of the principles of the fusion protein, assembly and application of the present invention. DETAILED DESCRIPTION

[0062] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0063] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0064] To facilitate understanding of the present technology, certain terms and phrases are defined below.

[0065] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0066] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0067] Homology: In biological phylogeny, two or more structures are said to be homologous if they have a common ancestor.

[0068] Antibody affinity refers to the binding strength between the antibody's antigen binding cluster and the antigenic determinant cluster of the antigen, or the binding force between the antibody and the antigen epitope or antigenic determinant cluster. It is essentially a non-covalent force that includes the attraction between amino acids, hydrogen bonds, hydrophobic forces, etc.

[0069] Some embodiments of the present invention relate to a protein having at least a serum albumin fragment, which can be combined with a hydrophobic degradable polymer through hydrophobic interaction. In the present invention, it is albumin, i.e., serum albumin, which can be at least one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, and chicken ovalbumin.

[0070] In some preferred embodiments, the protein is a protein derived from human serum albumin having at least three or four hydrophobic domains, the protein having a hydrophobic region is a protein derived from human serum albumin having at least five hydrophobic domains, or the above-mentioned protein with a complete hydrophobic domain after substitution, deletion and / or addition of one or more amino acids, more preferably, a protein derived from human serum albumin having six or seven hydrophobic domains.

[0071] In the present invention, single-chain variable fragment (scFv) refers to a brief part of the immunoglobulin Fab fragment that is artificially expressed through genetic recombination technology and retains the specific binding to the antigen. It is composed of a heavy chain and a light chain, and the two chains are connected by a short polypeptide hinge. scFv retains the characteristics of antigen recognition and binding, and at the same time, its molecule is small and can be recombined with a polypeptide or protein to form a fusion protein.

[0072] The single-chain antibody is derived from a functional fragment of an antibody and comprises at least one light chain variable region (VL) and / or heavy chain variable region (VH) of an antibody.

[0073] The single-chain antibody is derived from human, mouse or other species, which is consistent with the species of serum albumin.

[0074] The connecting peptide can be a peptide sequence conventionally used to connect polypeptides, which can connect two polypeptides and fold them naturally into the desired structure. It is usually a short peptide with a hydrophobicity and a certain degree of stretchability. The purpose of the present invention is to separate the two fused proteins to alleviate the mutual interference between the two. The connecting peptide can be flexible or rigid. In certain embodiments, a flexible connecting peptide may be advantageous, which can connect two protein / polypeptide components and maintain their respective activities and functions. In some of the embodiments, in the fusion protein, the protein in the hydrophobic region and the immunomodulatory antibody functional fragment are connected by a connecting peptide. In some embodiments, the connecting peptide, for example, uses [GlyGlyGlyGlySer]n, where n is an integer of 0-4, more preferably 1, 2, 3. When n is 0, it means that the related fragments are directly connected.

[0075] Some embodiments of the present invention relate to hydrophobic degradable polyesters and their derivatives, which are currently known degradable biomaterials, and also include new degradable biomaterials produced by further research and development in the future, which can be combined with the hydrophobic region of the serum albumin part. The polyester is an aliphatic polyester or its derivatives.

[0076] The present invention constructs and expresses SpyCatcher003-SA fusion protein, and prepares nanoparticles by combining it with a hydrophobic high molecular polymer; at the same time, fusion protein is constructed by combining SpyTag003 with exogenous ScFv and the like; the nanoparticles are incubated with the fusion protein containing SpyTag003, and the fusion protein can be uniformly combined on the surface of the nanoparticles through isopeptide bonds to form a bi-multispecific antibody.

[0077] The related sequences used in the following examples.

[0078] SpyCatcher003:VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT(SEQ ID No.11)

[0079] SpyTag003:RGVPHIVMVDAYKRYK(SEQ ID No.12)

[0080] SEQ ID No.1

[0081] SpyCatcher003-HSA

[0082] VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT GGGGSGGGGSGGGGS DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE MPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHHHHHH

[0083] SEQ ID No.2

[0084] HSA-SpyCatcher003DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL

[0085] AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKKCCAAA

[0086] DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV

[0087] SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP

[0088] CFSALEVDETYVPKEFNAETTFFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVM

[0089] DDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSVTTLSGLS

[0090] GEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISTGHVKDFYLY

[0091] PGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTHHHHHHHHHHSEQ ID No.3

[0092] αHER2 ScFv-SpyTag003

[0093] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGCAPKLLIYSASFLYSGVPSRFSGSRS

[0094] GTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGSGGSGGCPPCGGGGSEVQLVE

[0095] SGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA

[0096] DTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGCGTLVTVSS GGGGSGGGGSGGGG

[0097] S RGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHH

[0098] SEQ ID No.4

[0099] SpyTag003-αHER2 ScFv

[0100] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDV

[0101] NTAVAWYQQKPGCAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY

[0102] TTPPTFGQGTKVEIKGGGSGGSGGCPPCGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNI

[0103] KDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE

[0104] DTAVYYCSRWGGDGFYAMDYWGCGTLVTVSSEQKLISEEDLNSAVDHHHHHHSEQ ID No.5

[0105] αCD3 ScFv-SpyTag003

[0106] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTP

[0107] ARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVGGGGSGGGGSGG

[0108] GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNN

[0109] YATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQ

[0110] GTLVTVSSGGGGSGGGGSGGGGSRGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHHSEQ IDNo.6

[0111] SpyTag003-αCD3 ScFv

[0112] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAV

[0113] TTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYC

[0114] ALWYSNLWVFGGGTKLTVGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASG

[0115] FTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMN

[0116] SLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSEQKLISEEDLNSAVDHHHHHHSEQ IDNo.7

[0117] α4-1BB ScFv-SpyTag003

[0118] EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNY

[0119] SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSSGGGGS

[0120] GGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDK

[0121] NRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVLGGGGS

[0122] GGGGSGGGGSRGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHHSEQ ID No.8

[0123] SpyTag003-α4-1BB ScFv

[0124] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYSF

[0125] STYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASD

[0126] TAMYYCARGYGIFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASI

[0127] TCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIPERFSGSNSGNTATLTISGTQAMDE

[0128] ADYYCATYTGFGSLAVFGGGTKLTVLEQKLISEEDLNSAVDHHHHHHSEQ ID No.9

[0129] αCD22 ScFv-SpyTag003

[0130] DVQVTQSPSSLSASVGDRVTITCRSSQSLANSYGNTFLSWYLHKPGKAPQLLIYGISNRFSG

[0131] VPDRFSGSGSGTDFTLTISSLQPEDFATYYCLQGTHQPYTFGQGTKVEIKGGGGSGGGGSGG

[0132] GGSEVQLVQSGAEVKKPGASVKVSCKASGYRFTNYWIHWVRQAPGQGLEWIGGINPGNN

[0133] YATYRRKFQGRVTMTADTSTSTVMELSSLRSEDTAVYYCTREGYGNYGAWFAYWGQGTLV

[0134] TVSSGGGGSGGGGSGGGGSRGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHHSEQ ID No.10

[0135] SpyTag003-αCD22 ScFv

[0136] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSDVQVTQSPSSLSASVGDRVTITCRSSQSL

[0137] ANSYGNTFLSWYLHKPGKAPQLLIYGISNRFSGVPDRFSGSGSGTDFTLTISSLQPEDFATYY

[0138] CLQGTHQPYTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKA

[0139] SGYRFTNYWIHWVRQAPGQGLEWIGGINPGNNYATYRRKFQGRVTMTADTSTSTVMELSS

[0140] LRSEDTAVYYCTREGYGNYGAWFAYWGQGTLVTVSSEQKLISEEDLNSAVDHHHHHH

[0141] Raw materials and their sources used in the examples:

[0142] SpyCatcher003-HSA, αHER2 ScFv-SpyTag003, αCD3 ScFv-SpyTag003, α4-1BBScFv-SpyTag003, and αCD22 ScFv-SpyTag003 were transiently expressed in HEK-293F cells and purified by AKTA purification instrument. The flexible link in the fusion protein is (GGGGS)3.

[0143] Polylactic acid PLA134K, a L-polylactic acid with a molecular weight of 134,000 Da and an ester-terminated end cap, was purchased from Jinan Daigang Biotechnology Co., Ltd.

[0144] Dichloromethane: purchased from Guangzhou Chemical Reagent Factory.

[0145] pXC17.4αHER2 ScFv-SpyTag003 was commissioned to be synthesized by GenScript Biopharmaceuticals Co., Ltd. pXC17.4SpyCatcher003-HSA was commissioned to be synthesized by GenScript Biopharmaceuticals Co., Ltd.

[0146] PEI40000: Polysciences, purchased from Chenxue Biotechnology (Guangzhou) Co., Ltd.

[0147] HEK-293F cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0148] BT-474 cells were purchased from Xiamen Yimo Biotechnology Co., Ltd.

[0149] HT-29 cells were purchased from Guangzhou Zhenzhi Biotechnology Co., Ltd.

[0150] HEK CD02 culture medium: purchased from Shanghai Youyi Biotechnology Co., Ltd.

[0151] HER2 antigen: ACRO, purchased from Guangzhou Zhijianlian Biotechnology Co., Ltd.

[0152] ELISA reagents: purchased from Beijing Sino Biological Technology Co., Ltd.

[0153] Mouse anti-Myc tag antibody: Guangzhou Juyan Biotechnology Co., Ltd.

[0154] anti HSAantibody; purchased from Beijing Sino Biological Technology Co., Ltd.

[0155] anti-HIS antibody; purchased from Beijing Sino Biological Technology Co., Ltd.

[0156] Protein L: purchased from Beijing Sino Biological Technology Co., Ltd.

[0157] Cy5-NHS dye: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0158] Cy3-NHS dye: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0159] FITC-NHS dye: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0160] CellTrace Far Red dye: purchased from ThermoFisher Technology Co., Ltd.

[0161] Example 1 Construction and expression of SpyCatcher003-HSA fusion protein

[0162] 1. Construction of recombinant plasmid

[0163] Synthesize the gene sequence corresponding to the target protein sequence in the following order:

[0164] The target protein sequence is SpyCatcher003-Link-HSA-10*HIS, named SpyCatcher003-HSA, and the specific sequence is shown in SEQ ID No: 1.

[0165] The signal peptide sequence was added to the N-terminus of the gene sequence corresponding to the above protein sequence, the start codon was added to the N-terminus, the stop codon was added to the C-terminus, and the full-length gene sequence was cloned into the pXC17.4 vector, and HindIII and EcoRI restriction sites and signal peptides were added upstream and downstream. The synthesis of the above plasmid was entrusted to GenScript Biotech Co., Ltd., and the plasmid was named pXC17.4 SpyCatcher003-HSA. The schematic diagram of the recombinant vector is shown in the figure below. Figure 1 As shown in A, the 10*HIS sequence contained therein is used for purification.

[0166] 2. Expression and purification of fusion protein

[0167] According to conventional methods, the above synthetic plasmid was transformed into Escherichia coli, and a single colony was picked and expanded.

[0168] The plasmid was extracted by culture and then transiently transfected into HEK-293F cells.

[0169] First, HEK-293F cells were expanded to 4E6 cells / mL with a total culture volume of 100 mL; 200 μg pXC17.4 SpyCatcher003-HSA was diluted to 5 mL with 0.15 M sodium chloride solution, and 400 μg PEI 40000 was diluted to 5 mL with 0.15 M sodium chloride solution; then the PEI40000 dilution was slowly added to the plasmid dilution solution and mixed, and allowed to stand for 15 minutes; finally, the above mixture was slowly poured into the cell solution, and then placed in a CO2 incubator for culture at 37°C, 8% CO2, and 130 rpm. After 7 days of batch culture, the supernatant was collected after centrifugation at 5000g for 15min. The expressed fusion protein had a 10*HIS tag at its C-terminus, which could be purified by affinity chromatography through specific binding with NI, and finally collected in a solution containing 200mM imidazole. Then, a 30kDa ultrafiltration tube was used for multiple ultrafiltrations, and the ultrafiltration solution was replaced with an aqueous solution at 3000g and 4°C, and the molecular weight of the target fusion protein was characterized by SDS-PAGE after purification, as shown in FIG. Figure 2 The SDS-PAGE results showed that the molecular weight of SpyCatcher003-HSA fusion protein was 80 kDa+, which was consistent with the expected 80.91 kDa. The structural domain was preliminarily characterized by Western Blot. Figure 3 The results showed that it contained HSA and HIS domains, which was consistent with expectations. Finally, the purity of the target protein was preliminarily characterized by HPLC. Figure 4 The results showed that its purity was higher than 95%, which met the requirements of subsequent experiments.

[0170] Example 2 Expression and purification of αHER2 ScFv-SpyTag003

[0171] 1. Construction of recombinant plasmid

[0172] Synthesize the gene sequence corresponding to the target protein sequence in the following order:

[0173] The target protein sequence is αHER2 ScFv-Link-SpyTag003-Myc-6*HIS, named αHER2 ScFv-SpyTag003,

[0174] The specific sequence is shown in SEQ ID No: 3.

[0175] The signal peptide sequence was added to the N-terminus of the gene sequence corresponding to the above protein sequence, the start codon was added to the N-terminus, the stop codon was added to the C-terminus, and the full-length gene sequence was cloned into the pXC17.4 vector. HindIII and EcoRI restriction sites were added upstream and downstream, respectively. The 6*HIS tag was used for purification, and the Myc tag was used for subsequent characterization tags. The synthesis of the above plasmid was entrusted to GenScript Biotech Co., Ltd., and the plasmid was named pXC17.4αHER2 ScFv-SpyTag003. The schematic diagram of the recombinant vector is shown in the figure. Figure 1 As shown in B.

[0176] 2. Expression and purification of fusion protein

[0177] The above synthetic plasmid was transformed into Escherichia coli, and a single colony was picked and expanded for plasmid extraction. After extraction, HEK-293F cells were transiently transformed. First, HEK-293F cells were expanded to 4E6 cells / mL, and the total culture volume was 100mL; 100μg pXC17.4αHER2 ScFv-SpyTag003 was diluted to 5mL with 0.15M sodium chloride solution, and 200μg PEI40000 was diluted to 5mL with 0.15M sodium chloride solution; then the PEI40000 dilution was slowly added to the plasmid dilution solution and mixed, and allowed to stand for 15min; finally, the above mixture was slowly poured into the cell solution, and then placed in a CO2 incubator for culture, and the culture conditions were 37°C, 8% CO2, and 130rpm. After 7 days of batch culture, the supernatant was collected after centrifugation at 5000g for 15min. The expressed fusion protein had a 6*HIS tag at its C-terminus, which could be purified by affinity chromatography through specific binding with NI. It was finally collected in a solution containing 200mM imidazole, and then ultrafiltered multiple times using a 30kDa ultrafiltration tube. The ultrafiltration solution was replaced with PBS solution at 3000g and stored after sterilization. After the target fusion protein was purified, its molecular weight was characterized by SDS-PAGE, such as Figure 5 The results showed that the molecular weight of pXC17.4αHER2 ScFv-SpyTag003 fusion protein was 30kDa+, which was consistent with the expected 31.44kDa. The structural domain was preliminarily characterized by Western Blot. Figure 6 The results showed that it contained ScFv and HIS domains, which was consistent with expectations. The purity of the target protein was then preliminarily characterized by HPLC. Figure 7 The results showed that its purity was higher than 90%, which met the requirements of subsequent experiments.

[0178] 3. Characterization of affinity between fusion protein and antigen by ELISA

[0179] The affinity of αHER2 ScFv-SpyTag003 fusion protein to the target antigen HER2 was characterized by enzyme-linked immunosorbent assay (ELISA). Human HER2 antigen was diluted to 1 μg / mL with Coatingbuffer, 100 μL / well, incubated at 37°C for 2h or overnight at 4°C, and washed three times with PBST. Blocked with 200 μL / well, 1% protein-free blocking solution, washed three times with PBST. The fusion protein αHER2 ScFv-SpyTag003 was diluted with PBST to 20 μg / mL, 2 μg / mL, 200 ng / mL, 20 ng / mL, 2 ng / mL, 0.2 ng / mL, 0.02 ng / mL, 0.002 ng / mL, 100 μL / well, incubated at 37°C for 2h, and washed six times with PBST. Mouse anti-Myc tag antibody (HRP) was used as the secondary antibody and washed six times with PBST. ELISA colorimetric solution was used, and the reaction was carried out in the dark for 10-20 minutes, and then terminated with 2M sulfuric acid. The absorbance at 450-630 nm was detected by an ELISA reader. The affinity curve of αHER2ScFv-SpyTag003 is shown in Figure 8 , its Kd = 0.29nM. The Kd value reflects the affinity of the compound for the target. The smaller the value, the stronger the affinity. The results showed that the expressed αHER2 ScFv-SpyTag003 had a strong affinity with the target antigen, indicating that the fusion of SpyTag003 would not affect the affinity of the antibody ScFv.

[0180] Example 3 Characterization of the binding of SpyCatcher003-HSA to αHER2 ScFv-SpyTag003

[0181] The two expressed fusion proteins (SpyCatcher003-HSA and αHER2 ScFv-SpyTag003) were mixed in different ratios to explore the binding of the two proteins. The molar ratios were 1:2, 1:1 and 2:1, respectively. The mixed system was PBS buffer with a pH of 7.4. After mixing for 30 minutes, SDS-PAGE was performed. The SDS-PAGE results are shown in the figure. Fig. 9 As shown in the figure, when the molar ratio of the two is 1:1, the two can completely react to generate a new protein at 112kDa. When the ratio is not 1:1, but 2:1 or 1:2, there will be residual protein residues. At the same time, the structure after binding was characterized by Western Blot using anti-HSA, anti-HIS, and anti-ScFv. The results are shown in the figure. Fig.10 It shows that the two can be fully combined in a 1:1 situation.

[0182] Example 4 Nanoparticles (NPSCHSA ) assembly and characterization

[0183] 1. NP SCHSA Preparation

[0184] Use dichloromethane as solvent to prepare PLLA at a concentration of 10 mg / mL 134k The solution was used as the oil phase, and a 5 mg / mL SpyCatcher003-HSA fusion protein aqueous solution was used as the water phase. 100 μL of the oil phase and 1 mL of the water phase were placed in a 5 mL centrifuge tube and emulsified using an ultrasonic cell disruptor. The probe diameter of the ultrasonic disruptor was 3 mm, the amplitude was 40%, the ultrasound was continued for 5 seconds and then stopped for 2 seconds, and the total ultrasound time was 2 minutes. After the ultrasound, the emulsion was transferred to a rotary evaporator and the dichloromethane was removed in the order of 400 / 350 / 300 / 250 / 200 / 150 / 100 / 75 / 50 / 25 mbar for 2 minutes each. After the evaporation, the fusion protein aliphatic polyester nanoassembly was collected. The hydrated diameter of the nanoassembly was detected using a nanoparticle size analyzer, and the corresponding NP SCHSA Particle size distribution diagram Fig.11 As shown, the particle size is about 100 nm and the PDI is about 0.09, indicating that it has excellent dispersibility.

[0185] 2. Scanning electron microscopy characterization of NPs SCHSA

[0186] NP SCHSA The concentration was diluted to 9, and when the ATTN value detected by the nanoparticle size instrument was 9, 2 μL was added to the silicon wafer, and after the water evaporated, gold was sprayed on it, and then it was observed under a scanning electron microscope. The electron microscope results were as follows: Fig.12 As shown, the nanoparticles are independent and full spherical.

[0187] 3. NP SCHSA Stability assessment

[0188] The nanoparticles were placed in aqueous solution and PBS solution and stored at 37°C with shaking. The particle size and PDI were measured at 0, 2, 4, 8, 24, 36, 60, 96, 144, 180, and 252 hours. The results are as follows: Fig.13 As shown, there is no obvious change in particle size and PDI for up to ten days, indicating that the nanoparticles prepared by the present invention have good stability.

[0189] Example 5 Rapid preparation and characterization of nanomedicine

[0190] 1. Preparation of Nanomedicine

[0191] NP SCHSAMixed with αHER2 ScFv-SpyTag003 fusion protein at a molar ratio of 3:1 in PBS solution at room temperature, and the mixed proteins were uniformly named NP SCHSA&αHER2-ScFv Then the particle size, PDI and potential were tested by nanoparticle size analyzer. The results are as follows Fig.14 As shown, after binding to the fusion protein, the overall particle size increases by 30 nm, and the potential also increases to a certain extent due to the binding of the fusion protein, which is consistent with expectations.

[0192] 2. Nanoflow cytometry characterization of NPs SCHSA Binding αHER2 ScFv-SpyTag003 fusion protein

[0193] The fluorescent molecule labeled with Cy5 on the SpyCatcher003-HSA fusion protein was named SpyCatcher003-HSA Cy5 At the same time, the αHER2 ScFv-SpyTag003 fusion protein was labeled with AF488 fluorescent molecules and named αHER2ScFv-SpyTag003 AF488 . Then use SpyCatcher003-HSA Cy5 The fusion protein is prepared according to the particle preparation method in Example 4 to prepare fluorescent nanoparticles NP SCHSA-Cy5 , and then combined it with αHER2 ScFv-SpyTag003 AF488 The cells were incubated in PBS for 1 hour, centrifuged at 15,000 rpm for 45 minutes, the supernatant was removed, the particles were resuspended in PBS, and finally characterized by nanoflow cytometry. The experimental results are shown in Fig.15 As shown, nanoparticles NP SCHSA-Cy5 Able to interact with αHER2ScFv-SpyTag003 AF488 Effective binding, αHER2ScFv-SpyTag003 can be detected on nearly 90% of the particle surface AF488 .

[0194] 3. HPLC characterization of NPs SCHSA Binding ratio of αCD22 ScFv-SpyTag003 fusion protein

[0195] NP SCHSA The cells were co-incubated with αCD22 ScFv-SpyTag003 at a molar ratio of 6:1, 3:1, 2:1 and 1:1. After incubation at room temperature for 1 h, samples were taken to detect the amount of free αCD22 ScFv-SpyTag003. The experimental results are shown in Fig. 20 As shown, when the molar ratio is 1-6:1, the corresponding nanoparticles can be obtained, but they are not NPs. SCHSAThe more the amount, the better. When the ratio is (2-3):1, there is very little free αCD22 ScFv-SpyTag003. Especially when the ratio is 2:1, there is almost no free αCD22 ScFv-SpyTag003 in the system. Therefore, this ratio can be used for subsequent binding.

[0196] Example 6 αHER2 ScFv-SpyTag003 binding enhances nanoparticle targeting

[0197] 1. Flow cytometry characterization of αHER2 ScFv-SpyTag003 covalently bound NP SCHSA Can promote nanoparticle binding to HER2 ++ BT-474 cells

[0198] The αHER2 ScFv-SpyTag003 fusion protein was labeled with FITC fluorescent molecules and named αHER2ScFv-SpyTag003. FITC NP SCHSA-Cy5 Binding αHER2 ScFv-SpyTag003 FITC The particles after fusion protein are named NP SCHSA-Cy5&αHER2-FITC .

[0199] BT474 cells were cultured until the confluence was 80%, the supernatant was removed, and the cells were digested with trypsin and counted; 50W cells were transferred to a 1.5mL EP tube, and the NPs prepared in advance were added according to different experimental purposes. SCHSA-Cy5 or NP SCHSA-Cy5&αHER2-FITC After incubation on ice for 15 min, centrifuge at 300 g and 4 °C for 5 min, and then analyze the fluorescence intensity of the particles bound to the cell surface by flow cytometry. Fig.16 As shown, αHER2 ScFv-SpyTag003 was added FITC After binding to the cell surface, the fluorescence intensity of Cy5 increased significantly, indicating that NP SCHSA-Cy5 It can bind to the cell surface through αHER2 ScFv-SpyTag003.

[0200] 2. Confocal characterization of αHER2 ScFv-SpyTag003 covalently bound NPs SCHSA Improved nanoparticle binding to HER2++ BT-474 cells

[0201] BT-474 cells were cultured until the confluence was 80%, the supernatant was removed, and the cells were digested with trypsin and counted; 20W cells were transferred to a 1.5mL EP tube, and the NPs prepared in advance were added according to different experimental purposes. SCHSA-Cy5 or NP SCHSA-Cy5&αHER2-FITCAfter incubation on ice for 15 min, centrifuge at 300 g and 4 °C for 5 min, and then characterize the fluorescence of the cell surface by confocal microscopy. Fig.17 As shown, without adding αHER2 ScFv-SpyTag003 FITC When NPs were detected on the cell surface SCHSA-Cy5 The signal of αHER2 ScFv-SpyTag003 was FITC After that, strong FITC and Cy5 signals were observed on the cell surface, indicating that NP SCHSA-Cy5 It can bind to the cell surface through αHER2 ScFv-SpyTag003.

[0202] Example 7 αHER2 ScFv-SpyTag003 and αCD3 ScFv-SpyTag003 combined with nanoparticles enhances the binding effect of immune cells on tumor cells

[0203] 1. Confocal characterization of bispecific NPs SCHSA&αHER2 / CD3 Promotes binding of effector and target cells

[0204] Combine αHER2 ScFv-SpyTag003 and αCD3 ScFv-SpyTag003 with NP SCHSA Co-incubation to obtain NP SCHSA&αHER2 / CD3 , used for subsequent experiments.

[0205] HT-29 cells were cultured to a confluence of 80%, the supernatant was removed, and the cells were digested with trypsin and resuspended in 1640 culture medium; 1100w / mL HT-29 cells were transferred to a sterile centrifuge tube, 1μL CSFE dye was added, and the cells were treated at 37°C for 15min, and then ten times the volume of DMEM+10% FBS was added to incubate for 5min to terminate the staining, and the supernatant was removed by centrifugation at 800rpm, and 10W cells were inoculated into a confocal dish and cultured for 24 hours.

[0206] Take 500w of T cells, centrifuge at 400g for 10min, resuspend the cells with 1mL 1640 serum-free medium, add 1μL CellTrace Far Red dye for staining, treat at 37℃ for 15min, add ten times the volume of DMEM+10% FBS and incubate for 5min to terminate the staining, centrifuge at 400g for 10min, remove the supernatant, take 50W cells and inoculate them into the corresponding confocal dish, add appropriate amount of PBS, 30μg / mL of free αHER2 ScFv-SpyTag003 and αCD3ScFv-SpyTag003 in equal proportions, co-culture for 4H, then gently aspirate the supernatant, rinse gently with PBS three times, and finally characterize the binding of the two cells by confocal microscopy. Fig.18As shown, compared with the other two groups, the addition of NP SCHSA&αHER2 / CD3 Significantly increased the ratio of effector-target cell binding.

[0207] Example 8 HPLC Characterization of NPs SCHSA Can bind three different ScFv-SpyTag003 simultaneously

[0208] Three different α4-1BB ScFv-SpyTag003 (SEQ ID No: 7), αHER2 ScFv-SpyTag003 (SEQ ID No: 5), and αCD22 ScFv-SpyTag003 (SEQ ID No: 9) were stained with Cy5, Cy3, and FITC fluorescent molecules, respectively, and then NPs were SCHSA The three fluorescently labeled ScFv-SpyTag003 were incubated in PBS at a molar ratio of 6:1:1:1. After overnight incubation at 4°C, the particles and the peak time of the fluorescent molecules were detected by the ultraviolet and fluorescence channels of HPLC. The experimental results are shown in Fig.19 As shown, the ultraviolet peak of the nanoparticles and the peak times of three different fluorescent molecules are consistent, indicating that the nanoparticles can bind to three different ScFv-SpyTag003 and pass through the detector at the same time, indicating that the nanoparticles can simultaneously bind to multiple fusion proteins containing SpyTag003, providing guidance for the preparation of multi-specific antibodies.

[0209] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A fusion protein composition, characterized in that: The fusion protein composition includes a fusion protein A of SpyCatcher003-albumin, and a fusion protein B of at least one protein-SpyTag003; The fusion protein A is formed by the fusion of SpyCatcher003 protein and albumin; The fusion protein B is formed by fusion of a protein and SpyTag003, and the protein is selected from at least one single-chain antibody.

2. The fusion protein composition according to claim 1, characterized in that The albumin is any one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, and chicken ovalbumin.

3. The fusion protein composition according to claim 1, characterized in that The single-chain antibody is at least any one of the antibodies targeting the following antigens: antibodies targeting HER2, CD137L, CD137, 4-1BBL, 4-1BB, OX40L, OX40, ICOSL, ICOS, CD86, CD80, CD28, LFA3, CD2, CTLA4, PDL1, PD1, CD70, CD27, GAL9, TIM3, CD111, CD96, CD112, CD226, CD115, CD113, TIGIT, CD39, CD73, CD47, SIRPα, TNFα, IL1β, IL6, TGFβ, IL-10, IL-12 derived from humans or other species.

4. The fusion protein composition according to claim 1, characterized in that The fusion protein B is one, or two, or three or more; each different fusion protein B has a different single-chain antibody.

5. The fusion protein composition according to claim 1, characterized in that: In the fusion protein A, the albumin is linked to the N-terminus or C-terminus of SpyCatcher003 via a flexible connecting peptide; and / or in the fusion protein B, the protein is linked to the N-terminus or C-terminus of SpyTag003 via a flexible connecting peptide.

6. The fusion protein composition according to claim 5, characterized in that: The residue of the connecting peptide is [GGGGS]n, or (EAAAK)n, wherein n is any integer, preferably, n is an integer of 1-4.

7. A gene encoding the fusion protein composition according to any one of claims 1 to 6, or a plasmid vector containing a gene encoding the recombinant fusion protein composition according to any one of claims 1 to 6, wherein: The coding genes include a coding gene for fusion protein A and a coding gene for fusion protein B.

8. The coding gene according to claim 7, characterized in that The coding gene of the fusion protein A is shown in any one of SEQ ID No: 1 and SEQ ID No: 2; and / or The encoding gene of the fusion protein B is shown in any one of SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9 and SEQ ID No:

10.

9. An engineered bacterium, protoplast, or in vitro cloning system containing the coding gene or plasmid vector according to claim 7 or 8.

10. A mono / bi / multi-specific immunological drug, characterized in that: The immune drug is obtained by mixing a nanoassembly with at least one fusion protein B in the fusion protein composition described in any one of claims 1 to 6 and connecting them through covalent bonds. The nanoassembly is formed by the fusion protein A in the fusion protein composition described in any one of claims 1 to 6 and a hydrophobic degradable polyester or a derivative thereof through hydrophobic interaction.

11. The mono / bi / multi-specific immunological drug according to claim 10, characterized in that: The degradable polyester is an aliphatic polyester, or an aliphatic polyester modified with polyethylene glycol.

12. The mono / bi / multi-specific immunological drug according to claim 11, characterized in that: The aliphatic polyester includes, but is not limited to, at least one of polylactide, polyglycolide, poly(lactide-co-glycolide), and polycaprolactone.

13. The mono / bi / multi-specific immunological drug according to claim 12, characterized in that: The block molecular weight of the polylactide is 1 kDa to 1,100 kDa; or the block molecular weight of the poly(lactide-co-glycolide) is 1 kDa to 1,100 kDa, wherein the ratio of lactide to glycolide is in the range of 95 / 5 to 50 / 50.

14. The mono / bi / multi-specific immunological drug according to claim 10, characterized in that: The ratio of the amount of the fusion protein A to the amount of the fusion protein B is (3-1):

1. Most preferably, the ratio of the amount of the fusion protein A to the amount of the fusion protein B is 1:

1.

15. A method for preparing the mono / bi / multi-specific immunological drug according to claim 10, characterized in that: The following steps are involved: (1) mixing the fusion protein A with water or physiological saline at a concentration of 0.5 to 20 mg / mL to obtain an aqueous phase; mixing the hydrophobic degradable polyester and its derivatives with an organic solvent to obtain an oil phase at a concentration of 0.5 to 10 mg / mL; (2) preparing an oil-in-water emulsion from the aqueous solution and the oil solution of step (1); (3) separating and purifying the emulsion to obtain a nanoassembly; (4) Mixing the nanoassembly with at least one of the fusion proteins B at room temperature to obtain the drug.

16. The preparation method according to claim 15, characterized in that: The organic solvent is at least one of chloroform, dichloromethane and ethyl acetate.

17. The preparation method according to claim 15, characterized in that: The concentration of the recombinant fusion protein A in the aqueous phase is 1-10 mg / ml; and / or, the concentration of the hydrophobic degradable polyester and its derivatives in the oil phase is 1-10 mg / ml; and / or, the weight ratio of the hydrophobic degradable polyester and its derivatives to the fusion protein A is 1:1-10, preferably 1:5-10, and / or, The volume ratio of the water phase to the oil phase is 1-10:1, preferably 5-10:

1.

18. The preparation method according to claim 15, characterized in that: The molar ratio of the nanoassembly to the fusion protein B is (1-10):1, preferably the molar ratio of the nanoassembly to the fusion protein B is (1-6):1, and more preferably (2-3):

1.

19. Use of the immune drug according to any one of claims 10 to 14 in the preparation of products for tumor immunotherapy, autoimmune disease treatment, inflammation treatment, and infection treatment.

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