A bispecific T cell adapter and its application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a bispecific T cell adapter and its application. The present invention constructs an exosome scaffold protein, including a recognition sequence sorted into the exosome, wherein the recognition sequence is a chimeric late domain (cLD); the chimeric late domain is a composite motif including the late domain of Ebola virus, HIV virus and Rous sarcoma virus. The present invention establishes a new EVs surface antibody display platform; and using the established antibody display platform, scFv targeting tumor antigens and scFv targeting CD3 are co-displayed on the surface of EVs, and exosomes are transformed into bispecific T cell adapters, which can activate the killing potential of T cells, thereby achieving the purpose of targeted killing of tumor cells, while interacting with target cells and functional cells, thereby enhancing the killing effect of T cells on target cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bispecific T cell engager and its application. Background Art

[0002] Tumor immunotherapy has become the fourth largest tumor treatment after surgery / radiotherapy and chemotherapy. In recent years, immune checkpoint inhibitors (ICIs) have been widely used in tumor treatment, and antibody drugs targeting PD-1 / PD-L1 have significantly prolonged the survival of some tumor patients. However, it is worth noting that the overall response rate of immune checkpoint inhibitors is not high, and 70% to 80% of patients still experience disease progression after treatment. Therefore, the development of the next generation of immunotherapy drugs has become a hot topic in the field of tumor immunotherapy.

[0003] Bispecific antibodies (BsAb) are one of the representatives of the next generation of immunotherapy. BsAb refers to an antibody molecule that can specifically bind to two antigen epitopes at the same time and is constructed by gene recombination, chemical coupling or quadruple hybridoma. Among them, bispecific T-cell engager (BiTE) is a BsAb that targets T cells and tumor antigens at the same time. Binatumomab is the first bispecific antibody approved for marketing, used to treat relapsed or refractory B-cell acute lymphoblastic leukemia. Clinical data show that 42.9% of patients achieve complete remission after two treatments. Mechanistically, blinatumomab binds to the B cell surface antigen CD19 at one end and the T cell surface antigen CD3 at the other end, connecting malignant B cells with T cells. After CD3 is activated, T cells release cytotoxic cytokines, thereby specifically killing tumor cells.

[0004] Exosomes (EVs) are phospholipid bilayer membrane vesicles with a diameter of 30 to 120 nm secreted by cells. EVs can effectively transport biologically active substances such as RNA and proteins to target cells, thereby mediating information transmission between cells and participating in many processes of cell life activities and disease occurrence and development. Exosomes have excellent carrying capacity, low immunogenicity, good tissue compatibility and high stability. Therefore, exosomes are regarded as potential next-generation drug delivery carriers.

[0005] In view of the excellent pharmacological properties of exosomes, researchers have proposed that specific target proteins (such as antibodies) can be displayed on the surface of EVs membranes through genetic engineering, which can transform EVs into targeted and therapeutic drug carriers, such as those used to activate the body's anti-tumor immunity. Compared with antibodies alone, the advantages of this strategy are: increasing the tissue penetration of antibody drugs (such as the blood-brain barrier); increasing the number of antibody molecules per unit area to better activate or target target proteins; and using EVs to load drugs to achieve the effect of combined treatment. At present, the technical platforms used for EVs surface protein display include the Smart-Exos platform and the PTGFRN platform. For example, Shi et al. established the Smart-Exos platform, which connects the anti-EGFR (αEGFR) single-chain variable fragment (scFv) with the anti-CD3 single-chain antibody (αCD3 scFv) through a (G3S)4 linker and fuses it with the transmembrane domain of the PDGFR (Platelet-derived growth factor receptor) protein. The plasmid vector is then stably expressed in HEK293T cells to extract the derived αEGFR×αCD3 EVs. The results showed that αEGFR×αCD3 Smart-Exos can specifically connect T cells with EGFR + tumor cells and simultaneously activate T cells against EGFR + Killing of tumor cells. Except for the Smart-Exos platform and the PTGFRN platform, there are still few reports on technical platforms for displaying antibodies on the surface of EVs. Therefore, the construction and development of new platform technologies for displaying antibodies on the surface of EVs will provide new tools for EVs-based tumor immunotherapy. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a bispecific T cell engager and its application. The present invention establishes a new EVs surface antibody display platform; and uses the established antibody display platform to co-display scFv targeting tumor antigens and scFv targeting CD3 on the surface of EVs, transforming exosomes into bispecific T cell engagers (bispecific T-cell engager exosomes, BiTExos), activating T cell anti-tumor immunity, such as Figure 1 shown.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention constructs an exosome scaffold protein, which includes a recognition sequence sorted to exosomes, wherein the recognition sequence is a chimeric late domain (cLD); the chimeric late domain is a composite motif including the late domains of Ebola virus, HIV virus and Rous sarcoma virus.

[0009] The exosome scaffold protein of the present invention can be specifically sorted into EVs by extracting the key "information code" in the Late Domain and retaining the part that interacts with the endosomal sorting and transport complex family proteins (ESCRTs), thereby obtaining a specific exosome sorting motif, and then constructing an exosome sorting motif based on the late domain for antibody display, which can be used as a general platform technology for the display of target proteins on the surface of EVs.

[0010] As a preferred embodiment of the exosome scaffold protein of the present invention, the amino acid sequence of the recognition sequence is shown in SEQ ID NO: 1.

[0011] As a preferred embodiment of the exosome scaffold protein of the present invention, its amino acid sequence is shown in SEQ ID NO: 2.

[0012] In a second aspect, the present invention constructs a LEAP scaffold protein (referred to as LEAP scaffold protein, L ate domain-based e xosomal a ntibody surface display p latform), which includes CD8 protein signal peptide, myc tag protein, hinge region and transmembrane domain of CD8 protein, endoplasmic reticulum shuttle sequence, the chimeric late domain (including the late domain of Ebola virus, HIV virus and Rous sarcoma virus), endoplasmic reticulum export sequence and Foldon motif.

[0013] The single-fragment antibody scFv is inserted between the myc tag protein and the hinge region and transmembrane domain of the CD8 protein; the fluorescent protein is inserted between the hinge region and transmembrane domain of the D8 protein and the endoplasmic reticulum shuttle sequence.

[0014] The specific sequence is: MALPVTALLLPLALLLHAARPEQKLISEEDL-(single-fragment antibody scFv insertion position)-TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG TCGVLLLSLVITLYCLEGKSRITSEGEYIPLDQIDINVGTPGGS-(fluorescent protein insertion position)-PTAPPPEYPTAPPPEYGPLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYP LASLRSLFGSDPSSQTASAPPPPYVGTASAPPPPYVGTSFCYENEVGSFCYENEVTRGGGGSGPRGYIPEAPRDGQCYVRCDGEWVLLSTFLG.

[0015] In a third aspect, the present invention establishes an EVs surface antibody display platform, including the exosome scaffold protein or the LEAP scaffold protein.

[0016] In a fourth aspect, the present invention establishes a bispecific T cell engager, wherein the bispecific T cell engager is an exosome in which a scFv targeting a tumor antigen and a scFv targeting CD3 are displayed together on the surface of a vesicle; the scFv targeting CD3 is fused to the exosome scaffold protein or the LEAP scaffold protein.

[0017] The bispecific T cell engager of the present invention can engage T cells and tumor cells, and can "take a two-pronged approach" to activate the killing potential of T cells, thereby achieving the purpose of targeted killing of tumor cells, while interacting with target cells and functional cells, thereby enhancing the killing effect on target cells.

[0018] In a fifth aspect, the present invention applies the exosome scaffold protein and the LEAP scaffold protein to the display of target proteins on the surface of EVs.

[0019] In a sixth aspect, the present invention applies the EVs surface antibody display platform to the display of target proteins on the EVs surface.

[0020] In a seventh aspect, the present invention uses the bispecific T cell engager in the preparation of anti-tumor immune drugs or preparations.

[0021] As a preferred embodiment of the application described in the present invention, the anti-tumor immune drug or preparation is an anti-tumor immune drug or preparation that specifically activates T cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] A new EVs surface antibody display platform has been established. The exosome scaffold protein of the present invention can be specifically sorted into EVs. By extracting the key "information code" in the Late Domain and retaining the part that interacts with the endosomal sorting and transport complex family proteins (ESCRTs), a specific exosome sorting motif is obtained, and then an exosome sorting motif based on the late domain for antibody display is constructed, which can be used as a general platform technology for the display of target proteins on the surface of EVs.

[0024] The present invention utilizes the established antibody display platform to co-display scFv targeting tumor antigens and scFv targeting CD3 on the surface of EVs, transforming exosomes into bispecific T cell adapters, which can specifically activate the anti-tumor immunity of T cells. The bispecific T cell adapter of the present invention can connect T cells and tumor cells, and can "take a two-pronged approach" to activate the killing potential of T cells, thereby achieving the purpose of targeted killing of tumor cells, while interacting with target cells and functional cells, thereby enhancing the killing effect on target cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the process of the present invention; in the figure, the single fragment antibodies of αPD-L1 and αCD3 are displayed on the surface of the exosome membrane through the LEAP scaffold protein, so that the modified engineered exosomes bridge PD-L1 + Tumor cells and T cells, and activate T cell anti-tumor immunity.

[0026] Figure 2 In ab, a is a schematic diagram of the plasmid vector construction design; in 2b, d, and f, Western Blot results show that with the continuous superposition of Late domain and Foldon motif, the interaction between the fusion protein and TSG101, ALIX, and WWP2 becomes closer. Figure 2 The cLD-containing fusion proteins in c, e, and f recruit TSG101, ALIX, and WWP2 to migrate to the plasma membrane. Figure 2 hWesternBlot results showed that with the continuous superposition of Late domain and Foldon motif (FD), the signal of mCh in EVs continued to increase; Figure 2 i, j show that Lyn-mCh-cLD co-localizes with CD63 on multivesicular bodies and plasma membranes. + EVs are secreted extracellularly.

[0027] Figure 3a and b show the optimization process of LEAP scaffold protein. As shown in the figure, after the endoplasmic reticulum shuttle sequence (vector G) and the endoplasmic reticulum export sequence (vector H) were fused to vector F, its plasma membrane targeting was significantly increased, and the co-localization with CD9 was significantly increased. The design of vector H was selected as the preferred design and named LEAP scaffold protein. The figure shows the fusion of a single fragment antibody (scFv) of αPD-L1 with the LEAP scaffold protein, and the fusion protein is called αPD-L1-LEAP. Figure 3 ce shows the difference between αPD-L1-LEAP and cLD-removed αPD-L1-LEAP ΔcLD ), the interaction between αPD-L1-LEAP and TSG101, ALIX, and WWP2 was closer, and αPD-L1-LEAP was more likely to be sorted into EVs ( Figure 3 f).

[0028] Figure 4 a is a schematic diagram of the LEAP scaffold protein. The scFvs of αPD-L1 and αCD3 were fused to the LEAP scaffold protein and inserted between the myc and CD8 transmembrane domains to form αPD-L1-LEAP or αCD3-LEAP. αPD-L1-LEAP was expressed in HEK293T cells, and the derived αPD-L1 EVs were collected and co-incubated with target cells. Figure 4 c, d show that αPD-L1 EVs target and bind to PD-L1 + on target cells, but not on CD19 + target cells binding. Figure 4 e and 4f show that after αCD3 EVs were co-incubated with Jurkat T cells, their calcium influx increased and the expression of the activation marker CD6 increased.

[0029] Figure 5 a shows that αPD-L1-LEAP and αCD3-LEAP were co-expressed in HEK293T cells, and the derived EVs were extracted and named αPD-L1×αCD3 Bispecific Exosomes (BiTExos). T cells and CAL-27 cells with high expression of PD-L1 were co-incubated with PBS, αPD-L1 EVs, αCD3 EVs or αPD-L1×αCD3 BiTExos, respectively. Figure 5 The results of bd showed that in the αPD-L1×αCD3 BiTExos treatment group, T cell activation indicators CD25, CD69, degranulation marker CD107a and cytokine levels increased significantly, and T cell proliferation and division increased significantly ( Figure 5 e).

[0030] Figure 6a shows that T cells (red fluorescent marker) were co-cultured with MDA-MB-468 cells with low PD-L1 expression, or CAL-27 and MDA-MB-231 cells with high PD-L1 expression, and PBS, αPD-L1EVs, αCD3 EVs or αPD-L1×αCD3BiTExos were added respectively. It can be seen that αPD-L1×αCD3 BiTExos effectively binds T cells to PD-L1 + The tumor cells are cross-linked together. Figure 6 be shows that T cells were co-cultured with a variety of different tumor cells, and then PBS, αPD-L1 EVs, αCD3 EVs or αPD-L1×αCD3 BiTExos were added respectively. It can be seen that the αPD-L1×αCD3 BiTExos treatment group can significantly activate T cells to kill tumor cells.

[0031] Figure 7 a Schematic diagram showing the construction of αEGFR-LEAP and αCD19-LEAP. Figure 7 b shows that αEGFR-LEAP and αCD19-LEAP colocalize with mCh-CD63 in HEK293T producer cells. Figure 7 cd shows that αEGFR EVs target and bind to EGFR + target cells without CD19 + cells combined. Figure 7 ef shows that αCD19 EVs target and bind to CD19 + target cells without CD19 - cells combined. DETAILED DESCRIPTION

[0032] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0034] Example 1: Construction of a novel exosome sorting motif based on Late Domain

[0035] After the virus infects the cell, the viral protein can "hostage" the endosomal sorting complexes required for transport (ESCRTs) in the cell through its late domain to help it bud and release. The late domain is a short and conserved sequence in a variety of retroviral proteins. The virus uses its late domain to interact with the ESCRTs family proteins of the host cell to help it bud and release from the infected cell. For example, the PTAP motif in the late domain of the HIV virus can interact with the UEV domain of the TSG101 protein, and the LYP(x)nL motif can interact with the V domain of the ALIX protein. In the budding stage, the HIV virus recruits TSG101 and ALIX to the budding site through the PTAP and LYP(x)nL motifs. TSG101 and ALIX promote the outward convex deformation and shearing of the plasma membrane, helping the HIV virus bud and release. In the genesis of exosomes, the ESCRTs family is widely involved in the sorting, transport and secretion of exosome-related proteins.

[0036] The present invention is constructed as Figure 2 The five vectors shown in a first fuse the membrane targeting peptide "MGCIKSKRKDK" derived from the tyrosine protein kinase Lyn with mCherry (mCh), and then fuse the Late Domains from different viruses to the C-terminus of Lyn-mCh in sequence, namely: 1) p6 domain from HIV virus (LQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ); 2) p2b domain of Rous sarcoma virus (PPPPYVGTASAPPPPYVG); 3) Late Domain of Ebola virus (PTAPPPEYPTAPPPEY), and this composite motif is called chimeric late domain (cLD). The Foldon motif (GYIPEAPRDGQCYVRCDGEWVLLSTFLG) is further fused to promote the trimerization of the fusion protein, and the cLD and Foldon motif are connected by a connecting peptide (TRGGGGSGPR). The above plasmids were transfected into HEK293T cells. Figure 2 The Western Blot results in b, d, and f show that with the continuous superposition of the Late domain and Foldon motif, the interaction between the fusion protein and TSG101, ALIX, and WWP2 becomes closer. Figure 2The cLD-containing fusion protein in c, e, and f recruits TSG101, ALIX, and WWP2 to migrate to the plasma membrane. EVs from different transfection groups were extracted for Western Blot detection. The results showed that with the continuous superposition of Late Domain and Foldon motifs, the mCh signal in EVs continued to increase ( Figure 2 h). Lyn-mCh-cLD was co-transfected with pHluorin_M153R-CD63 and EGFP-CD63 into HEK293T cells. Confocal fluorescence imaging results showed that Lyn-mCh-LD and EGFP-CD63 co-localized in multivesicular bodies and on the plasma membrane ( Figure 2 i) At the same time, CD63 secreted into the extracellular space was labeled with pHluorin_M153R. + Bright mCh signals can also be seen in EVs ( Figure 2 j) The above results show that the cLD motif can be used as an "identification code" for protein sorting into exosomes. Proteins containing the cLD motif can be sorted into EVs and secreted into the extracellular space with EVs.

[0037] The amino acid sequence of the cLD motif (SEQ ID NO: 1) is as follows:

[0038] PTAPPPEYPTAPPPEYGPLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKEL YPLASLRSLFGSDPSSQTASAPPPPYVGTASAPPPPYVG.

[0039] The amino acid sequence of the cLD motif fused to the Foldon motif (SEQ ID NO: 2) is as follows:

[0040] PTAPPPEYPTAPPPEYGPLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKEL YPLASLRSLFGSDPSSQTASAPPPPYVGTASAPPPPYVGTRGGGGSGPRGYIPE APRDGQCYVRCDGEWVLLSTFLG.

[0041] Example 2: scFv fused with LEAP can be displayed on the surface of exosome membrane

[0042] Proteins containing cLD motifs can be sorted into EVs. In order to verify whether it is possible to establish a synthetic engineered exosome scaffold protein based on the cLD motif, so that the single chain variable fragment (scFv) fused to it can be displayed on the surface of the exosome membrane. Figure 3 The plasmid vector shown in a replaces the signal activation domain (CD3ζ) of the first-generation chimeric antigen receptor (CAR) with a cLD motif and a Foldon motif, and adds an endoplasmic reticulum shuttle sequence and an endoplasmic reticulum export sequence, respectively, to promote its targeted binding to the plasma membrane.

[0043] The design of the vector is: CD8 protein signal peptide (MALPVTALLLPLALLLHAARP) - Myc tag (EQKLISEEDL) - (single fragment antibody insertion position) - CD8 hinge region and transmembrane domain (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLA GTCGVLLLSLVITLYC) - (fluorescent protein insertion position) - endoplasmic reticulum shuttle sequence (KSRITSEGEYIPLDQIDINV), late domains of Ebola virus, HIV virus and Rous sarcoma virus (PTAPPPEYPTAPPPEYGPLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELY PLASLRSLFGSDPSSQTASAPPPPYVGTASAPPPPYVG), the endoplasmic reticulum export sequence (FCYENEVGSFCYENEV) and the Foldon motif (GYIPEAPRDGQCYVRCDGEWVLLSTFLG), and this structure is called LEAP scaffold protein ( L ate domain-based e xosomal a ntibody surface display p latform).

[0044] The amino acid sequence of the LEAP scaffold protein is as follows:

[0045] MALPVTALLLPLALLLHAARPEQKLISEEDL-(single fragment antibody insertion position)-TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG TCGVLLLSLVITLYCLEGKSRITSEGEYIPLDQIDINVGTPGGS-(fluorescent protein insertion position)-PTAPPPEYPTAPPPEYGPLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYP LASLRSLFGSDPSSQTASAPPPPYVGTASAPPPPYVGTSFCYENEVGSFCYENE VTRGGGGSGPRGYIPEAPRDGQCYVRCDGEWVLLSTFLG.

[0046] αPD-L1-LEAP can interact with ALIX, TSG101 and WWP2 proteins ( Figure 3 ce) and were efficiently sorted into EVs ( Figure 3 f). After fusion with LEAP scaffold protein, αPDL1 scFv can be sorted to the surface of EVs. Figure 4 c, d show that αPD-L1 EVs target and bind to PD-L1 + target cells, but not CD19+ target cells. Figure 4 e and 4f show that after fusion with LEAP scaffold protein, αCD3 scFv can be sorted to the surface of EVs. After αCD3 EVs are co-incubated with Jurkat T cells, their calcium ion influx increases and the expression of activation marker CD6 increases.

[0047] Example 3: αPDL1×αCD3 BiTExos activate T cell anti-tumor immunity

[0048] The activation indexes and cytokine levels were detected to further prove the activation effect of αPDL1×αCD3BiTExos on T cell anti-tumor immunity. Cal27 was co-incubated with PBMCs, and PBS, αPD-L1 EVs, αCD3EVs or αPD-L1×αCD3 BiTExos were added respectively. The flow cytometry results showed that the T cell activation indexes CD25, CD69, degranulation marker CD107a and cytokine significantly increased in the αPD-L1×αCD3 BiTExos treatment group, and T cell proliferation and division significantly increased ( Figure 5 e)

[0049] . Figure 6a shows that T cells (red fluorescent marker) were co-cultured with MDA-MB-468 cells with low PD-L1 expression, or CAL-27 and MDA-MB-231 cells with high PD-L1 expression, and PBS, αPD-L1EVs, αCD3 EVs or αPD-L1×αCD3BiTExos were added respectively. It can be seen that αPD-L1×αCD3 BiTExos effectively binds T cells to PD-L1 + The tumor cells are cross-linked together. Figure 6 be shows that T cells were co-cultured with a variety of different tumor cells, and then PBS, αPD-L1 EVs, αCD3 EVs or αPD-L1×αCD3 BiTExos were added. It can be seen that the αPD-L1×αCD3 BiTExos treatment group can significantly activate T cells to kill tumor cells. The above results further illustrate that αPD-L1×αCD3 BiTExos can activate T cell anti-tumor immunity.

[0050] Example 4: LEAP scaffold protein can display αEGFR and αCD19 on the surface of EVs

[0051] Confocal and flow cytometry detection revealed that both αEGFR EVs and αCD19 EVs targeted and bound to target cells expressing the corresponding antigens without off-target binding, further demonstrating that the LEAP scaffold protein is widely compatible with a variety of single-fragment antibodies and can be displayed on the surface of EVs ( Figure 7 ).

[0052] The present invention establishes a new EVs surface antibody display platform; and uses the established antibody display platform to co-display scFv targeting tumor antigens and scFv targeting CD3 on the surface of EVs, transforming exosomes into bispecific T cell engagers that can specifically activate T cells' anti-tumor immunity.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An exosome scaffold protein, characterized in that The exosome scaffold protein includes a recognition sequence sorted to exosomes, wherein the recognition sequence is a chimeric late domain; the chimeric late domain is a composite motif including the late domains of Ebola virus, HIV virus and Rous sarcoma virus.

2. The exosome scaffold protein according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

3. The exosome scaffold protein according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

2.

4. A LEAP scaffold protein, characterized in that It includes CD8 protein signal peptide, myc tag protein, hinge region and transmembrane domain of CD8 protein, endoplasmic reticulum shuttle sequence, chimeric late domain according to claim 1 or 2, endoplasmic reticulum export sequence and Foldon motif in sequence.

5. An EVs surface antibody display platform, characterized in that: It comprises the exosome scaffold protein according to any one of claims 1 to 3 or the LEAP scaffold protein according to claim 4.

6. A bispecific T cell engager, characterized in that: The bispecific T cell engager is an exosome that is co-displayed on the surface of the vesicle by a single fragment antibody targeting a tumor antigen and a scFv targeting CD3; the scFv targeting CD3 is fused to the exosome scaffold protein described in any one of claims 1 to 3 or the LEAP scaffold protein described in claim 4.

7. Use of the exosome scaffold protein according to any one of claims 1 to 3 and the LEAP scaffold protein according to claim 4 in the display of target proteins on the surface of EVs.

8. Use of the EVs surface antibody display platform described in claim 5 in the display of target proteins on the surface of EVs.

9. Use of the bispecific T cell engager according to claim 6 in the preparation of anti-tumor immune drugs or preparations.

10. The use according to claim 9, characterized in that: The anti-tumor immune drug or preparation is an anti-tumor immune drug or preparation that specifically activates T cells.

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