Bispecific antibody modified vesicles, bispecific antibodies, nucleic acids, recombinant vectors, genetically engineered cells, methods and applications

Abstract

The present invention relates to a bispecific antibody modified vesicle, which comprises a vesicle and a bispecific antibody bound to the surface of the vesicle; wherein the bispecific antibody is bound to a transmembrane domain on the surface of the vesicle; the bispecific antibody comprises a first antigen binding domain and a second antigen binding domain; the first antigen binding domain is a CD19 antigen binding domain or a HER2 antigen binding domain, and the second antigen binding domain is a CD3 antigen binding domain. The bispecific antibody modified vesicle can significantly prolong the half-life of the bispecific antibody drug and increase patient compliance. It also relates to a bispecific antibody, a nucleic acid, a recombinant vector, a genetically engineered cell, a method for preparing the genetically engineered cell, a method for preparing the bispecific antibody modified vesicle, an application, a pharmaceutical composition, and a method for testing the binding density of the bispecific antibody in the bispecific antibody modified vesicle.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and further to the field of antibody technology, and in particular to a bispecific antibody-modified vesicle, a bispecific antibody, a nucleic acid, a recombinant vector, a genetically engineered cell, a method for preparing a genetically engineered cell, a method for preparing a bispecific antibody-modified vesicle, an application, a pharmaceutical composition, and a method for testing the binding density of a bispecific antibody in a bispecific antibody-modified vesicle. Background Art

[0002] Bispecific antibodies (BsAb) are a class of biological agents with great prospects in the treatment of cancer. BsAb is composed of two antibody single-chain variable fragments connected by a peptide chain, which can recognize target cells and effector cells respectively, and can act as a bridge between the two. For example, Bispecific T cell engager (BiTE) is a type of bispecific antibody. Its effector cell recognition end can effectively activate T cells by binding to CD3e on the surface of T cells, and mediate the killing effect of T cells on tumor cells that is independent of the MHC complex. For example, Blinatumomab is a BiTE that combines CD3 on T cells and CD19 on target cells. It is the first bispecific immunotherapy drug approved by the FDA. It has been shown to be effective for CD19+ B-cell precursor ALL (acute lymphoblastic leukemia) and is currently used for the second-line treatment of patients with Philadelphia chromosome-negative relapsed or refractory ALL. Compared with chimeric antigen receptor T cell (CAR-T) therapy, the aforementioned BsAb immunotherapy can guide T cells to tumor cells, and bispecific immunotherapy drugs can be produced as finished drugs, which is easy to control the dosage and reduces the patient's personal medication costs. However, BiTEs and other biologics based on bispecific antibodies are protein preparations with short half-lives. They are inevitably lost during use. In clinical practice, they require continuous administration by injection pumps, which is not convenient, and the patient's medication process is long and has poor compliance. Based on this, new bispecific antibody drugs are urgently needed to be developed. Summary of the invention

[0003] Based on this, the purpose of the present invention includes providing a bispecific antibody modified vesicle, which can significantly prolong the half-life of the bispecific antibody drug, reduce the pressure of the injection pump required for continuous administration in the clinic, and increase patient compliance. The present invention also provides bispecific antibodies, nucleic acids, recombinant vectors, genetically engineered cells, methods for preparing genetically engineered cells, methods for preparing bispecific antibody modified vesicles, applications (such as applications in the preparation of drugs), pharmaceutical compositions, and methods for testing the binding density of bispecific antibodies in bispecific antibody modified vesicles.

[0004] In a first aspect of the present invention, a bispecific antibody (BsAb)-modified vesicle is provided, comprising a vesicle and a bispecific antibody (BsAb) bound to the surface of the vesicle;

[0005] Wherein, the bispecific antibody is bound to the membrane structure on the surface of the vesicle; the bispecific antibody comprises a first antigen binding domain and a second antigen binding domain; the first antigen binding domain is a CD19 antigen binding domain or a HER2 antigen binding domain, and the second antigen binding domain is a CD3 antigen binding domain.

[0006] In some embodiments, the binding density of the bispecific antibody on the surface of the vesicle is that the average number of AsAbs bound per square micrometer of the vesicle surface is greater than 100; and / or the diameter of the bispecific antibody-modified vesicle is 80 to 1000 nm, preferably with an average diameter of 100 to 200 nm.

[0007] In some embodiments, any of the bispecific antibodies is independently a BiTE antibody.

[0008] In some embodiments, any one of the bispecific antibodies independently comprises the first antigen binding domain, a connecting peptide, the second antigen binding domain, a hinge region and a transmembrane domain connected in series; wherein the transmembrane domain is embedded in the phospholipid bilayer on the surface of the vesicle and bound.

[0009] In some embodiments, the bispecific antibody-modified vesicle satisfies any one, any multiple partial or all of the following features:

[0010] (a1) the first antigen-binding domain comprises an anti-CD19 single-chain antibody variable region whose amino acid sequence is as shown in SEQ ID No.: 1, or comprises an anti-HER2 single-chain antibody variable region whose amino acid sequence is as shown in SEQ ID No.: 2;

[0011] (a2) the second antigen-binding domain comprises an anti-CD3 single-chain antibody variable region with an amino acid sequence as shown in SEQ ID No.: 3;

[0012] (a3) the amino acid sequence of the connecting peptide is shown in SEQ ID No.: 4;

[0013] (a4) the amino acid sequence of the hinge region is shown in SEQ ID No.: 5; and

[0014] (a5) The amino acid sequence of the transmembrane domain is shown in SEQ ID No.:6.

[0015] In some embodiments, the vesicles are derived from one or more of mammalian cells and insect cells.

[0016] In some embodiments, the vesicles are derived from one or more of HEK-293T cells, Nalm6 cells, and RAW264.7 cells.

[0017] In a second aspect of the present invention, a bispecific antibody is provided, which is as defined in the first aspect of the present invention.

[0018] In a third aspect of the present invention, a nucleic acid is provided, wherein the nucleotide sequence of the nucleic acid comprises a nucleotide sequence in at least one of the following groups:

[0019] (i) a nucleotide sequence encoding the bispecific antibody according to the second aspect of the present invention; and

[0020] (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i);

[0021] Preferably, the nucleic acid is DNA, mRNA or a combination thereof.

[0022] In some embodiments, the upstream of the coding sequence of the bispecific antibody also includes a nucleotide sequence encoding a signal peptide. Preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID No.:7.

[0023] In the fourth aspect of the present invention, a recombinant vector is provided, wherein the recombinant vector contains one or more of the coding sequence of the bispecific antibody according to the second aspect of the present invention and the nucleotide sequence of the nucleic acid according to the third aspect of the present invention.

[0024] In some embodiments, the recombinant vector is one or more of a lentiviral vector, a retroviral vector, a transient transfection vector, and an adenovirus vector.

[0025] In a fifth aspect of the present invention, a genetically engineered cell is provided, wherein the genetically engineered cell is capable of endogenously secreting vesicles, and the genome of the genetically engineered cell is integrated with one or more of the coding sequence of the bispecific antibody of the second aspect of the present invention and the nucleotide sequence of the nucleic acid of the third aspect of the present invention, or the genetically engineered cell contains a recombinant vector, and the recombinant vector contains one or more of the coding sequence of the bispecific antibody of the second aspect of the present invention and the nucleotide sequence of the nucleic acid of the third aspect of the present invention;

[0026] Wherein, the surface of the vesicle has a phospholipid bilayer.

[0027] In a sixth aspect of the present invention, a method for preparing a genetically engineered cell is provided, comprising the following steps:

[0028] Providing a mother cell capable of endogenously secreting vesicles and providing a recombinant vector; wherein the surface of the vesicle has a phospholipid bilayer; and the recombinant vector contains one or more of the coding sequence of the bispecific antibody described in the second aspect of the present invention and the nucleotide sequence of the nucleic acid described in the third aspect of the present invention;

[0029] The recombinant vector is transformed or transfected into the mother cell.

[0030] In some embodiments, the concentration of the mother cells is 1×10 5 / mL~1×10 6 / mL, and the infection index MOI is 1-10.

[0031] In some embodiments, the mother cell is selected from one or more of a mammalian cell and an insect cell;

[0032] Wherein, the mammalian cells include one or more of HEK-293T cells, Nalm6 cells and RAW264.7 cells.

[0033] In a seventh aspect of the present invention, a method for preparing a bispecific antibody-modified vesicle is provided, comprising the following steps:

[0034] Provided is a genetically engineered cell according to the fifth aspect of the present invention or a genetically engineered cell prepared by the preparation method according to the sixth aspect of the present invention; wherein the genetically engineered cell comprises a first coding sequence of the bispecific antibody defined in the first aspect of the present invention, and further comprises a second coding sequence located upstream of the first coding sequence, wherein the second coding sequence encodes a signal peptide;

[0035] The genetically engineered cells are cultured to obtain the bispecific antibody-modified vesicles.

[0036] In some embodiments, the preparation method has one or more of the following characteristics:

[0037] (t1) the step of culturing the genetically engineered cells comprises the following process: the genetically engineered cells secrete vesicles under the exogenous stimulation condition, synthesize bispecific antibodies with a signal peptide at the C-terminus, the C-terminus of the bispecific antibody is embedded into the phospholipid bilayer on the surface of the genetically engineered cells under the mediation of the signal peptide, the genetically engineered cells secrete vesicles in a budding manner, and the signal peptide at the C-terminus of the bispecific antibody is removed;

[0038] (t2) The genetically engineered cells are cultured under ultraviolet light, the wavelength of the ultraviolet light is 280 to 315 nm, and the irradiation intensity is 300 to 400 J / m 2 ;and,

[0039] (t3) The amino acid sequence of the signal peptide is shown in SEQ ID No.:7.

[0040] In the eighth aspect of the present invention, provided is the use of the bispecific antibody-modified vesicles described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the genetically engineered cells described in the fifth aspect of the present invention, the genetically engineered cells prepared by the preparation method described in the sixth aspect of the present invention, or the bispecific antibody-modified vesicles prepared by the preparation method described in the seventh aspect of the present invention in the preparation of drugs for preventing and / or treating tumors or cancer.

[0041] In the ninth aspect of the present invention, a pharmaceutical composition is provided, comprising at least one of the bispecific antibody-modified vesicles described in the first aspect of the present invention and the bispecific antibody-modified vesicles prepared by the preparation method described in the seventh aspect of the present invention; the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0042] In the tenth aspect of the present invention, a method for testing the binding density of the bispecific antibody in the bispecific antibody-modified vesicle according to the first aspect of the present invention is provided, characterized in that it comprises the following steps:

[0043] Obtain a standard curve of fluorescence value-concentration of the fluorescent marker;

[0044] Preparing a mixed solution A containing the bispecific antibody-modified vesicles, wherein each bispecific antibody unit carries one of the fluorescent markers;

[0045] The concentration C of the bispecific antibody unit in the mixed solution A is calculated using the standard curve of the fluorescence value-concentration of the fluorescent marker. BsAb ; and using nanoparticle tracking analysis method to determine the concentration C of the bispecific antibody modified vesicles in the mixed solution A BsAb-EV ;

[0046] According to the C BsAb and the C BsAb-EV The average number of bispecific antibody units N bound to a bispecific antibody modified vesicle is calculated. BsAb@EV .

[0047] In some embodiments, the method for testing the binding density of the bispecific antibody in the bispecific antibody-modified vesicles further comprises the following steps: using a nanoparticle tracking analysis method to determine the average particle size D of the bispecific antibody-modified vesicles. BsAb-EV , combined with the N BsAb@EVThe number of bispecific antibody units bound per unit surface area of ​​the bispecific antibody modified vesicles was calculated as follows: BsAb@EV .

[0048] The bispecific antibody-modified vesicles provided in the first aspect of the present invention and the bispecific antibody-modified vesicles prepared in the seventh aspect of the present invention can significantly prolong the half-life of the bispecific antibody drug, reduce the pressure of continuous administration of the injection pump required clinically, increase patient compliance, and further reduce the economic burden on patients.

[0049] The bispecific antibody-modified vesicles provided in the first aspect of the present invention and the bispecific antibody-modified vesicles prepared in the seventh aspect of the present invention can also encapsulate other drug ingredients, thereby effectively overcoming the bottleneck of BiTEs and other bispecific antibody-based biological preparations that only mobilize one type of immune cells, and can provide effective drug preparations (drug compositions) or medicines for the treatment of tumors that are currently difficult to completely treat with single therapies, especially solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0051] Figure 1 Schematic diagram of the structure of a bispecific antibody (BsAb) modified vesicle (BsAb-EV) in one embodiment of the present invention;

[0052] Figure 2 Schematic diagram of the structure of two bispecific antibody-modified vesicles according to one embodiment of the present invention, wherein (A) corresponds to CD19×CD3 BiTE, and (B) corresponds to HER2×CD3 BiTE;

[0053] Figure 3 This is a schematic diagram of the structure of the plasmid used in constructing the CD19×CD3 BiTE lentiviral packaging vector in one embodiment of the present invention;

[0054] Figure 4 This is a schematic diagram of the structure of the plasmid used to construct the HER2×CD3 BiTE lentiviral packaging vector in one embodiment of the present invention;

[0055] Figure 5 This is a result diagram of flow cytometry sorting of bispecific antibody-modified vesicles in one embodiment of the present invention, wherein a lentivirus was used to infect a HEK-293T cell line;

[0056] Figure 6 Images (A, C, E) and particle distribution curves (B, D, F) of nanoparticle tracking analysis (NTA) for structural identification of vesicles in one embodiment of the present invention; the abscissa is the particle diameter (nm), and the ordinate is the particle concentration (particles / mL, i.e., the number of particles per milliliter); wherein A and B correspond to wt EV (unmodified bispecific antibody), C and D correspond to CD19 BiTE-EV, and E and F correspond to HER2 BiTE-EV;

[0057] Figure 7 Transmission electron microscopy (TEM) images of bispecific antibody-modified vesicles prepared using HEK-293T cell lines in one embodiment of the present invention, (A) corresponds to wild-type vesicles (wt-EV) not bound to bispecific antibodies, (B) corresponds to CD19 BiTE-EV, and (C) corresponds to HER2 BiTE-EV; wherein the scale bars shown in (A), (B) and (C) are all 100 nm;

[0058] Figure 8 The results of in vitro T cell activation test of bispecific antibody-modified vesicles prepared using HEK-293T cell line in one embodiment of the present invention;

[0059] Fig. 9 The specific killing effect test results of the bispecific antibody-modified vesicle CD19 BiTE-EV in one embodiment of the present invention, using Nalm6 cells as target cells;

[0060] Fig.10 The specific killing effect test results of the bispecific antibody modified vesicle CD19 BiTE-EV in one embodiment of the present invention, using Raji cells as target cells; the ratio of effector cells to target cells E:T in (A) is 5:1, and the E:T in (B) is 1:1;

[0061] Fig.11 This is a graph showing the specific killing test results of the bispecific antibody-modified vesicle HER2 BiTE-EV on target cells in one embodiment of the present invention, using BT474 cells and MDA-MB-231 cells as target cells respectively;

[0062] Fig.12 This is a tumor cell imaging test result of the in vivo targeting effect of the bispecific antibody-modified vesicles in one embodiment of the present invention;

[0063] Fig.13 This is a flow cytometry test result diagram of the uptake of bispecific antibody-modified vesicles by Nalm6 cells in one embodiment of the present invention;

[0064] Fig.14This is a quantitative analysis diagram of the uptake of bispecific antibody-modified vesicles by Nalm6 cells in one embodiment of the present invention, wherein the abscissa represents the co-incubation time (time after coculture) and the ordinate represents the standardized mean fluorescence intensity;

[0065] Fig.15 A comparison diagram of the results of preparing bispecific antibody-modified vesicles with and without ultraviolet irradiation in one embodiment of the present invention;

[0066] Fig.16 Nanoparticle tracking analysis (NTA) diagram of bispecific antibody-modified vesicles prepared by Nalm6 cell line (A) and RAW264.7 cell line (B) in one embodiment of the present invention; the abscissa is particle diameter (nm), and the ordinate is particle concentration (particles / mL);

[0067] Fig.17 Transmission electron microscopy (TEM) images ((A) and (B)) and microscope photographs ((C) and (D)) of bispecific antibody-modified vesicles prepared using Nalm6 cell line and RAW264.7 cell line in one embodiment of the present invention, after ultraviolet irradiation for 1 hour; wherein, the scale bars shown in (A) and (B) are both 250 nm; the scale bars shown in (A) and (B) are both 100 μm;

[0068] Fig.18 is the fluorescence intensity-concentration standard curve of green fluorescent protein (GFP);

[0069] Fig.19 This is a graph showing the specific killing effect test results of the bispecific antibody-modified vesicles CD19 BiTE-EV prepared from the Nalm6 cell line and the RAW264.7 cell line in one embodiment of the present invention, using the Raji cell line; the ordinate is the specific apoptosis rate (% specific lysis). DETAILED DESCRIPTION

[0070] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present invention. In addition, in the following description, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0071] Unless otherwise defined, all technical and scientific terms used herein 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 herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0072] the term

[0073] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0074] The terms "and / or", "or / and", and "and / or" used in this article have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and the combination of A and B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0075] In the present application, when at least three features are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it is equivalent to the expression "having one or more features", for example, "TA, and / or, TB, and / or, TC, and / or, TD" is equivalent to "having one or more of the following features: TA, TB, TC and TD".

[0076] In the present invention, "plurality", "multiple" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0077] As used herein, "combinations thereof" and the like include all suitable combinations of any two or more of the listed items.

[0078] Herein, the “suitable” mentioned in “suitable combination”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0079] Herein, "preferred" and "better" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. If multiple "preferred" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "preferred" item is independent.

[0080] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0081] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0082] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0083] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0084] In the present invention, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows for broadly including quantitative intervals such as percentage intervals, ratio intervals, and ratio intervals.

[0085] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0086] In the present invention, the term "room temperature" generally refers to 4°C to 35°C, preferably 20°C ± 5°C. In some embodiments of the present invention, room temperature refers to 20°C to 30°C.

[0087] In the present invention, when it comes to the unit of the data range, if there is a unit only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 3-5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0088] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0089] In the present invention, the term "antibody" is a protein that binds to a specific antigen, and it generally refers to all proteins and protein fragments containing a complementarity-determining region (CDR), especially full-length antibodies or antibody functional fragments. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies, and the term "antibody functional fragment" is a substance that contains part or all of the antibody CDR, which lacks at least one amino acid present in the full-length chain but is still able to specifically bind to the antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including complete antibodies) for binding to a given epitope. In some embodiments, antibodies or antibody functional fragments have the function of specifically recognizing and binding to tumor cell surface antigens, and this binding is usually specific. In some embodiments, the antibody functional fragment comprises a single heavy chain (or a portion of a heavy chain) and a single light chain (or a portion of a light chain). In other embodiments, the antibody functional fragment comprises at least one heavy chain (or a portion of a heavy chain) and an equal number of light chains (or a portion of a light chain). In other embodiments, the antibody functional fragment comprises at least one heavy chain variable region and at least one light chain variable region, and further, the number of heavy chain variable regions and the number of light chain variable regions are equal. In some embodiments, the antibody functional fragment comprises two heavy chains (or a portion of a heavy chain) and two light chains (or a portion of a light chain). Antibody functional fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic cleavage or chemical cleavage of antigen binding molecules (including complete antibodies).

[0090] In the present invention, the term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed "US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the circumstances, the term "CDR" is used to refer to a region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope. In another specific embodiment, CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by IMGT.

[0091] In the present invention, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen.

[0092] In the present invention, variants of antibodies are also within the scope of the protection claimed in the present invention, such as sequences having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with each CDR or framework (FR), or light chain variable region (VL) and / or heavy chain variable region (VH) described in the present invention, or amino acid or nucleotide sequence of the full length of the antibody. In some embodiments, the variant of the antibody includes at least the above 6 CDRs; in other embodiments, the variant of the antibody includes at least one heavy chain variable region and one light chain variable region; in other embodiments, the variant of the antibody includes at least one heavy chain and one light chain; in other embodiments, the variant form contains two identical light chains (or sub-portions thereof) and two identical heavy chains (or sub-portions thereof). In some embodiments, the variant of the antibody is obtained by conservative modification or conservative substitution or replacement of the antibody sequence provided by the present invention. "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that the sequence can be changed without changing the biological activity of the protein. It should be understood that in the present invention, at least the biological activity of specifically binding to the target antigen is not changed. It is known to those skilled in the art that, in general, single amino acid replacement in a non-essential region of a polypeptide does not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, 4th edition). In addition, replacement of amino acids with similar structure or function substantially destroys the biological activity.

[0093] In a first aspect of the present invention, a bispecific antibody-modified vesicle is provided, comprising a vesicle and a bispecific antibody bound to the surface of the vesicle;

[0094] Wherein, the bispecific antibody binds to the transmembrane domain on the surface of the vesicle;

[0095] Furthermore, the bispecific antibody comprises a first antigen binding domain and a second antigen binding domain; the first antigen binding domain is a CD19 antigen binding domain or a HER2 antigen binding domain, and the second antigen binding domain is a CD3 antigen binding domain.

[0096] In the present invention, the term "bispecific antibody", which can be referred to as BsAb, refers to an artificial antibody containing two specific antigen binding sites, which can build a bridge between target cells and functional molecules (or effector cells) to stimulate a directed immune response.

[0097] In the present invention, the term "vesicle" refers to a class of ordered molecular assemblies with a closed double-layer structure that spontaneously form when dispersed in water. Unless otherwise specified, the "vesicle" used in the present invention can be an endogenous vesicle or an artificially constructed vesicle. Endogenous extracellular vesicles (EV) are phospholipid bilayer particle structures secreted by cells, and their size can be between (but not limited to) 150nm and 1000nm. In normal physiological processes, extracellular vesicles can transport a variety of active biomolecules, such as lipids, DNA, RNA and proteins, and play a communication role between cells. As a delivery carrier, vesicles have circulatory stability in vivo, and can effectively cross tissue barriers, cell barriers and intracellular barriers to transport "cargo" and induce functional changes in target cells. However, vesicles lack targeting when administered systemically, so it is difficult to avoid off-target effects.

[0098] In the present invention, unless otherwise specified, "bispecific antibody-modified vesicles" are nanoparticles stably bound by the transmembrane domain of the bispecific antibody being embedded in the phospholipid bilayer of the vesicle.

[0099] In the present invention, “bispecific antibody-modified vesicles” and “BsAb-EVs” have the same meaning and can be used interchangeably.

[0100] In the present invention, "CD19" is mainly present on the surface of normal and tumor B cells and follicular dendritic cells.

[0101] In the present invention, "HER2" mainly exists in various solid tumor cells, such as breast cancer, lung cancer, etc.

[0102] In the present invention, "CD3" is mainly present on the surface of T cells.

[0103] The bispecific antibody-modified vesicles provided in the first aspect of the present invention and the bispecific antibody-modified vesicles prepared in the seventh aspect of the present invention can significantly prolong the half-life of the bispecific antibody drug, reduce the pressure of continuous administration of the injection pump required clinically, increase patient compliance, and further reduce the economic burden on patients.

[0104] In some embodiments, the binding density of the bispecific antibody on the surface of the vesicle is greater than 100 AsAbs bound per square micrometer of the vesicle surface. The average number of AsAbs bound to the surface of the vesicles per square micrometer can also be greater than 200, greater than 250, greater than 300, greater than 400, greater than 500, 100-1000, 200-1000, 300-1000, 400-1000, 100-800, 200-800, 300-800, 400-800, 100-600, 200-600, 300-600, 400-600, etc., and can also be selected from an interval consisting of any two of the following values: 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000. The binding density of the bispecific antibody on the surface of the vesicle can be obtained by the test method for the binding density of the bispecific antibody in the bispecific antibody-modified vesicle described in the tenth aspect of the present invention. Taking fluorescent protein (GFP) as an example, the binding density of the bispecific antibody on the surface of the vesicle can be obtained by the test method for the binding density of the bispecific antibody in the bispecific antibody-modified vesicle described in the tenth aspect of the present invention. Fig.18 The standard curve shown aids in the calculation.

[0105] In some embodiments, the bispecific antibody modified vesicles may contain other drug components, which may be recorded as the second drug component. These solutions can effectively solve the limitation that the current BsAb (including but not limited to BiTE) can only mobilize one type of immune cells. In the present invention, the second drug component can be selected from chemotherapeutic drugs (alkylating agents, antimetabolites, anthracyclines, etc.), small molecule targeted drugs, innate immune agonists, etc.

[0106] In some embodiments, any of the bispecific antibodies is independently a BiTE antibody. In this case, the bispecific antibody (BsAb) modified vesicle (BsAb-EV) can be referred to as BiTE-EV.

[0107] In the present invention, the terms "BiTE" and "Bispecific T cell engager" have the same meaning and can be used interchangeably. BiTE is a type of bispecific antibody, and its effector cell recognition end can effectively activate T cells by binding to CD3e on the surface of T cells, and mediate the killing effect of T cells on tumor cells that is independent of the MHC complex.

[0108] In some embodiments, any of the bispecific antibodies independently comprises a first antigen binding domain, a connecting peptide, a second antigen binding domain, a hinge region and a transmembrane domain connected in series in sequence; wherein the transmembrane domain is embedded in the phospholipid bilayer on the surface of the vesicle and binds. The sequence structures of the connecting peptide, the hinge region and the transmembrane domain in different bispecific antibodies may be the same or different.

[0109] In the present invention, the transmembrane domain can be embedded in the phospholipid bilayer on the surface of the vesicle to achieve the binding of the bispecific antibody to the vesicle. Taking the bispecific antibody as BiTE as an example, reference can be made to Figure 1 .

[0110] In some embodiments, the diameter of the bispecific antibody-modified vesicle may be 80 to 1000 nm, further 100 to 800 nm, further 100 to 500 nm, further 100 to 400 nm, further 100 to 250 nm, further 100 to 200 nm. Figure 6 , Fig.16 The diameter of the bispecific antibody-modified vesicle can also be selected from any one or two of the following intervals: 80 nm, 100 nm, 120 nm, 150 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, etc.

[0111] In some embodiments, the average diameter of the bispecific antibody-modified vesicles can be 100-400 nm, further can be 100-250 nm, further can be 100-200 nm, and can also be selected from any one or two of the following intervals: 80 nm, 100 nm, 120 nm, 150 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, etc.

[0112] In the present application, the size of the bispecific antibody-modified vesicles refers to the average diameter only when specifically indicated.

[0113] In some embodiments, the bispecific antibody-modified vesicle satisfies any one, any multiple (such as 2, 3, 4) partial or all of the following characteristics:

[0114] (a1) the first antigen-binding domain comprises an anti-CD19 single-chain antibody variable region whose amino acid sequence is as shown in SEQ ID No.: 1, or comprises an anti-HER2 single-chain antibody variable region whose amino acid sequence is as shown in SEQ ID No.: 2;

[0115] (a2) the second antigen-binding domain comprises an anti-CD3 single-chain antibody variable region with an amino acid sequence as shown in SEQ ID No.: 3;

[0116] (a3) the amino acid sequence of the connecting peptide is shown in SEQ ID No.: 4;

[0117] (a4) the amino acid sequence of the hinge region is shown in SEQ ID No.: 5; and

[0118] (a5) The amino acid sequence of the transmembrane domain is shown in SEQ ID No.:6.

[0119] In some embodiments, the first antigen binding domain comprises an anti-CD19 single-chain antibody variable region (Anti-CD19 scFv) whose amino acid sequence is shown in SEQ ID No.: 1, or an anti-HER2 single-chain antibody variable region (Anti-HER2 scFv) whose amino acid sequence is shown in SEQ ID No.: 2.

[0120] SEQ ID No.:1(Anti-CD19 scFv):

[0121] DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGG SQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS

[0122] SEQ ID No.:2(Anti-HER2 scFv):

[0123] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0124] In some embodiments, the second antigen-binding domain comprises an anti-CD3 single-chain antibody variable region having an amino acid sequence as shown in SEQ ID No.:3.

[0125] SEQ ID No.: 3 (Anti-CD3 scFv):

[0126] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0127] In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID No.:4.

[0128] SEQ ID No.: 4 (GS linker): GGGS.

[0129] In some embodiments, the amino acid sequence of the hinge region is as shown in SEQ ID No.:5.

[0130] SEQ ID No.: 5 (CD8 Hinge):

[0131] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0132] In some embodiments, the amino acid sequence of the transmembrane domain is as shown in SEQ ID No.:6.

[0133] SEQ ID No.: 6: IYIWAPLAGTCGVLLLSLVITLYC.

[0134] In some embodiments, the vesicles are derived from one or more of mammalian cells, insect cells, etc.

[0135] In some embodiments, the vesicles are derived from one or more of HEK-293T cells, Nalm6 cells, and RAW264.7 cells.

[0136] In some embodiments, the vesicles are derived from HEK-293T cells.

[0137] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE (which can be recorded as CD19×CD3 BiTE) with an amino acid sequence as shown in SEQ ID No.: 8. In this case, the bispecific antibody-modified vesicle can be recorded as CD19×CD3BiTE-EV.

[0138] SEQ ID No.: 8 (CD19×CD3 BiTE, including transmembrane domain):

[0139] .

[0140] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE (which can be recorded as HER2×CD3 BiTE) with an amino acid sequence as shown in SEQ ID No.: 9. In this case, the bispecific antibody-modified vesicle can be recorded as HER2×CD3BiTE-EV.

[0141] SEQ ID No.: 9 (HER2×CD3 BiTE, including transmembrane domain):

[0142] .

[0143] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE containing a signal peptide whose amino acid sequence is shown in SEQ ID No.: 10 (which can be recorded as SP-CD19×CD3 BiTE).

[0144] SEQ ID No.: 10 (SP-CD19×CD3 BiTE, including signal peptide and transmembrane domain):

[0145] .

[0146] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE containing a signal peptide whose amino acid sequence is shown in SEQ ID No.: 11 (which can be recorded as SP-HER2×CD3 BiTE).

[0147] SEQ ID No.: 11 (SP-HER2×CD3 BiTE, including signal peptide and transmembrane domain):.

[0148] In a second aspect of the present invention, a bispecific antibody is provided, which is as defined in the first aspect of the present invention.

[0149] In some embodiments, the bispecific antibody comprises a first antigen binding domain and a second antigen binding domain; the first antigen binding domain is a CD19 antigen binding domain or a HER2 antigen binding domain, and the second antigen binding domain is a CD3 antigen binding domain.

[0150] In some embodiments, the bispecific antibody is a BiTE antibody.

[0151] In some embodiments, any of the bispecific antibodies independently comprises a first antigen binding domain, a connecting peptide, a second antigen binding domain, a hinge region and a transmembrane domain connected in series in sequence; wherein the transmembrane domain can be embedded in the phospholipid bilayer on the surface of the vesicle and bind. wherein the sequence structures of the connecting peptide, the hinge region and the transmembrane domain in different bispecific antibodies may be the same or different.

[0152] In some embodiments, the first antigen binding domain comprises an anti-CD19 single-chain antibody variable region (Anti-CD19 scFv) whose amino acid sequence is shown in SEQ ID No.: 1, or an anti-HER2 single-chain antibody variable region (Anti-HER2 scFv) whose amino acid sequence is shown in SEQ ID No.: 2.

[0153] In some embodiments, the second antigen-binding domain comprises an anti-CD3 single-chain antibody variable region having an amino acid sequence as shown in SEQ ID No.:3.

[0154] In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID No.:4.

[0155] In some embodiments, the amino acid sequence of the hinge region is as shown in SEQ ID No.:5.

[0156] In some embodiments, the amino acid sequence of the transmembrane domain is as shown in SEQ ID No.:6.

[0157] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE containing a signal peptide whose amino acid sequence is shown in SEQ ID No.: 10 (which can be recorded as SP-CD19×CD3 BiTE, including a signal peptide and a transmembrane domain).

[0158] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE containing a signal peptide whose amino acid sequence is shown in SEQ ID No.: 11 (which can be recorded as SP-HER2×CD3 BiTE, including a signal peptide and a transmembrane domain).

[0159] In some embodiments, the N-terminus of the bispecific antibody does not include a signal peptide, which can be obtained by synthesizing a signal peptide in a bispecific antibody containing a signal peptide.

[0160] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE having an amino acid sequence as shown in SEQ ID No.: 8 (which can be recorded as CD19×CD3 BiTE, including a transmembrane domain).

[0161] In some embodiments, the bispecific antibody-modified vesicle comprises a BiTE having an amino acid sequence as shown in SEQ ID No.: 9 (which can be recorded as HER2×CD3 BiTE, including a transmembrane domain).

[0162] In a third aspect of the present invention, a nucleic acid is provided, wherein the nucleotide sequence of the nucleic acid comprises a nucleotide sequence in at least one of the following groups:

[0163] (i) a nucleotide sequence encoding the bispecific antibody according to the second aspect of the present invention; and

[0164] (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i);

[0165] In some preferred embodiments, the nucleic acid is DNA, mRNA or a combination thereof.

[0166] In some embodiments, a nucleotide sequence encoding a signal peptide is also included upstream of the coding sequence of the bispecific antibody, wherein the sequence structure of the signal peptide in different nucleic acids may be the same or different.

[0167] In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID No.:7.

[0168] SEQ ID No.:7: MALPVTALLLPLALLLHAARP.

[0169] In some embodiments, the nucleotide sequence of the nucleic acid is as shown in SEQ ID No.:12, corresponding to the coding sequence of SEQ ID No.:10.

[0170] Nucleotide sequence of SEQ ID No.: 12 (SP-CD19×CD3 BiTE):

[0171]

[0172] In some embodiments, the nucleotide sequence of the nucleic acid is as shown in SEQ ID No.:13, corresponding to the coding sequence of SEQ ID No.:11.

[0173] SEQ ID No.: 13 nucleotide sequence (SP-HER2×CD3 BiTE):

[0174]

[0175] In the fourth aspect of the present invention, a recombinant vector is provided, wherein the recombinant vector contains one or more of the coding sequence of the bispecific antibody according to the second aspect of the present invention and the nucleotide sequence of the nucleic acid according to the third aspect of the present invention.

[0176] In the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, etc.; non-limiting examples include yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P-derived artificial chromosomes (PAC); non-limiting examples of bacteriophages include lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40), etc. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as one or more of enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0177] In the present invention, "recombinant vector" refers to an artificially constructed vector.

[0178] In some embodiments, the recombinant vector is one or more of a lentivirus vector, a retrovirus vector, a transient transfection vector, and an adenovirus vector.

[0179] In a fifth aspect of the present invention, a genetically engineered cell is provided, wherein the genetically engineered cell is capable of endogenously secreting vesicles, and the genome of the genetically engineered cell is integrated with one or more of the coding sequence of the bispecific antibody of the second aspect of the present invention and the nucleotide sequence of the nucleic acid of the third aspect of the present invention, or the genetically engineered cell contains a recombinant vector, and the recombinant vector contains one or more of the coding sequence of the bispecific antibody of the second aspect of the present invention and the nucleotide sequence of the nucleic acid of the third aspect of the present invention;

[0180] The surface of the vesicle has a phospholipid bilayer, so that the bispecific antibody can be embedded through the transmembrane domain to achieve stable binding.

[0181] In the present invention, the "genetically engineered cells" herein may refer to genetically engineered cells obtained through genetic modification.

[0182] In some embodiments, the genetically engineered cell is a host cell that can be used to synthesize the antibody of the first aspect of the present invention.

[0183] In some embodiments, the host cell is a eukaryotic cell.

[0184] In some embodiments, the host cell is a mammalian cell.

[0185] In some embodiments, host cells suitable for expressing antibodies of the invention include, but are not limited to, mammalian cells such as NS0, Sp2 / 0, CHO, COS, HEK, fibroblasts, and myeloma cells. Human cells can be used, thus allowing the molecules to be modified with human glycosylation patterns. Other eukaryotic cell lines can also be used.

[0186] In some embodiments, the host cell is a mammalian cell or an avian cell.

[0187] In some embodiments, the host cell is a fish cell or an amphibian cell, such as the host cell type described in patent document CN105018527A.

[0188] In some embodiments, the host cell is a rodent cell, such as a cell derived from a rat, a mouse, or a hamster.

[0189] In some embodiments, the host cell is a primate cell, such as a human cell.

[0190] In some embodiments, the host cell is a primary cell, such as a tumor cell, a hepatocyte, a cardiomyocyte, a neuron, an endothelial cell, a stem cell, or the like.

[0191] In some embodiments, the genetically engineered cells are derived from HEK-293T cells (human embryonic kidney 293 cells).

[0192] In some embodiments, the genetically engineered cells are derived from one or more of HEK-293T cells (human embryonic kidney 293 cells), Nalm6 cells, and RAW264.7 cells.

[0193] In a sixth aspect of the present invention, a method for preparing a genetically engineered cell is provided, comprising the following steps:

[0194] Providing a mother cell capable of endogenously secreting vesicles and providing a recombinant vector; wherein the surface of the vesicle has a phospholipid bilayer; and the recombinant vector contains one or more of the coding sequence of the bispecific antibody described in the second aspect of the present invention and the nucleotide sequence of the nucleic acid described in the third aspect of the present invention;

[0195] The recombinant vector is transformed or transfected into the mother cell.

[0196] In some embodiments, the mother cell is selected from one or more of mammalian cells, insect cells, and the like.

[0197] In some embodiments, the mammalian cells may include one or more of HEK-293T cells, Nalm6 cells, RAW264.7 cells, and the like.

[0198] In some embodiments, the mother cell is a HEK-293T cell. Reference may be made to some specific embodiments below.

[0199] In some embodiments, the mother cell is an insect cell. Insect cells are eukaryotic cells derived from insects, which can be cultured in large quantities in vitro and can produce extracellular vesicles.

[0200] In some embodiments, the recombinant vector is a lentiviral vector. In this case, the step of transforming or transfecting the recombinant vector into the mother cell corresponds to transfecting the recombinant vector into the mother cell.

[0201] In some embodiments, the concentration of the mother cells can be 1×10 5 / mL~1×10 6 100,000 to 1,000,000 cells / mL (i.e., 100,000 to 1,000,000 cells / mL), further 100,000 to 800,000 cells / mL, further 100,000 to 600,000 cells / mL, further 100,000 to 500,000 cells / mL, further 100,000 to 400,000 cells / mL, further 100,000 to 200,000 cells / mL. A non-limiting example of the concentration of the mother cells is 1×10 5 / mL, 2×10 5 / mL, 3×10 5 / mL, 4×10 5 / mL, 5×10 5 / mL, 6×10 5 / mL, 7×10 5 / mL, 8×10 5 / mL, 9×10 5 / mL, 1×10 6 Pieces / mL, etc.

[0202] In some embodiments, the infection index MOI is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The infection index MOI can be selected from 1-8, further selected from 1-5, further selected from 1-4, and further selected from 1-3.

[0203] In some embodiments, the concentration of the mother cells is 1×10 5 / m~1×10 6 / mL, and the infection index MOI is 1-10.

[0204] In a seventh aspect of the present invention, a method for preparing a bispecific antibody-modified vesicle is provided, comprising the following steps:

[0205] Provided is a genetically engineered cell according to the fifth aspect of the present invention or a genetically engineered cell prepared by the preparation method according to the sixth aspect of the present invention; wherein the genetically engineered cell comprises a first coding sequence of the bispecific antibody defined in the first aspect of the present invention, and further comprises a second coding sequence located upstream of the first coding sequence, wherein the second coding sequence encodes a signal peptide;

[0206] The genetically engineered cells are cultured to obtain the bispecific antibody-modified vesicles. The step of culturing the genetically engineered cells includes the following process: the genetically engineered cells secrete vesicles under the exogenous stimulation conditions, synthesize bispecific antibodies with a signal peptide at the C-terminus, the C-terminus of the bispecific antibody is embedded in the phospholipid bilayer on the surface of the genetically engineered cells under the mediation of the signal peptide, the genetically engineered cells secrete vesicles in a budding manner, and the signal peptide at the C-terminus of the bispecific antibody is removed.

[0207] In some embodiments, taking the bispecific antibody as BiTE as an example, the preparation method can also refer to Figure 1 .

[0208] In some embodiments, the step of culturing the genetically engineered cells can be performed under exogenous stimulation.

[0209] In some embodiments, the exogenous stimulation condition is selected from one or more of ultraviolet light and the use of serum-free culture medium.

[0210] In some embodiments, the preparation method is carried out under ultraviolet light irradiation. Since ultraviolet irradiation can be lethal to cells, ultraviolet irradiation is usually not performed during the process of viral vector packaging. In the present invention, it was unexpectedly found that the yield of BsAb-EV can be significantly improved by ultraviolet irradiation. The inventor speculates that this is because when cells are in a dying state due to ultraviolet irradiation, the cell membrane can wrap the available materials in the cell into a large number of vesicles and release them outside the cell. By selecting the appropriate irradiation intensity, the production efficiency can be improved while ensuring a certain cell activity.

[0211] In some embodiments, the exogenous stimulation condition is ultraviolet light. Those skilled in the art can reasonably select the wavelength and irradiation time of ultraviolet light according to the needs of maintaining cell activity and improving the yield of BsAb-EV. In some embodiments, the wavelength of ultraviolet light is 280 to 315 nm. In other embodiments, the ultraviolet light irradiation time can be 0.5 to 2 hours, and non-limiting examples include 1 hour.

[0212] In some embodiments, the UV irradiation intensity is 0 to 400 J / m 2 (may include or exclude the left endpoint 0), and further may be 0 to 350 J / m 2 (may include or exclude the left endpoint 0), and further may be 50 to 400 J / m 2 , and further can be 100~400J / m 2 , and further can be 200~400J / m 2 , and further can be 250~400J / m 2 , and further can be 300~400J / m 2 , some non-limiting examples are 50 J / m 2 、100J / m 2 、150J / m 2 、200J / m 2 , 250J / m 2 、300J / m 2 、350J / m 2 wait.

[0213] In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID No.:7.

[0214] In some embodiments, the signal peptide is further removed. When the preprotein containing the signal peptide is transferred to the endoplasmic reticulum, signal peptidase (SPase) is responsible for cutting the signal peptide from the preprotein to allow the mature protein to fold correctly.

[0215] In the eighth aspect of the present invention, provided is the use of the bispecific antibody-modified vesicles described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the genetically engineered cells described in the fifth aspect of the present invention, the genetically engineered cells prepared by the preparation method described in the sixth aspect of the present invention, or the bispecific antibody-modified vesicles prepared by the preparation method described in the seventh aspect of the present invention, and further, their use in the preparation of drugs for preventing and / or treating tumors or cancers.

[0216] In the present invention, "prevention" includes aspects such as prevention, treatment, and adjuvant therapy. As used herein, "prevention" means to alleviate, slow the progression, attenuate, prevent, or maintain an existing disease or condition. "Prevention" also includes curing one or more symptoms of a disease or condition, preventing its development, or alleviating it to a certain extent. In the present invention, "prevention" has the same meaning as "prevention and / or treatment", "prevention, treatment, or prevention and treatment", and can be used interchangeably.

[0217] In the present invention, "drug" includes any agent, compound, composition or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects of "drug" in vivo is not particularly limited, and it can be a systemic effect or only a local effect. The activity of the "drug" is not particularly limited, and it can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.

[0218] In the ninth aspect of the present invention, a pharmaceutical composition is provided, comprising at least one of the bispecific antibody-modified vesicles described in the first aspect of the present invention and the bispecific antibody-modified vesicles prepared by the preparation method described in the seventh aspect of the present invention; the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0219] In the present invention, the "composition" may be a combination of a plurality of substances, and further, may be used in combination or may be a mixture of the combination.

[0220] In the present invention, "pharmaceutical composition" refers to a composition that has the effect of preventing and treating diseases or symptoms and can be used as a medicine or can be used to prepare a medicine.

[0221] In some embodiments, the pharmaceutical composition is a drug.

[0222] In the present invention, "drug" refers to a directly administrable pharmaceutical preparation, which usually has a prescribed usage and dosage.

[0223] In the present invention, "pharmaceutically acceptable" refers to those ligands, materials, compositions and / or dosage forms that are suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0224] In the present invention, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the phrase "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with drug administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0225] In the present invention, non-limiting examples of the "carrier" in the "pharmaceutically acceptable carrier" include, but are not limited to, mannitol, sorbitol, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium ethylenediaminetetraacetate (disodium EDTA), calcium sodium EDTA, carbonates, acetates, phosphates of monovalent alkali metals or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, oligofructose, dextran, glycine, starch, sucrose, dextrin (such as maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, etc.

[0226] In some embodiments, the pharmaceutical preparation may be a liquid preparation. Liquid preparations refer to preparations containing a liquid phase, such as solutions, suspensions, emulsions, etc., without limitation.

[0227] In some embodiments, depending on the different modes of administration, the pharmaceutical preparation can be an oral preparation, an injection, a drop, a patch, a tube feeding preparation, etc.

[0228] It should be understood that the drug contains a therapeutically effective amount of the bispecific antibody-modified vesicles of the present invention.

[0229] In the present invention, "therapeutically effective amount" refers to the amount of the active pharmaceutical ingredient that will cause a biological or medical response in an individual for a disease, disorder and / or symptom, for example, the amount of Akkermansia muciniphila of the present invention that brings a pharmacologically positive effect to an individual, wherein the pharmacologically positive effect includes but is not limited to reducing or inhibiting the activity of an enzyme or protein or improving symptoms, alleviating symptoms, slowing down or delaying the progression of a disease, or preventing a disease.

[0230] In the present invention, "subject" refers to a patient who takes the pharmaceutical composition.

[0231] In the present invention, "subject" is an animal, preferably a mammal, more preferably a human, and the subject includes but is not limited to a patient with a disease, illness and / or symptom. The subject in the present invention is preferably a mammal. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0232] In some embodiments, the subject is a mammal.

[0233] In some embodiments, the pharmaceutical composition is suitable for humans or other mammals. In the present invention, "other mammals" are not humans, and non-limiting examples of "other mammals" include cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, etc., and further include mice or rats; non-limiting examples of "other mammals" include primates.

[0234] In some embodiments, the subject is a human, mouse, or rat.

[0235] In the present invention, "patient" refers to an animal, preferably a mammal, such as a human, a mouse, or a rat.

[0236] Different subjects may result in different selection ranges of other ingredients in the aforementioned pharmaceutical composition.

[0237] In some embodiments, the administration of the drug includes, but is not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection.

[0238] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, specifically, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices. For example, the suspension may contain a suspending agent, specifically, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methylate and agar or mixtures of these substances.

[0239] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0240] It is to be understood that the drugs of the embodiments of the present invention can be added with different pharmaceutically acceptable carriers to prepare suitable clinical dosage forms, which include but are not limited to the dosage forms described above.

[0241] In the tenth aspect of the present invention, a method for testing the binding density of the bispecific antibody in the bispecific antibody-modified vesicle according to the first aspect of the present invention is provided, characterized in that it comprises the following steps:

[0242] Obtain a standard curve of fluorescence value-concentration of the fluorescent marker;

[0243] Preparing a mixed solution A containing the bispecific antibody-modified vesicles, wherein each bispecific antibody unit carries one of the fluorescent markers;

[0244] The concentration C of the bispecific antibody unit in the mixed solution A is calculated using the standard curve of the fluorescence value-concentration of the fluorescent marker. BsAb ; and using the nanoparticle tracking analysis (NTA) method to determine the concentration C of the bispecific antibody modified vesicles in the mixed solution A BsAb-EV ;

[0245] According to the C BsAb and the C BsAb-EVThe average number of bispecific antibody units N bound to a bispecific antibody modified vesicle is calculated. BsAb@EV .

[0246] In some embodiments, the method for testing the binding density of the bispecific antibody in the bispecific antibody-modified vesicles further comprises the following steps: using a nanoparticle tracking analysis method to determine the average particle size D of the bispecific antibody-modified vesicles. BsAb-EV , combined with the N BsAb@EV The number of bispecific antibody units bound per unit surface area of ​​the bispecific antibody modified vesicles was calculated as follows: BsAb@EV .

[0247] In some embodiments, the fluorescent marker can be selected from green fluorescent protein (GFP), red fluorescent protein (RFP), and luciferase.

[0248] In some embodiments, the fluorescence intensity-concentration standard curve of green fluorescent protein (GFP) is as follows: Fig.18 .

[0249] In some embodiments, a coding sequence of a fluorescent protein can be added to the nucleic acid of the third aspect of the present invention, so that the bispecific antibody contains the fluorescent protein and is fluorescently labeled.

[0250] Some specific embodiments are provided below.

[0251] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manuals of this area (such as "Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)" etc.) or normal conditions, and can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0252] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0253] In the following examples, unless otherwise specified, "room temperature" refers to 20°C to 30°C.

[0254] Example 1. Preparation of bispecific antibody modified vesicles (BsAb-EV)

[0255] 1.1. Sequence design of bispecific antibodies

[0256] For reference Figure 2 The structure shown.

[0257] The CD19×CD3 BiTE with an amino acid sequence as shown in SEQ ID No.: 8 was selected, and the corresponding signal peptide with an amino acid sequence as shown in SEQ ID No.: 7. The amino acid sequence of the SP-CD19×CD3 BiTE containing the signal peptide is shown in SEQ ID No.: 10, and its coding sequence is shown in SEQ ID No.: 12.

[0258] The HER2×CD3 BiTE with an amino acid sequence as shown in SEQ ID No.: 9 was selected, and the corresponding signal peptide with an amino acid sequence as shown in SEQ ID No.: 7. The amino acid sequence of the SP-HER2×CD3 BiTE containing the signal peptide is shown in SEQ ID No.: 11, and its coding sequence is shown in SEQ ID No.: 13.

[0259] 1.2. Lentiviral packaging

[0260] (1) Plasmid purification and cell culture: 18 to 24 hours before transfection, 2.5 × 10 6 Place HEK-293T cells in a 10 cm dish and incubate at 37°C overnight. The cells should reach 65% to 70% confluence within 24 hours.

[0261] (2) Transfection of HEK-293T cells: Add the transfer vector and packaging plasmid to Opti-MEM and mix; vortex for 10 seconds, and then incubate the mixture at room temperature for 15 minutes; add the mixture dropwise to the culture dish and return the culture dish to the 37°C cell culture incubator.

[0262] (3) Collecting viral supernatant: After incubation for 12 to 18 hours, replace the culture medium and continue culturing for 48 hours.

[0263] (4) Transfer the cell culture supernatant to a 15 mL centrifuge tube. Centrifuge at 3000 × g for 15 minutes and filter the supernatant. The plasmid map can be found at Figure 3 and Figure 4 .

[0264] Figure 3 This is the plasmid map used to construct the CD19×CD3 BiTE lentiviral packaging vector.

[0265] Figure 4 This is the plasmid map used to construct the HER2×CD3 BiTE lentiviral packaging vector. The promoter is human elongation factor 1alpha (HEF1α).

[0266] The nucleotide sequence of the promoter HEF1α used in the embodiments of the present application is as follows:

[0267] CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAA

[0268] GTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAA

[0269] CTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCG

[0270] TATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAAC

[0271] ACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTT

[0272] GCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGG

[0273] GTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTG

[0274] CTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCT

[0275] TCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGC

[0276] TGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTG

[0277] GTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATG

[0278] TTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCA

[0279] AGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGG

[0280] CGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGG

[0281] CCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGA

[0282] GTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCA

[0283] CTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCG

[0284] TCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGG

[0285] AGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTT

[0286] GAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCA

[0287] TTTCAGGTGTCGTGAGCTAGC(SEQ ID No.:14)。

[0288] Example 2. Preparation of Bispecific Antibody-Modified Vesicles

[0289] 2.1. Experimental Example

[0290] (1) Cell line establishment. HEK-293T cell line was established by infecting HEK-293T cell line with lentivirus, and high expression clones were isolated by flow cytometry. The mCherry strong positive cell sorting was adopted, and the mCherry red fluorescent protein expression sequence was connected to the 5' end of BiTE in BiTE-EV through the 3' end of T2A sequence. T2A sequence will cause ribosome jumping during protein translation in cells, so that BiTE-EV and mCherry fluorescent protein can be expressed in 1:1 ratio in HEK-293T cells, and they are independent and non-interfering with each other.

[0291] The T2A sequence is GSGEGRGSLLTCGDVEENPGP (SEQ ID No.: 15).

[0292] (2) Stimulate the mother cells to produce vesicles: UVB (280-315 nm) irradiates the cells for 1 h at an intensity of 300 J / m 2 , and continue to culture in a 37°C incubator for 18 h. After culture, aspirate the supernatant containing cell debris and vesicles for separation.

[0293] (3) Centrifuge at 1500 rpm for 5 min at 4°C to separate dead cells and cell debris, and collect the supernatant for the next centrifugation step.

[0294] (4) Centrifuge at 18,000 × g for 1 h at 4°C. Collect the precipitate, which is the vesicles, and resuspend it in PBS or serum-free medium.

[0295] The HEK-293T cell line is infected with a lentivirus containing the coding sequence of SP-CD19×CD3 BiTE, and the resulting bispecific antibody-modified vesicles can be recorded as CD19×CD3 BiTE-EV, or CD19 BiTE EV-293T or CD19BiTE-EV or CD19BiTE-EV.

[0296] The HEK-293T cell line is infected with a lentivirus containing the coding sequence of SP-HER2×CD3 BiTE, and the resulting bispecific antibody-modified vesicles can be referred to as HER2×CD3 BiTE-EV, or HER2 BiTE EV-293T or HER2BiTE-EV or HER2BiTE-EV.

[0297] 2.2. Comparative Example

[0298] Steps (2), (3), and (4) in 2.1. are performed using HEK-293T cell lines that have not been infected with lentivirus. The resulting vesicles can be referred to as wt HEK-293T or wt-EV.

[0299] The results of flow cytometry sorting of bispecific antibody modified vesicles can be found in Figure 5 As can be seen from the figure, the sorted BiTE HEK-293T cells are cell clones that stably express BiTE.

[0300] 2.3. Vesicle structure identification

[0301] 2.3.1. NTA nanoparticle tracking analysis

[0302] Test method: Track and analyze the Brownian motion of each particle, and calculate the hydrodynamic diameter and concentration of the nanoparticles using the Stockes-Einstein equation. Instrument name: ZetaView (ParticleMetrix).

[0303] NTA nanoparticle tracking analysis results can be found in Figure 6 and Table 1, Table 2, and Table 3.

[0304] Table 1.

[0305] parameter wt HEK-293T CD19 BiTE EV-293T HER2 BiTE EV-293T Mean fluorescence intensity n / a 52633 59842

[0306] Among them, "n / a" means Not applicable.

[0307] Table 2. Peak analysis

[0308] sample Particle size (nm) Concentration (number of particles / mL) Volume percentage EV 155.2 <![CDATA[1.9E+7 (i.e., 1.9×10 7 )]]> 99.9％ CD19BiTE-EV 166.9 3.2E+7 99.9％ HER2 BiTE-EV 145.2 9.1E+6 99.9％

[0309] Table 3. X value analysis

[0310]

[0311] In the present invention, the X value in the NTA analysis can represent the particle size distribution of the particles. Among them, X10, X50, and X90 respectively represent the particle sizes corresponding to the cumulative number distribution percentages of the particles reaching 10%, 50%, and 90%; that is, X50 can represent the median of the particle size. The Mean value in the NTA analysis represents the average value of the particle size, and unless otherwise specified, refers to the average value of the number of particles.

[0312] In the present invention, unless otherwise specified, the "particle size" in NTA analysis refers to "diameter".

[0313] Test results analysis

[0314] According to Table 1, it can be seen that the bispecific antibody CD19×CD3 BiTE or HER2×CD3 BiTE was successfully linked to the surface of HEK-293T cells.

[0315] According to Table 2, it can be seen that the peak particle size and particle number of extracellular vesicles modified with bispecific antibodies CD19×CD3 BiTE or HER2×CD3 BiTE are not significantly different from those of unmodified extracellular vesicles.

[0316] According to Table 3, it can be seen that the particle size distribution and uniformity of the extracellular vesicles modified with the bispecific antibody CD19×CD3 BiTE or HER2×CD3 BiTE are not significantly different from those of the unmodified extracellular vesicles.

[0317] 2.3.2. Transmission electron microscopy (TEM) analysis

[0318] Test method:

[0319] Using a Pelco Easyglow instrument, place a 5 μL volume of sample on a thin carbon grid and discharge for 2 minutes. Take 5 μL of freshly prepared 2% uranyl acetate staining solution, place it on the sample holding grid, and incubate with the sample on the grid for 2 minutes. Use filter paper to absorb excess sample and stain, leaving a thin layer of stain particles on the grid. Repeat the staining process again and let the grid dry until imaging.

[0320] Transmission electron microscopy (TEM) was collected with a Tecnai T12 transmission electron microscope (TEM) at 100KeV. Images were recorded on a GatanOneview 4Kx4K camera. Each image was collected by exposing the sample for 4 seconds and a total of 100 dose-graded images were collected into a single micrograph. Data were collected at a focus of -1.5 microns to 2 microns at 30K-40K magnification (30,000-40,000 times magnification).

[0321] The results of TEM analysis can be found in Figure 7 , (A) corresponds to wild-type vesicles (wt-EV) without bispecific antibody, (B) corresponds to CD19 BiTE-EV, and (C) corresponds to HER2 BiTE-EV. Figure 7 It can be seen that the BiTE-modified vesicles still have complete phospholipid membrane structure, and their size and morphology are not significantly different from those of wild-type vesicles (wt-EV).

[0322] 2.3.3. Analysis of binding density of bispecific antibodies on the surface of bispecific antibody-modified vesicles

[0323] Test method: Construct BiTE-TM-GFP fusion protein, the N-terminus of green fluorescent protein (GFP) is directly connected to the C-terminus of the transmembrane domain TM. Obtain the standard curve of fluorescence value-concentration of the fluorescent marker by using GFP fluorescent protein standard ( Fig.18); preparing a mixed solution A containing bispecific antibody-modified vesicles, wherein each bispecific antibody unit carries a fluorescent marker; and calculating the concentration C of the bispecific antibody unit in the mixed solution A using a standard curve of the fluorescence value-concentration of the fluorescent marker. BsAb ; and nanoparticle tracking analysis (NTA) was used to determine the concentration C of the bispecific antibody modified vesicles in the mixture A BsAb-EV ;

[0324] According to C BsAb and C BsAb-EV The average number of bispecific antibody units N bound to a bispecific antibody modified vesicle was calculated. BsAb@EV .

[0325] Test result: N BsAb@EV＝ C BsAb / C BsAb-EV

[0326] For the detailed process, please refer to Section 5.3 of Example 5 below.

[0327] 2.4. Functional testing

[0328] 2.4.1. In vitro T cell activation experiment

[0329] Test method: Different concentrations of BiTE-EV were added to NF-κB / Jurkat / GFP cultured in vitro TM Transcriptional Reporter Cell Line cells. Among them, NF-κB / Jurkat / GFP TM Transcriptional Reporter Cell Line is a transcriptional reporter cell line that can cause cells to express GFP fluorescent protein after Jurkat T cells are activated.

[0330] The test results can be found in Figure 8 . Figure 8 The results of in vitro T cell activation tests of bispecific antibody-modified vesicles showed that BiTE-EVs can effectively activate Jurkat T cells, and the activation effect is concentration-dependent.

[0331] 2.4.2.CD19 BiTE-EV specific killing effect test

[0332] (1) Test method: Nalm6 cells were used as target cells. Nalm6 is a human B-cell precursor leukemia cell line that highly expresses CD19 on its surface. The CD19 gene in wild-type Nalm6 cells was knocked out using CRISPR Cas9 technology to obtain the Nalm6-CD19KO cell line as a negative control. Using this group of cell lines, the specific killing effect of CD19 BiTE EV was detected by a luciferase-based cytotoxicity assay. The target cells were co-cultured with human peripheral blood T cells for 24 hours at a specified E:T ratio. The lysis percentage was calculated according to the formula: 100×(experimental well reading-spontaneous) / (maximum-spontaneous). Among them, spontaneous is the fluorescence intensity released by the target cells cultured alone, and maximum is the fluorescence intensity released after the target cells were dissolved with 5% SDS. The cytotoxicity results are the mean ± SD of 3 technical replicates.

[0333] In the present invention, unless otherwise specified, E:T refers to the ratio of the number of effector cells to target cells. In this example, the effector cells are human peripheral blood T cells, and the target cells are Nalm6 cells.

[0334] The test results can be found in Fig. 9 . Fig. 9 The results of the specific killing effect test of the bispecific antibody modified vesicle CD19 BiTE-EV. Fig. 9 It can be seen that under different E:T conditions, CD19 BiTE-EV can mediate T cells to effectively kill target (CD19) positive Nalm6 tumor cells, and this effect is concentration gradient dependent; CD19BiTE-EV will not mediate T cells to kill target negative (CD19-) Nalm6-CD19KO tumor cells, and its mediated tumor killing effect is highly specific.

[0335] (2) Test method: Raji cells were used as target cells. Raji is a human Burkitt lymphoma cell line that highly expresses CD19 on its surface. The CD19 gene in wild-type Raji cells was knocked out using CRISPR Cas9 technology to obtain the Raji-CD19KO cell line as a negative control. Using this group of cell lines, the specific killing effect of CD19BiTE EV was detected using a GFP-based real-time cytotoxicity test. The X-axis represents the time axis, and the Y-axis represents the standardized green fluorescence intensity, which reflects the number of target cells. Spontanous is a group in which target cells are cultured alone.

[0336] The test results can be found in Fig.10 . Fig.10The results of the specific killing effect test of the bispecific antibody modified vesicle CD19 BiTE-EV. Fig.10 It can be seen that under the condition of E:T=5:1, the tumor cells in the Spontanous group, wt-EV treatment group and Media treatment group continued to proliferate during the observation time until the growth density limit was reached (reaching the plateau phase). In the BiTE-EV low-dose group (low), medium-dose group (medium), and high-dose group (high), the number of tumor cells decreased over time until it could not be detected.

[0337] Under the condition of E:T=1:1, similarly, the tumor cells in the Spontanous group, wt-EV treatment group, and Media treatment group continued to proliferate during the observation period until the growth density limit was reached (reaching the plateau phase). However, the tumor proliferation rate of the low-dose group (low) given BiTE-EV was significantly inhibited, and the number of tumor cells in the medium-dose group (medium) and the high-dose group (high) decreased over time, and the high-dose group decreased to undetectable levels.

[0338] 2.4.3. HER2 BiTE-EV specific killing effect test

[0339] Test method: BT474 cells were used as target cells. BT474 is a human-derived breast cancer cell line with high expression of HER2 on the surface. MDA-MB-231 is a human-derived breast cancer cell line with low expression of HER2 on the surface. Using this group of cell lines, the specific killing effect of HER2 BiTE EV was detected by a luciferase-based cytotoxicity assay. The target cells were co-cultured with human peripheral blood T cells for 24 hours at a specified E:T ratio. The lysis percentage was calculated according to the formula: 100×(experimental well reading-spontaneous) / (maximum-spontaneous). Among them, spontaneous is the fluorescence intensity released by the target cells cultured alone, and maximum is the fluorescence intensity released after the target cells were dissolved with 5% SDS. The cytotoxicity results are the mean ± SD of 3 technical replicates.

[0340] The test results can be found in Fig.11 . Fig.11 The results of the specific killing effect test of the bispecific antibody modified vesicle HER2 BiTE-EV. Fig.11It can be seen that under different E:T conditions, HER2 BiTE-EV can mediate T cells to effectively kill target (HER2)-positive BT474 tumor cells, and this effect is concentration gradient-dependent; HER2 BiTE-EV will not mediate T cells to kill target-negative (HER2-) MDA-MB-231 tumor cells, and its mediated tumor killing effect is highly specific.

[0341] The literature (Hoseini SS, et al. J Immunother Cancer 2020; 8: e001626.doi: 10.1136 / jitc-2020-001626) records the in vitro data of anti CD19 BiTE, which shows that under the condition of E:T = 10:1, the maximum tumoricidal effect was achieved at a high concentration of 0.01-0.1 nM, and the killing efficiency of the target cell Nalm6 was only about 60%.

[0342] 2.5. In vivo targeting test

[0343] Test method: Use 1×10 6 Raji cells formed tumors subcutaneously in the abdomen of NSG mice. The imaging results of tumor cells are as follows Fig.12 After imaging, mice were given 5×10 6 Human peripheral blood T cells and 9.8×10 8 Figure 3. Tumor cell targeting in vivo by bispecific antibody-modified vesicles 48 and 72 hours after injection of T cells and vesicles. Fig.12 As shown, CD19BiTE EVs can be seen to accumulate in tumors.

[0344] 2.6. Test of the absorption rate of bispecific antibody-modified vesicles by target cells

[0345] Test method: wt EV or CD19 BiTE EV was stained with Cell Trace Violet and co-cultured with Nalm6 target cells, and the uptake of vesicles by Nalm6 cells was detected at 1h, 3h, 6h, and 12h.

[0346] The flow cytometer test results and quantitative analysis results are as follows Fig.13 and Fig.14 As shown, CD19 BiTE EVs were taken up at a significantly higher rate than wt EVs. Results are the mean ± SD of three technical replicates. Statistical analysis was performed using two-way ANOVA, ****P < 0.0001.

[0347] Example 3. Investigation of UV irradiation conditions

[0348] Preparation method: Inoculate 5×10 6 Wild-type HEK-293T cells were cultured in RPMI1640 medium supplemented with 10% Exosome-Depleted FBS at 37°C and 5% CO2 for 24 h (denoted as 0 h), and then treated with UV irradiation or without UV irradiation. After treatment, the cells were placed back at 37°C and 5% CO2 for 18 h, and the vesicles in the supernatant were collected.

[0349] Irradiation conditions: UVB (ultraviolet light type B), wavelength 280-315nm, irradiation intensity 300J / m 2 , irradiation time 1h.

[0350] Experimental results:

[0351] Fig.15 The figure is a comparison of the results of the bispecific antibody modified vesicles in this example with and without UV irradiation. The experimental group UVB irradiation group and the control group without UVB irradiation were set up respectively. The experimental group prepared and irradiated cells according to the above conditions, and the control group used the same cell preparation method without irradiation. After collecting the vesicles, NTA and TEM were used for analysis. The quantitative results of NTA single samples are shown in Tables 4-6. According to the test and analysis results, UVB irradiation has no significant effect on the particle size and distribution of the produced vesicles. In terms of yield, the vesicle yield in the UVB irradiation group is significantly increased. In this example, after UV irradiation, the vesicle yield is 3.67 times that of the group without UVB irradiation ((4.03×10 10 ) / (1.10×10 10 )). According to the TEM test results (not provided with figures), it is further shown that UVB irradiation has no significant effect on the vesicle morphology.

[0352] Table 4.

[0353]

[0354] Table 5.

[0355] Processing conditions Particle size (nm) Volume percentage UV irradiation 154.6 100％ No UV exposure 146.6 100％

[0356] Table 6. X-value analysis of NTA test

[0357] Processing conditions Mean(nm) X10(nm) Median(X50)(nm) X90(nm) UV irradiation 171.4 98.2 156.5 253.9 No UV exposure 160.7 102.3 148.5 227.5

[0358] Example 4. Different mother cells

[0359] 4.1. Preparation of Nalm6 cell line

[0360] The recombinant vector (CD19×CD3 BiTE lentiviral packaging vector constructed in Example 1) was transfected into Nalm6 mother cells by lentivirus. After 48 hours of transfection, the membrane fusion BiTE protein and the co-expressed fluorescent protein could be detected by flow cytometry. Stable mother cell lines expressing BiTE were obtained by flow cytometry. The lentiviral infection conditions were: cell concentration 1×10 5 ~1×10 6 / mL, infection index MOI = 1-10.

[0361] 4.2. Preparation of RAW264.7 cell line

[0362] The preparation method is basically the same as 4.1, except that the mother cell type is changed to RAW264.7 cell line.

[0363] 4.3. Preparation of BiTE-modified vesicles (BiTE-EVs)

[0364] The method of Example 2 was used to prepare CD19×CD3 BiTE-modified vesicles (CD19 BiTE-EVs) under ultraviolet irradiation conditions using the Nalm6 cell line prepared in 4.1 and the RAW264.7 cell line prepared in 4.2.

[0365] Ultraviolet irradiation conditions: UVB, wavelength 280-315nm, irradiation intensity 300J / m 2 , irradiation time 1h.

[0366] After 1 hour of UV irradiation, the cells were observed under a microscope. Fig.17 As shown in (C) and (D) in the figure, the cells were then placed back in the 37°C, 5% CO2 conditions for 16 h, and the vesicles in the supernatant were collected and subjected to NTA and TEM tests.

[0367] 4.4. Particle size and morphology characterization

[0368] (1) NTA test analysis

[0369] The particle size analysis was performed using the NTA test method. The results can be found at Fig.16 and Table 7.

[0370] Fig.16 Nanoparticle tracking analysis (NTA) images of bispecific antibody-modified vesicles prepared for the Nalm6 cell line (A) and RAW264.7 cell line (B) in this example.

[0371] Table 7. NTA test results

[0372] Mother cells Source(Species) Diameter Nalm6 Homo sapiens 158.5nm RAW264.7 Mus musculus 151.2nm

[0373] (2) TEM characterization

[0374] Fig.17 Transmission electron microscopy (TEM) images of bispecific antibody-modified vesicles prepared using Nalm6 cell line (A) and RAW264.7 cell line (B) in this example.

[0375] According to Table 7, Fig.16 and Fig.17 The results show that the particle sizes of the vesicles produced by these two different mother cells are similar.

[0376] 4.4. Investigation of killing effect

[0377] Raji cells were used as target cells. Raji is a human Burkitt lymphoma cell line that highly expresses CD19 on its surface. The CD19 gene in wild-type Raji cells was knocked out using CRISPR Cas9 technology to obtain the Raji-CD19KO cell line as a negative control. The ratio of effector cells to target cells, E:T, is 5:1. Nalm6 and RAW264.7 cell lines were added as mother cells to produce CD19BiTE vesicles (CD19BiTE-EVs), and the concentrations are shown in Table 8. The specific killing effect of CD19BiTE-EVs was detected by a GFP-based real-time cytotoxicity assay. The horizontal axis (X-axis) represents the time axis, and the vertical axis (Y-axis) represents the standardized green fluorescence intensity (i.e., reflecting the number of target cells). The test results are consistent with Fig.10 Similarly, in this example, the Spontanous group was also set up as a target cell culture group.

[0378] Table 8.

[0379]

[0380] Fig.19 The specific killing effect test results of the bispecific antibody modified vesicles CD19 BiTE-EV prepared for the Nalm6 cell line and the RAW264.7 cell line, using the Raji cell line. It can be seen that when the vesicles derived from different mother cells are modified with BiTE-EV targeting the CD19 antigen, the specific killing effect on tumors is similar when the BiTE concentration is close.

[0381] Example 5. Calculation of binding density of bispecific antibodies

[0382] Experimental methods

[0383] Step (a): obtaining a standard curve of the fluorescence value-concentration of the fluorescent marker GFP by using a GFP fluorescent protein standard solution (0 nM, 1 nM, 3 nM, 5 nM, 8 nM), and obtaining a corresponding fitting equation by using binomial curve fitting;

[0384] Step (b): Taking CD19×CD3 BiTE as an example, construct the amino acid sequence of BiTE-TM-GFP fusion protein (connect the N-terminus of fluorescent marker protein GFP to the C-terminus of transmembrane domain TM so that each BiTE unit carries a GFP molecule). Prepare bispecific antibody modified vesicles (denoted as BiTE-TM-GFP-EV) according to the method of Example 3 (after UVB irradiation for 1 hour, culture in a 37°C incubator for 18 hours, and then collect vesicles), and prepare PBS mixed solution A containing bispecific antibody modified vesicles.

[0385] Step (c): Calculate the concentration C of the bispecific antibody unit (BiTE unit) in the mixed solution A using the standard curve fitting equation of the fluorescence value-concentration of GFP BsAb .

[0386] Step (d): Determine the concentration C of the bispecific antibody-modified vesicles in the mixed solution A by using nanoparticle tracking analysis (NTA) BsAb-EV .

[0387] Step (e): According to C BsAb and C BsAb-EV The average number of bispecific antibody units N bound to a bispecific antibody modified vesicle was calculated. BsAb@EV＝ C BsAb / C BsAb-EV .

[0388] 5.2. Conversion process

[0389] Step (a): Obtain a standard curve of the fluorescence value-concentration of the fluorescent marker GFP using GFP fluorescent protein standards (0 nM, 1 nM, 3 nM, 5 nM, 8 nM), such as Fig.18 and as shown in Table 9.

[0390] The fitting equation of fluorescence value and GPF concentration is: y = 16.668x 2 -12.75x (Formula I); Goodness of fit R 2 =

[0391] 0.9988. Where y is the fluorescence intensity (in RFU, relative fluorescence unit) and x is the concentration (in nM).

[0392] Table 9.

[0393] Concentration (nM) 0 1 3 5 8 Fluorescence intensity (RFU) 0 36 116 221 349

[0394] Step (b): Prepare bispecific antibody-modified vesicles according to the steps in Example 3, and determine the concentration C of bispecific antibody-modified vesicles in mixed solution A by NTA method. BsAb-EV =1.1×10 9 particles / mL.

[0395] Step (a): Take 10 μL of mixed solution A, which contains 1.1×10 EVs. 7 The fluorescence intensity is detected, and the concentration C of the bispecific antibody unit in the mixed solution A is calculated using the standard curve fitting equation (Formula I) of the fluorescence value-concentration of the fluorescent marker GFP. BsAb , as shown in Table 10, the results of three repeated independent experimental groups were used.

[0396] Step (d): Calculate the average number of bispecific antibody units bound to a bispecific antibody-modified vesicle:

[0397] N BsAb@EV＝ C BsAb / C BsAb-EV .

[0398] The results are shown in Table 10.

[0399] Step (e): From the Avogadro constant (6.02×10 23 / mol) to obtain the number of BiTE molecules contained in a unit amount of BiTE-EV = N BsAb@EV × Avogadro constant, the results are shown in Table 10.

[0400] Table 10.

[0401]

[0402] 5.3. Application of the assay method for measuring the binding density of bispecific antibodies

[0403] 5.3.1. Using Nalm6 cells as target cells

[0404] Fig. 9 In the example (Example 2), the number of EV particles was obtained by NTA detection. The results are shown in Table 11.

[0405] Table 11.

[0406]

[0407] In Table 11, n / a means Not applicable.

[0408] 5.3.2. Using Raji cells as target cells

[0409] Fig.10 In the example (Example 2), the relevant samples and relevant test results in Example 2 were used for analysis, and the number of EV particles was obtained by NTA detection. The results are shown in Table 12.

[0410] Table 12.

[0411]

[0412] In Table 12, n / a means Not applicable.

[0413] Table 13. Sequence summary

[0414]

[0415]

[0416] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0417] The above embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solution provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A bispecific antibody (BsAb) modified vesicle, characterized in that: The invention comprises a vesicle and a bispecific antibody bound to the surface of the vesicle; the bispecific antibody is composed of a first antigen binding domain, a connecting peptide, a second antigen binding domain, a hinge region and a transmembrane domain connected in series in sequence; wherein the bispecific antibody is bound to the membrane structure on the surface of the vesicle, and the transmembrane domain is embedded in the phospholipid bilayer on the surface of the vesicle and bound; Wherein, the first antigen binding domain is a CD19 antigen binding domain, and the second antigen binding domain is a CD3 antigen binding domain; The first antigen-binding domain comprises an anti-CD19 single-chain antibody variable region whose amino acid sequence is shown in SEQ ID No.: 1; the second antigen-binding domain comprises an anti-CD3 single-chain antibody variable region whose amino acid sequence is shown in SEQ ID No.: 3; the amino acid sequence of the connecting peptide is shown in SEQ ID No.: 4; the amino acid sequence of the hinge region is shown in SEQ ID No.: 5; and the amino acid sequence of the transmembrane domain is shown in SEQ ID No.:

6.

2. The bispecific antibody-modified vesicle according to claim 1, wherein: The binding density of the bispecific antibody on the vesicle surface is that the average number of AsAbs bound per square micrometer of the vesicle surface is greater than 100; and / or the diameter of the bispecific antibody-modified vesicle is 80 to 1000 nm.

3. The bispecific antibody-modified vesicle according to claim 2, wherein: The average diameter of the bispecific antibody-modified vesicles is 100 to 200 nm.

4. The bispecific antibody-modified vesicle according to claim 2, characterized in that: The bispecific antibody is a BiTE antibody.

5. The bispecific antibody-modified vesicle according to any one of claims 1 to 4, characterized in that: The vesicles are derived from one or more of mammalian cells and insect cells.

6. The bispecific antibody-modified vesicle according to claim 5, characterized in that: The vesicles are derived from one or more of HEK-293T cells, Nalm6 cells and RAW264.7 cells.

7. A genetically engineered cell, characterized in that: The genetically engineered cell is capable of endogenously secreting vesicles, and the genome of the genetically engineered cell is integrated with one or more of a coding sequence of a bispecific antibody and a nucleotide sequence of a nucleic acid, or the genetically engineered cell contains a recombinant vector, and the recombinant vector contains one or more of a coding sequence of a bispecific antibody and a nucleotide sequence of a nucleic acid; Wherein, the bispecific antibody is as defined in the bispecific antibody-modified vesicle according to any one of claims 1 to 4; the nucleotide sequence of the nucleic acid comprises a nucleotide sequence in at least one of the following groups: (i) a nucleotide sequence encoding the bispecific antibody; and (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i); Wherein, the surface of the vesicle has a phospholipid bilayer.

8. The genetically engineered cell according to claim 7, characterized in that The nucleic acid is DNA, mRNA or a combination thereof.

9. The genetically engineered cell according to claim 7, characterized in that A nucleotide sequence encoding a signal peptide is also included upstream of the coding sequence of the bispecific antibody.

10. The genetically engineered cell according to claim 9, characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID No.:

7.

11. The genetically engineered cell according to claim 7, characterized in that: The recombinant vector is one or more of a retroviral vector, a transient transfection vector and an adenoviral vector; wherein the retroviral vector includes a lentiviral vector.

12. The genetically engineered cell according to claim 7, characterized in that The genetically engineered cells are selected from one or more of mammalian cells and insect cells.

13. A method for preparing genetically engineered cells, characterized in that: The steps include: Providing a mother cell capable of endogenously secreting vesicles and providing a recombinant vector; wherein the surface of the vesicle has a phospholipid bilayer; the recombinant vector contains one or more of a coding sequence of a bispecific antibody and a nucleotide sequence of a nucleic acid; wherein the bispecific antibody is as defined in the bispecific antibody-modified vesicle according to any one of claims 1 to 4; the nucleotide sequence of the nucleic acid comprises a nucleotide sequence in at least one of the following groups: (i) a nucleotide sequence encoding the bispecific antibody; and (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i); The recombinant vector is transformed or transfected into the mother cell.

14. The method for preparing genetically engineered cells according to claim 13, characterized in that: The concentration of the mother cells is 1×10 5 / mL ~ 1×10 6 / mL, and the infection index MOI is 1~10.

15. The method for preparing genetically engineered cells according to claim 13, characterized in that: The mother cell is selected from one or more of mammalian cells and insect cells; Wherein, the mammalian cells include one or more of HEK-293T cells, Nalm6 cells and RAW264.7 cells.

16. A method for preparing a bispecific antibody-modified vesicle, characterized in that: The steps include: Provide the genetically engineered cell of claim 12 or the genetically engineered cell prepared by the preparation method of any one of claims 13 to 15; wherein the genetically engineered cell comprises a first coding sequence of the bispecific antibody in the bispecific antibody-modified vesicle of any one of claims 1 to 4, and further comprises a second coding sequence located upstream of the first coding sequence, wherein the second coding sequence encodes a signal peptide; The genetically engineered cells are cultured to obtain the bispecific antibody-modified vesicles.

17. The method for preparing a bispecific antibody-modified vesicle according to claim 16, characterized in that: Possess one or more of the following characteristics: (t1) the step of culturing the genetically engineered cells comprises the following process: the genetically engineered cells secrete vesicles under exogenous stimulation conditions, synthesize bispecific antibodies with a signal peptide at the C-terminus, the C-terminus of the bispecific antibody is embedded into the phospholipid bilayer on the surface of the genetically engineered cells under the mediation of the signal peptide, the genetically engineered cells secrete vesicles in a budding manner, and the signal peptide at the C-terminus of the bispecific antibody is removed; (t2) The genetically engineered cells are cultured under ultraviolet light, the wavelength of the ultraviolet light is 280-315 nm, and the irradiation intensity is 300-400 J / m 2 ;and, (t3) The amino acid sequence of the signal peptide is shown in SEQ ID No.:

7.

18. A use of the bispecific antibody-modified vesicle according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating precursor B-cell leukemia or Burkitt's lymphoma, or A use of the genetically engineered cell according to any one of claims 7 to 12 in the preparation of a medicament for preventing and / or treating precursor B-cell leukemia or Burkitt's lymphoma, or A use of a genetically engineered cell prepared by the preparation method according to any one of claims 13 to 15 in the preparation of a drug for preventing and / or treating precursor B-cell leukemia or Burkitt's lymphoma, or A use of the bispecific antibody-modified vesicles prepared by the preparation method according to claim 16 or 17 in the preparation of a drug for preventing and / or treating precursor B-cell leukemia or Burkitt's lymphoma.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises at least one of the bispecific antibody-modified vesicles according to any one of claims 1 to 6 and the bispecific antibody-modified vesicles prepared by the preparation method according to claim 16 or 17; the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

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