Bispecific antibody targeting CD3 and FAP and application thereof

Abstract

The invention discloses a bispecific antibody targeting CD3 and FAP and an application of the bispecific antibody. The bispecific antibody comprises a CD3 antigen binding domain and an FAP antigen binding domain. The bispecific antibody or the vesicle / exosome containing the bispecific antibody has remarkable tumor cell toxicity and can effectively remove fibroblasts around a mouse wound.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceutical technology, and further to the field of antibody technology, and in particular to a bispecific antibody targeting CD3 and FAP and a modified vesicle or exosome thereof, 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 and a modified vesicle or exosome thereof, an application, and a pharmaceutical composition. Background Art

[0002] Tissue repair after surgery goes through three stages: inflammation, proliferation of granulation tissue, and remodeling of maturation and scar formation. Many risk factors can cause uncontrollable scar fibrosis around the surgery, leading to severe tissue adhesions and complications. For example, peritoneal adhesions after abdominal and pelvic surgery, epidural scar adhesions after spinal surgery, etc. Current treatment and prevention methods include controlling inflammation, biomaterial barriers, and inhibiting fibroblasts. Summary of the invention

[0003] Based on this, it is necessary to provide at least a bispecific antibody targeting CD3 and FAP and its application.

[0004] In a first aspect of the present application, a bispecific antibody is provided, comprising a CD3 antigen binding domain and a FAP antigen binding domain.

[0005] The CD3 antigen binding domain comprises a heavy chain variable region (CD3 VH) and a light chain variable region (CD3 VL); the CD3 VH comprises one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, respectively, and the CD3 VL comprises one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, respectively;

[0006] The FAP antigen binding domain comprises a heavy chain variable region (FAP VH) and a light chain variable region (FAP VL); the FAP VH comprises one or more of HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, respectively, and the FAP VL comprises one or more of LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

[0007] In the second aspect of the present application, 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:

[0008] (i) a nucleotide sequence encoding the bispecific antibody of the first aspect; and

[0009] (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i).

[0010] In the third aspect of the present application, a recombinant vector is provided, wherein the recombinant vector comprises the nucleic acid as described in the second aspect.

[0011] In a fourth aspect of the present application, a genetically engineered cell is provided, comprising one or more of the nucleic acid described in the second aspect and the recombinant vector described in the third aspect.

[0012] In a fifth aspect of the present application, a method for preparing the genetically engineered cell as described in the fourth aspect is provided, the method comprising the following steps:

[0013] Providing a mother cell capable of endogenously secreting at least one of vesicles and exosomes and providing the recombinant vector as described in the third aspect;

[0014] The recombinant vector is transformed or transfected into the mother cell.

[0015] In the sixth aspect of the present application, a bispecific antibody-modified vesicle or exosome as described in the first aspect is provided, wherein the vesicle or exosome is selected from one or both of the bispecific antibody-modified vesicle and the bispecific antibody-modified exosome.

[0016] In a seventh aspect of the present application, there is provided a method for preparing the vesicle or exosome as described in the sixth aspect, comprising the following steps:

[0017] Cultivating the genetically engineered cell as described in the fourth aspect to obtain a culture fluid;

[0018] The vesicles or exosomes are isolated from the culture fluid.

[0019] In an eighth aspect of the present application, there is provided use of the bispecific antibody as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant vector as described in the third aspect, the genetically engineered cell as described in the fourth aspect, or the vesicle or exosome as described in the sixth aspect in the preparation of a drug for preventing and treating fibrosis.

[0020] In the ninth aspect of the present application, a pharmaceutical composition is provided, comprising the bispecific antibody as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant vector as described in the third aspect, the genetically engineered cell as described in the fourth aspect, and at least one of the vesicles or exosomes as described in the sixth aspect, and a pharmaceutically acceptable carrier.

[0021] In one embodiment of the present application, in vitro experiments showed that the bispecific antibody of the present application or the vesicles / exosomes containing the same had significant tumor cell toxicity; in vivo experiments showed that it could effectively clear the fibroblasts around the wounds of mice.

[0022] In the tenth aspect of the present application, a method for treating fibrosis in a subject is provided, the method comprising: administering to the subject the bispecific antibody as described in the first aspect, the genetically engineered cells as described in the fourth aspect, or the vesicles or exosomes as described in the sixth aspect, or the pharmaceutical composition as described in the ninth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the implementation methods and examples 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 use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0024] Figure 1 This is a schematic diagram of the structure of a bispecific antibody in one embodiment of the present application; the connecting lines between different functional regions represent that the different functional regions are operably connected; wherein, "hinge" represents a hinge.

[0025] Figure 2 The figure shows the transfection efficiency of NIH / 3T3 transfected with the lentivirus prepared with structure 9 (A) and the GPF expression efficiency after sorting (B) in one embodiment of the present application.

[0026] Figure 3 This is a scanning electron microscope (SEM) image of the FAP BiTE EV produced using structure 9 in one embodiment of the present application; wherein, “particles” represents particles and “Diameter” represents diameter.

[0027] Figure 4The vector map in one embodiment of the present application is shown; wherein, "region" represents region, "partial gapsequence" represents partial gap sequence, "promoter" represents promoter, and "hinge" represents hinge.

[0028] Figure 5 The cytotoxicity of the FAP BiTE protein prepared with structures 4-7 as the active ingredient in one embodiment of the present application is shown; wherein "Concentration" represents concentration and "cytotoxicity" represents cytotoxicity.

[0029] Figure 6 The in vivo verification process in one embodiment of the present application is shown; wherein, "mouse" represents mice.

[0030] Figure 7 The subcutaneous tumor size of mice and the survival curve of mice in one embodiment of the present application are shown; "RelativeTumor volume" represents the relative tumor volume, "Days after tumor inoculation" represents the number of days after tumor inoculation, "Probability of Survival" represents the possibility of survival, and "Time" represents the time.

[0031] Figure 8 In one embodiment of the present application, CD25 + The proportion of T cells; "activated" represents activated; where, **p=0.005.

[0032] Fig. 9 A standard curve prepared using green fluorescent protein (GFP) as a standard substance in one embodiment of the present application is shown; wherein “Concentration” represents concentration. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0036] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0037] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0038] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0039] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0040] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0041] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0042] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0043] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the 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.

[0044] In the present application, 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 and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which 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 range 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 broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0045] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0046] In the present application, the term "antibody" is a protein that binds to a specific antigen, and it generally refers to all proteins and protein fragments that contain 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).

[0047] In this application, 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.

[0048] In the present application, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen.

[0049] In the present application, variants of antibodies are also within the scope of protection claimed in the present application, 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 application, 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 in the present application. "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 application, 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 is substantially unlikely to destroy the biological activity.

[0050] The inventors creatively combined the CD3 antigen binding domain and the FAP antigen binding domain to prepare a bispecific antibody and vesicles and exosomes modified with a specific form of the bispecific antibody BiTE, and applied the specific form of the bispecific antibody to the treatment of diseases, especially fibrosis, and showed good therapeutic effects, becoming a first in this field.

[0051] In a first aspect of the present application, a bispecific antibody is provided, comprising a CD3 antigen-binding domain and a FAP antigen-binding domain, wherein the CD3 antigen-binding domain comprises a heavy chain variable region (CD3 VH) and a light chain variable region (CD3VL); the CD3 VH comprises one or more of HCDR1, HCDR2 and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, respectively, and the CD3 VL comprises one or more of LCDR1, LCDR2 and LCDR3, whose amino acid sequences are shown in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, respectively;

[0052] The FAP antigen binding domain comprises a heavy chain variable region (FAP VH) and a light chain variable region (FAP VL); the FAP VH comprises one or more of HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, respectively, and the FAP VL comprises one or more of LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

[0053] In this application, the term "bispecific antibody", which can be abbreviated 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.

[0054] In some embodiments, the CD3 VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, respectively, and the CD3 VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13, respectively;

[0055] The FAP VH comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, respectively, and the FAP VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

[0056] In some embodiments, the CD3 antigen binding domain comprises a VH with an amino acid sequence as shown in SEQ ID NO: 1, and comprises a VL with an amino acid sequence as shown in SEQ ID NO: 45;

[0057] The FAP antigen binding domain comprises a VH having an amino acid sequence as shown in SEQ ID NO:15, and comprises a VL having an amino acid sequence as shown in SEQ ID NO:17.

[0058] The sequence information of CDR is shown in Table 1 below.

[0059] Table 1

[0060]

[0061] In the present application, the FAP VL, FAP VH, CD3 VH and CD3 VL of the bispecific antibody can be operably linked in any order, as long as the resulting bispecific antibody can at least have the ability to simultaneously target FAP and CD3.

[0062] Exemplarily, in some embodiments, from N-terminus to C-terminus, the bispecific antibody comprises FAP VL, FAP VH, CD3 VH and CD3 VL that are operably linked in sequence.

[0063] In other embodiments, from N-terminus to C-terminus, the bispecific antibody comprises CD3 VH, FAP VL, FAP VH and CD3 VL operably linked in sequence.

[0064] In other embodiments, from N-terminus to C-terminus, the bispecific antibody comprises CD3 VH, FAPVH, FAP VL and CD3 VL operably linked in sequence.

[0065] In other embodiments, from N-terminus to C-terminus, the bispecific antibody comprises FAP VH, FAP VL, CD3 VH and CD3 VL operably linked in sequence.

[0066] In other embodiments, from N-terminus to C-terminus, the bispecific antibody comprises FAP VL, FAP VH, CD3 VH and CD3 VL operably linked in sequence.

[0067] Unless otherwise specified, the term "operably linked" in the present application refers to the functional relationship between different regions of a protein (e.g., a bispecific antibody), such as CD3 VH, FAP VH, FAP VL, and CD3 VL; wherein the different regions are linked to produce a protein.

[0068] Different regions, such as CD3 VH, FAP VH, FAP VL and CD3 VL, can be connected by a flexible connecting peptide (or linker). An exemplary flexible connecting peptide may be GGGGS, etc.

[0069] Without wishing to be bound by any theory, it is believed that the bispecific antibody prepared by connecting different VH and VL in the above manner can better target CD3 and FAP, thereby exerting its ability to kill target cells.

[0070] In some embodiments, the amino acid sequence of the bispecific antibody comprises the sequence shown in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39.

[0071] In some embodiments, the amino acid sequence of the bispecific antibody is shown in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39.

[0072] The bispecific antibody in the bispecific antibody of the present application is a humanized antibody or a human antibody.

[0073] In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTE).

[0074] In this application, 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.

[0075] In some embodiments, the BiTE further comprises a CD8 hinge and a CD8 transmembrane region. In some embodiments, the BiTE comprises, from the N-terminus to the C-terminus, a bispecific antibody as defined above, a CD8 hinge and a CD8 transmembrane region.

[0076] In the present application, 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.

[0077] The CD8 hinge in the present application may be a CD8 hinge commonly used in the art. In some embodiments, the amino acid sequence of the CD8 hinge comprises the sequence shown in SEQ ID NO:41.

[0078] The CD8 transmembrane region in the present application may be a CD8 transmembrane region commonly used in the art. In some embodiments, the amino acid sequence of the CD8 transmembrane region comprises a sequence as shown in SEQ ID NO:43.

[0079] In some embodiments, the BiTE further comprises GFP. In some embodiments, the amino acid sequence of GFP is shown in SEQ ID NO:31.

[0080] In the second aspect of the present application, 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:

[0081] (i) a nucleotide sequence encoding the bispecific antibody of the first aspect; and

[0082] (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i).

[0083] In some embodiments, the nucleotide sequence of the nucleic acid comprises a sequence as shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 or SEQ ID NO:40.

[0084] In the third aspect of the present application, a recombinant vector is provided, wherein the recombinant vector comprises the nucleic acid as described in the second aspect.

[0085] In the present application, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a 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 described in the present application 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.).

[0086] In this application, the term "recombinant vector" refers to an artificially constructed vector.

[0087] In this application, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) is operatively inserted and the genetic element is expressed. A vector can be, for example, a plasmid, a cosmid, a virus (e.g., a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus), an RNA vector, or a linear or circular DNA or RNA molecule, which can include chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acid molecules. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked.

[0088] 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.

[0089] In a fourth aspect of the present application, a genetically engineered cell is provided, comprising one or more of the nucleic acid described in the second aspect and the recombinant vector described in the third aspect.

[0090] In the present application, the term "genetically engineered cell" may refer to a genetically engineered cell obtained through genetic modification.

[0091] In some embodiments, the host cell of the genetically engineered cell can be used to synthesize the bispecific antibody described in the first aspect of this application.

[0092] In some embodiments, the genetically engineered cells are capable of endogenously secreting at least one of vesicles and exosomes, wherein the vesicles have a phospholipid bilayer structure.

[0093] In some embodiments, the host cell is a eukaryotic cell.

[0094] In some embodiments, the host cell is a mammalian cell.

[0095] In some embodiments, host cells suitable for expressing the bispecific antibodies described herein 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.

[0096] In some embodiments, the host cell is a mammalian cell or an avian cell.

[0097] 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.

[0098] In some embodiments, the host cell is a rodent cell, such as a cell derived from a rat, a mouse, or a hamster.

[0099] In some embodiments, the host cell is a primate cell, such as a human cell.

[0100] 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.

[0101] In some embodiments, the host cell is derived from HEK-293T cells (human embryonic kidney 293 cells).

[0102] In some embodiments, the host cell is derived from one or more of HEK-293T cells (human embryonic kidney 293 cells), CHO cells, Nalm6 cells, NIH / 3T3 cells and RAW264.7 cells.

[0103] In a fifth aspect of the present application, a method for preparing the genetically engineered cell as described in the fourth aspect is provided, the method comprising the following steps:

[0104] Providing a mother cell capable of endogenously secreting at least one of vesicles and exosomes and providing the recombinant vector as described in the third aspect;

[0105] The recombinant vector is transformed or transfected into the mother cell.

[0106] In some embodiments, the mother cell is selected from one or more of mammalian cells, insect cells, and the like.

[0107] In some embodiments, the mammalian cells may include one or more of HEK-293T cells, Nalm6 cells, RAW264.7 cells, and the like.

[0108] In some embodiments, the mother cell is a HEK-293T cell. Reference may be made to some specific embodiments below.

[0109] 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.

[0110] 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.

[0111] In some embodiments, the concentration of the mother cells can be 1×10 5 / mL~5×10 6 The concentration of the mother cells may be 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.

[0112] In the sixth aspect of the present application, a bispecific antibody-modified vesicle or exosome as described in the first aspect is provided, wherein the vesicle or exosome is selected from one or both of the bispecific antibody-modified vesicle and the bispecific antibody-modified exosome.

[0113] In this application, the term "vesicle" refers to a class of molecular ordered assemblies with a closed double-layer structure that spontaneously form when dispersed in water. Unless otherwise specified, the "vesicle" used in this application 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.

[0114] In the present application, unless otherwise specified, "bispecific antibody-modified vesicles" are nanoparticles stably bound by the transmembrane domain of the bispecific antibody embedded in the phospholipid bilayer of the vesicle.

[0115] In this application, “bispecific antibody-modified vesicles” and “BsAb-EVs” have the same meaning and can be used interchangeably.

[0116] In some embodiments, the binding density of the bispecific antibody on the surface of the vesicle is such that the average number of bispecific antibodies bound to the surface of each vesicle or exosome is at least (1 to 6)×10 3 The binding density of the bispecific antibody on the surface of the vesicle can be measured by conventional testing methods in the art. Exemplarily, the method may include the following steps:

[0117] Obtain a standard curve of the fluorescence value-concentration of the fluorescent marker (e.g., using green fluorescent protein (GFP) as a standard, using an ELISA reader to detect the relative fluorescence intensity (RFU) and draw a standard curve);

[0118] Preparing a mixed solution A containing the bispecific antibody-modified vesicles, wherein each bispecific antibody unit carries one of the fluorescent markers;

[0119] 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 ;

[0120] 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 .

[0121] The fluorescent marker used in the above steps may also be red fluorescent protein (RFP), luciferase and other fluorescent markers commonly used in the art.

[0122] In some embodiments, a fluorescent protein can be fused to the C-terminus of the bispecific antibody of the first aspect of the present application, so that the bispecific antibody contains a fluorescent protein and is fluorescently labeled. Exemplarily, the BiTE in the first aspect has an amino acid sequence as shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 or SEQ ID NO:40.

[0123] In some embodiments, the diameter of the bispecific antibody modified vesicle can be 20nm to 300nm, further can be 50nm to 250nm, and further can be 100nm to 150nm. In some embodiments, the diameter of the bispecific antibody modified vesicle is 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, or a range or value between any two values.

[0124] In some embodiments, the bispecific antibody-modified vesicles may contain other drug components, which may be referred to as the second drug component.

[0125] 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.

[0126] In some embodiments, the vesicles are derived from one or more of mammalian cells, insect cells, etc.

[0127] In some embodiments, the vesicles are derived from one or more of HEK-293T cells, Nalm6 cells, and RAW264.7 cells.

[0128] In some embodiments, the vesicles are derived from HEK-293T cells.

[0129] In a seventh aspect of the present application, there is provided a method for preparing the vesicle or exosome as described in the sixth aspect, comprising the following steps:

[0130] Cultivating the genetically engineered cell as described in the fourth aspect to obtain a culture fluid;

[0131] The vesicles or exosomes are isolated from the culture fluid.

[0132] In some embodiments, the vesicle is isolated using a method comprising the steps of:

[0133] The culture fluid is centrifuged, filtered, and centrifuged at a high speed of more than 100,000 g to remove any one or more of dead cells, cell debris, macromolecular proteins, and non-exosome extracellular microvesicles to prepare a supernatant;

[0134] The supernatant is clarified, and the clarified supernatant is treated by tangential flow filtration technology to separate the vesicles.

[0135] In some embodiments, the exosomes are isolated using a method comprising the steps of:

[0136] Centrifuge the culture solution at 300 g to 500 g for 5 min to 10 min, filter, and prepare a supernatant;

[0137] The supernatant is centrifuged at 15000g to 18000g for 1h to 1.5h, the precipitate is collected, and the exosomes are isolated.

[0138] In an eighth aspect of the present application, there is provided use of the bispecific antibody as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant vector as described in the third aspect, the genetically engineered cell as described in the fourth aspect, or the vesicle or exosome as described in the sixth aspect in the preparation of a drug for preventing and treating a disease.

[0139] In some embodiments, the disease is selected from the group consisting of fibrosis, scar, and tumor.

[0140] In some embodiments, the fibrosis is peri-surgical scar fibrosis induced by surgical operation.

[0141] In the present application, "prevention" includes aspects such as prevention, treatment, and adjuvant therapy. As used herein, "prevention" refers to alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or condition. "Prevention" also includes curing one or more symptoms of a disease or condition, preventing its development, or alleviating to a certain extent. In the present application, "prevention" has the same meaning as "prevention and / or treatment", "prevention, treatment, or prevention and treatment", and can be used interchangeably.

[0142] In this application, "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.

[0143] In the ninth aspect of the present application, a pharmaceutical composition is provided, comprising the bispecific antibody as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant vector as described in the third aspect, the genetically engineered cell as described in the fourth aspect, and at least one of the vesicles or exosomes as described in the sixth aspect, and a pharmaceutically acceptable carrier.

[0144] In the present application, a "composition" may be a combination of multiple substances, and further, may be used in combination, or may be a mixture of the combination.

[0145] In the present application, "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.

[0146] In some embodiments, the pharmaceutical composition is a drug.

[0147] In the present application, "drug" refers to a pharmaceutical preparation that can be administered directly, which usually has a prescribed usage and dosage.

[0148] In this application, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, used in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0149] In this application, "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.

[0150] In the present application, 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.

[0151] 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.

[0152] 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.

[0153] In some embodiments, the pharmaceutically acceptable carrier includes one or more of a buffer, an adjuvant, a diluent, or a combination thereof.

[0154] It should be understood that the drug contains a therapeutically effective amount of the bispecific antibody-modified vesicles of the present application.

[0155] In the present application, "therapeutically effective amount" refers to the amount of a pharmaceutically active 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 application that brings a pharmacologically positive effect to an individual, wherein the pharmacologically positive effect includes but is not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression or preventing disease, etc.

[0156] In the present application, "subject" refers to a patient who takes a pharmaceutical composition.

[0157] In the present application, "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 application 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.

[0158] In some embodiments, the subject is a mammal.

[0159] In some embodiments, the pharmaceutical composition is suitable for humans or other mammals. In the present application, "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.

[0160] In some embodiments, the subject is a human, mouse, or rat.

[0161] In the present application, "patient" refers to an animal, preferably a mammal, such as a human, a mouse, or a rat.

[0162] Different subjects may result in different selection ranges of other ingredients in the aforementioned pharmaceutical composition.

[0163] In some embodiments, the administration of the drug includes, but is not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection.

[0164] 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.

[0165] 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.

[0166] It is to be understood that the drugs of the embodiments of the present application 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.

[0167] Some examples are provided below.

[0168] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0169] Example 1 Construction of BiTE structure

[0170] Based on anti-hCD3 VH, anti-hCD3 VL, anti-hFAP VH and anti-hFAP VL, structures 1 to 4 were constructed according to different connection orders.

[0171] hCD8 hinge-hCD8 TM and hCD8 hinge-hCD8 TM-GFP were added to the C-termini of structures 1 to 4, respectively, to construct structures 5 to 12.

[0172] The structural diagrams of structures 3 to 12 are shown in Figure 1 The amino acid sequences and nucleic acid sequence numbers of structures 3 to 12 and the elements constituting them are shown in Table 2.

[0173] Table 2

[0174] Components / Structure Amino acid sequence Nucleic acid sequence anti-hCD3 VH SEQ ID NO:1 SEQ ID NO:2 anti-hCD3 VL SEQ ID NO:45 SEQ ID NO:46 anti-hFAP VH SEQ ID NO:15 SEQ ID NO:16 anti-hFAP VL SEQ ID NO:17 SEQ ID NO:18 anti-hCD3 HCDR1 SEQ ID NO:3 SEQ ID NO:4 anti-hCD3 HCDR2 SEQ ID NO:5 SEQ ID NO:6 anti-hCD3 HCDR3 SEQ ID NO:7 SEQ ID NO:8 anti-hCD3 LCDR1 SEQ ID NO:9 SEQ ID NO:10 anti-hCD3 LCDR2 SEQ ID NO:11 SEQ ID NO:12 anti-hCD3 LCDR3 SEQ ID NO:13 SEQ ID NO:14 anti-hFAP HCDR1 SEQ ID NO:19 SEQ ID NO:20 anti-hFAP HCDR2 SEQ ID NO:21 SEQ ID NO:22 anti-hFAP HCDR3 SEQ ID NO:23 SEQ ID NO:24 anti-hFAP LCDR1 SEQ ID NO:25 SEQ ID NO:26 anti-hFAP LCDR2 SEQ ID NO:27 SEQ ID NO:28 anti-hFAP LCDR3 SEQ ID NO:29 SEQ ID NO:30 hCD8 hinge SEQ ID NO:41 SEQ ID NO:42 hCD8 TM SEQ ID NO:43 SEQ ID NO:44 Structure 1 SEQ ID NO:33 SEQ ID NO:34 Structure 2 SEQ ID NO:35 SEQ ID NO:36 Structure 3 SEQ ID NO:37 SEQ ID NO:38 Structure 4 SEQ ID NO:39 SEQ ID NO:40 GFP SEQ ID NO:31 SEQ ID NO:32

[0175] Example 2 BiTE EV / exo preparation process

[0176] i. Establishment of mother cells

[0177] The plasmid containing structures 5-12 constructed in Example 1 is used to edit primary cells or cell lines, and the editing method can be stable or transient.

[0178] Next, the lentivirus prepared with structure 6 (see Figure 4 ) is used to transfect the mouse embryonic fibroblast cell line NIH / 3T3 as an example to describe the specific process of establishing mother cells:

[0179] The culture density of NIH / 3T3 is 1e6 / T175, and the transfection conditions are 3e4 cells in a 24-well plate, 1mL system; 100μL lentivirus + 400μL D10F transfection after 24h; subculture after 24h. Figure 2 As shown in A.

[0180] To further increase the density of bispecific antibodies on the surface of each EV / exo, high-expressing mother cell clones were further sorted by flow cytometry. The sorting conditions were as follows: 1e7 cells were prepared and resuspended in 300 μL of 1% FBS / PBS containing 2% bispecific antibodies, and the top 10% of cells in the MFI of the GFP channel were sorted by flow cytometry. The GFP expression efficiency after sorting was as follows Figure 2 As shown in B.

[0181] The parent cell line was named NIH / 3T3 FAP BiTE , the mother cell line can continuously secrete BiTE EVs and exo.

[0182] ii. Mother cells produce EV / exo

[0183] NIH / 3T3 FAP BiTE For EV production, 4e6 mother cells were cultured in a 15 cm cell culture dish at 37°C and 5% CO. 2 Culture statically. After 30 hours, replace the supernatant with serum-free DMEM, open the culture dish under sterile conditions, irradiate with UV for 1 hour, continue to culture for 4 hours, and then collect the cell culture supernatant. First, centrifuge at 300g for 10 minutes, then filter with a 0.22μm filter or filter membrane, and then centrifuge at 18000g, 4℃, for 1 hour. The precipitate is FAP BiTE EV.

[0184] NIH / 3T3 FAP BiTE Taking the production of exo as an example, 4e6 mother cells were cultured in a 15cm cell culture dish, and the supernatant was replaced with serum-free DMEM after 30 hours. The cell culture supernatant was collected after 24 hours. When collecting, the culture medium should be gently aspirated to avoid disturbing the cell layer to reduce the mixing of cell debris. First, centrifuge at 300g for 10 minutes, centrifuge at 2000g for 10 minutes to remove dead cells, and centrifuge at 10000g for 30 minutes to remove cell debris and large molecular proteins, filter with a 0.22μm filter or filter membrane, and then centrifuge at 100000g for 2 hours for further purification. Finally, the extracellular vesicles in the clarified culture supernatant are purified using tangential flow filtration (TFF) technology.

[0185] Structural characterization:

[0186] Nanoparticle Tracking Analysis (NTA) and scanning electron microscopy (SEM) were used to detect the FAP BiTE EV produced using structure 6. The results are shown in Figure 3 , showing that the average particle size is 20nm~300nm and the morphology is consistent.

[0187] Take the anti-hFAP BiTE EV and anti-hFAP BiTE exo produced by structure 12 as an example.

[0188] Green fluorescent protein (GFP) was used as the standard, and the relative fluorescence intensity (RFU) was detected using an ELISA reader, and a standard curve was drawn ( Fig. 9 ).

[0189] NTA was used to detect the number of EV or exosomes.

[0190] Sample 1 is anti-hFAP BiTE EV. The number of EV particles contained in the 100μL sample is 3E9 and the BiTE content is 8pmol.

[0191] Number of BiTE molecules in sample 1 = 8 × 10 -12 mol×6.022×10 23 molecules / mol = 4.818 × 10 12

[0192] The number of BiTE molecules on the surface of a single EV particle is: 4.818×10 12 ÷3 9 =1.61 3 indivual

[0193] Sample 2 is anti-hFAP BiTE exo. The number of exo particles contained in the 100 μL sample is 1E9 and the BiTE content is 5 pmol.

[0194] Number of BiTE molecules in sample 2 = 5 × 10 -12 mol×6.022×10 23 molecules / mol = 3.011 × 10 12

[0195] The number of BiTE molecules on the surface of a single EV particle is: 3.011×10 12 ÷1 9 =3.01 3 indivual.

[0196] Example 3 BiTE protein preparation process

[0197] Lentiviral packaging:

[0198] (1) Cell culture preparation: 18 to 24 hours before transfection, culture 8×10 6 Place HEK-293T cells in a T175 culture flask and incubate at 37°C overnight. The cells should reach 70% to 80% confluence within 24 hours.

[0199] (2) Transfection of HEK-293T cells: Prepare solution A and solution B. Solution A includes 3 ml culture medium + 30 μL HEPES. After thorough vortexing, add 90 μL lipo2000 and incubate at room temperature for 10 min. Solution B includes 43 μg packaging plasmid + 15 μg target plasmid. After 10 minutes, mix solution A and solution B and incubate at room temperature for 25-30 min. After 30 min, add the mixture dropwise to the cell culture flask and return the culture flask to the 37°C cell culture incubator.

[0200] (3) Collecting viral supernatant: After incubation for 12 to 18 hours, replace the culture medium and continue culturing for 48 hours.

[0201] (4) Transfer the cell culture supernatant to a 15 mL or 50 mL centrifuge tube, centrifuge at 3000 × g for 15 minutes, and filter the supernatant through a filter.

[0202] Protein preparation process

[0203] After the plasmids of structures 1 to 4 were packaged into lentivirus according to the above steps, they were used to infect cell lines (HEK293, purchased from ATCC) (1e6 HEK293 cells were cultured in a 6-well cell culture plate, 200 μL of lentivirus was added, and the cells were incubated at 37°C and 5% CO 2 After 24 hours, the medium was changed. The cells were the edited HEK293 cell line, which could continuously secrete BiTE protein. The cell culture supernatant was collected and the effective concentration of BiTE was increased by protein purification.

[0204] Experimental example: FAP BiTE protein / EV / exo mobilizes T cells to specifically kill target cells

[0205] i. In vitro validation

[0206] Mouse or human cell lines expressing FAP were used as target cells (K562-muFAP, K562-huFAP) to verify the function of FAP BiTE protein / EV / exo prepared in Examples 2 and 3. Specifically, peripheral blood T cells from mice or humans were co-cultured with tumor cells.

[0207] Taking a 96-well plate as an example, the co-culture conditions are: 10,000 target cells / well, 100,000 T cells / well, different concentrations of FAP BiTE protein / EV / exo were added to each well, and the total volume was 200 μL. The specific killing effect was detected by a luciferase-based cytotoxicity assay. The results are shown in the figure. Figure 5 The active ingredients are shown to be FAP BiTE proteins prepared from structures 1 to 4, and the volumes are divided into -, +, ++, +++ (0, 1 nM, 5 nM, 10 nM).

[0208] ii. In vivo validation

[0209] The applicant found that during the repair of mouse tissue damage, FAP + Fibroblasts are enriched around wounds. Taking structure 6 as an example, FAP BiTE EVs produced can effectively clear FAP + Fibroblasts. Collect and flow cytometry sort FAP cells from lymphoma patients + Fibroblasts (1e6) were cultured in vitro and then mixed with Raji cell line (1e6) and inoculated subcutaneously into NCG of immunodeficient mice. After inoculation of cells, human peripheral blood T cells (2e6) were given, and then FAP BiTE EV (1E9particle) or corresponding control (wt EV) were continuously given. The experimental process is as follows Figure 6 The subcutaneous tumor size of the two groups of mice and the survival curve of the mice are shown in Figure 7 As shown, the growth rate of subcutaneous tumors in mice treated with FAP BiTE EV was significantly inhibited and the survival time was significantly prolonged.

[0210] In the above experiments, peripheral blood cells of mice were collected and CD25 + The proportion of T cells (activated T cells) in the FAP BiTE EV group was significantly increased ( Figure 8 ).

[0211] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments 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.

[0212] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A bispecific antibody, characterized in that: It comprises a CD3 antigen binding domain and a FAP antigen binding domain, The CD3 antigen binding domain comprises a heavy chain variable region (CD3 VH) and a light chain variable region (CD3 VL); the CD3 VH comprises one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, respectively, and the CD3 VL comprises one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, respectively; The FAP antigen binding domain comprises a heavy chain variable region (FAP VH) and a light chain variable region (FAP VL); the FAPVH comprises one or more of HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, respectively, and the FAP VL comprises one or more of LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

2. The bispecific antibody according to claim 1, characterized in that The CD3 VH comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, respectively, and the CD3 VL comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13, respectively; The FAP VH comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, respectively, and the FAP VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

3. The bispecific antibody according to claim 2, characterized in that The CD3 antigen binding domain comprises a VH with an amino acid sequence as shown in SEQ ID NO: 1, and comprises a VL with an amino acid sequence as shown in SEQ ID NO: 45; The FAP antigen binding domain comprises a VH having an amino acid sequence as shown in SEQ ID NO:15, and comprises a VL having an amino acid sequence as shown in SEQ ID NO:

17.

4. The bispecific antibody according to any one of claims 1 to 3, wherein The bispecific antibody satisfies any one of the following features (i) to (iv): (i) from the N-terminus to the C-terminus, the bispecific antibody comprises FAP VL, FAP VH, CD3 VH and CD3 VL that are operably linked in sequence; (ii) from the N-terminus to the C-terminus, the bispecific antibody comprises CD3 VH, FAP VL, FAP VH and CD3 VL that are operably linked in sequence; (iii) from the N-terminus to the C-terminus, the bispecific antibody comprises CD3 VH, FAPVH, FAP VL and CD3 VL that are operably linked in sequence; from the N-terminus to the C-terminus, the bispecific antibody comprises FAP VH, FAP VL, CD3 VH and CD3 VL that are operably linked in sequence; (iv) from the N-terminus to the C-terminus, the bispecific antibody comprises FAP VL, FAPVH, CD3 VH and CD3 VL that are operably linked in sequence; Optionally, the amino acid sequence of the bispecific antibody comprises a sequence as shown in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:

39.

5. The bispecific antibody according to any one of claims 1 to 4, wherein It is a murine antibody, a humanized antibody or a human antibody.

6. The bispecific antibody according to any one of claims 1 to 5, wherein It is a bispecific T cell engager (BiTE).

7. The bispecific antibody according to claim 6, characterized in that The BiTE also comprises a CD8 hinge and a CD8 transmembrane region; Wherein, the amino acid sequence of the CD8 hinge optionally comprises the sequence shown in SEQ ID NO:41; the amino acid sequence of the CD8 transmembrane region optionally comprises the sequence shown in SEQ ID NO:

43.

8. A nucleic acid, characterized in that 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 according to any one of claims 1 to 7; and (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i); Optionally, the nucleic acid is DNA, mRNA or a combination thereof.

9. The nucleic acid according to claim 8, characterized in that The nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 or SEQ ID NO:

40.

10. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid according to claim 8 or 9; Optionally, the recombinant vector is one or more of a lentiviral vector, a retroviral vector, a transient transfection vector and an adenoviral vector.

11. A genetically engineered cell, characterized in that: It comprises one or more of the nucleic acid according to claim 8 or 9 and the recombinant vector according to claim 10; Optionally, the nucleic acid is integrated into the genome of the genetically engineered cell.

12. The genetically engineered cell according to claim 11, wherein It is capable of endogenously secreting at least one of vesicles and exosomes; Optionally, the vesicle has a phospholipid bilayer structure.

13. The genetically engineered cell according to claim 11 or 12, characterized in that The genetically engineered cells are selected from one or more of mammalian cells and insect cells; Optionally, the mammalian cells are selected from one or more of HEK-293T cells, Nalm6 cells, NIH / 3T3 cells and RAW264.7 cells.

14. The method for preparing a genetically engineered cell according to any one of claims 11 to 13, characterized in that: The steps include: Providing a mother cell capable of endogenously secreting at least one of vesicles and exosomes and providing the recombinant vector as claimed in claim 10; Transforming or transfecting the recombinant vector into the mother cell; Wherein, the vesicle optionally has a phospholipid bilayer structure.

15. A bispecific antibody-modified vesicle or exosome according to any one of claims 1 to 7, characterized in that: The vesicle or exosome is selected from one or both of the vesicle modified with the bispecific antibody and the exosome modified with the bispecific antibody; Optionally, the bispecific antibody is bound to the surface of the vesicle or the exosome; Optionally, the vesicles or the exosomes are secreted from the genetically engineered cells according to any one of claims 11 to 13; Optionally, any of the bispecific antibodies is independently a BiTE; Optionally, the binding density of the bispecific antibody on the surface of the vesicle or the exosome is at least (1-6)×10 3 indivual; Optionally, the diameter of the vesicle or the exosome is 20 nm to 300 nm.

16. A method for preparing vesicles or exosomes according to claim 15, characterized in that: It includes the following steps: Cultivating the genetically engineered cell according to any one of claims 11 to 13 to obtain a culture solution; isolating the vesicles or exosomes from the culture fluid; Optionally, the vesicles are isolated using a method comprising the steps of: The culture fluid is centrifuged, filtered, and centrifuged at a high speed of more than 100,000 g to remove any one or more of dead cells, cell debris, macromolecular proteins, and non-exosome extracellular microvesicles to prepare a supernatant; Clarifying the supernatant, treating the clarified supernatant with tangential flow filtration technology to separate the vesicles; Optionally, the exosomes are isolated using a method comprising the following steps: Centrifuge the culture solution at 300 g to 500 g for 5 min to 10 min, filter, and prepare a supernatant; The supernatant is centrifuged at 15000g-18000g for 1h-1.5h, the precipitate is collected, and the exosomes are separated.

17. Use of the bispecific antibody according to any one of claims 1 to 7, the nucleic acid according to claim 8 or 9, the recombinant vector according to claim 10, the genetically engineered cell according to any one of claims 11 to 13, or the vesicle or exosome according to claim 15 in the preparation of a drug for preventing and treating a disease; Optionally, the disease is selected from the group consisting of fibrosis, scar and tumor; in, The fibrosis may optionally be peri-surgical scar fibrosis induced by surgery.

18. A pharmaceutical composition, characterized in that It comprises the bispecific antibody according to any one of claims 1 to 7, the nucleic acid according to claim 8 or 9, the recombinant vector according to claim 10, the genetically engineered cell according to any one of claims 11 to 13, and at least one of the vesicle or exosome according to claim 15, and a pharmaceutically acceptable carrier; Optionally, the pharmaceutically acceptable carrier comprises one or more of a buffer, an adjuvant, a diluent or a combination thereof.

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