Bispecific antibodies and uses thereof

Abstract

There is provided a bispecific antigen binding molecule or fragment thereof comprising a first antigen binding domain and a second antigen binding domain wherein the first antigen binding domain is for specific binding to a cell surface antigen; the second antigen binding domain is used for specifically binding to vesicular oral membrane virus glycoprotein (VSV-G). The bispecific antigen binding molecule or the fragment thereof can efficiently mediate VSV-G lentivirus to infect cells which do not express a VSV-G receptor LDLR (Low Density Lipoprotein Receptor).

Description

Bispecific antibodies and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2023101060920 filed on February 13, 2023, and this application cites the full text of the above-mentioned Chinese patent application. Technical Field

[0003] The present disclosure belongs to the field of immunology, and specifically relates to a bispecific antibody and its application. Background Art

[0004] In recent years, gene therapy has entered a golden age of rapid growth. Gene therapy aims to cure diseases or enhance disease resistance by replacing defective genes or adding new ones, and holds promise for treating a range of conditions, including cancer, cystic fibrosis, heart disease, diabetes, hemophilia, and HIV. With the increasing maturity of delivery systems used in gene therapy, the barriers to drug translation are being overcome. The most common delivery system is viral vectors, which can enter cells by specifically recognizing cell surface receptors and deliver their genetic material to targeted cells. To overcome potential risks, researchers are continuously selecting and refining viral vectors to ensure patient safety and maximize delivery efficiency. To date, the US Food and Drug Administration (FDA) has approved eight gene therapy approaches using three different types of viral vectors: adeno-associated virus (AAV), lentiviral vectors, and herpes simplex virus-based vectors. As of February 2022, 25 viral vector-based treatments are in late-stage development, with an additional 120 in Phase II trials. Of all three types of viral vectors, lentiviral vectors are the second most commonly utilized, used in 30% of gene therapies, second only to adeno-associated viral vectors.

[0005] As a member of the retroviral family, lentivirus has many characteristics that make it suitable for gene therapy: the lentivirus exogenous gene loading capacity can reach 10kb; lentivirus can integrate genes into the target cell genome to achieve stable expression, and can infect not only dividing cells but also non-dividing cells; lentivirus causes a low immune response in target cells.

[0006] When a lentivirus infects a host cell, the main steps include binding to the host cell, fusing with the host cell membrane, releasing structural proteins, enzyme proteins, and the viral core, reverse transcription of the viral RNA under the action of the reverse transcriptase, and forming a pre-integration complex with the integrase. After the pre-integration complex enters the cell nucleus, the integrase catalyzes its integration into the host genome, and the exogenous gene pre-promoter drives its expression in the cytoplasm. Among them, the interaction between the viral surface glycoprotein and the host cell surface receptor protein can be used to achieve infection of specific cells. The first and most widely used glycoprotein is the vesicular stomatitis virus glycoprotein (VSV-G) because they have a wide range of tropism, produce high titers of lentivirus, and have high stability.

[0007] The low-density lipoprotein receptor (LDL-R) is the primary cell surface entry receptor for VSV-G pseudotyped lentivirus. The LDL-R is a 36-sided, single-chain glycoprotein composed of 839 amino acid residues and containing five functional domains. It is widely distributed on the cell membranes of tissues throughout the body, including hepatocytes, vascular smooth muscle cells, monocytes, and macrophages. The LDL-R extracellular domain consists of a ligand-binding domain, an epidermal growth factor precursor homology domain, and a C-terminal domain rich in O-linked oligosaccharides. The ligand-binding domain consists of seven cysteine-rich repeats (CR1-CR7). VSV-G can independently bind to two distinct CR domains (CR2 and CR3). Although LDLR is expressed in nearly all tissues, its expression on the surface of resting T cells, B cells, and hematopoietic stem cells (HSCs) is extremely low, resulting in very low infection efficiency in these cells. Whether in scientific research experiments or clinical applications such as CAR-T cell immunotherapy, the transformation and utilization of these cells through lentiviruses are greatly limited.

[0008] Summary of the Invention

[0009] To address this issue, the present disclosure provides a novel technology that uses bispecific molecules to mediate gene delivery independent of the VSV-G glycoprotein receptor LDLR.

[0010] In one aspect of the present disclosure, a bispecific antigen-binding molecule or a fragment thereof is provided, comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain is used to specifically bind to a cell surface antigen; and the second antigen-binding domain is used to specifically bind to vesicular stomatitis virus glycoprotein (VSV-G).

[0011] In another aspect, the present disclosure provides a bispecific antigen binding molecule or fragment thereof, comprising:

[0012] a first polypeptide comprising VH, CH1, CH2, CH3, CR2, and CR3; and a second polypeptide comprising VL and CL;

[0013] Among them, VH is the heavy chain variable region of the first antigen-binding domain, VL is the light chain variable region of the first antigen-binding domain, and the first antigen-binding domain is used to specifically bind to cell surface antigens; CH1, CH2 and CH3 are the first, second and third constant regions of the IgG molecule, respectively, CL is the light chain constant region; CR2 and CR3 are the second and third CR domains of the low-density lipoprotein receptor (LDL-R).

[0014] In another aspect, the present disclosure further provides a bispecific antigen-binding molecule or a fragment thereof, comprising:

[0015] A first antigen-binding domain comprising an scFv comprising VH and VL; and a second antigen-binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for connecting the scFv and the second antigen-binding domain;

[0016] Among them, the first antigen binding domain is used to specifically bind to cell surface antigens; VH is the heavy chain variable region, VL is the light chain variable region; CH2 and CH3 are the second and third constant regions of the IgG molecule respectively; CR2 and CR3 are the second and third CR domains of the low-density lipoprotein receptor (LDL-R).

[0017] In another aspect, the present disclosure further provides a bispecific antigen-binding molecule or a fragment thereof, comprising:

[0018] A first antigen-binding domain comprising VHH, and a second antigen-binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for connecting the VHH and the second antigen-binding domain;

[0019] Among them, the first antigen binding domain is used to specifically bind to cell surface antigens; CH2 and CH3 are the second and third constant regions of IgG molecules respectively; CR2 and CR3 are the second and third CR domains of low-density lipoprotein receptor (LDL-R).

[0020] In another aspect, the present disclosure further provides a bispecific antigen-binding molecule or a fragment thereof, comprising:

[0021] A first antigen-binding domain comprising a natural ligand of a cell surface protein, and a second antigen-binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for linking the natural ligand and the second antigen-binding domain;

[0022] Among them, the first antigen binding domain is used to specifically bind to cell surface antigens; CH2 and CH3 are the second and third constant regions of IgG molecules respectively; CR2 and CR3 are the second and third CR domains of low-density lipoprotein receptor (LDL-R).

[0023] In another aspect, the present disclosure also provides a polynucleotide encoding the bispecific antigen-binding molecule or a fragment thereof provided by the present disclosure.

[0024] In another aspect, the present disclosure also provides a recombinant vector comprising the polynucleotide provided by the present disclosure.

[0025] In another aspect, the present disclosure further provides a host cell comprising the polynucleotide or recombinant vector provided by the present disclosure.

[0026] In another aspect, the present disclosure also provides methods of producing the bispecific antigen binding molecules or fragments thereof of the present disclosure.

[0027] In another aspect, the present disclosure also provides a method for transducing a target gene into a subject using a lentiviral vector, comprising administering the bispecific antigen-binding molecule or fragment thereof provided by the present disclosure to the subject.

[0028] Beneficial effects of the present disclosure:

[0029] (1) This disclosure designs a novel bispecific molecule that can efficiently mediate VSV-G lentiviral infection of cells that do not express the VSV-G receptor LDLR. In this disclosure, the antigen-binding domain for specifically binding to cell surface antigens can be widely replaced to achieve specific targeting of different types of cells.

[0030] (2) The inventors also found that for some cells that are difficult to infect (low or no expression of LDLR), such as primary cells, the use of bispecific molecules can greatly improve the efficiency of target gene transduction, thereby greatly increasing the probability of target gene integration into the host cell genome, providing a favorable approach for constructing stable cell lines, overexpressing, knocking down or knocking down specific genes, and exploring gene functions.

[0031] (3) Due to the many advantages of lentiviral vectors, they have become the main vector for infecting T cells, especially for the production of CAR-T cells. However, on the one hand, viral production is difficult to scale up, and on the other hand, the limited number of T cells and the restriction of activation efficiency further increase the difficulty of CAR-T cell production. The bispecific molecules disclosed in the present invention enhance the infection efficiency of lentivirus on T cells, which can reduce the requirements for T cell number and virus titer when preparing CAR-T cells, and can further reduce the production cost of CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1A to 1F show schematic structural diagrams of the bispecific antigen-binding molecules disclosed herein; one end for binding to cells can be designed as a Fab format, scFv, single-domain antibody VHH, or a natural ligand of a cell surface-specific membrane protein; one end for binding to the VSV-G pseudotyped lentivirus is the CR2 and CR3 domains of the human low-density lipoprotein receptor (LDLR), which can also be replaced with the CR2 and CR3 domains of the low-density lipoprotein receptor (LDLR) of porcine, equine, or bovine species; the two binding domains can be connected via the Fc of an antibody to increase valency, or directly connected using a flexible short linker such as GSG.

[0033] Figure 2 shows a schematic diagram of cell infection using bispecific antigen-binding molecules. First, a specific bispecific molecule is incubated with cells at a certain concentration at 4°C to allow the bispecific molecule to bind to specific proteins on the cell surface. The cells are then centrifuged to remove free bispecific molecules from the supernatant. The cells are then resuspended with a certain titer of VSV-G pseudotyped lentivirus, and the VSV-G protein on the virus surface binds to the CR2-CR3 domain of the LDLR. The bispecific molecule not only shortens the physical distance between the cell and the virus, but also, after being stimulated by the bispecific molecule, the cell surface proteins undergo endocytosis, allowing the viral particles to be encapsulated and entered into the cell. The low pH in the endosome promotes conformational changes in the VSV-G protein, which fuses with the membrane and releases genetic material into the cell. This is then reverse transcribed and further integrated into the cell genome, achieving long-term gene transduction. The process starting from endocytosis is consistent with traditional VSV-G pseudotyped lentivirus infection through the LDLR receptor.

[0034] Figure 3 shows the results of flow cytometry detection of LDLR expression on the surface of different types of cells. By using flow cytometry antibodies against human low-density lipoprotein receptor LDLR, the expression level was detected in different cell lines. Compared with commonly used cell lines, the LDLR expression level of human primary T cells that were not activated by CD3 / CD28 antibodies was lower. After CD3 / CD28 antibody stimulation, LDLR expression was upregulated.

[0035] Figure 4 shows the comparative analysis results of the infection efficiency of VSV-G pseudotyped GFP lentivirus of equivalent titers in different cell types. Different cell types were infected with the same titer of VSV-G pseudotyped GFP lentivirus, and the proportion of GFP-positive cells was examined by flow cytometry.

[0036] FIG5 shows the expression results of different bispecific molecules verified by SDS-PAGE. By verifying the expression of the bispecific molecules using SDS-PAGE, it was found that the protein molecular weights of the different bispecific molecules all met the theoretical sizes.

[0037] Figure 6 shows a bar chart of the binding ratios of bispecific molecules to different cell surface membrane proteins. By incubating different cell types with different bispecific molecules and using a fluorescent secondary antibody that binds to the Fc region of the bispecific molecule to detect binding, it was demonstrated that the expressed protein can bind to specific cells through the primary binding domain.

[0038] Figure 7 shows a bar chart of the binding percentages of different bispecific molecules to 293T cells overexpressing VSV-G protein. By incubating 293T cells overexpressing VSV-G protein with different bispecific molecules and quantifying binding using a fluorescent secondary antibody that binds to the Fc region of the bispecific molecules, it was demonstrated that the expressed proteins can bind to VSV-G protein through the CR2-CR3 domain of the human low-density lipoprotein receptor (LDLR).

[0039] 8A-C show the flow cytometric detection of the enhancement effect of bispecific molecules (targeting HLA) on the infection efficiency of lentiviral-infected Ramos cells.

[0040] Figures 9A-C show the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting HLA) on the infection efficiency of lentivirus-infected Jurkat T cells.

[0041] Figure 10A-C shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting HLA) on the infection efficiency of lentivirus-infected human peripheral blood mononuclear cells.

[0042] Figure 11A-C shows the flow cytometric detection of the enhancement effect of bispecific molecules (targeting CD19) on the infection efficiency of lentiviral-infected Ramos cells.

[0043] FIG12 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD20) on the infection efficiency of lentiviral-infected Ramos cells.

[0044] FIG13 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD45) on the infection efficiency of lentiviral-infected Ramos cells.

[0045] FIG14 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD40) on the infection efficiency of lentiviral-infected Ramos cells.

[0046] FIG15 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD3) on the infection efficiency of lentivirus-infected Jurkat T cells.

[0047] FIG16 shows the flow cytometric detection of the enhancement effect of bispecific molecules (targeting CD3) on the infection efficiency of lentivirus-infected unstimulated human primary T cells.

[0048] FIG17 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD3) on the infection efficiency of lentiviral-infected human primary T cells activated by CD3 / CD28 antibodies.

[0049] FIG18 shows the flow cytometric detection of the enhancement effect of bispecific molecules (targeting CD4) on the infection efficiency of lentivirus-infected unstimulated human primary T cells. DETAILED DESCRIPTION

[0050] In order to make this disclosure more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0051] I. Definition

[0052] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to specific compositions or method steps, as these compositions or method steps may vary. As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a / kind" and "the" include plural reference objects. The term "a" (or "a"), and the terms "one or more" and "at least one" are used interchangeably herein.

[0053] Furthermore, the use of "and / or" herein should be understood as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this disclosure relates. For example, the Coneise Dictionary of Biomedicine and Molecular Biology, Juo, PeiShow, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary for many of the terms used in this disclosure.

[0055] Units, prefixes, and symbols are all expressed in their SI (International System of Units) accepted form. Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the direction of amino to carboxyl groups. The subheadings provided herein are not limitations of the various aspects, which can be obtained by reference to this specification as a whole. Therefore, the terms defined below are more fully defined by reference to the specification in their entirety.

[0056] It should be understood that when an aspect is described with the language "comprising," other similar aspects described with respect to "consisting of" and / or "consisting essentially of" are also provided.

[0057] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides are referred to by their universally recognized one-letter codes.

[0058] As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and their derivatives, such as fragments.

[0059] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above substances through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab)2, and Fv fragments), single-chain variable fragments (scFv), disulfide-stabilized scFv, multispecific antibodies such as bispecific antibodies generated from at least two complete antibodies and / or antigen-binding fragments thereof, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigenic determinant portion of an antibody, and any other modified immunoglobulin molecules comprising an antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0060] Conventional immunoglobulins are tetramers composed of two heavy chains and two light chains, with a combined molecular weight of approximately 150 kDa. A typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region comprises three domains (CH1, CH2, and CH3). Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. These VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FWs). Each VH and VL comprises three CDRs and four FWs arranged from amino terminus to carboxyl terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host cells or factors, including different cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0061] The term "antigen binding domain" refers to the site of an antigen binding molecule, i.e., one or more amino acid residues, that provides interaction with the antigen. For example, the antigen binding domain of an antibody comprises amino acid residues from the complementary determining regions (CDRs). Examples of antigen binding domains include, but are not limited to, Fab, Fab', F(ab')2, and single-chain Fv (scFv) fragments.

[0062] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-peptide linker-VH or can include VH-peptide linker-VL.

[0063] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM. The corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0064] In the case of IgG, IgA, and IgD antibodies, the constant region comprises three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a segment of variable length rich in proline and cysteine. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0065] The term "Fc" is used herein to define the C-terminal region of an immunoglobulin heavy chain, i.e., the two polypeptide chains that form a dimer comprising the C-terminal constant region of an immunoglobulin heavy chain that can stabilize its own association. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, e.g., an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0066] In members of the Camelidae family, a significant proportion of serum antibodies are homodimeric IgG with a molecular weight of approximately 80 kD (Hamers-Casterman et al., 1993, Nature, 363, 446-448). These heavy chain immunoglobulins (Ig) contain three domains, the variable region of which is referred to as VHH (variable domain of heavy chain of heavy-chain antibody). Recombinant VHH (approximately 12 to 14 kD) constitutes a complete antigen-binding domain and exhibits a broad antigen-binding spectrum. Their hypervariable regions are enlarged and exhibit unique properties, such as the replacement of three to four hydrophobic framework residues (that interact with conventional antibody VL) with more hydrophilic amino acids. To stabilize the enlarged CDRs, VHHs may have additional disulfide bonds between CDR1 and CDR3 in dromedary camels and between CDR2 and CDR3 in llamas (Harmsen and De Haard, 2007, Appl Microbiol Biotechnol., 77, 13-22; Muyldermans, 2001, J Biotechnol., 74, 277-302). The enlarged CDR3 loop can adopt a convex conformation, whereas the conventional paratope is constrained to a concave or planar structure (Muyldermans, 2001, J Biotechnol., 74, 277-302). These features allow VHH to recognize unique epitopes that are poorly immunogenic for conventional antibodies (Lafaye, 2009, Mol Immuno., 46, 695-704; Wernery, 2001, J Vet Med B Infect Dis Vet Public Health., 48, 561-568). Although VHH is defined as a monovalent antibody, excluding any avidity effect by default, the biological activity measured as IC50 in vitro can be similar to that of conventional bivalent antibody molecules (Thys et al., 2010, Antiviral Res., 87, 257-264). In the present disclosure, "VHH antibody", "heavy chain variable region (VHH)", "VHH domain" and "nanobody VHH" can be used interchangeably.

[0067] "Monoclonal antibody" refers to a homogeneous antibody population that is capable of highly specific recognition and binding to a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants.

[0068] The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, single-chain variable fragment (scFv), fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made by any number of means, including but not limited to, by hybridomas, phage selection, recombinant expression, and transgenic animals (e.g., expression of human antibodies in transgenic mice).

[0069] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that has been engineered to contain minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody is a human immunoglobulin in which residues from a CDR are replaced with residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) with desired specificity, affinity, and ability (Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-327; Verhoeyen et al., 1988, Science, 239: 1534-1536). In some instances, the FW residues of a human immunoglobulin are replaced with corresponding residues from an antibody of a non-human species with desired specificity and / or affinity and / or ability.

[0070] The humanized antibody can be further modified by refining and optimizing antibody specificity and / or affinity and / or ability by replacing the other residues in the FW district and / or in the non-human residue of replacement.Usually, the humanized antibody will basically comprise all at least one and typically two or three variable domains, these variable domains contain all or substantially all CDR districts corresponding to non-human immunoglobulin, and all or substantially all FW districts are those of human immunoglobulin consensus sequences.The humanized antibody can also comprise at least a portion of immunoglobulin constant region or domain (Fc), typically at least a portion of human immunoglobulin.The example of the method for producing humanized antibodies is described in U.S. Patent number 5,225,539 or 5,639,641.

[0071] The term "human antibody" means an antibody produced by a person or an antibody with an amino acid sequence corresponding to an antibody produced by a person using any technology known in the art (for example, recombinant expression in cultured cells or expression in transgenic animals). Therefore, the term human antibody also encompasses antibodies with an amino acid sequence corresponding to an antibody initially produced by a person (or its engineered variants or derivatives) but expressed in a non-human system (for example, produced by chemical synthesis; recombinant expression in microorganisms, mammalian or insect cells; or expressed in an animal subject). Therefore, antibodies obtained from a human subject or from a human cell (for example, a hybridoma or cell line expressing a recombinant antibody or its fragment) and subsequently expressed in an animal such as a mouse are considered to be human antibodies. This definition of human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polynucleotides, such as, for example, antibodies comprising mouse light chain and human heavy chain polynucleotides.

[0072] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more animal species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity and / or affinity and / or capacity, while the constant regions are homologous to sequences in an antibody derived from another species (usually human) to avoid eliciting an immune response in these species.

[0073] The term "bispecific" means that the antigen binding molecule is able to specifically bind to two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen binding molecule is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0074] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, or a complete extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof, can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen or cell or preparation containing the antigen can be used to generate antibodies specific for the antigenic determinant. The antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the aforementioned antigens can be used alone or in combination with one or more immunogenicity-enhancing adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0075] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. Epitopes generally exist in a unique spatial conformation and include at least 3-15 amino acids. Methods for determining the epitope bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0076] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear, cyclic, or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers, such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA-mediated amino additions such as arginylation, ubiquitination, or any other manipulation such as conjugation to a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0077] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. As used herein, "amino acid substitution" or "substitution" or "replacement" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution S32A refers to the replacement of serine at position 32 with alanine.

[0078] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J, 17, 323-329 (2000)) and traditional binding assays (Heeley Endocr, Res, 28, 217-229 (2002)).

[0079] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding portion and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). Therefore, equivalent affinities can comprise different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0080] As used herein, the term "valency" refers to the presence of a specified number of antigen-binding regions in an antigen-binding molecule. Thus, for example, the term "tetravalent bispecific antigen-binding molecule" refers to a bispecific antigen-binding molecule having four antigen-binding regions, each of which can bind to the same or different antigens; in some examples, for example, "bivalent for a target" refers to the presence of two antigen-binding regions for a specific target in the bispecific antigen-binding molecule. In other examples, for example, a CD3 monovalent bispecific antigen-binding molecule refers to the presence of one antigen-binding region for CD3 in the bispecific antigen-binding molecule.

[0081] In some examples of the present disclosure, the antibodies are bivalent for both the cell surface antigen and VSV-G. In other examples, the antibodies may not be bivalent for both the cell surface antigen and VSV-G, such as monovalent, trivalent, or tetravalent; the valencies of the antibodies for the cell surface antigen and VSV-G may be the same or different.

[0082] It should be noted that the division of the CDRs and FRs in the antibody variable regions of the present disclosure is determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any naming system based on the antibody sequences of the present disclosure are clearly within the scope of the present disclosure.

[0083] The term "sequence identity" or "sequence similarity" or "sequence homology" refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences (and introducing gaps, if necessary) to obtain maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed to determine percent amino acid sequence identity using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0084] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells with cytotoxins. Specific high-affinity IgG antibodies directed against the surface of the target cells "arm" the cytotoxic cells and are required for this killing. The lysis of the target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement. It is contemplated that, in addition to antibodies, other proteins containing an Fc region (specifically, Fc fusion proteins) that have the ability to specifically bind to target cells bearing antigen will be able to achieve cell-mediated cytotoxicity. For simplicity, the cell-mediated cytotoxicity resulting from the activity of Fc fusion proteins is also referred to herein as ADCC activity.

[0085] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and a polymer thereof in single-stranded or double-stranded form. Unless explicitly limited, the term "nucleic acid" or "polynucleotide" also includes nucleic acids containing analogs of known natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as sequences explicitly indicated.

[0086] The term "vector" means a construct capable of delivering, and in some aspects, expressing, in a host cell, one or more genes or one or more sequences of interest. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors linked to a cationic condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0087] In this disclosure, unless otherwise expressly provided, "activation", "stimulation" and "treatment" for cells or receptors may have the same meaning, for example, a cell or receptor is activated, stimulated or treated with a ligand. "Ligand" includes natural and synthetic ligands, such as cytokines, cytokine variants or analogs, mutant proteins and binding compounds derived from antibodies (such as antibodies and binding fragments thereof). "Ligand" also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response" or "reaction", such as the response of a cell, tissue, organ or organism, includes changes in biochemical or physiological behavior, such as changes in the concentration, density, adhesion or migration of some components within a biological compartment (such as a tissue, cell, organelle, etc.), gene expression rate or differentiation state, which changes can be associated with activation, stimulation or treatment.

[0088] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, without regard to the number of generations. Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the initially transformed cells are included herein. Host cells are any type of cell system that can be used to produce the bispecific antigen binding molecules of the present disclosure. Host cells include cultured cells, such as mammalian cultured cells, such as Jurkat cells, PBMC cells, A375 cells, U251 cells, and U87 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.

[0089] The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0090] The term "stable transfection" or "stable transfection" refers to the introduction and integration of exogenous nucleic acid, DNA or RNA, into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated the foreign DNA into its genomic DNA.

[0091] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. The antibodies or antigen-binding fragments of the present invention are engineered to incorporate one or more human FR regions into non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website (http: / / imgt.cines.fr) or from The Immunoglobulin Facts Book (2001) ISBN: 012441351.

[0092] The engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0093] II. Bispecific Antigen Binding Molecules or Fragments thereof

[0094] In one aspect of the present disclosure, a bispecific antigen-binding molecule (also referred to as a bispecific antibody in the present disclosure) or a fragment thereof is provided, which comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain is used to specifically bind to a cell surface antigen; and the second antigen-binding domain is used to specifically bind to vesicular stomatitis virus glycoprotein (VSV-G).

[0095] In the present disclosure, the first antigen-binding domain for specifically binding to a cell surface antigen can be widely replaced to achieve specific targeting of different types of cells.

[0096] In some embodiments, the cells can be cells that underexpress low-density lipoprotein receptor (LDL-R); as used herein, the terms "underexpression" and "low expression level" are interchangeable and should be understood to mean a decrease of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 50%, 80%, 100% or more compared to a "control" or "threshold value." For example, Student's T-tests can be repeated for genes with at least one expression intensity below the threshold to determine significance.

[0097] In some embodiments, the cell may be a cell that does not express low-density lipoprotein receptor (LDL-R).

[0098] In some embodiments, the cells include but are not limited to T cells, B cells, NK cells, and hematopoietic stem cells (HSCs).

[0099] In some embodiments, the T cells and B cells are primary cells.

[0100] In some embodiments, the T cells and B cells are resting cells, which have lower LDL-R expression levels than activated cells.

[0101] In some embodiments, the cell surface antigen is selected from B cell surface antigen, NK cell surface antigen, hematopoietic stem cell surface antigen or T cell surface antigen; exemplarily, the cell surface antigen is selected from any one of CD3, CD19, CD45, CD20, CD34, CD40, CD56, CD16, CD133, CD147, CD123, CD138, CD22, CD30, CD33, CD38, CD70, CD4, CD5, CD8A&CD8B, and CD7.

[0102] In some embodiments, the second antigen binding domain comprises the CR2 and CR3 domains of the low-density lipoprotein receptor (LDL-R).

[0103] In some embodiments, the CR2 and CR3 domains are of human, equine, or porcine origin.

[0104] In some embodiments, the second antigen binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence shown in SEQ ID NO.1.

[0105] In some embodiments, the amino acid sequence of the second antigen binding domain is: VTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPV (SEQ ID NO. 1).

[0106] In some embodiments, the amino acid sequence of the second antigen-binding domain has one or more amino acid substitutions, insertions and / or deletions compared to SEQ ID NO.1, and the altered second antigen-binding domain has an activity of specific binding to vesicular stomatitis virus glycoprotein (VSV-G) comparable to or improved than that of the wild type.

[0107] In some embodiments, "several" means 5 or less, more preferably 3 or less, and most preferably 2 or less. For example, the amino acid sequence of the second antigen-binding domain has 5, 4, 3, 2 or 1 amino acid residue substitutions, insertions and / or deletions compared to SEQ ID NO. 1.

[0108] In some embodiments, the CR2 and CR3 domains act as single domain antibodies that specifically bind to vesicular stomatitis virus glycoprotein.

[0109] In some embodiments, the bispecific antibody binds to cell surface antigens so that the cell surface is coated with the bispecific antibody molecules. During viral infection, the virus contacts the cell by recognizing the CR2 and CR3 domains on the bispecific antibody. Specific molecules on the cell surface, such as CD19, CD3, and CD45, undergo clathrin-dependent endocytosis after being bound by the specific antibody. This endocytosis process mediates the entry of the virus into the target cell, ultimately achieving gene delivery.

[0110] In some embodiments, the first antigen binding domain comprises a heavy chain (HC) variable region (VH) and / or a light chain (LC) variable region (VL).

[0111] As mentioned in the present disclosure, the first antigen-binding domain for specifically binding to a cell surface antigen may comprise the heavy chain variable region and / or light chain variable region of any disclosed antibody against the cell surface antigen. Exemplarily, it may be the heavy chain variable region and / or light chain variable region of an antibody against any of the above-mentioned cell surface antigens, such as but not limited to CD3, CD19, CD45, CD20, CD34, CD40, CD56, CD16, CD133, CD147, CD123, CD138, CD22, CD30, CD33, CD38, CD70, CD4, CD5, CD8A & CD8B, CD7, and HLA.

[0112] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence shown in SEQ ID NOs. 87-95;

[0113] and / or

[0114] A light chain variable region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO. 96-104.

[0115] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 87; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 96. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD19.

[0116] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 88; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 97. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD3 (OKT3). In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 89; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 98. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD45.

[0117] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 90; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 99. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD34.

[0118] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 91; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 100. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD20.

[0119] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 92; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 101. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD40.

[0120] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO. 93; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO. 102. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD4.

[0121] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 94; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO. 103. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen CD3 (UCHT1).

[0122] In some embodiments, the first antigen-binding domain may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO. 95; and a light chain variable region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO. 104. In some embodiments, the first antigen-binding domain may specifically bind to the cell surface antigen HLA.

[0123] In some embodiments, the first antigen binding domain may further comprise a heavy chain (HC) constant region (CH) or a fragment thereof and / or a light chain (LC) constant region (CL) or a fragment thereof.

[0124] In some embodiments, the heavy chain constant region or fragment thereof is an IgG constant region; in some embodiments, the heavy chain constant region or fragment thereof includes at least the first constant region CH1 of IgG.

[0125] In some embodiments, the light chain constant region is a kappa constant region or a lambda constant region.

[0126] In some embodiments, the first antigen binding domain comprises at least one of Fab, Fab', F(ab')2, scFv, or VHH.

[0127] In some embodiments, the first antigen binding domain is a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or an affinity-optimized antibody.

[0128] In some embodiments, the first antigen binding domain further comprises an Fc domain.

[0129] In some embodiments, the Fc domain is of murine or human origin.

[0130] In some embodiments, the Fc domain further comprises at least one mutation that can reduce or enhance the ADCC activity of the bispecific antibody.

[0131] In some embodiments, the Fc domain comprises a modification that promotes binding of the two chains of the Fc domain; in some embodiments, the Fc structure comprises a Knob chain and a Hole chain.

[0132] In some embodiments, the second antigen binding domain is covalently linked to the carboxyl terminus of the heavy chain and / or light chain of the first antigen binding domain;

[0133] In some embodiments, the second antigen binding domain is covalently linked to the amino terminus of the heavy chain and / or light chain of the first antigen binding domain;

[0134] In some embodiments, the second antigen binding domain is covalently embedded in the polypeptide chain of the heavy chain and / or light chain of the first antigen binding domain.

[0135] In some embodiments, when both the heavy chain and the light chain of the first antigen-binding domain are connected to the second antigen-binding domain, the connection sites of the second antigen-binding domain to the light chain and the heavy chain may be the same or different, for example, one second antigen-binding domain is connected to the carboxyl terminus of the heavy chain, and the other second antigen-binding domain may be connected to the carboxyl terminus, amino terminus, or embedded in the polypeptide chain of the light chain; or one second antigen-binding domain is connected to the amino terminus of the heavy chain, and the other second antigen-binding domain may be connected to the carboxyl terminus, amino terminus, or embedded in the polypeptide chain of the light chain, etc.

[0136] In some embodiments, the second antigen-binding domain is connected to the heavy chain and / or light chain of the first antigen-binding domain via a peptide linker; in some embodiments, the peptide linker is selected from L1, L2, L3 or L4, wherein the sequence of L1 is (G4S)x, where x is any integer from 1 to 6 (SEQ ID NO.7), the sequence of L2 is (G4S)xA, where x is any integer from 1 to 6 (SEQ ID NO.8), the sequence of L3 is EPKSSDKTHTCPPCP (SEQ ID NO.9), and the sequence of L4 is DKTHTCPPCP (SEQ ID NO.10); preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

[0137] In some embodiments, the bispecific antigen binding molecule is tetravalent or hexavalent.

[0138] In some embodiments, the bispecific antigen binding molecule is bivalent or tetravalent for two targets.

[0139] In some embodiments, exemplary molecular structures of the bispecific antigen-binding molecules are shown in Figures 1A to 1F.

[0140] In one example, as shown in FIG1A , the first antigen-binding domain of the antibody has a Fab structure, and the second antigen-binding domain is connected to the C-terminus of the heavy chain.

[0141] In one example, as shown in FIG1B , the first antigen-binding domain of the antibody has a Fab structure and an Fc domain, and the second antigen-binding domain is connected to the C-terminus of the heavy chain (ie, the C-terminus of the heavy chain CH3).

[0142] The antibody shown in FIG1A and FIG1B is a tetravalent antibody that is bivalent for both cell surface antigens and VSV-G.

[0143] In other examples, the second antigen-binding domain can also be connected to the C-terminus of the light chain (such as the light chain CL).

[0144] In another example, as shown in Figure 1C, the first antigen-binding domain of the antibody comprises a Fab structure and an Fc domain, wherein the C-termini of both the light and heavy chains are connected to a second antigen-binding domain. In this case, the antibody is bivalent for cell surface antigens and tetravalent for VSV-G. The inventors have discovered that bispecific antibodies with this structure have significantly improved binding to VSV-G.

[0145] The antibody shown in FIG1C is hexavalent, including bivalent antibodies to cell surface antigens and tetravalent antibodies to VSV-G.

[0146] In one example, as shown in FIG1D , the first antigen-binding domain of the antibody has an scFv structure and an Fc domain, and the second antigen-binding domain is connected to the C-terminus of the Fc domain (ie, heavy chain CH3).

[0147] In one example, as shown in FIG1E , the first antigen-binding domain of the antibody is a VHH antibody, and the second antigen-binding domain is connected to the C-terminus of the first antigen-binding domain.

[0148] The antibody shown in FIG. 1D is bivalent and monovalent for both cell surface antigens and VSV-G.

[0149] In one example, as shown in FIG1F , the first antigen-binding domain of the antibody has a ligand structure and an Fc domain, and the second antigen-binding domain is connected to the N-terminus of the Fc domain.

[0150] The antibody shown in Figure IF is tetravalent and is bivalent for both cell surface antigens and VSV-G.

[0151] In the above examples, the second antigen-binding domain can be connected to the end of the first antigen-binding domain via a peptide linker.

[0152] In some embodiments, the bispecific antigen binding molecule or fragment thereof comprises:

[0153] a first polypeptide comprising VH, CH1, CH2, CH3, CR2, and CR3; and a second polypeptide comprising VL and CL;

[0154] VH is the heavy chain variable region, VL is the light chain variable region; CH1, CH2 and CH3 are the first, second and third constant regions of the IgG molecule, respectively, CL is the light chain constant region; CR2 and CR3 are the second and third CR domains of the low-density lipoprotein receptor (LDL-R).

[0155] In some embodiments, as shown in FIG1B , the first polypeptide comprises VH, CH1, CH2, CH3, CR2, and CR3 in N-terminal to C-terminal order; the second polypeptide comprises VL and CL in N-terminal to C-terminal order;

[0156] In some embodiments, the two polypeptides associate to form an antigen binding site that targets cell surface antigens; one end recognizes specific cell surface antigens (such as CD3 on the surface of T cells, CD19 on the surface of B cells, etc.) through the variable region of the antibody, and at the other end of the Fc, it binds to the VSV-G glycoprotein of the virus by coupling the CR2 and CR3 domains of LDLR.

[0157] In some embodiments, the bispecific antigen-binding molecule is tetravalent. For example, it comprises an antibody that can bind to four antigen-binding sites of two different antigens (cell surface antigen and VSV-G).

[0158] In some embodiments, the bispecific antigen binding molecule is bivalent for two targets (cell surface antigen and VSV-G).

[0159] In some embodiments, the first polypeptide comprises a peptide linker between CH3 and CR2; preferably, the peptide linker is selected from L1, L2, L3 or L4, wherein the sequence of L1 is shown in SEQ ID NO.7, the sequence of L2 is shown in SEQ ID NO.8, the sequence of L3 is shown in SEQ ID NO.9, and the sequence of L4 is shown in SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

[0160] In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO. 2; the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO. 3;

[0161] In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence shown in SEQ ID NO.4; the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence shown in SEQ ID NO.5.

[0162] In some embodiments, as shown in Figure 1C, the second polypeptide further comprises CR2 and CR3 in the N-terminal to C-terminal sequence. In some embodiments, the CR2 and CR3 are linked to the carboxyl terminus of CL.

[0163] In some embodiments, the second polypeptide further comprises a peptide linker between CL and CR2; preferably, the peptide linker is selected from L1, L2, L3 or L4; wherein the sequence of L1 is shown in SEQ ID NO.7, the sequence of L2 is shown in SEQ ID NO.8, the sequence of L3 is shown in SEQ ID NO.9, and the sequence of L4 is shown in SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

[0164] In some embodiments, the bispecific antigen binding molecule or fragment thereof comprises:

[0165] A first antigen-binding domain comprising an scFv comprising VH and VL; and a second antigen-binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for connecting the scFv and the second antigen-binding domain;

[0166] VH is the heavy chain variable region, VL is the light chain variable region; CH2 and CH3 are the second and third constant regions of the IgG molecule respectively; CR2 and CR3 are the second and third CR domains of the low-density lipoprotein receptor (LDL-R).

[0167] In some embodiments, as shown in FIG1D , the bispecific antigen-binding molecule or fragment thereof comprises scFv, CH2, CH3, CR2, and CR3 in order from N-terminus to C-terminus; the scFv comprises VH and VL;

[0168] In some embodiments, the bispecific antigen binding molecule is tetravalent.

[0169] In some embodiments, the bispecific antigen binding molecule is bivalent for two targets.

[0170] In some embodiments, the bispecific antigen-binding molecule or fragment thereof comprises a peptide linker between scFv and CH2; preferably, the linker is selected from L1, L2, L3 or L4, wherein the sequence of L1 is shown in SEQ ID NO.7, the sequence of L2 is shown in SEQ ID NO.8, the sequence of L3 is shown in SEQ ID NO.9, and the sequence of L4 is shown in SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

[0171] In some embodiments, the bispecific antigen-binding molecule or fragment thereof comprises a peptide linker between CH3 and CR2; preferably, the peptide linker is selected from L1, L2, L3 or L4, wherein the sequence of L1 is shown in SEQ ID NO.7, the sequence of L2 is shown in SEQ ID NO.8, the sequence of L3 is shown in SEQ ID NO.9, and the sequence of L4 is shown in SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

[0172] In some embodiments, the bispecific antigen binding molecule or fragment thereof comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO.6.

[0173] In some embodiments, the bispecific antigen binding molecule or fragment thereof comprises:

[0174] A first antigen-binding domain comprising a VHH antibody; and a second antigen-binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for connecting the VHH and the second antigen-binding domain;

[0175] Among them, CH2 and CH3 are the second and third constant regions of IgG molecules, respectively; CR2 and CR3 are the second and third CR domains of low-density lipoprotein receptor (LDL-R).

[0176] In some embodiments, as shown in FIG. 1E , the bispecific antigen-binding molecule or fragment thereof comprises VHH, CR2, and CR3 in order from N-terminus to C-terminus.

[0177] In some embodiments, the bispecific antigen binding molecule is bivalent.

[0178] In some embodiments, the bispecific antigen binding molecule is monovalent for both targets.

[0179] In some embodiments, the bispecific antigen-binding molecule or fragment thereof comprises a peptide linker between VHH and CR2; preferably, the linker is selected from L1, L2, L3 or L4, wherein the sequence of L1 is shown in SEQ ID NO.7, the sequence of L2 is shown in SEQ ID NO.8, the sequence of L3 is shown in SEQ ID NO.9, and the sequence of L4 is shown in SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

[0180] In some embodiments, the bispecific antigen binding molecule or fragment thereof comprises:

[0181] A first antigen-binding domain comprising a natural ligand of a cell surface protein, and a second antigen-binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for linking the natural ligand and the second antigen-binding domain;

[0182] Among them, the first antigen binding domain is used to specifically bind to cell surface antigens; CH2 and CH3 are the second and third constant regions of IgG molecules respectively; CR2 and CR3 are the second and third CR domains of low-density lipoprotein receptor (LDL-R).

[0183] In some embodiments, as shown in FIG1F , the bispecific antigen-binding molecule or fragment thereof comprises CR2, CR3, CH2, CH3 and a natural ligand in order from N-terminus to C-terminus.

[0184] In some embodiments, the natural ligand is CD40 ligand. In some embodiments, the amino acid sequence of the CD40 ligand is shown in SEQ ID NO. 106.

[0185] In some embodiments, the bispecific antigen binding molecule is tetravalent.

[0186] In some embodiments, the bispecific antigen binding molecule is bivalent for two targets.

[0187] In some embodiments, the bispecific antigen-binding molecule or fragment thereof comprises a peptide linker between CR3 and CH2; preferably, the linker is selected from L1, L2, L3 or L4, wherein the sequence of L1 is shown in SEQ ID NO.7, the sequence of L2 is shown in SEQ ID NO.8, the sequence of L3 is shown in SEQ ID NO.9, and the sequence of L4 is shown in SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

[0188] In some embodiments, knowing the sequence information of the heavy chain variable region (VH) and / or light chain variable region (VL) of the first antigen-binding domain, the sequence information of the second antigen-binding domain (CR2 and CR3), and the connection relationship between the first antigen-binding domain and the second antigen-binding domain, a person skilled in the art can obtain the full-length sequence of the antibody based on the conventional sequences of CH1, CH2, CH3, CL, etc. in the prior art (for example, directly replacing the VH and VL in the full-length sequence of the antibody provided in the present disclosure with the heavy chain and / or light chain variable regions (VH and / or VL) of the antigen-binding domain that binds to other target cell surface antigens to obtain a bispecific antibody that binds to other target cell surface antigens), as well as the nucleic acid sequence encoding the antibody.

[0189] In another aspect, the present disclosure provides the use of the bispecific antigen-binding molecules or fragments thereof of the present disclosure for mediated receptor-independent lentiviral vector gene transduction; preferably, the object of the gene transduction is a cell that lowly expresses or does not express low-density lipoprotein receptor; preferably, the object of the gene transduction is selected from T cells, B cells, NK cells or hematopoietic stem cells; preferably, the T cells and the B cells are primary cells; preferably, the T cells and the B cells are resting cells.

[0190] III. Preparation of Bispecific Antigen Binding Molecules or Fragments thereof

[0191] The bispecific antibodies of the present disclosure can be prepared according to methods known in the art. For example, the bispecific antibodies of the present disclosure can be produced using hybridoma methods such as those described by Kohler and Milstein (1975) Nature 256:495.

[0192] Using the hybridoma method, mice, hamsters or other suitable host animals are immunized as described above to induce the production of lymphocytes through antibodies, and these lymphocytes are specifically bound to the immune antigen. Lymphocytes can also be immunized in vitro. After immunity, these lymphocytes are separated and fused with suitable myeloma cell lines using, for example, polyethylene glycol, to form hybridoma cells that can then be selected to leave from unfused lymphocytes and myeloma cells. As determined by immunoprecipitation, immunoblotting or by in vitro binding assay (for example, radioimmunoassay (RIA); Enzyme-linked immunosorbent assay (ELISA)), the specific hybridoma of the production monoclonal antibody for the selected antigen can then be bred using standard methods (Goding, monoclonal antibodies: principles and practice (Monoclonal Antibodies: Principles and Practice), American Academic Press, 1986) in vitro culture or in vivo as ascites tumors in animals. These monoclonal antibodies can then be purified from the culture medium or ascites for the above-mentioned polyclonal antibodies.

[0193] The bispecific antibodies of the present disclosure can also be prepared using recombinant DNA methods as described in U.S. Patent No. 4,816,567. Such as by using RT-PCR of oligonucleotide primers of the gene encoding antibody heavy chain and light chain using specific amplification, the polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, and their sequences are determined using conventional procedures. The polynucleotides encoding the separation of heavy chain and light chain are then cloned into suitable expression vectors, which enter host cells such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not produce immunoglobulin proteins in addition during transfection, and monoclonal antibodies are produced by these host cells. Similarly, recombinant antibodies of a desired species, or molecules comprising antigen-binding fragments thereof, can be isolated from phage display libraries expressing the CDRs of the desired species as described (McCafferty et al., Nature 348:552-554 (1990); Clarkson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991)).

[0194] Recombinant DNA technology can be used to further modify one or more polynucleotides encoding the bispecific antibodies of the present disclosure in a variety of different ways to produce alternative bispecific antibodies of the present disclosure. In some aspects, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be replaced with (1) those of a human antibody, for example, to produce a chimeric antibody or (2) a non-immunoglobulin polypeptide to produce a fusion antibody. In some aspects, the constant region is truncated or removed to produce a desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0195] In certain aspects, the bispecific antibodies of the present disclosure are human antibodies or antigen-binding fragments thereof. Human antibodies can be directly prepared using different techniques known in the art. Immortalized human B lymphocytes isolated from immune individuals producing antibodies against target antigens can be produced (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 47: 86-95 (1991); and U.S. Patent No. 5,750,373). One or more cDNAs encoding antibodies in immortalized human B lymphocytes can then be prepared and inserted into expression vectors and / or heterologous host cells for use in expressing non-naturally occurring recombinant forms of antibodies.

[0196] Likewise, the bispecific antibodies or antigen-binding fragments thereof of the present disclosure can be selected from a phage library that expresses human antibodies or fragments thereof as fusion proteins with heterologous phage proteins, as described, e.g., in Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et al., Proc. Nat. Acad. Sci. USA 95:6157-6162 (1998); Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol. Biol. 222:581 (1991). Techniques for generating and using antibody phage libraries are also described in U.S. Patent Nos. 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963, each of which is incorporated by reference in its entirety.

[0197] Affinity maturation strategies and chain shuffling strategies (Marx et al., BioTechnology 10:779-783 (1992), which reference is incorporated by reference in its entirety) are known in the art and can be used to generate high-affinity human antibodies or antigen-binding fragments thereof.

[0198] In some aspects, the bispecific antibodies of the present disclosure can be humanized antibodies. Methods for engineering, humanizing or resurfacing non-human antibodies or human antibodies can also be used and these methods are well known in the art. Humanized, resurfaced or similarly engineered antibodies can have one or more amino acid residues from non-human sources such as, but not limited to, mice, rats, non-human primates or other mammals. These non-human amino acid residues are often replaced by residues referred to as "import" residues, which are typically taken from the "input" variable, constant or other domains of known human sequences. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or improve binding, affinity, on (on) rate, off (off) rate, avidity, specificity, half-life, or any other suitable feature known in the art. Typically, CDR residues are directly and primarily involved in influencing antigen (such as cell surface antigen) binding. Therefore, part or all of the non-human or human CDR sequence is maintained and the non-human sequence of the variable and constant regions can be replaced with human or other amino acids. In certain aspects, human CDRs are inserted into non-human antibody scaffolds to allow for the production of antibodies with reduced immunogenicity in animal model systems, eg, "murinized" antibodies.

[0199] The bispecific antibodies of the present disclosure can optionally be humanized, resurfaced, or engineered, wherein high affinity for cell surface antigens and other favorable biological properties are retained. To achieve this goal, humanized (or human) or engineered antibodies and resurfaced antibodies can optionally be prepared by a method of analyzing the parental sequences and different conceptual humanized and engineered products using three-dimensional models of the parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are well known to those skilled in the art.

[0200] Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to a first target (such as CD3, CD19, CD45, etc.) and / or a second target (VSV-G). In this way, framework residues can be selected and combined from the consensus and input sequences to achieve the desired antibody characteristics, such as increased affinity for one or more target antigens.

[0201] Humanization, resurfacing, or engineering of the bispecific antibodies of the present disclosure can be performed using any known method, such as, but not limited to, those described in the following references: Jones et al., Nature 321:522 (1986); Reichman et al., Nature 332:323 (1988); Weingarten et al., Science 239:1534 (1988), Sims et al., J. Immunol. 151:2296 (1993); Josiah and Leske, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,639,641; 5,7 23,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585, 089; 5,225,539; 4,816,567; 7,557,189; 7,538,195; and 7,342,110; WO 90 / 14443; WO 90 / 14424; WO 90 / 14430; and EP 229 246, each of which is incorporated herein by reference in its entirety, including the references cited therein.

[0202] In certain aspects, fragments of the bispecific antibodies of the present disclosure are provided. Different techniques are known for producing antibody fragments. Traditionally, these fragments are derived by proteolytic digestion of intact antibodies (e.g., Morimoto et al., J. Biochem. Biophy. Methods 24: 107-117 (1993); Brennan et al., Science, 229: 81 (1985)). In certain aspects, the bispecific antibody fragments of the present disclosure are recombinantly produced. All Fab, Fv, and scFv antibody fragments can be expressed in E. coli or other host cells and secreted therefrom, thereby allowing the production of large quantities of these fragments. Such antibody fragments can also be isolated from the antibody phage libraries discussed above. These antibody fragments can also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for producing antibody fragments will be clear to skilled practitioners.

[0203] Techniques suitable for producing single-chain antibodies (scFvs) can be found in, for example, U.S. Patent No. 4,946,778. In addition, methods suitable for constructing Fab expression libraries can be found in, for example, Huse et al., Science 246: 1275-1281 (1989), to allow rapid and efficient identification of monoclonal Fab fragments with desired specificity for cell surface antigens (e.g., CD3, CD19, CD45, etc.), or derivatives, fragments, analogs, or homologs thereof. Antibody fragments can be produced by techniques in the art, including, but not limited to: (a) F(ab')2 fragments produced by pepsin digestion of antibody molecules; (b) Fab fragments produced by reducing the disulfide bridges of F(ab')2 fragments; (c) Fab fragments produced by treating antibody molecules with papain and a reducing agent; and (d) Fv fragments.

[0204] In some respects, especially in the case of antibody fragment, antibody or its Fab can be modified to increase its serum half-life.This can be achieved, for example, in the following manner: by the appropriate region in mutant antibody or antibody fragment, the salvage receptor binding epitope is incorporated into the antibody or antibody fragment or by incorporating the epitope into the peptide tag of the antibody or antibody fragment that is subsequently fused to either end or in the middle (for example, by DNA or peptide synthesis) or by YTE mutation.Other methods (for example, being conjugated to heterologous molecules such as PEG) that increase the serum half-life of antibody or its Fab are well known in the art.

[0205] It should be noted that in some aspects, bispecific antibody molecules can be engineered to directly fuse the CH3 domain to the hinge region of the antibody or its fragment of the corresponding modification. In other constructs, a peptide spacer can be inserted between the CH2 and / or CH3 domains of the hinge region and the modification. For example, a compatible construct can be expressed in which the CH2 domain is missing and the remaining CH3 domain (modified or unmodified) is connected to the hinge region using a 5-20 amino acid spacer. Such a spacer can be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible or ensure that the hinge region remains flexible. However, it should be noted that the amino acid spacer can be shown to be immunogenic in some cases and to induce an undesirable immune response for the construct. Therefore, in some aspects, any spacer added to the construct will be relatively non-immunogenic, or even omitted generally, to maintain the biochemical quality of the modified antibody's hope.

[0206] The bispecific antibodies of the present disclosure can be expressed from one or more vectors. For example, in some embodiments, the first polypeptide is expressed by a vector and the second polypeptide is expressed by a second vector. In some embodiments, the first polypeptide and the second polypeptide are expressed by a vector. In some embodiments, the expression efficiency of the polypeptide is enhanced and / or increased by using only one vector. In some embodiments, the production efficiency of the bispecific antibody is enhanced and / or increased by expressing only two polypeptides. In some embodiments, the production efficiency of the bispecific antibody is enhanced and / or increased by expressing only two polypeptides compared to expressing three or more polypeptides. In some embodiments, the formation of active antigen binding sites is enhanced and / or increased by expressing only two polypeptides. In some embodiments, the formation of active antigen binding sites is enhanced and / or increased by expressing two polypeptides compared to expressing three or more polypeptides. In some embodiments, the production efficiency of the bispecific antibody is enhanced and / or increased by forming homodimer molecules compared to forming heterodimer molecules. In some embodiments, the stability of the bispecific antibody is enhanced and / or increased by forming homodimer molecules compared to forming heterodimer molecules.

[0207] In another aspect, the present disclosure provides a polynucleotide encoding the bispecific antigen binding molecule of the present disclosure or a fragment thereof.

[0208] The polynucleotides disclosed herein can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand. In some aspects, the DNA is a cDNA used to produce non-naturally occurring recombinant antibodies.

[0209] In some aspects, these polynucleotides are isolated. In some aspects, these polynucleotides are pure basically. In some aspects, these polynucleotides comprise the coding sequence of the mature polypeptide (for example, as the leader sequence of the secretory sequence of the control polypeptide from cell transportation) that is fused in the same reading frame with the polynucleotide (natural or heterologous) that for example assists in expressing and secreting polypeptide from host cell. The polypeptide with leader sequence is a preprotein and can have the leader sequence cracked by the host cell to form the mature form of polypeptide. These polynucleotides can also encode the preprotein that adds other 5 ' amino acid residues as mature protein. In some aspects, these polynucleotides are changed into and use for optimizing codon for a certain host cell.

[0210] In certain aspects, these polynucleotides include the coding sequence of the mature anti-antibody molecule fused in the same reading frame as the heterologous marker sequence, which allows, for example, purification of the encoded polypeptide. For example, the marker sequence can be, for example, a hexa-histidine (His6) tag supplied by a pQE-9 vector to provide purification of the mature polypeptide of the marker in the presence of a bacterial host. In other aspects, the marker sequence can be, for example, a hemagglutinin (HA) tag derived from an influenza hemagglutinin protein when using a mammalian host (e.g., COS-7 cells).

[0211] The present disclosure further relates to variants of the polynucleotides, eg, encoding the bispecific antibody fragments, analogs, and derivatives of the present disclosure.

[0212] In some aspects, these polynucleotide variants may be expressed in coding regions, noncoding regions, or both. In some aspects, these polynucleotide variants may be expressed in a silent manner, such as by changing the codons in human mRNA to produce a specific host, such as those preferred by bacterial hosts such as escherichia coli.

[0213] In some aspects, the DNA sequence encoding the bispecific antibody of the present disclosure can be constructed by chemical synthesis, for example, using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and those codons of the host cell that will be conducive to producing the recombinant polypeptide of interest. Standard methods can be applicable to the separation polynucleotide sequence of the isolated polypeptide of interest of the synthetic coding. For example, a complete amino acid sequence can be used to construct a gene for reverse translation. In addition, a DNA oligomer containing the nucleotide sequence of the specific isolated polypeptide of the coding can be synthesized. For example, several small oligonucleotides of the part of the desired polypeptide can be synthesized and then connected. Each oligonucleotide typically contains 5 ' or 3 ' overhangs for complementary assembly.

[0214] In case assembling (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequence of encoding the interested specific isolated polypeptide will be inserted in the expression vector and operably connected to the expression control sequence that is applicable to the host of hope expressing protein. Suitable assembling can for example be confirmed by nucleotide sequencing, restriction map and expressing biologically active polypeptide in applicable host. As well known in the art, in order to obtain the high expression level of transfected gene in host, this gene must be operably connected to functional transcription and translation expression control sequence in the expression host of selection.

[0215] In another aspect, the present disclosure provides a recombinant vector comprising the polynucleotide of the present disclosure.

[0216] In certain aspects, recombinant vectors are replicable DNA constructs having polypeptide chains encoding the disclosed antibodies and / or antigen-binding fragments thereof, synthetic or cDNA-derived DNA fragments operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit typically comprises an assembly of: (1) a genetic element or elements that regulate gene expression, such as a transcriptional promoter or enhancer, (2) a structure or coding sequence that is transcribed into mRNA and translated into a protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Such regulatory elements may include an operator sequence that controls transcription.

[0217] The ability to replicate in the host and the selection gene that promotes identification of transformants, which are usually given by the origin of replication, can be additionally incorporated. When the DNA regions are functionally related to each other, they are operably connected. For example, if the DNA of a signal peptide (secretion leader) is expressed as a precursor that participates in polypeptide secretion, it is operably connected to the DNA of the polypeptide; if the transcription of the promoter control sequence, it is operably connected to the coding sequence; or if the ribosome binding site is positioned to allow translation, it is operably connected to the coding sequence. Structural elements intended for use in yeast expression systems include leader sequences that can achieve protein translation through extracellular secretion of the host cell. Alternatively, in the case of expressing a recombinant protein without using a leader or transport sequence, it can include a methionine residue at the N-terminus. This residue can optionally be subsequently cracked from the expressed recombinant protein to provide a final product.

[0218] The selection of expression control sequences and expression vectors will depend on the selection of the host. Various expression host / vector combinations can be adopted. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9, and derivatives thereof, plasmids with a wider host range, such as M13 and filamentous single-stranded DNA phage.

[0219] In another aspect, the present disclosure provides a host cell comprising a polynucleotide or recombinant vector of the present disclosure. This includes prokaryotes, yeast, insects, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or bacteria. Higher eukaryotic cells include established mammalian cell lines as described below. Cell-free translation systems can also be used. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosures of which are hereby incorporated by reference.

[0220] Additional information on methods of protein production, including antibody production, can be found in, for example, Guan Gong Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823, each of which is hereby incorporated by reference in its entirety.

[0221] Different mammalian or insect cell culture systems can also be advantageously used to express the bispecific antibody molecules or antigen-binding fragments thereof of the present disclosure. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 monkey kidney cell line described by Gluzman (Cell 23: 175, 1981); and other cell lines, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), NSO, HeLa, and BHK cell lines. Mammalian expression vectors can contain non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. The baculovirus system for producing heterologous proteins in insect cells is reviewed by Lucko W and Summers, Biotechnology 6:47 (1988).

[0222] The antibody or its Fab produced by the host of conversion can be purified according to any suitable method.Such standard method includes, for example, chromatography (for example, ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility or any other standard technique by protein purification.Affinity tags such as hexa-histidine, maltose binding domain, influenza capsid sequence, glutathione S-transferase etc. can be attached to protein to allow simple purification by passing through on appropriate affinity column.For example, proteolysis, nuclear magnetic resonance or x-ray crystallography can also be used to physically characterize isolated proteins.

[0223] For example, can first use commercially available protein concentration filter, for example Millipore ultrafiltration unit concentrates the supernatant from the system that recombinant protein is secreted into substratum.After the concentration step, concentrate can be applied to the purification matrix that is applicable to.Alternatively, can adopt anion exchange resin, for example matrix or substrate with side chain diethylaminoethyl (DEAE) group.These matrices can be acrylamide, agarose, dextran, cellulose or are usually used in other types of protein purification.Alternatively, can adopt cation exchange step.

[0224] Suitable cation exchangers include different insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps using hydrophobic RP-HPLC media (e.g., silica gel with side chain methyl or other aliphatic groups) can be used to further purify the antibody molecule. Some or all of the aforementioned purification steps can also be used in different combinations to provide a homogeneous recombinant protein.

[0225] Bispecific antibodies or antigen-binding fragments thereof produced in bacterial culture can be isolated, for example, by initial extraction from a cell pellet, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in the expression of recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents.

[0226] Methods known in the art for purifying antibodies and other proteins also include, for example, those described in US Patent Publication Nos. US20080312425, US20080177048, and US20090187005, each of which is hereby incorporated by reference in its entirety.

[0227] In another aspect, the present disclosure provides a method for transducing a target gene into a subject using a lentiviral vector, comprising administering a bispecific antigen binding molecule or a fragment thereof of the present disclosure to the subject.

[0228] The lentiviral vector in the present disclosure can encode exogenous genes in its genomic RNA. Recombinant viral vectors containing exogenous genes can be obtained by inserting exogenous genes into the genome of the lentiviral vector. The "exogenous gene" mentioned in the present disclosure can be any target gene that needs to be expressed in the cell, and can be a gene encoding a natural protein, or a protein modified by deleting, replacing or inserting amino acid residues in the natural protein. The exogenous gene can be inserted into any target position in the non-coding region of the protein in the viral genome. The target gene is transferred into the target cell through the lentiviral vector and expressed in the target cell; further, the expression of the target gene in the target cell can play a role in preventing and or treating diseases. Those skilled in the art can select the target gene carried by the lentiviral vector as needed, and the present disclosure is not limited here.

[0229] Example

[0230] Plasmid vector construction

[0231] (1) For the CD19scfv-VSV-G bispecific antibody, the vector construction includes inserting the DNA encoding the anti-human CD19 scFv fragment before the DNA encoding the human antibody Fc segment, inserting the DNA fragment encoding the human LDL-R CR2-CR3 after the antibody Fc segment, and inserting the three connected sequences into an expression vector that can be expressed in mammalian cells. The mammalian cell expression vector MG-HSP-v1 contains a CMV promoter that can initiate downstream gene expression in mammalian cells.

[0232] The scFv fragment of anti-human CD19 was optimized according to the article (DOI:10.1182 / blood-2010-04-281931) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain a DNA sequence and synthesize it. Its nucleotide sequence is shown in SEQ ID NO.56. The DNA encoding the human antibody Fc segment was optimized according to the protein database UniProt (P01857·IGHG1_HUMAN) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain a DNA sequence and synthesize it. Its nucleotide sequence is shown in SEQ ID NO.57. The DNA sequence of CR2-CR3 was optimized using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) according to the protein database UniProt (P01130·LDLR_HUMAN) and synthesized. The nucleotide sequence is shown in SEQ ID NO. 58.

[0233] The three fragments were ligated using overlap extension polymerase chain reaction (EER). The primers used are listed in Table 1. The expression vector MG-HSP-v1 (nucleotide sequence shown in SEQ ID NO. 55) was linearized by treatment with BsaI restriction endonuclease (NEB, Cat# R3733L).

[0234] Table 1

[0235] Add the following ingredients to a 1.5 mL Eppendorf tube:

[0236] Mix the above ingredients and let them stand on ice for 5 minutes. Add 1 μl of Exonuclease III (Takara, Cat#2170A), mix thoroughly with a pipette, and let them stand on ice for 60 minutes. Add 1 μl of 0.5M EDTA (Invitrogen, Cat#11568896), mix thoroughly, heat at 65°C for 5 minutes, and let them stand on ice for 5 minutes. After centrifugation, add the entire mixture to 100 μl of DH5α, incubate on ice for 20 minutes, heat shock at 42°C for 45 seconds, and incubate on ice for 2 minutes. Add antibiotic-free LB medium and pre-shake for 60 minutes before spreading onto plates containing ampicillin. After 16 hours, pick a single clone, amplify it, and extract the plasmid after sequencing.

[0237] (2) For CD3 (OKT3) -VSV-G, CD3 (UCHT1) -VSV-G, CD4-VSV-G, CD20-VSV-G, CD40-VSV-G, CD45-VSV-G, and HLA-VSV-G bispecific antibodies, two vectors need to be constructed. In one, the DNA encoding the VH fragment of anti-human CD3 (OKT3), CD3 (UCHT1), CD4, CD20, CD40, CD45, or HLA is inserted before the DNA encoding the CH1-Fc segment of the human antibody, and the DNA fragment encoding the CR2-CR3 of the human LDL-R is inserted after the antibody Fc segment. The three segments are connected and the complete sequence is inserted into an expression vector that can be expressed in mammalian cells. Among them, the mammalian cell expression vector MG-HSP.v2 (synthetic) contains a CMV promoter, which can initiate downstream gene expression in mammalian cells. Second, DNA encoding the VL segment of an antibody against human CD3 (OKT3), CD3 (UCHT1), CD4, CD20, CD40, CD45, or HLA is inserted before DNA encoding the CL segment of a human antibody. The two linked sequences are then inserted into an expression vector capable of expression in mammalian cells. The mammalian cell expression vector, MG-LSP.V1 (synthetic), contains a CMV promoter, which can drive downstream gene expression in mammalian cells.

[0238] Table 2

[0239] The VH and VL fragments of the anti-human CD3 (OKT3) were optimized according to the article (DOI: 10.1073 / pnas.0402295101) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain DNA sequences and synthesize them (wherein, the VH DNA sequence is shown as SEQ ID NO. 60, and the VL DNA sequence is shown as SEQ ID NO. 67). The VH and VL fragments of the anti-human CD3 (UCHT1) were optimized according to the article (DOI: 10.1073 / pnas.0407359101) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain DNA sequences and synthesize them (wherein, the VH DNA sequence is shown as SEQ ID NO. 61, and the VL DNA sequence is shown as SEQ ID NO.68); the VH and VL fragments of the anti-human CD4 fragments were optimized according to the patent (https: / / patents.google.com / patent / CN101245108B / en) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain DNA sequences and synthesized (wherein, the VH DNA sequence is shown as SEQ ID NO.62, and the VL DNA sequence is shown as SEQ ID NO.69); the VH and VL fragments of the anti-human CD20 fragments were optimized according to the patent (https: / / patents.google.com / patent / CN101245108B / en) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain DNA sequences and synthesized (wherein, the VH DNA sequence is shown as SEQ ID NO.63, and the VL DNA sequence is shown as SEQ ID No. 70); the VH and VL fragments of the anti-human CD40 fragment were prepared according to the patent (https: / / patents.google.com / patent / EP3925977A1 / en) and optimized using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) were optimized to obtain DNA sequences and synthesized (wherein, the VH DNA sequence is shown in SEQ ID NO.64, and the VL DNA sequence is shown in SEQ ID NO.71); the VH and VL fragments of anti-human CD45 were optimized according to the database (GenBank: QES69311.1) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain DNA sequences and synthesized (wherein, the VH DNA sequence is shown in SEQ ID NO.65, and the VL DNA sequence is shown in SEQ ID NO.72); the VH and VL fragments of anti-human HLA were optimized according to the article (DOI: DOI: 10.1016 / 0092-8674(78)90296-9) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain DNA sequences and synthesized (wherein, the VH DNA sequence is shown in SEQ ID NO.66, and the VL DNA sequence is shown in SEQ ID NO.73); the DNA encoding the human antibody CH1-Fc segment was optimized according to the protein database UniProt (P01857·IGHG1_HUMAN) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain the DNA sequence and synthesize it (its nucleotide sequence is shown in SEQ ID NO.74); the DNA encoding the human antibody CL segment was optimized according to the protein database UniProt (P01834·IGKC_HUMAN) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain the DNA sequence and synthesize it (its nucleotide sequence is shown in SEQ ID NO.75); the DNA encoding the human LDL-R CR2-CR3 is the same as (1).

[0240] The desired fragments were ligated using overlap extension polymerase chain reaction. The primers used are listed in Table 3. The expression vectors MG-HSP-v2 (nucleotide sequence shown in SEQ ID NO. 59) or MG-LSP-v1 (nucleotide sequence shown in SEQ ID NO. 76) were linearized by treating them with BsaI restriction endonuclease (NEB, Cat# R3733L).

[0241] Table 3

[0242] For CD3 (OKT3) (or CD3 (UCHT1), CD4, CD20, CD40, CD45, HLA)-VH-IgG1-CH1-Fc-LDLR-CR2 / 3 plasmid, add each component to a 1.5 mL Eppendorf tube according to the following ratio:

[0243] For CD3 (OKT3) (or CD3 (UCHT1), CD4, CD20, CD40, CD45, HLA)-VL-CL plasmid, add the following components to a 1.5 mL Eppendorf tube:

[0244] Mix the above ingredients and let them stand on ice for 5 minutes. Add 1 μl of Exonuclease III (Takara, Cat#2170A), mix thoroughly with a pipette, and let them stand on ice for 60 minutes. Add 1 μl of 0.5M EDTA (Invitrogen, Cat#11568896), mix thoroughly, heat at 65°C for 5 minutes, and let them stand on ice for 5 minutes. After centrifugation, add the entire mixture to 100 μl of DH5α, incubate on ice for 20 minutes, heat shock at 42°C for 45 seconds, and incubate on ice for 2 minutes. Add antibiotic-free LB medium and pre-shake for 60 minutes before spreading onto plates containing ampicillin. After 16 hours, pick a single clone, amplify it, and extract the plasmid after sequencing.

[0245] (3) For the CD40L-VSV-G bispecific antibody, the vector construction includes inserting the DNA fragment encoding the CR2-CR3 of the human LDL-R before the Fc segment of the antibody, inserting the DNA fragment encoding the human CD40 ligand after the DNA encoding the human antibody Fc segment, and inserting the three connected sequences into an expression vector that can be expressed in mammalian cells. The mammalian cell expression vector MG-HSP-v1 contains a CMV promoter that can initiate downstream gene expression in mammalian cells.

[0246] The fragment of human CD40 ligand was optimized according to the protein database UniProt (P29965·CD40L_HUMAN) using the codon optimization tool of GenScript (https: / / www.genscript.com / gensmart-free-gene-codon-optimization.html) to obtain the DNA sequence and synthesize the nucleotide sequence as shown in SEQ ID NO.; the DNA encoding the human antibody Fc segment is the same as (1), and the DNA encoding the human LDL-R CR2-CR3 is the same as (1).

[0247] The three fragments were ligated using overlap extension polymerase chain reaction (EER). The primers used are listed in Table 1. The expression vector MG-HSP-v1 (nucleotide sequence shown in SEQ ID NO. 55) was linearized by treatment with BsaI restriction endonuclease (NEB, Cat# R3733L).

[0248] Table 4

[0249] Add the following ingredients to a 1.5 mL Eppendorf tube:

[0250] Mix the above ingredients and let them stand on ice for 5 minutes. Add 1 μl of Exonuclease III (Takara, Cat#2170A), mix thoroughly with a pipette, and let them stand on ice for 60 minutes. Add 1 μl of 0.5M EDTA (Invitrogen, Cat#11568896), mix thoroughly, heat at 65°C for 5 minutes, and let them stand on ice for 5 minutes. After centrifugation, add the entire mixture to 100 μl of DH5α, incubate on ice for 20 minutes, heat shock at 42°C for 45 seconds, and incubate on ice for 2 minutes. Add antibiotic-free LB medium and pre-shake for 60 minutes before spreading onto plates containing ampicillin. After 16 hours, pick a single clone, amplify it, and extract the plasmid after sequencing.

[0251] Example 1 Preparation of bispecific antibody molecules

[0252] (1) Day -2: Expi-293F cells were cultured in suspension in Union293 cell culture medium (supplemented with 1% GlutaMax) in a 37°C incubator with 5% CO2 and a rotation speed of 180 rpm. Ensure that the newly recovered cells have been cultured for three or more generations and have a viability of ≥90%. Culture and expand Epi293F cells until the day before transfection (Day -1) when the cell density should reach approximately 3-5×106 / mL.

[0253] (2) Day 1: Final density 2.5-3×10 6 Inoculate cells at 1% 4% CO / mL and allow the cells to grow overnight.

[0254] (3) Day 0: Determine the viable cell density and viability. The viable cell density and viability should reach 4.5-5.5×10 6 / mL and ≥95%, then transfection can be performed. Dilute the cells to 3×10 6 viable cells / mL, gently shake the culture flask to mix the cells. Prepare the transfection mixture in a sterile 15mL centrifuge tube (taking a 30mL expression system as an example): Opti-MEM Reduced Serum Medium 3mL Total expression plasmid (light chain: heavy chain = 2.5:1) 24μg Polyethylenimine (PEI) transfection reagent 96μg

[0255] Use a pipette to gently blow the mixture evenly and let it stand at room temperature for 10 minutes. Add 27 mL of 3×10 6 / mL of Expi-293F cells, and then add the mixture dropwise to the cells in the culture dish, shake gently to mix, and place the cells in a 37°C incubator containing 5% CO2 for 24 hours.

[0256] (4) Day 1: Add 300 μL of 300 mM valproic acid (VPA, Sigma cat#P4543; prepared with water and filtered through a 0.22 μM filter) and 270 μL of 45% glucose (Sigma cat#G8769) to the cells, shake gently to mix, and culture the cells in a 37°C incubator containing 5% CO2.

[0257] (5) Day 5: Protein collection. Transfer the cells to a 50 mL centrifuge tube and centrifuge at 4000 rpm for 10 minutes at 4°C. Transfer the supernatant to a new 50 mL centrifuge tube.

[0258] (6) Mix 400 μL of Protein G purification medium (Genscript cat#L00664) and add 1 mL of 1× PBS. Centrifuge at 600 g for 10 minutes at 4°C and discard the supernatant. Repeat the washing process three times to remove the ethanol in the storage solution. Add the washed Protein G purification medium to the supernatant obtained in step (5) and incubate at 4°C with rotation for 2-4 hours.

[0259] (7) Prepare the gravity column for purification. Place a hydrophilic frit of appropriate size into a 3 mL affinity chromatography column tube and pre-wet the frit with equilibration / wash solution (25 mM Tris, 150 mM NaCl; pH 7.2). Transfer the supernatant after incubation to the column tube. Use gravity to allow unbound proteins and impurities to flow out with the culture medium supernatant. The Protein G purification medium bound to the target protein accumulates in the column tube under the action of the frit. Slowly add 10 column volumes of equilibration / wash solution to the column tube to wash the Protein G purification medium. Slowly add 1 mL of eluent (0.1 M glycine, pH 2-3) and collect the eluent at the outlet (add 1 / 10 volume of neutralization solution (1 M Tris, pH 7.5-9) in advance). Measure the protein concentration. Repeat the elution step until the protein concentration of the effluent is undetectable.

[0260] (8) Use a 50KD ultrafiltration tube to replace the protein solvent from the eluent with 1× PBS and concentrate to 0.5 mg / mL. Filter with a 0.22 μM filter and store in a 4°C refrigerator for short-term storage or at -20°C for extended storage.

[0261] Among them, the heavy chain sequence of the CD3-VSV-G bispecific antibody is shown in SEQ ID NO.2, and the light chain sequence is shown in SEQ ID NO.3; the heavy chain sequence of the CD45-VSV-G bispecific antibody is shown in SEQ ID NO.4, and the light chain sequence is shown in SEQ ID NO.5; the CD19scfv-VSV-G bispecific antibody sequence is shown in SEQ ID NO.6.

[0262] Example 2 VSV-G pseudotyped lentivirus packaging

[0263] (1) Day 0: Trypsinize HEK293T cells and collect a certain amount of cells in a six-well plate. The cell mass should be controlled to reach a confluence of about 70-80% at the time of transfection on the second day to increase transfection efficiency. Place the cells in a 37°C incubator with 5% CO2 for 8-24 hours.

[0264] (2) Day 1: Transfection can begin when cells are fully attached and have reached an appropriate density. 2 hours before transfection, remove the HEK293T cell culture medium using a vacuum pump and replace with 1.5 mL of fresh DMEM medium (supplemented with 10% FBS (inactivated by heating at 56°C for 30 minutes) and 1% penicillin-streptomycin, referred to as DMEM complete medium below). Prepare the plasmid mixture for transfection in a sterile 1.5 mL Eppendorf tube. Prepare each reaction according to the following formula:

[0265] Gently pipette the mixture until smooth and allow it to stand at room temperature for 10 minutes. After this time, add the mixture dropwise to the cells in the culture dish, gently shaking in a figure-of-ten (X) or figure-of-eight (E8) pattern to mix thoroughly. After incubating the cells in a 37°C incubator with 5% CO₂ for 8 hours, replace the culture medium with 2 mL of fresh complete DMEM.

[0266] (3) Day 3: 48 hours after transfection, collect the culture medium of HEK293T cells in a sterile Eppendorf tube. Centrifuge the culture medium at 500 g for 10 minutes at 4°C to remove cell debris and impurities. Then, filter the culture medium using a syringe and filter to obtain VSV-G pseudotyped lentivirus carrying GFP fluorescent protein mRNA. Aliquot the filtered virus solution and store it at -80°C until use.

[0267] Example 3: Enhancement of lentiviral infection of Ramos cells using bispecific molecules (targeting HLA)

[0268] (1) Day 1: Target Ramos cells were cultured in IMDM medium (the medium was supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, and 1% GlutaMAX, and IMDM complete medium was used below). Before infection, target cells were harvested and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 300 g for 3 minutes at 4°C, and the culture medium was discarded.

[0269] (2) Use pre-chilled IMDM medium (supplemented with 2% FBS) to grade the dilution of the HLA-VSV-G bispecific molecule to a final concentration of 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, and 0.00 μg / mL, respectively. (At the same time, use 2.5 μg / mL of an antibody targeting only HLA (without the LDLR-CR2-CR3 domain) as a control.) Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS) to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 300 g for 3 minutes at 4°C and discard the medium.

[0270] (3) Resuspend the cells again in 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS), centrifuge at 300 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0271] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 0.7 using pre-chilled IMDM complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the cell surface has already bound the bispecific molecule) with 0.5 mL of the virus mixture. Gently pipette to mix, then inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0272] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 300 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of IMDM complete medium preheated to 37°C and inoculate them into a 24-well plate. Check the cell status and density daily and replace with fresh medium or increase the cell culture volume if necessary to ensure that the cell density does not exceed 3 × 10 6 pieces / mL.

[0273] (7) Day 4: 60 hours after infection, the infection efficiency was detected by flow cytometry, and uninfected cells were used as controls. The results are shown in Figures 8A to 8C.

[0274] Example 4: Enhancement of lentiviral infection of Jurkat T cells using bispecific molecules (targeting HLA)

[0275] (1) Day 1: Jurkat T cells were cultured in RPMI-1640 medium (supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, 1% GlutaMAX, and 0.1% 2-mercaptoethanol (55 mM). RPMI-1640 complete medium is used below. Before infection, target cells were harvested and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 300 g for 3 minutes at 4°C, and the culture medium was discarded.

[0276] (2) Use pre-chilled RPMI-1640 medium (supplemented with 2% FBS) to grade the HLA-VSV-G bispecific molecule to a final concentration of 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, and 0.00 μg / mL, respectively. (An antibody targeting only HLA (without the LDLR-CR2-CR3 domain) at 2.5 μg / mL is used as a control.) Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled RPMI-1640 medium (supplemented with 2% FBS) to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 300 g for 3 minutes at 4°C and discard the medium.

[0277] (3) Resuspend the cells again in 1 mL of pre-chilled RPMI-1640 medium (supplemented with 2% FBS), centrifuge at 300 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0278] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 1 using pre-chilled RPMI-1640 complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the cell surface has already bound the bispecific molecule) in 0.5 mL of the virus mixture. Gently pipette to mix, then inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0279] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 300 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of RPMI-1640 complete medium preheated to 37°C and inoculate them into a 24-well plate. Check the cell status and density every day. If necessary, replace the culture medium with fresh medium or expand the cell culture volume to ensure that the cell density does not exceed 3 × 10 6 pieces / mL.

[0280] (6) Day 4: 60 hours after infection, the infection efficiency was detected by flow cytometry, and uninfected cells were used as controls. The results are shown in Figures 9A to 9C.

[0281] Example 5: Enhancing Lentivirus Infection of Human Peripheral Blood Mononuclear Cells Using Bispecific Molecules (Targeting HLA)

[0282] (1) Day 1: Thaw frozen human peripheral blood mononuclear cells (PBMCs), resuspend and count. Add 1×10 6PBMC cells were cultured and supplemented with a final volume of 2 mL of complete culture medium (X-VIVO 15 serum-free medium, 2% FBS (inactivated by heating at 56°C for 30 minutes), referred to as complete culture medium below), and IL-15 (5 ng / mL) and IL-7 (10 ng / mL) were added to the culture medium.

[0283] (2) Day 2: Before infection, target cells were collected and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 500 g for 3 minutes at 4°C, and the culture medium was discarded.

[0284] (2) Dilute the HLA-VSV-G bispecific molecule with pre-chilled complete medium to a final concentration of 1.25 and 0.00 μg / mL, respectively (use an antibody targeting only HLA (without the LDLR-CR2-CR3 domain) at 2.5 μg / mL as a control). Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled complete medium to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 500 g for 3 minutes at 4°C and discard the medium.

[0285] (3) Resuspend the cells again in 1 mL of pre-chilled complete medium, centrifuge at 500 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0286] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 1 using pre-chilled complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the bispecific molecule has already bound to the cell surface) with 0.3 mL of the virus mixture. Add IL-15 (5 ng / mL) and IL-7 (10 ng / mL). Mix thoroughly by gently pipetting and inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0287] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 500 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of complete culture medium preheated to 37°C, add IL-15 (5 ng / mL) and IL-7 (10 ng / mL), and seed into a 24-well plate. Check the cell status and density daily and replace with fresh culture medium or increase the cell culture volume if necessary.

[0288] (6) Day 4: 72 hours after infection, the infection efficiency was detected by flow cytometry, and uninfected cells were used as a control. The results are shown in Figure 10.

[0289] Example 6: Enhancement of lentiviral infection of Ramos cells using bispecific molecules (targeting CD19)

[0290] (1) Day 1: Target Ramos cells were cultured in IMDM medium (the medium was supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, and 1% GlutaMAX, and IMDM complete medium was used below). Before infection, target cells were harvested and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 300 g for 3 minutes at 4°C, and the culture medium was discarded.

[0291] (2) Dilute the bispecific molecule CD19scfv-VSV-G with pre-chilled IMDM medium (supplemented with 2% FBS) to a final concentration of 5 or 0 μg / mL. Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS) to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 300 g for 3 minutes at 4°C and discard the medium.

[0292] (3) Resuspend the cells again in 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS), centrifuge at 300 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0293] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 1 using pre-chilled IMDM complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the cell surface has already bound the bispecific molecule) with 0.5 mL of the virus mixture. Gently pipette to mix, then inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0294] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 300 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of IMDM complete medium preheated to 37°C and inoculate them into a 24-well plate. Check the cell status and density every day. Replace the culture medium or increase the cell culture volume if necessary to ensure that the cell density does not exceed 3 × 10 6 pieces / mL.

[0295] (6) Day 4: 72 hours after infection, the infection efficiency was detected by flow cytometry, and uninfected cells were used as a control. The results are shown in Figure 11.

[0296] Example 7: Enhancement of lentiviral infection of Ramos cells using bispecific molecules (targeting CD20)

[0297] (1) Day 1: Target Ramos cells were cultured in IMDM medium (the medium was supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, and 1% GlutaMAX, and IMDM complete medium was used below). Before infection, target cells were harvested and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 300 g for 3 minutes at 4°C, and the culture medium was discarded.

[0298] (2) Dilute the bispecific molecule CD20-VSV-G with pre-chilled IMDM medium (supplemented with 2% FBS) to a final concentration of 5 or 0 μg / mL. Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS) to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 300 g for 3 minutes at 4°C and discard the medium.

[0299] (3) Resuspend the cells again in 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS), centrifuge at 300 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0300] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 1 using pre-chilled IMDM complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the cell surface has already bound the bispecific molecule) with 0.5 mL of the virus mixture. Gently pipette to mix, then inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0301] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 300 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of IMDM complete medium preheated to 37°C and inoculate them into a 24-well plate. Check the cell status and density every day. Replace the culture medium or increase the cell culture volume if necessary to ensure that the cell density does not exceed 3 × 10 6 pieces / mL.

[0302] (6) Day 4: 60 hours after infection, the infection efficiency was detected by flow cytometry. The results are shown in Figure 12.

[0303] Example 8: Enhancement of lentiviral infection of Ramos cells using bispecific molecules (targeting CD45)

[0304] (1) Day 1: Target Ramos cells were cultured in IMDM medium (the medium was supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, and 1% GlutaMAX, and IMDM complete medium was used below). Before infection, target cells were harvested and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 300 g for 3 minutes at 4°C, and the culture medium was discarded.

[0305] (2) Dilute the bispecific molecule CD40-VSV-G with pre-chilled IMDM medium (supplemented with 2% FBS) to a final concentration of 5 or 0 μg / mL. Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS) to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 300 g for 3 minutes at 4°C and discard the medium.

[0306] (3) Resuspend the cells again in 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS), centrifuge at 300 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0307] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 1 using pre-chilled IMDM complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the cell surface has already bound the bispecific molecule) with 0.5 mL of the virus mixture. Gently pipette to mix, then inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0308] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 300 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of IMDM complete medium preheated to 37°C and inoculate them into a 24-well plate. Check the cell status and density every day. Replace the culture medium or increase the cell culture volume if necessary to ensure that the cell density does not exceed 3 × 10 6 pieces / mL.

[0309] (6) Day 4: 72 hours after infection, the infection efficiency was detected by flow cytometry. The results are shown in Figure 13.

[0310] Example 9: Enhancement of lentiviral infection of Ramos cells using bispecific molecules (targeting CD40)

[0311] (1) Day 1: Target Ramos cells were cultured in IMDM medium (the medium was supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, and 1% GlutaMAX, and IMDM complete medium was used below). Before infection, target cells were harvested and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 300 g for 3 minutes at 4°C, and the culture medium was discarded.

[0312] (2) Dilute the bispecific molecule CD40-VSV-G or CD40L-VSV-G in pre-chilled IMDM medium (supplemented with 2% FBS) to a final concentration of 5 μg / mL (using cells without antibody treatment as a control). Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS) to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 300 g for 3 minutes at 4°C and discard the medium.

[0313] (3) Resuspend the cells again in 1 mL of pre-chilled IMDM medium (supplemented with 2% FBS), centrifuge at 300 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0314] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 1 using pre-chilled IMDM complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the cell surface has already bound the bispecific molecule) with 0.5 mL of the virus mixture. Gently pipette to mix, then inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0315] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 300 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of IMDM complete medium preheated to 37°C and inoculate them into a 24-well plate. Check the cell status and density every day. Replace the culture medium or increase the cell culture volume if necessary to ensure that the cell density does not exceed 3 × 10 6 pieces / mL.

[0316] (6) Day 4: 60 hours after infection, the infection efficiency was detected by flow cytometry. The results are shown in Figure 14.

[0317] Example 10: Enhancement of lentiviral infection of Jurkat T cells using bispecific molecules (targeting CD3)

[0318] (1) Day 1: Jurkat T cells were cultured in RPMI-1640 medium (supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, 1% GlutaMAX, and 0.1% 2-mercaptoethanol (55 mM). RPMI-1640 complete medium is used below. Before infection, target cells were harvested and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 300 g for 3 minutes at 4°C, and the culture medium was discarded.

[0319] (2) Dilute the bispecific molecule CD3(OKT3)-VSV-G with pre-chilled RPMI-1640 medium (supplemented with 2% FBS) to a final concentration of 5 or 0 μg / mL. Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled RPMI-1640 medium (supplemented with 2% FBS) to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 300 g for 3 minutes at 4°C and discard the medium.

[0320] (3) Resuspend the cells again in 1 mL of pre-chilled RPMI-1640 medium (supplemented with 2% FBS), centrifuge at 300 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0321] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 2 using pre-chilled RPMI-1640 complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the cell surface has already bound the bispecific molecule) with 0.5 mL of the virus mixture. Gently pipette to mix, then inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0322] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 300 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of RPMI-1640 complete medium preheated to 37°C and inoculate them into a 24-well plate. Check the cell status and density every day. If necessary, replace the culture medium with fresh medium or expand the cell culture volume to ensure that the cell density does not exceed 3 × 10 6 pieces / mL.

[0323] (6) Day 4: 60 hours after infection, the infection efficiency was detected by flow cytometry. The results are shown in Figure 15.

[0324] Example 11: Enhancement of lentiviral infection of unstimulated human primary T cells using bispecific molecules (targeting CD3)

[0325] (1) Day 1: Thaw frozen human peripheral blood mononuclear cells (PBMCs), resuspend and count. Add 1×10 6 PBMC cells were cultured and supplemented with a final volume of 2 mL of complete culture medium (X-VIVO 15 serum-free medium, 2% FBS (inactivated by heating at 56°C for 30 minutes), referred to as complete culture medium below), and IL-15 (5 ng / mL) and IL-7 (10 ng / mL) were added to the culture medium.

[0326] (2) Day 2: Before infection, target cells were collected and counted, and a specific number of cells were collected into sterile 1.5 mL Eppendorf tubes (approximately 5 × 10 cells per infection reaction). 5 cells), centrifuged at 500 g for 3 minutes at 4°C, and the culture medium was discarded.

[0327] (2) Dilute the CD3(OKT3)-VSV-G or CD3(UCHT1)-VSV-G bispecific molecule with pre-chilled complete medium to a final concentration of 5 μg / mL (using cells without antibody treatment as a control). Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled complete medium to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 500 g for 3 minutes at 4°C and discard the medium.

[0328] (3) Resuspend the cells again in 1 mL of pre-chilled complete medium, centrifuge at 500 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0329] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 5 using pre-chilled complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the bispecific molecule has already bound to the cell surface) with 0.5 mL of the virus mixture. Add IL-15 (5 ng / mL) and IL-7 (10 ng / mL). Mix by gently pipetting and inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0330] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 500 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of complete culture medium preheated to 37°C, add IL-15 (5 ng / mL) and IL-7 (10 ng / mL), and inoculate into a 24-well plate. Check the cell status and density daily and replace with fresh culture medium or increase the cell culture volume if necessary.

[0331] (6) Day 4: 72 hours after infection, the infection efficiency was detected by flow cytometry. The results are shown in Figure 16.

[0332] Example 12: Enhancement of lentiviral infection of CD3 / CD28 antibody-activated human primary T cells using bispecific molecules (targeting CD3)

[0333] (1) Day 1: Dilute CD3 and CD28 antibodies in sterile water to a final concentration of 1 μg / mL. Use a pipette to add 0.5 mL (= 0.5 μg antibody) of antibody solution per well to a non-tissue culture treated 24-well plate and incubate in an incubator at 37°C for 4 hours. Aspirate the antibody solution and add 1 mL of complete culture medium (X-VIVO 15 serum-free culture medium, 2% FBS (heated at 56°C for 30 minutes to inactivate), referred to as complete culture medium below) to each well and incubate in an incubator at 37°C for 15 to 30 minutes. During this period, the frozen PBMCs were revived and resuspended for counting. Aspirate the culture medium and add 1×10 6 PBMC cells were added and the final volume was 2 mL of complete culture medium. PBS was added to the empty wells to prevent the evaporation of culture medium.

[0334] (2) Day 2: Add IL-15 (5 ng / mL) and IL-7 (10 ng / mL).

[0335] (3) Day 4: Before infection, target cells were collected and counted. A specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 × 105 cells per infection reaction), centrifuged at 500 g for 3 min at 4°C, and the culture medium was discarded.

[0336] (2) Dilute the CD3(OKT3)-VSV-G or CD3(UCHT1)-VSV-G bispecific molecule with pre-chilled complete medium to a final concentration of 5 μg / mL (using cells without antibody treatment as a control). Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled complete medium to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 500 g for 3 minutes at 4°C and discard the medium.

[0337] (3) Resuspend the cells again in 1 mL of pre-chilled complete medium, centrifuge at 500 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0338] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 5 using pre-chilled complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the bispecific molecule has already bound to the cell surface) with 0.5 mL of the virus mixture. Add IL-15 (5 ng / mL) and IL-7 (10 ng / mL). Mix by gently pipetting and inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0339] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 500 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of complete culture medium preheated to 37°C, add IL-15 (5 ng / mL) and IL-7 (10 ng / mL), and inoculate into a 24-well plate. Check the cell status and density daily and replace with fresh culture medium or increase the cell culture volume if necessary.

[0340] (6) Day 4: 72 hours after infection, the infection efficiency was detected by flow cytometry. The results are shown in Figure 17.

[0341] Example 13: Enhancement of lentiviral infection of unstimulated human primary T cells using bispecific molecules (targeting CD4)

[0342] (1) Day 1: Thaw frozen human peripheral blood mononuclear cells (PBMCs), resuspend and count. Add 1×10 6 PBMC cells were cultured and supplemented with a final volume of 2 mL of complete culture medium (X-VIVO 15 serum-free medium, 2% FBS (inactivated by heating at 56°C for 30 minutes), referred to as complete culture medium below), and IL-15 (5 ng / mL) and IL-7 (10 ng / mL) were added to the culture medium.

[0343] (2) Day 2: Before infection, target cells were collected and counted. A specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 × 105 cells per infection reaction), centrifuged at 500 g for 3 minutes at 4°C, and the culture medium was discarded.

[0344] (2) Dilute the CD4 bispecific molecule with pre-chilled complete medium to a final concentration of 5 μg / mL (using a control without antibody treatment). Resuspend the cells centrifuged in step 1 with 50 μL of the mixture and incubate in a 4°C refrigerator for 15 minutes. After incubation, add 1 mL of pre-chilled complete medium to the cell suspension to dilute the concentration of the bispecific molecule in the system. Centrifuge at 500 g for 3 minutes at 4°C and discard the medium.

[0345] (3) Resuspend the cells again in 1 mL of pre-chilled complete medium, centrifuge at 500 g for 3 minutes at 4°C, and discard the medium. Repeat this step once more.

[0346] (4) Prepare a VSV-G pseudotyped lentivirus mixture for infection. Dilute the virus to an MOI of approximately 5 using pre-chilled complete medium. Resuspend the cells collected by centrifugation in step (3) (at this point, the bispecific molecule has already bound to the cell surface) with 0.5 mL of the virus mixture. Add IL-15 (5 ng / mL) and IL-7 (10 ng / mL). Mix by gently pipetting and inoculate into a 24-well plate. Centrifuge at 1800 g for 45 minutes at 37°C (acceleration 9, deceleration 5). After centrifugation, place the cells in a 37°C incubator containing 5% CO2 and culture.

[0347] (5) Day 2: 24 hours after infection, gently pipette the cells and transfer them to a sterile 1.5 mL Eppendorf tube. Centrifuge at 500 g for 3 minutes at room temperature and discard the culture medium. Resuspend the cells in 1 mL of complete culture medium preheated to 37°C, add IL-15 (5 ng / mL) and IL-7 (10 ng / mL), and inoculate into a 24-well plate. Check the cell status and density daily and replace with fresh culture medium or increase the cell culture volume if necessary.

[0348] (6) Day 4: 72 hours after infection, the infection efficiency was detected by flow cytometry. The results are shown in Figure 18.

[0349] As can be seen from Figures 8 to 18, when Jurkat T cells, Ramos cells, and human primary T cells (unstimulated or stimulated with CD3 / CD28 antibodies) were infected with equal amounts of VSV-G pseudotyped lentivirus, the use of corresponding bispecific antibodies could improve the infection efficiency of VSV-G pseudotyped lentivirus. Among them, the use of HLA-VSV-G bispecific antibody increased the infection efficiency of Ramos cells from 4.5% to 53.8% (Figure 8); the use of HLA-VSV-G bispecific antibody increased the infection efficiency of Jurkat T cells from 4.5% to 53.8% (Figure 8); The infection efficiency of T cells was increased from 38.0% to 75.7% (Figure 9); the infection efficiency of human peripheral blood mononuclear cells was increased from 0.052% to 62.7% using the HLA-VSV-G bispecific antibody (Figure 10); the infection efficiency of Ramos cells was increased from 21.4% to 59.4% using the CD29scFv-VSV-G bispecific antibody (Figure 11); the infection efficiency of Ramos cells was increased from 21.4% to 59.4% using the CD20-VSV-G bispecific antibody (Figure 12). The infection efficiency of Ramos cells was increased from 34.6% to 68.2% (Figure 12); the CD45-VSV-G bispecific antibody increased the infection efficiency of Ramos cells from 34.6% to 58.1% (Figure 13); the CD40-VSV-G or CD40L-VSV-G bispecific antibodies increased the infection efficiency of Ramos cells from 34.6% to 42.9% or 51.7% (Figure 14); the CD3-VSV-G bispecific antibody increased the infection efficiency of Jurkat cells from 34.6% to 42.9% or 51.7% (Figure 15); The T cell infection efficiency was increased from 66.7% to a maximum of 81.9% (Figure 15); the use of CD3 (OKT3)-VSV-G or CD3 (UCHT1)-VSV-G bispecific antibodies increased the infection efficiency of unstimulated human primary T cells from 9.19% to 33.46 or 30.61% (Figure 16); the use of CD3 (OKT3)-VSV-G or CD3 (UCHT1)-VSV-G bispecific antibodies increased the infection efficiency of CD3 / CD28 antibody-activated human primary T cells from 16.0% to 40. 7% or 23.0% (Figure 17); the CD4-VSV-G bispecific antibody increased the infection efficiency of unstimulated human primary T cells from 9.19% to 43.05% or 43.05% (Figure 18); by comparing the staining of T cell surface specific proteins in Figure 18 with that in Figure 16, it can be seen that after lentiviral infection mediated by the bispecific molecule targeting CD3, the expression of CD3 on the cell surface is downregulated; the lentiviral infection mediated by the bispecific molecule targeting CD4 leads to the downregulation of CD4 expression on the cell surface, which further indicates that the lentivirus enters the cell through the action of the bispecific molecule.

[0350] In addition, by analyzing the mean fluorescence intensity (MFI) results in Figures 8 to 18, it was found that the bispecific antibodies disclosed herein not only improved the efficiency of viral infection, but also increased the expression level of the target gene (GFP) in cells. Among them, the use of the HLA-VSV-G bispecific antibody increased the expression level of the target protein in Ramos cells from 49038 to a maximum of 165640 (Figure 8); the use of the HLA-VSV-G bispecific antibody increased the expression level of the target protein in Jurkat cells from 49038 to 165640 (Figure 8); The target protein expression level of T cells increased from 8689 to a maximum of 17143 (Figure 9); the HLA-VSV-G bispecific antibody increased the target protein expression level of human peripheral blood mononuclear cells from 227 to 4489 (Figure 10); the CD29scFv-VSV-G bispecific antibody increased the target protein expression level of Ramos cells from 5238 to 11043 (Figure 11); the CD20-VSV-G bispecific antibody increased the target protein expression level of Ramos cells from 127289 to 163324 (Figure 12); the CD3-VSV-G bispecific antibody increased the target protein expression level of Jurkat cells from 127289 to 163324 (Figure 13); The expression level of the target protein on T cells increased from 18338 to 32697 (Figure 15); the use of CD3 (OKT3)-VSV-G or CD3 (UCHT1)-VSV-G bispecific antibodies increased the expression level of the target protein on unstimulated human primary T cells from 10264 to 58730 or 34160 (Figure 16); the use of CD3 (OKT3)-VSV-G or CD3 (UCHT1)-VSV-G bispecific antibodies increased the expression level of the target protein on human primary T cells activated by CD3 / CD28 antibodies from 225907 to 453000 or 337512 (Figure 17); the use of CD4-VSV-G bispecific antibodies increased the expression level of the target protein on unstimulated human primary T cells from 10264 to 22693 (Figure 18).

[0351] This indicates that the bispecific antibody disclosed herein can improve the infection efficiency and expression efficiency of VSV-G pseudotyped lentivirus in different cells.

Claims

1. A bispecific antigen-binding molecule or a fragment thereof, comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain is used to specifically bind to a cell surface antigen; and the second antigen-binding domain is used to specifically bind to vesicular stomatitis virus glycoprotein (VSV-G).

2. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The first antigen binding domain comprises a heavy chain (HC) variable region (VH) and / or a light chain (LC) variable region (VL).

3. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The first antigen binding domain further comprises a heavy chain (HC) constant region (CH) or a fragment thereof and / or a light chain (LC) constant region (CL) or a fragment thereof; preferably, the heavy chain constant region or a fragment thereof is an IgG constant region; and / or the light chain constant region is a κ constant region or a λ constant region.

4. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The first antigen binding domain comprises at least one of Fab, Fab', F(ab')2, scFv, a natural ligand of a cell surface protein or VHH.

5. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The first antigen binding domain is a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or an affinity-optimized antibody.

6. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The first antigen binding domain further comprises an Fc domain; preferably, the Fc domain comprises at least one mutation that can reduce or enhance the ADCC activity of the bispecific antibody.

7. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The cell surface antigen is selected from B cell surface antigen, NK cell surface antigen, hematopoietic stem cell surface antigen or T cell surface antigen; preferably, the cell surface antigen is selected from any one of CD3, CD19, CD45, CD20, CD34, CD40, CD56, CD16, CD133, CD147, CD123, CD138, CD22, CD30, CD33, CD38, CD70, CD4, CD5, CD8A&CD8B, CD7, and HLA.

8. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The second antigen binding domain comprises the CR2 and CR3 domains of the low density lipoprotein receptor (LDL-R).

9. The bispecific antigen-binding molecule or fragment thereof according to claim 8, wherein: The second antigen binding domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO.

1.

10. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: (a) the second antigen-binding domain is covalently linked to the carboxyl terminus of the heavy chain of the first antigen-binding domain; (b) the second antigen-binding domain is covalently linked to the amino terminus of the heavy chain of the first antigen-binding domain; or, (c) the second antigen-binding domain is covalently embedded in the polypeptide chain of the heavy chain of the first antigen-binding domain; and / or (d) the second antigen-binding domain is covalently linked to the carboxyl terminus of the light chain of the first antigen-binding domain; (e) the second antigen-binding domain is covalently linked to the amino terminus of the light chain of the first antigen-binding domain; or, (f) the second antigen-binding domain is covalently embedded in the polypeptide chain of the light chain of the first antigen-binding domain.

11. The bispecific antigen-binding molecule or fragment thereof according to claim 1, wherein: The second antigen-binding domain is connected to the heavy chain and / or light chain of the first antigen-binding domain via a peptide linker; preferably, the peptide linker is selected from L1, L2, L3 or L4, wherein the sequence of L1 is as shown in SEQ ID NO.7, the sequence of L2 is as shown in SEQ ID NO.8, the sequence of L3 is as shown in SEQ ID NO.9, and the sequence of L4 is as shown in SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

12. A bispecific antigen-binding molecule or a fragment thereof, comprising: A first polypeptide comprising VH, CH1, CH2, CH3, CR2 and CR3; and a second polypeptide comprising VL and CL; Among them, VH is the heavy chain variable region of the first antigen binding domain, VL is the light chain variable region of the first antigen binding domain, and the first antigen binding domain is used to specifically bind to cell surface antigens; CH1, CH2 and CH3 are the first, second and third constant regions of IgG molecules, respectively, CL is the light chain constant region; CR2 and CR3 are the second and third CR domains of the low-density lipoprotein receptor (LDL-R).

13. The bispecific antigen binding molecule or fragment thereof according to claim 12, wherein the first polypeptide comprises VH, CH1, CH2, CH3, CR2 and CR3 in order from N-terminus to C-terminus; and the second polypeptide comprises VL and CL in order from N-terminus to C-terminus; Preferably, the CR2 and CR3 domains comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO.

1.

14. The bispecific antigen-binding molecule or fragment thereof according to claim 12, wherein the two polypeptides associate to form an antigen-binding site targeting a cell surface antigen; preferably, the cell surface antigen is selected from a B cell surface antigen, a NK cell surface antigen, a hematopoietic stem cell surface antigen or a T cell surface antigen; preferably, the cell surface antigen is selected from any one of CD3, CD19, CD45, CD20, CD34, CD40, CD56, CD16, CD133, CD147, CD123, CD138, CD22, CD30, CD33, CD38, CD70, CD4, CD5, CD8A & CD8B, CD7, and HLA.

15. The bispecific antigen binding molecule or fragment thereof according to claim 12, wherein the bispecific antigen binding molecule is tetravalent or hexavalent; preferably, the bispecific antigen binding molecule is divalent or tetravalent for two targets.

16. The bispecific antigen binding molecule or fragment thereof according to claim 13, wherein the first polypeptide comprises a peptide linker between CH3 and CR2; preferably, the peptide linker is selected from L1, L2, L3 or L4, wherein, The sequence of L1 is shown as SEQ ID NO.7, the sequence of L2 is shown as SEQ ID NO.8, the sequence of L3 is shown as SEQ ID NO.9, and the sequence of L4 is shown as SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

17. The bispecific antigen binding molecule or fragment thereof of claim 12, wherein the first polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO.2; the second polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO.3; or The first polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO.4; the second polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO.

5.

18. The bispecific antigen binding molecule or fragment thereof according to claim 13, wherein the second polypeptide further comprises CR2 and CR3 domains in the order from N-terminus to C-terminus; preferably, the second polypeptide further comprises a peptide linker between CL and CR2; preferably, the peptide linker is selected from L1, L2, L3 or L4; wherein, The sequence of L1 is shown as SEQ ID NO.7, the sequence of L2 is shown as SEQ ID NO.8, the sequence of L3 is shown as SEQ ID NO.9, and the sequence of L4 is shown as SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

19. A bispecific antigen-binding molecule or a fragment thereof, comprising: A first antigen binding domain comprising an scFv comprising VH and VL; and a second antigen binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for connecting the scFv and the second antigen binding domain; Among them, the first antigen binding domain is used to specifically bind to cell surface antigens; VH is the heavy chain variable region, VL is the light chain variable region; CH2 and CH3 are the second and third constant regions of the IgG molecule respectively; CR2 and CR3 are the second and third CR domains of the low-density lipoprotein receptor (LDL-R).

20. The bispecific antigen binding molecule or fragment thereof according to claim 19, comprising scFv, CH2, CH3, CR2 and CR3 in order from N-terminus to C-terminus; the scFv comprises VH and VL; Preferably, the CR2 and CR3 domains comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO.

1.

21. The bispecific antigen binding molecule or fragment thereof according to claim 20, wherein the scFv targets a cell surface antigen; preferably, the cell surface antigen is selected from a B cell surface antigen, a NK cell surface antigen, a hematopoietic stem cell surface antigen or a T cell surface antigen; preferably, the cell surface antigen is selected from any one of CD3, CD19, CD45, CD20, CD34, CD40, CD56, CD16, CD133, CD147, CD123, CD138, CD22, CD30, CD33, CD38, CD70, CD4, CD5, CD8A&CD8B, CD7, and HLA.

22. The bispecific antigen binding molecule or fragment thereof according to any one of claims 19 to 21, wherein the bispecific antigen binding molecule is tetravalent.

23. The bispecific antigen binding molecule or fragment thereof according to any one of claims 19 to 21, wherein the bispecific antigen binding molecule is bivalent towards two targets.

24. The bispecific antigen binding molecule or fragment thereof according to claim 20, comprising a peptide linker between scFv and CH2; preferably, the peptide linker is selected from L1, L2, L3 or L4, wherein, The sequence of L1 is shown as SEQ ID NO.7, the sequence of L2 is shown as SEQ ID NO.8, the sequence of L3 is shown as SEQ ID NO.9, and the sequence of L4 is shown as SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

25. The bispecific antigen binding molecule or fragment thereof according to claim 20, comprising a peptide linker between CH3 and CR2; preferably, the peptide linker is selected from L1, L2, L3 or L4, wherein, The sequence of L1 is shown as SEQ ID NO.7, the sequence of L2 is shown as SEQ ID NO.8, the sequence of L3 is shown as SEQ ID NO.9, and the sequence of L4 is shown as SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

26. The bispecific antigen binding molecule or fragment thereof of claim 19, comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO.

6.

27. A bispecific antigen binding molecule or a fragment thereof, comprising: A first antigen-binding domain comprising VHH, and a second antigen-binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for connecting the VHH and the second antigen-binding domain; Among them, the first antigen binding domain is used to specifically bind to cell surface antigens; the CH2 and CH3 are the second and third Constant region; CR2 and CR3 are the second and third CR domains of the low-density lipoprotein receptor (LDL-R).

28. The bispecific antigen binding molecule or fragment thereof of claim 27, comprising VHH, CR2 and CR3 in order from N-terminus to C-terminus; preferably, the CR2 and CR3 domains comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO.

1.

29. The bispecific antigen-binding molecule or fragment thereof according to claim 27, wherein: The cell surface antigen is selected from B cell surface antigen, NK cell surface antigen, hematopoietic stem cell surface antigen or T cell surface antigen; preferably, the cell surface antigen is selected from any one of CD3, CD19, CD45, CD20, CD34, CD40, CD56, CD16, CD133, CD147, CD123, CD138, CD22, CD30, CD33, CD38, CD70, CD4, CD5, CD8A&CD8B, CD7, and HLA.

30. The bispecific antigen binding molecule or fragment thereof according to claim 27, wherein the bispecific antigen binding molecule is bivalent; preferably, the bispecific antigen binding molecule is monovalent for two targets.

31. The bispecific antigen binding molecule or fragment thereof according to claim 28, comprising a peptide linker between VHH and CR2; preferably, the linker is selected from L1, L2, L3 or L4, wherein, The sequence of L1 is shown as SEQ ID NO.7, the sequence of L2 is shown as SEQ ID NO.8, the sequence of L3 is shown as SEQ ID NO.9, and the sequence of L4 is shown as SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

32. A bispecific antigen binding molecule or a fragment thereof, comprising: A first antigen binding domain comprising a natural ligand of a cell surface protein, and a second antigen binding domain comprising CR2 and CR3; and optionally, CH2 and CH3 domains for connecting the natural ligand and the second antigen binding domain; Among them, the first antigen binding domain is used to specifically bind to cell surface antigens; CH2 and CH3 are the second and third constant regions of IgG molecules respectively; CR2 and CR3 are the second and third CR domains of low-density lipoprotein receptor (LDL-R).

33. The bispecific antigen binding molecule or fragment thereof of claim 32, comprising CR2, CR3, CH2, CH3 and a natural ligand in order from N-terminus to C-terminus; preferably, the CR2 and CR3 domains comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO.

1.

34. The bispecific antigen binding molecule or fragment thereof according to claim 32, wherein: The cell surface antigen is selected from B cell surface antigen, NK cell surface antigen, hematopoietic stem cell surface antigen or T cell surface antigen; preferably, the cell surface antigen is selected from any one of CD3, CD19, CD45, CD20, CD34, CD40, CD56, CD16, CD133, CD147, CD123, CD138, CD22, CD30, CD33, CD38, CD70, CD4, CD5, CD8A&CD8B, CD7, and HLA.

35. The bispecific antigen binding molecule or fragment thereof according to claim 32, wherein the bispecific antigen binding molecule is tetravalent; preferably, the bispecific antigen binding molecule is bivalent for two targets.

36. The bispecific antigen binding molecule or fragment thereof according to claim 33, comprising a peptide linker between CR3 and CH2; preferably, the linker is selected from L1, L2, L3 or L4, wherein The sequence of L1 is shown as SEQ ID NO.7, the sequence of L2 is shown as SEQ ID NO.8, the sequence of L3 is shown as SEQ ID NO.9, and the sequence of L4 is shown as SEQ ID NO.10; preferably, the L1 peptide linker is G4S (SEQ ID NO.11) or (G4S)3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G4S)3A (SEQ ID NO.13) or (G4S)4A (SEQ ID NO.14).

37. Use of the bispecific antigen binding molecule or fragment thereof according to any one of claims 1-36 for mediated receptor-independent lentiviral vector gene transduction; preferably, the object of gene transduction is a cell that low-expresses or does not express low-density lipoprotein receptor; preferably, the object of gene transduction is selected from T cells, B cells, NK cells or hematopoietic stem cells; preferably, the T cells and the B cells are primary cells.

38. A polynucleotide encoding the bispecific antigen binding molecule or fragment thereof according to any one of claims 1 to 36; or, a recombinant vector comprising the polynucleotide; or, a host cell comprising the polynucleotide or the recombinant vector.

39. A method of producing the bispecific antigen binding molecule or fragment thereof of any one of claims 1-36.

40. A method for transducing a target gene into a subject using a lentiviral vector, comprising administering to the subject the bispecific antigen binding molecule or fragment thereof of any one of claims 1-36.