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WO2024169990A1 - 双特异性抗体及其应用 - Google Patents

双特异性抗体及其应用 Download PDF

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Publication number
WO2024169990A1
WO2024169990A1 PCT/CN2024/077450 CN2024077450W WO2024169990A1 WO 2024169990 A1 WO2024169990 A1 WO 2024169990A1 CN 2024077450 W CN2024077450 W CN 2024077450W WO 2024169990 A1 WO2024169990 A1 WO 2024169990A1
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Prior art keywords
antigen
seq
sequence
fragment
cell surface
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PCT/CN2024/077450
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English (en)
French (fr)
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黄德力
陈丁瑞
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浙江大学绍兴研究院
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Publication of WO2024169990A1 publication Critical patent/WO2024169990A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure belongs to the field of immunology, and specifically relates to a bispecific antibody and its application.
  • Gene therapy achieves the purpose of curing diseases or improving the ability to resist diseases by replacing defective genes or adding new genes. It is expected to treat a series of diseases such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia, AIDS, etc.
  • diseases such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia, AIDS, etc.
  • the most common delivery system is viral vectors, because viruses can enter cells by specifically recognizing cell surface receptors and deliver the genetic material they carry to targeted cells. In order to overcome potential risks, researchers continue to select and improve viral vectors to ensure patient safety and maximize delivery efficiency. So far, the U.S.
  • FDA Food and Drug Administration
  • AAV adeno-associated virus
  • lentiviral vectors are the second most commonly used vector, used in 30% of gene therapies, second only to adeno-associated viral vectors.
  • lentivirus As a member of the retroviridae family, lentivirus has many characteristics that make it suitable for gene therapy: the lentivirus exogenous gene loading capacity can reach 10kb; the 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; the lentivirus induces a low immune response in target cells.
  • the main steps include binding to the host cell, releasing structural proteins, enzyme proteins and the virus core after fusing with the host cell membrane, reverse transcription of the viral RNA under the action of reverse transcriptase and forming a pre-integration complex with the integrase, and 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.
  • 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.
  • VSV-G vesicular stomatitis virus glycoprotein
  • Low-density lipoprotein receptor is the main entry receptor of VSV-G pseudotyped lentivirus on the cell surface.
  • the low-density lipoprotein receptor is a 36-sided single-chain glycoprotein composed of 839 amino acid residues and 5 functional domains. It is widely distributed on the cell membrane surface of various tissues in the body, such as hepatocytes, vascular smooth muscle cells, monocytes, and macrophages.
  • the extracellular domain of the low-density lipoprotein receptor includes a ligand-binding domain, an epidermal growth factor precursor homology domain, and an O-linked oligosaccharide-rich domain at the C-terminus.
  • the ligand-binding domain consists of 7 cysteine-rich repeat sequences (CR1-CR7). VSV-G can independently bind to two different CR domains (CR2 and CR3). Although LDLR is expressed in almost all tissues, its expression level on the surface of resting T cells, B cells, or hematopoietic stem cells (HSC) is extremely low, resulting in very low infection efficiency of these cells. Whether in scientific research experiments or clinical use such as CAR-T cell immunotherapy, the transformation and utilization of these cells through lentivirus is greatly limited.
  • the present disclosure provides a new technology using bispecific molecules to mediate gene delivery independent of the VSV-G glycoprotein receptor LDLR.
  • a bispecific antigen-binding molecule or a fragment thereof 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).
  • VSV-G vesicular stomatitis virus glycoprotein
  • the present disclosure provides 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;
  • 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
  • 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).
  • the present disclosure also provides 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;
  • 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).
  • LDL-R low-density lipoprotein receptor
  • the present disclosure also provides 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;
  • 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).
  • LDL-R low-density lipoprotein receptor
  • the present disclosure further provides 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;
  • 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).
  • LDL-R low-density lipoprotein receptor
  • the present disclosure also provides a polynucleotide encoding the bispecific antigen-binding molecule or a fragment thereof provided by the present disclosure.
  • the present disclosure also provides a recombinant vector comprising the polynucleotide provided by the present disclosure.
  • the present disclosure also provides a host cell, which comprises the polynucleotide or recombinant vector provided by the present disclosure.
  • the present disclosure also provides a method for producing the bispecific antigen binding molecules or fragments thereof of the present disclosure.
  • the present disclosure also provides a method of transducing a target gene into a subject using a lentiviral vector, comprising administering the bispecific antigen-binding molecule or a fragment thereof provided by the present disclosure to the subject.
  • the present disclosure designs a novel bispecific molecule that can efficiently mediate VSV-G lentivirus infection of cells that do not express the VSV-G receptor LDLR.
  • the antigen binding domain used to specifically bind to cell surface antigens can be widely replaced to achieve specific targeting of different types of cells.
  • 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 other gene function studies.
  • lentiviral vectors 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.
  • virus 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, and can reduce the requirements for the number of T cells and virus titer when preparing CAR-T cells, which can further reduce the production cost of CAR-T cells.
  • FIGS 1A to 1F show schematic diagrams of the structures of the bispecific antigen-binding molecules of the present invention; wherein the end for binding to cells can be designed to be in the form of Fab, scFv, single-domain antibody VHH or a natural ligand of a cell surface-specific membrane protein; the end for binding to the VSV-G pseudotype lentivirus is the CR2 and CR3 domains of the human low-density lipoprotein receptor (LDLR), which can also be replaced by the CR2 and CR3 domains of the low-density lipoprotein receptor (LDLR) of porcine, horse, or bovine species; the two binding domains can be connected via the Fc of an antibody to increase the valency, or directly connected using a flexible short linker such as GSG.
  • LDLR human low-density lipoprotein receptor
  • FIG. 2 shows a schematic diagram of using bispecific antigen-binding molecules to infect cells.
  • a specific bispecific molecule is incubated with cells at a certain concentration at 4°C to allow the bispecific molecule to bind to the specific protein on the cell surface.
  • the cells are centrifuged to remove the free bispecific molecules in the supernatant.
  • the cells are resuspended with a certain titer of VSV-G pseudotype lentivirus, and the VSV-G protein on the surface of the virus binds to the CR2-CR3 domain of LDLR.
  • the bispecific molecule not only shortens the physical distance between the cell and the virus, but also the protein on the cell surface undergoes endocytosis after being stimulated by the bispecific molecule, so that the virus particles are wrapped into the cell, and the low pH value in the endosome promotes the conformational change of the VSV-G protein, which fuses with the membrane and releases the genetic material into the cell. Transcribed and further integrated into the cell's genome to achieve long-term gene transduction; the process starting from endocytosis is consistent with the infection of traditional VSV-G pseudotyped lentivirus through the LDLR receptor.
  • Figure 3 shows the results of flow cytometry detection of LDLR expression on the surface of different types of cells.
  • flow cytometry antibodies against human low-density lipoprotein receptor LDLR 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.
  • FIG4 shows the comparative analysis results of the infection efficiency of VSV-G pseudotype GFP lentivirus with equivalent titers on different types of cells, by infecting different types of cells with VSV-G pseudotype GFP lentivirus with the same titer, and detecting the proportion of GFP-positive cells by flow cytometry.
  • FIG5 shows the expression results of different bispecific molecules verified by SDS-PAGE.
  • Figure 6 shows a bar chart of the binding ratio of bispecific molecules to different cell surface membrane proteins.
  • Figure 7 shows a bar chart of the binding ratio of different bispecific molecules to 293T cells overexpressing VSV-G protein.
  • 8A-C show the flow cytometry detection of the enhancement effect of bispecific molecules (targeting HLA) on the infection efficiency of lentivirus-infected Ramos cells.
  • Figure 9A-C shows the flow cytometric enhancement of the infection efficiency of lentivirus-infected Jurkat T cells by bispecific molecules (targeting HLA).
  • 10A-C show 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.
  • Figure 11A-C shows the flow cytometry detection of the enhancement effect of bispecific molecules (targeting CD19) on the infection efficiency of lentivirus-infected Ramos cells.
  • FIG12 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD20) on the infection efficiency of lentivirus-infected Ramos cells.
  • FIG13 shows the flow cytometry detection of the enhancement effect of bispecific molecules (targeting CD45) on the infection efficiency of lentivirus-infected Ramos cells.
  • FIG14 shows the flow cytometry detection of the enhancement effect of bispecific molecules (targeting CD40) on the infection efficiency of lentivirus-infected Ramos cells.
  • Figure 15 shows the flow cytometric enhancement of the infection efficiency of lentivirus-infected Jurkat T cells by bispecific molecules (targeting CD3).
  • FIG. 16 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD3) on the infection efficiency of lentivirus-infected unstimulated human primary T cells.
  • FIG17 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD3) on the infection efficiency of lentivirus-infected human primary T cells activated by CD3/CD28 antibodies.
  • FIG. 18 shows the flow cytometric analysis of the enhancement effect of bispecific molecules (targeting CD4) on the infection efficiency of lentivirus-infected unstimulated human primary T cells.
  • 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 single-letter codes.
  • antigen binding molecule refers in its broadest sense to a molecule that specifically binds to an antigenic determinant.
  • antigen binding molecules are immunoglobulins and their derivatives, such as fragments.
  • 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.
  • 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 produced from at least two complete antibodies and/or their antigen-binding fragments, 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.
  • Conventional immunoglobulins are tetramers consisting of two heavy chains and two light chains with a combined molecular weight of about 150 kDa.
  • Typical antibodies contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region contains three domains (CH1, CH2, and CH3).
  • Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region contains a domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FWs).
  • CDRs complementary determining regions
  • FWs framework regions
  • Each VH and VL contains three CDRs and four FWs arranged from the amino terminus to the carboxyl terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of 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.
  • antigen binding domain refers to the site of an antigen binding molecule, i.e., one or more amino acid residues, which provide interaction with an antigen.
  • the antigen binding domain of an antibody comprises amino acid residues from a complementary determining region (CDR).
  • CDR complementary determining region
  • antigen binding domains include, but are not limited to, Fab, Fab', F(ab')2, and single-chain Fv (scFv) fragments.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • 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 may include VL-peptide linker-VH or may include VH-peptide linker-VL.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • IgG and IgA can be further divided into different subclasses, such as IgG can be divided into IgG1, IgG2, IgG3, 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 distinct types, called ⁇ and ⁇ , based on the amino acid sequence of their constant domains.
  • the constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain, CH4).
  • CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length.
  • Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.
  • Fc is used herein to define the C-terminal region of an immunoglobulin heavy chain, i.e., two polypeptide chains forming a dimer that include a C-terminal constant region of an immunoglobulin heavy chain that is capable of stabilizing its own association.
  • the term includes native sequence Fc regions and variant Fc regions.
  • 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 includes the IgG CH2 and IgG CH3 constant domains.
  • the numbering of amino acid residues in the Fc region or constant region is in accordance with 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.
  • VHH variable domain of heavy chain of heavy-chain antibody
  • VHH 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, while the conventional paratope is restricted 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).
  • VHH is defined as a monovalent antibody, any avidity effect is excluded by default, and 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).
  • VHH antibody “heavy chain variable region (VHH)”, “VHH domain” and “nanobody VHH” can be used interchangeably.
  • “Monoclonal antibody” refers to a homogeneous antibody population that is involved in highly specific recognition and binding to a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants.
  • 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 molecules comprising an antigen recognition site.
  • 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).
  • humanized antibody refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that is engineered to contain minimal non-human (e.g., mouse) sequences.
  • a humanized antibody is a human immunoglobulin in which residues from CDR are replaced with residues from CDRs of 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).
  • FW residues of a human immunoglobulin are replaced with corresponding residues from antibodies of non-human species with desired specificity and/or affinity and/or ability.
  • the humanized antibody can be further modified by replacing the other residues in the FW district and/or refining and optimizing antibody specificity and/or affinity and/or ability in the non-human residue replaced.
  • 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 regions corresponding to non-human immunoglobulins, and all or substantially all FW regions are those of human immunoglobulin consensus sequences.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or a domain (Fc), typically at least a portion of a human immunoglobulin.
  • Fc immunoglobulin constant region or a domain
  • human antibody means an antibody produced by a person or an antibody having an amino acid sequence corresponding to an antibody prepared by a person using any technique known in the art (e.g., recombinant expression in cultured cells or expression in transgenic animals). Therefore, the term human antibody also encompasses antibodies having an amino acid sequence corresponding to an antibody originally produced by a person (or its engineered variants or derivatives) but expressed in a non-human system (e.g., produced by chemical synthesis; recombinant expression in microorganisms, mammals or insect cells; or expressed in an animal subject).
  • a non-human system e.g., produced by chemical synthesis; recombinant expression in microorganisms, mammals or insect cells; or expressed in an animal subject.
  • antibodies obtained from a human subject or from a human cell e.g., a hybridoma or cell line expressing a recombinant antibody or its fragment
  • an animal such as a mouse
  • human antibodies include complete or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy chain and/or light chain polynucleotide, such as, for example, antibodies comprising a mouse light chain and a human heavy chain polynucleotide.
  • chimeric antibody refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more animal species.
  • the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one species of mammals (e.g., mice, rats, rabbits, etc.) with the desired specificity and/or affinity and/or capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually humans) in order to avoid eliciting an immune response in these species.
  • bispecific means that an antigen binding molecule is able to specifically bind to two different antigenic determinants.
  • a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant.
  • a bispecific antigen binding molecule is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.
  • an antigen refers to a substance recognized and specifically bound by an antibody or antibody binding fragment.
  • 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.
  • ECD extracellular domain
  • Peptides, proteins, glycoproteins, polysaccharides and lipids, parts 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 triggers an immune response. Any form of antigen or a cell or preparation containing the antigen can be used to generate antibodies specific to antigenic determinants.
  • Antigens can be isolated full-length proteins, cell surface proteins (e.g., immunized with cells expressing at least a portion of the antigen on their surface), or soluble proteins (e.g., immunized with only the ECD portion of the protein) or protein constructs (e.g., Fc antigens).
  • 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 may be genomic or non-genomic (e.g., cDNA) and may encode at least a portion of the ECD sufficient to elicit an immunogenic response.
  • Any vector may 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.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • An epitope can be composed of adjacent amino acids or by three nucleotides in a protein. The epitope formed by adjacent amino acids is usually retained after exposure to denaturing solvents, while the epitope formed by tertiary folding is usually lost after treatment with denaturing solvents. Epitopes usually 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, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
  • polypeptide polypeptide
  • peptide protein
  • the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the 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.
  • 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 addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with a labeling component.
  • amino acid refers to natural and/or non-natural or synthetic amino acids, including glycine and 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.
  • amino acid modification includes amino acid substitutions, insertions and/or deletions in a polypeptide sequence.
  • amino acid substitution or “substitution” or “replacement” herein means replacing an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
  • substitution S32A means that the serine at position 32 is replaced by alanine.
  • 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)).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • Binding 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).
  • binding affinity refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand).
  • the affinity of a molecule X to 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).
  • affinities can include 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).
  • valency refers to the presence of a specified number of antigen binding regions in an antigen binding molecule.
  • tetravalent bispecific antigen binding molecule refers to a bispecific antigen binding molecule having four antigen binding regions, the four antigen binding regions can bind to the same or different antigens; in some examples, for example, "bivalent for a target” refers to the inclusion 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 inclusion of an antigen binding region for CD3 in the bispecific antigen binding molecule.
  • the antibody is bivalent for both the cell surface antigen and VSV-G.
  • the antibody may not be bivalent for the cell surface antigen and VSV-G, such as monovalent, trivalent, tetravalent, etc.; the "valence" of the antibody for the cell surface antigen and VSV-G may be the same or different.
  • CDR and FR of the antibody variable region 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, based on the antibody sequence of the present disclosure, humanized antibodies containing one or more CDRs derived from any naming system are clearly maintained within the scope of the present disclosure.
  • sequence identity or “sequence similarity” or “sequence homology” refers to the percentage of identical amino acid residues in a candidate sequence and a reference polypeptide sequence, after the sequences are aligned (and gaps are introduced, if necessary) to obtain the maximum percentage sequence identity, and any conservative substitutions are not considered as part of the sequence identity.
  • Sequence alignments can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • MEGALIGN DNASTAR
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., natural killer (NK) cells, neutrophils, and macrophages
  • NK natural killer
  • macrophages cytotoxic cells
  • Specific high-affinity IgG antibodies directed against the surface of target cells "arm" the cytotoxic cells and are required for this killing. Lysis of the target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.
  • 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.
  • a specific nucleic acid sequence also implicitly includes variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences thereof, as well as sequences explicitly indicated.
  • vector means a construct capable of delivering, and in some aspects, expressing one or more genes or one or more sequences of interest in a host cell.
  • 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 connected to a cationic condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.
  • activation 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 related to activation, stimulation or treatment.
  • host cell refers 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 exactly the same as the parent cell in terms of 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, etc., as well as cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues.
  • 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, etc.
  • transfection 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.
  • stable transfection or “stable transfection” refers to the introduction and integration of foreign nucleic acid, DNA or RNA, into the genome of the transfected cell.
  • stable transfectant refers to a cell that has stably integrated the foreign DNA into its genomic DNA.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) (http://imgt.cines.fr) or from The Immunoglobulin Facts Book (2001) ISBN: 012441351).
  • the engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially 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 antibodies can be purified and collected by conventional techniques.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • a bispecific antigen-binding molecule also referred to as a bispecific antibody in the present disclosure
  • a fragment thereof 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).
  • VSV-G vesicular stomatitis virus glycoprotein
  • 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.
  • the cell may be a cell that does not express low-density lipoprotein receptor (LDL-R).
  • LDL-R low-density lipoprotein receptor
  • the cells include but are not limited to T cells, B cells, NK cells, hematopoietic stem cells (HSC).
  • the T cells and B cells are primary cells.
  • the T cells and B cells are resting cells. Compared to cells in an activated state, resting cells have lower LDL-R expression levels.
  • 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.
  • the second antigen binding domain comprises the CR2 and CR3 domains of the low density lipoprotein receptor (LDL-R).
  • LDL-R low density lipoprotein receptor
  • the CR2 and CR3 domains are of human, horse, or porcine origin.
  • 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.
  • amino acid sequence of the second antigen binding domain is: VTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPV (SEQ ID NO.1).
  • 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.
  • VSV-G vesicular stomatitis virus glycoprotein
  • “several” means 5 or less, more preferably 3 or less, and most preferably 2 or less.
  • 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.
  • the CR2 and CR3 domains act as single domain antibodies that specifically bind to vesicular stomatitis virus glycoprotein.
  • the bispecific antibody binds to cell surface antigens so that the cell surface is coated with the bispecific antibody molecules.
  • the virus contacts the cell by recognizing the CR2 and CR3 domains on the bispecific antibody, and specific molecules on the cell surface such as CD19, CD3, CD45, etc. undergo clathrin-dependent endocytosis after being bound by the specific antibody.
  • the endocytosis process mediates the virus to enter the target cell, ultimately achieving gene delivery.
  • the first antigen binding domain comprises a heavy chain (HC) variable region (VH) and/or a light chain (LC) variable region (VL).
  • HC heavy chain
  • LC light chain
  • the first antigen-binding domain for specifically binding to a cell surface antigen may comprise a heavy chain variable region and/or a light chain variable region of any disclosed antibody against the cell surface antigen.
  • it may be a heavy chain variable region and/or a 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.
  • 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. 87-95;
  • 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 with the sequence shown in SEQ ID NO.96-104.
  • the first antigen binding domain may comprise a heavy chain variable region having an amino acid sequence with 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.87; and a light chain variable region having an amino acid sequence with 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.
  • the first antigen binding domain can specifically bind to the cell surface antigen CD19.
  • the first antigen binding domain may include a heavy chain variable region having an amino acid sequence with 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.88; and a light chain variable region having an amino acid sequence with 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.97.
  • the first antigen binding domain may specifically bind to the cell surface antigen CD3 (OKT3).
  • the first antigen binding domain may comprise a heavy chain variable region having an amino acid sequence with 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.89; and a light chain variable region having an amino acid sequence with 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.98.
  • the first antigen binding domain may specifically bind to the cell surface antigen CD45.
  • the first antigen binding domain may comprise a heavy chain variable region having an amino acid sequence with 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.90; and a light chain variable region having an amino acid sequence with 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.99.
  • the first antigen binding domain may specifically bind to the cell surface antigen CD34.
  • the first antigen binding domain may comprise a heavy chain variable region having an amino acid sequence with 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.91; and a light chain variable region having an amino acid sequence with 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.100.
  • the first antigen binding domain may specifically bind to the cell surface antigen CD20.
  • the first antigen binding domain may comprise a heavy chain variable region having an amino acid sequence with 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.92; and a light chain variable region having an amino acid sequence with 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.101.
  • the first antigen binding domain may specifically bind to the cell surface antigen CD40.
  • the first antigen binding domain may include a heavy chain variable region having an amino acid sequence with 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.93; and a light chain variable region having an amino acid sequence with 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.102.
  • the first antigen binding domain may specifically bind to the cell surface antigen CD4.
  • the first antigen binding domain may comprise a heavy chain variable region having an amino acid sequence with 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.94; and a light chain variable region having an amino acid sequence with 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.103.
  • the first antigen binding domain may specifically bind to the cell surface antigen CD3 (UCHT1).
  • the first antigen binding domain may comprise a heavy chain variable region having an amino acid sequence with 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.95; and a light chain variable region having an amino acid sequence with 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.104.
  • the first antigen binding domain may specifically bind to the cell surface antigen HLA.
  • 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.
  • HC heavy chain
  • LC light chain
  • 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.
  • the light chain constant region is a kappa constant region or a lambda constant region.
  • the first antigen binding domain comprises at least one of Fab, Fab', F(ab')2, scFv or VHH.
  • the first antigen binding domain is a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or an affinity-optimized antibody.
  • the first antigen binding domain further comprises an Fc domain.
  • the Fc domain is of murine or human origin.
  • the Fc domain further comprises at least one mutation that can reduce or enhance the ADCC activity of the bispecific antibody.
  • the Fc domain comprises a modification that promotes the binding of two chains of the Fc domain; in some embodiments, the Fc structure comprises a Knob chain and a Hole chain.
  • 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;
  • 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
  • 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.
  • 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 another 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 another second antigen-binding domain may be connected to the carboxyl terminus, amino terminus, or embedded in the polypeptide chain of the light chain, etc.
  • 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 (G 4 S) x, x is any integer from 1 to 6 (SEQ ID NO.7), the sequence of L2 is (G 4 S) xA, 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 G 4 S (SEQ ID NO.11) or (G 4 S) 3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G 4 S) 3 A (SEQ ID NO.13) or (G 4 S) 4 A (SEQ ID NO.14).
  • the bispecific antigen binding molecule is tetravalent or hexavalent.
  • the bispecific antigen binding molecule is bivalent or tetravalent for two targets.
  • exemplary molecular structures of the bispecific antigen-binding molecules are shown in FIG. 1A to FIG. 1F .
  • 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.
  • 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).
  • the antibody shown in FIG. 1A and FIG. 1B is a tetravalent antibody that is bivalent to both the cell surface antigen and VSV-G.
  • the second antigen binding domain can also be connected to the C-terminus of the light chain (such as the light chain CL).
  • the first antigen-binding domain of the antibody has a Fab structure and an Fc domain, wherein the C-termini of the light chain and the heavy chain are both connected to a second antigen-binding domain, in which case the antibody is bivalent for cell surface antigens and tetravalent for VSV-G.
  • the inventors have found that the binding ability of the bispecific antibody of this structure to VSV-G is significantly improved.
  • the antibody shown in FIG. 1C is hexavalent, including two valent antibodies to cell surface antigens and four valent antibodies to VSV-G.
  • 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).
  • the first antigen-binding domain of the antibody is a VHH antibody
  • the second antigen-binding domain is connected to the C-terminus of the first antigen-binding domain.
  • the antibody shown in Figure ID is bivalent and monovalent for both cell surface antigens and VSV-G.
  • 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.
  • the antibody shown in Figure IF is tetravalent and is bivalent for both cell surface antigens and VSV-G.
  • the second antigen-binding domain can be connected to the end of the first antigen-binding domain via a peptide linker.
  • the bispecific antigen binding molecule or fragment thereof comprises:
  • a first polypeptide comprising VH, CH1, CH2, CH3, CR2 and CR3; and a second polypeptide comprising VL and CL;
  • 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).
  • the first polypeptide comprises VH, CH1, CH2, CH3, CR2 and CR3;
  • the second polypeptide comprises VL and CL in order from N-terminus to C-terminus;
  • 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.
  • specific cell surface antigens such as CD3 on the surface of T cells, CD19 on the surface of B cells, etc.
  • the bispecific antigen binding molecule is tetravalent.
  • it comprises an antibody that can bind to four antigen binding sites of two different antigens (cell surface antigen and VSV-G).
  • the bispecific antigen binding molecule is bivalent for two targets (cell surface antigen and VSV-G).
  • 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 G 4 S (SEQ ID NO.11) or (G 4 S) 3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G 4 S) 3 A (SEQ ID NO.13) or (G 4 S) 4 A (SEQ ID NO.14).
  • 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
  • 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;
  • 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.
  • the second polypeptide further comprises CR2 and CR3 in the N-terminal to C-terminal sequence.
  • the CR2 and CR3 are connected to the carboxyl terminus of CL.
  • 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 G 4 S (SEQ ID NO.11) or (G 4 S) 3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G 4 S) 3 A (SEQ ID NO.13) or (G 4 S) 4 A (SEQ ID NO.14).
  • 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.
  • the bispecific antigen binding molecule or fragment thereof comprises:
  • 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;
  • 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).
  • LDL-R low-density lipoprotein receptor
  • 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;
  • the bispecific antigen binding molecule is tetravalent.
  • the bispecific antigen binding molecule is bivalent for two targets.
  • 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 G 4 S (SEQ ID NO.11) or (G 4 S) 3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G 4 S) 3 A (SEQ ID NO.13) or (G 4 S) 4 A (SEQ ID NO.14).
  • 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,
  • 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 G 4 S (SEQ ID NO.11) or (G 4 S) 3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G 4 S) 3 A (SEQ ID NO.13) or (G 4 S) 4 A (SEQ ID NO.14).
  • 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
  • the bispecific antigen binding molecule or fragment thereof 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.6.
  • the bispecific antigen binding molecule or fragment thereof comprises:
  • 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;
  • 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).
  • LDL-R low-density lipoprotein receptor
  • the bispecific antigen-binding molecule or fragment thereof comprises VHH, CR2 and CR3 in the order from N-terminus to C-terminus.
  • the bispecific antigen binding molecule is bivalent.
  • the bispecific antigen binding molecule is monovalent for both targets.
  • 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 G 4 S (SEQ ID NO.11) or (G 4 S) 3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G 4 S) 3 A (SEQ ID NO.13) or (G 4 S) 4 A (SEQ ID NO.14).
  • 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,
  • the bispecific antigen binding molecule or fragment thereof comprises:
  • 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;
  • 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).
  • LDL-R low-density lipoprotein receptor
  • the bispecific antigen-binding molecule or fragment thereof comprises CR2, CR3, CH2, CH3 and a natural ligand in order from the N-terminus to the C-terminus.
  • the natural ligand is CD40 ligand.
  • the amino acid sequence of the CD40 ligand is shown in SEQ ID NO.106.
  • the bispecific antigen binding molecule is tetravalent.
  • the bispecific antigen binding molecule is bivalent for two targets.
  • 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 G 4 S (SEQ ID NO.11) or (G 4 S) 3 (SEQ ID NO.12); more preferably, the L2 peptide linker is (G 4 S) 3 A (SEQ ID NO.13) or (G 4 S) 4 A (SEQ ID NO.14).
  • 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,
  • knowing the sequence information of the heavy chain variable region (VH) and/or the 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 according to the conventional sequences of CH1, CH2, CH3, CL, etc.
  • 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
  • VH and/or VL heavy chain and/or light chain variable regions
  • 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 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; preferably, the T cells and the B cells are resting cells.
  • bispecific antibodies of the present disclosure can be prepared according to methods known in the art.
  • the bispecific antibodies of the present disclosure are produced using hybridoma methods such as those described by Kohler and Milstein (1975) Nature 256:495.
  • mice, hamsters, or other appropriate host animals are immunized as described above to induce the production of lymphocytes of antibodies, which will specifically bind to the immunizing antigen.
  • Lymphocytes can also be immunized in vitro. After immunization, 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 from unfused lymphocytes and myeloma cells.
  • hybridomas that produce monoclonal antibodies specifically for the selected antigen can then be propagated using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) in vitro cultures 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.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the bispecific antibodies of the present disclosure can also be prepared using a recombinant DNA method as described in U.S. Patent No. 4,816,567. Such as by using RT-PCR of oligonucleotide primers encoding the genes of the antibody heavy chain and light chain, the polynucleotides encoding the monoclonal antibody are separated from mature B cells or hybridoma cells, and their sequences are determined using conventional procedures.
  • the polynucleotides encoding the separation of the 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 when transfected, and monoclonal antibodies are produced by these host cells.
  • 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 when transfected, and monoclonal antibodies are produced by these host cells.
  • recombinant antibodies or molecules comprising antigen-binding fragments thereof of a desired species 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)).
  • Recombinant DNA technology can be used to further modify one or more polynucleotides encoding bispecific antibodies of the present disclosure in a variety of different ways to produce alternative bispecific antibodies of the present disclosure.
  • the constant domains of the light chain and heavy chain of a mouse monoclonal antibody can be replaced with (1) such as those of a human antibody to produce a chimeric antibody or (2) a non-immunoglobulin polypeptide to produce a fusion antibody.
  • 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.
  • the bispecific antibodies of the present disclosure are human antibodies or antigen-binding fragments thereof.
  • Human antibodies can be isolated using various techniques known in the art. Immortalized human B lymphocytes isolated from in vitro immunization or from an immunized individual producing antibodies against a target antigen can be generated (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. Pat. No. 5,750,373).
  • One or more cDNAs encoding the antibody in the immortalized human B lymphocytes can then be prepared and inserted into an expression vector and/or a heterologous host cell for use in expressing a non-naturally occurring recombinant form of the antibody.
  • 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, for example, in Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et al., Proc. 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 producing and using antibody phage libraries are also described in U.S. Pat. Nos.
  • Affinity maturation strategies and chain shuffling strategies are known in the art and can be used to generate high affinity human antibodies or antigen-binding fragments thereof.
  • the bispecific antibodies of the present disclosure may be humanized antibodies.
  • Methods for engineering, humanizing or resurfacing non-human antibodies or human antibodies may also be used and are well known in the art.
  • Humanized, resurfaced or similarly engineered antibodies may 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 "import" variable, constant or other domains of known human sequences. Such imported sequences may be used to reduce immunogenicity or reduce, enhance or improve binding, affinity, on rate, off rate, avidity, specificity, half-life, or any other suitable feature known in the art.
  • CDR residues are directly and primarily involved in influencing antigen (such as cell surface antigen) binding. Therefore, part or all of a non-human or human CDR sequence is maintained and the non-human sequences of the variable and constant regions may be replaced with human or other amino acids.
  • human CDRs are inserted into non-human antibody scaffolds to allow for the preparation of antibodies with reduced immunogenicity in animal model systems, eg, "murinized” antibodies.
  • the bispecific antibodies of the present disclosure may optionally be humanized, resurfaced, or engineered, wherein high affinity for cell surface antigens and other favorable biological properties are retained.
  • humanized (or human) or engineered antibodies and resurfaced antibodies may optionally be prepared by a method of analyzing 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 well known to those skilled in the art.
  • 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); Richman et al., Nature 332:323 (1988); Weinghing 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.
  • 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)).
  • 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, thus allowing the production of large amounts 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.
  • Techniques suitable for producing single-chain antibody scFv can be found in, for example, U.S. Pat. No. 4,946,778.
  • methods suitable for constructing Fab expression libraries can be found in, for example, Huse et al., Science 246: 1275-1281 (1989), so as to allow rapid and efficient identification of monoclonal Fab fragments with desired specificity for cell surface antigens (such as 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 bridge bonds of F(ab')2 fragments; (c) Fab fragments produced by treating antibody molecules with papain and a reducing agent, and (d) Fv fragments.
  • the antibody or antigen-binding fragment thereof may be modified so as to increase its serum half-life. This can be accomplished, for example, by incorporating a salvage receptor binding epitope into the antibody or antibody fragment by mutating the appropriate region in the antibody or antibody fragment or by incorporating the epitope into a peptide tag that is subsequently fused to the antibody or antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis), or by YTE mutation.
  • Other methods of increasing the serum half-life of an antibody or its antigen-binding fragment e.g., conjugated to a heterologous molecule such as PEG are well known in the art.
  • 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.
  • a peptide spacer can be inserted between the CH2 and/or CH3 domains of the hinge region and the modification.
  • a compatible construct can be expressed, wherein 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.
  • 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.
  • the amino acid spacer can be proven to be immunogenic in some cases and to cause an undesirable immune response for the construct. Therefore, in some aspects, any spacer added to the construct will be relatively non-immunogenic, or even generally omitted, in order to maintain the biochemical quality of the hope of the modified antibody.
  • the bispecific antibodies of the present disclosure can be expressed from one or more vectors.
  • the first polypeptide is expressed by a vector and the second polypeptide is expressed by a second vector.
  • the first polypeptide and the second polypeptide are expressed by a vector.
  • the expression efficiency of the polypeptide is enhanced and/or increased by using only one vector.
  • the production efficiency of the bispecific antibody is enhanced and/or increased by expressing only two polypeptides.
  • the production efficiency of the bispecific antibody is enhanced and/or increased by expressing only two polypeptides compared to expressing three or more polypeptides.
  • 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.
  • the present disclosure provides a polynucleotide encoding the bispecific antigen binding molecule of the present disclosure or a fragment thereof.
  • the polynucleotides disclosed herein may be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand.
  • the DNA is a cDNA used to produce a non-naturally occurring recombinant antibody.
  • these polynucleotides are isolated. In some aspects, these polynucleotides are substantially pure. In some aspects, these polynucleotides include the coding sequence of the mature polypeptide fused in the same reading framework as the polynucleotides (natural or heterologous) that assist in expressing and secreting polypeptides from host cells (for example, the leader sequence used as the secretory sequence for controlling polypeptide transportation from cells).
  • the polypeptide with the leader sequence is a preprotein and can have a leader sequence cracked by the host cell to form a mature form of the polypeptide.
  • These polynucleotides can also encode the preprotein as a mature protein plus an additional 5' amino acid residue. In some aspects, these polynucleotides are changed to optimize codon usage for a certain host cell.
  • 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.
  • the marker sequence can be, for example, supplied by a pQE-9 vector to provide a purified six-histidine (His6) tag fused to the mature polypeptide of the marker in the case of a bacterial host.
  • 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).
  • HA hemagglutinin
  • the present disclosure further relates to variants of the polynucleotides, eg, encoding the bispecific antibody fragments, analogs, and derivatives of the present disclosure.
  • polynucleotide variants can contain changes in coding regions, noncoding regions or both. In some respects, these polynucleotide variants contain changes that produce silent substitutions, additions or deletions, but do not change the properties or activity of the encoded polypeptide. In some respects, nucleotide variants are produced by replacing the silence caused by the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, for example, in order to optimize the codon expression of a specific host (codons in human mRNA are changed into those preferred by bacterial hosts such as Escherichia coli).
  • the DNA sequence encoding the bispecific antibody of the present disclosure can be constructed by chemical synthesis, for example, using an oligonucleotide synthesizer.
  • oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and those codons of the host cell in which the recombinant polypeptide of interest will be produced by selection. Standard methods can be applicable to the separation polynucleotide sequence of the separation polypeptide of interest of the synthesis coding. For example, a complete amino acid sequence can be used to construct a gene for reverse translation.
  • a DNA oligomer containing a nucleotide sequence encoding a specific separation polypeptide 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 a 5' or 3' overhang for complementary assembly.
  • the polynucleotide sequence encoding the specific isolated polypeptide of interest will be inserted into an expression vector and operably linked to an expression control sequence suitable for expressing the protein in the desired host. Suitable assembly can be confirmed, for example, by nucleotide sequencing, restriction maps, and expressing the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of the transfected gene in the host, the gene must be operably linked to a functional transcription and translation expression control sequence in the expression host of choice.
  • the present disclosure provides a recombinant vector comprising the polynucleotide of the present disclosure.
  • recombinant vectors are reproducible DNA constructs having polypeptide chains encoding antibodies of the present disclosure or/and 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.
  • Transcriptional units typically include the assembly of the following: (1) a genetic element or elements that have a regulatory effect on 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 transcription and translation initiation and termination sequences.
  • Such regulatory elements may include an operator sequence that controls transcription.
  • the ability to replicate in a host may additionally be incorporated.
  • they are functionally related to each other, they are operably connected.
  • the DNA of a signal peptide secretion leader
  • it is operably connected to the DNA of the polypeptide
  • the transcription of a promoter control sequence it is operably connected to the coding sequence
  • a 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 enable proteins to be secreted and translated by the host cell extracellularly.
  • a recombinant protein in the case of expressing a recombinant protein without a leader or transport sequence, it may include a methionine residue at the N-terminus. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors containing 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 E. coli, including pCR 1, pBR322, pMB9, and their derivatives, plasmids with a wider host range, such as M13, and filamentous single-stranded DNA phages.
  • the present disclosure provides a host cell comprising a polynucleotide or a recombinant vector of the present disclosure.
  • a host cell comprising a polynucleotide or a recombinant vector of the present disclosure.
  • 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, N.Y., 1985), and the relevant disclosures of this reference are hereby incorporated by reference.
  • 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.
  • 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.
  • 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).
  • the antibody or its Fab produced by the host of transformation can be purified according to any suitable method.
  • standard methods include, for example, chromatography (for example, ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility or any other standard techniques 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.
  • proteolysis, nuclear magnetic resonance or x-ray crystallography can also be used to physically characterize isolated proteins.
  • ⁇ concentration filters can be used first, for example Millipore ultrafiltration unit concentrates the supernatant from the system where the recombinant protein is secreted into the culture medium. After the concentration step, the concentrate can be applied to a suitable purification matrix.
  • an anion exchange resin can be adopted, for example a matrix or substrate with a side chain diethylaminoethyl (DEAE) group.
  • These matrices can be acrylamide, agarose, dextran, cellulose or other types that are usually used in protein purification.
  • a cation exchange step can be adopted.
  • Suitable cation exchangers include different insoluble matrices containing sulfopropyl or carboxymethyl groups.
  • 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
  • RP-HPLC media e.g., silica gel with side chain methyl or other aliphatic groups
  • Bispecific antibodies or antigen-binding fragments thereof of the present disclosure 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.
  • the present disclosure provides a method of 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.
  • the lentiviral vector in the present disclosure can encode an exogenous gene in its genomic RNA.
  • a recombinant viral vector containing an exogenous gene can be obtained by inserting an exogenous gene into the genome of the lentiviral vector.
  • the "exogenous gene" referred to in the present disclosure can be any target gene that needs to be expressed in a cell, and can be a gene encoding a natural protein, or a protein modified by deleting, replacing or inserting amino acid residues in a 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 a 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 the disease.
  • a 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 the disease.
  • 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.
  • the vector construction includes inserting the DNA encoding the scfv fragment of anti-human CD19 before the DNA encoding the Fc segment of the human antibody, inserting the DNA fragment encoding the CR2-CR3 of the human LDL-R after the Fc segment of the antibody, and inserting the three connected complete sequences into an expression vector that can be expressed in mammalian cells.
  • the mammalian cell expression vector MG-HSP-v1 contains a CMV promoter, which can initiate downstream gene expression in mammalian cells.
  • the scFv fragment of anti-human CD19 was optimized according to the article (DOI:10.1182/blood-2010-04-281931) by 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.56.
  • the DNA encoding the human antibody Fc segment was optimized according to the protein database UniProt (P01857 ⁇ IGHG1_HUMAN) by the codon optimization tool of GenScript (https://www.genscript.com).
  • GenScript https://www.genscript.com
  • GenScript https://www.genscript.com
  • GenScript https://www.genscript.com
  • GenScript https://www.genscript.com/gensmart-free-gene-codon-optimization.html
  • the DNA encoding human LDL-R CR2-CR3 was obtained and synthesized after optimization according to the protein database UniProt (P01130 ⁇ LDLR_HUMAN) using the codon optimization tool of GenScript (https://www.genscript.com/gensmart-free-gene-codon-optimization.html), and the nucleotide sequence is shown in SEQ ID NO.58.
  • the three fragments were linked together using overlap extension polymerase chain reaction.
  • the primers used are shown in Table 1.
  • the expression vector MG-HSP-v1 (whose nucleotide sequence is shown in SEQ ID NO.55) was treated with BsaI restriction endonuclease (NEB, Cat#R3733L) to linearize it.
  • 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 Fc segment of the antibody, and the three segments are connected and the complete sequence is inserted into an expression vector that can be expressed in mammalian cells.
  • the mammalian cell expression vector MG-HSP.v2 (synthetic) contains a CMV promoter, which can promote the expression of downstream genes in mammalian cells.
  • the mammalian cell expression vector MG-LSP.V1 contains a CMV promoter, which can initiate downstream gene expression in mammalian cells.
  • the VH and VL fragments of anti-human CD3 were optimized according to the article (DOI: 10.1073/pnas.0402295101) by 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.60, and the VL DNA sequence is shown in SEQ ID NO.67); the VH and VL fragments of anti-human CD3 (UCHT1) were optimized according to the article (DOI: 10.1073/pnas.0407359101) by 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.61, and the VL DNA sequence is shown in SEQ ID NO.68); the VH and VL
  • 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 synthesized (its nucleotide sequence is shown in SEQ ID NO.75), and the DNA encoding human LDL-R CR2-CR3 is the same as (1).
  • the desired fragments were linked together using overlap extension polymerase chain reaction.
  • the primers used are shown in Table 3.
  • the expression vector MG-HSP-v2 (whose nucleotide sequence is shown in SEQ ID NO.59) or MG-LSP-v1 (whose nucleotide sequence is shown in SEQ ID NO.76) was treated with BsaI restriction endonuclease (NEB, Cat#R3733L) to linearize it.
  • CD3(OKT3) or CD3(UCHT1), CD4, CD20, CD40, CD45, HLA)-VH-IgG1-CH1-Fc-LDLR-CR2/3 plasmid
  • CD3(OKT3) or CD3(UCHT1), CD4, CD20, CD40, CD45, HLA-VL-CL plasmid
  • the vector construction includes inserting the DNA fragment encoding the CR2-CR3 of human LDL-R before the Fc segment of the antibody, inserting the DNA fragment encoding the human CD40 ligand after the DNA encoding the Fc segment of the human antibody, 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, which can initiate downstream gene expression in mammalian cells.
  • the fragment of human CD40 ligand was optimized according to the protein database UniProt (P29965 ⁇ CD40L_HUMAN) by 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.
  • GenScript https://www.genscript.com/gensmart-free-gene-codon-optimization.html
  • the DNA encoding the human antibody Fc segment is the same as (1)
  • the DNA encoding the human LDL-R CR2-CR3 is the same as (1).
  • the three fragments were linked together using overlap extension polymerase chain reaction.
  • the primers used are shown in Table 1.
  • the expression vector MG-HSP-v1 (whose nucleotide sequence is shown in SEQ ID NO.55) was treated with BsaI restriction endonuclease (NEB, Cat#R3733L) to linearize it.
  • 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.
  • Example 2 VSV-G pseudotype lentivirus packaging
  • HEK293T cells were digested with trypsin and a certain amount of cells were collected and plated in a six-well plate. The cell amount should be controlled to reach a confluence of about 70-80% on the second day of transfection to increase transfection efficiency. The cells were placed in a 37°C incubator with 5% CO2 for 8-24 hours.
  • Example 3 Using bispecific molecules (targeting HLA) to enhance lentiviral infection of Ramos cells
  • 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, hereinafter referred to as IMDM complete medium). Before infection, target cells were collected and counted, and a specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 ⁇ 10 5 cells per infection reaction), centrifuged at 300 g for 3 minutes at 4°C, and the medium was discarded.
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 0.7 with pre-cooled IMDM complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), blow gently to mix, and then inoculate into a 24-well plate, and centrifuge at 1800g 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.
  • Example 4 Using bispecific molecules (targeting HLA) to enhance lentiviral infection of Jurkat T cells
  • Target Jurkat T cells were cultured in RPMI-1640 medium (the medium was supplemented with 10% FBS (heated at 56°C for 30 minutes to inactivate), 1% penicillin-streptomycin, 1% GlutaMAX, 0.1% 2-mercaptoethanol (55 mM)), hereinafter referred to as RPMI-1640 complete medium). Before infection, target cells were collected and counted, and a specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 ⁇ 10 5 cells per infection reaction), centrifuged at 300 g for 3 minutes at 4°C, and the medium was discarded.
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 1 with pre-cooled RPMI-1640 complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), blow gently to mix, and then inoculate into a 24-well plate, and centrifuge at 1800g 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.
  • Example 5 Using bispecific molecules (targeting HLA) to enhance lentiviral infection of human peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • Day 1 thaw frozen human peripheral blood mononuclear cells (PBMCs), resuspend and count.
  • PBMCs peripheral blood mononuclear cells
  • complete medium X-VIVO 15 serum-free medium, 2% FBS (heated at 56°C for 30 minutes to inactivate), referred to as complete medium below
  • IL-15 5 ng/mL
  • IL-7 10 ng/mL
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 1 with pre-cooled complete medium, resuspend the cells collected by centrifugation in step (3) with 0.3 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), add IL-15 (5 ng/mL) and IL-7 (10 ng/mL), blow gently to mix, and then inoculate into a 24-well plate, centrifuge at 1800g 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.
  • Example 6 Using bispecific molecules (targeting CD19) to enhance the infection of lentivirus to Ramos cells
  • 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, hereinafter referred to as IMDM complete medium). Before infection, target cells were collected and counted, and a specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 ⁇ 10 5 cells per infection reaction), centrifuged at 300 g for 3 minutes at 4°C, and the medium was discarded.
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 1 with pre-cooled IMDM complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), blow gently to mix, and then inoculate into a 24-well plate, and centrifuge at 1800g 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.
  • Example 7 Using bispecific molecules (targeting CD20) to enhance the infection of lentivirus to Ramos cells
  • 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, hereinafter referred to as IMDM complete medium). Before infection, target cells were collected and counted, and a specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 ⁇ 10 5 cells per infection reaction), centrifuged at 300 g for 3 minutes at 4°C, and the medium was discarded.
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 1 with pre-cooled IMDM complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), blow gently to mix, and then inoculate into a 24-well plate, and centrifuge at 1800g 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.
  • Example 8 Using bispecific molecules (targeting CD45) to enhance the infection of lentivirus to Ramos cells
  • 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, hereinafter referred to as IMDM complete medium). Before infection, target cells were collected and counted, and a specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 ⁇ 10 5 cells per infection reaction), centrifuged at 300 g for 3 minutes at 4°C, and the medium was discarded.
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 1 with pre-cooled IMDM complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), blow gently to mix, and then inoculate into a 24-well plate, and centrifuge at 1800g 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.
  • Example 9 Using bispecific molecules (targeting CD40) to enhance the infection of lentivirus to Ramos cells
  • 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, hereinafter referred to as IMDM complete medium). Before infection, target cells were collected and counted, and a specific number of cells were collected into a sterile 1.5 mL Eppendorf tube (approximately 5 ⁇ 10 5 cells per infection reaction), centrifuged at 300 g for 3 minutes at 4°C, and the medium was discarded.
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 1 with pre-cooled IMDM complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), blow gently to mix, and then inoculate into a 24-well plate, and centrifuge at 1800g 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.
  • Example 10 Using bispecific molecules (targeting CD3) to enhance lentiviral infection of Jurkat T cells
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 2 with pre-cooled RPMI-1640 complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), blow gently to mix, and then inoculate into a 24-well plate, and centrifuge at 1800g 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.
  • Example 11 Using bispecific molecules (targeting CD3) to enhance the infection of unstimulated human primary T cells by lentivirus
  • PBMCs peripheral blood mononuclear cells
  • Day 1 thaw frozen human peripheral blood mononuclear cells (PBMCs), resuspend and count.
  • PBMCs peripheral blood mononuclear cells
  • complete medium X-VIVO 15 serum-free medium, 2% FBS (heated at 56°C for 30 minutes to inactivate), referred to as complete medium below
  • IL-15 5 ng/mL
  • IL-7 10 ng/mL
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 5 with pre-cooled complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), add IL-15 (5 ng/mL) and IL-7 (10 ng/mL), blow gently to mix, and then inoculate into a 24-well plate, centrifuge at 1800g 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.
  • Example 12 Using bispecific molecules (targeting CD3) to enhance the infection of lentivirus on CD3/CD28 antibody-activated human primary T cells
  • 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
  • cryopreserved PBMCs are revived, resuspended and counted.
  • Aspirate the culture medium add 1 ⁇ 10 6 PBMC cells to each well, and add Fill the wells with complete culture medium to a final volume of 2 mL.
  • PBS can be added to empty wells to prevent evaporation of the culture medium.
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 5 with pre-cooled complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), add IL-15 (5 ng/mL) and IL-7 (10 ng/mL), blow gently to mix, and then inoculate into a 24-well plate, centrifuge at 1800g 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.
  • Example 13 Enhancement of lentiviral infection of unstimulated human primary T cells using bispecific molecules (targeting CD4)
  • PBMCs peripheral blood mononuclear cells
  • Day 1 thaw frozen human peripheral blood mononuclear cells (PBMCs), resuspend and count.
  • PBMCs peripheral blood mononuclear cells
  • complete medium X-VIVO 15 serum-free medium, 2% FBS (heated at 56°C for 30 minutes to inactivate), referred to as complete medium below
  • IL-15 5 ng/mL
  • IL-7 10 ng/mL
  • VSV-G pseudotype lentivirus mixture for infection, dilute the virus to an MOI of about 5 with pre-cooled complete medium, resuspend the cells collected by centrifugation in step (3) with 0.5 mL of the virus mixture (the bispecific molecules have been bound to the cell surface at this time), add IL-15 (5 ng/mL) and IL-7 (10 ng/mL), blow gently to mix, and then inoculate into a 24-well plate, centrifuge at 1800g 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.
  • the infection efficiency of T cells was increased from 66.7% to 81.9% (Figure 15).
  • the infection efficiency of unstimulated human primary T cells was increased from 9.19% to 33.46% or 30.61% ( Figure 16) by using CD3 (OKT3) -VSV-G or CD3 (UCHT1) -VSV-G bispecific antibodies.
  • the infection efficiency of CD3/CD28 antibody-activated human primary T cells was increased from 16.0% to 40.
  • the bispecific antibodies disclosed in the present invention not only improve the efficiency of viral infection, but also increase the expression level of the target gene (GFP) in the cell, wherein the use of the HLA-VSV-G bispecific antibody increased the target protein expression level of Ramos cells from 49038 to a maximum of 165640 (Figure 8); the use of the HLA-VSV-G bispecific antibody increased the target protein expression level of Jurkat T cells from 8689 to a maximum of 17143 ( Figure 9); the use of 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 use of the CD29scFv-VSV-G bispecific antibody increased the target protein expression level of Ramos cells from 5238 to 11043 ( Figure 11); the use of the CD20-VSV-G bispecific antibody increased the target protein expression level of Ramos cells from 5238 to 110
  • MFI mean fluorescence intensity
  • bispecific antibody disclosed herein can improve the infection efficiency and expression efficiency of VSV-G pseudotype lentivirus in different cells.

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Abstract

提供了一种双特异性抗原结合分子或其片段，其包含第一抗原结合结构域和第二抗原结合结构域，其中所述第一抗原结合结构域用于特异性结合细胞表面抗原；所述第二抗原结合结构域用于特异性结合水疱性口膜炎病毒糖蛋白(VSV-G)。所述的双特异性抗原结合分子或其片段可以高效地介导VSV-G慢病毒感染不表达VSV-G受体LDLR的细胞。

Description

双特异性抗体及其应用
相关申请的交叉引用
本申请要求于2023年2月13日提交的中国专利申请2023101060920号的优先权，本申请引用上述中国专利申请的全文。
技术领域
本公开属于免疫学领域，具体涉及一种双特异性抗体及其应用。
背景技术
近年来，基因治疗已经迈入了快速增长的黄金时代。基因治疗通过取代有缺陷的基因或增新基因来达到治愈疾病或提高抵抗疾病能力的目的，有望治疗如癌症、囊性纤维化、心脏病、糖尿病、血友病、艾滋病等一系列的疾病。随着递送系统在基因疗法中应用的逐渐成熟，基因治疗药物转化的壁垒被逐渐攻破，最常见的递送系统是病毒载体，因为病毒可以通过特异性识别细胞表面受体进入细胞，并将其携带的遗传物质递送到靶向细胞中。为了克服潜在的风险，研究人员不断选择和改良病毒载体，以确保患者的安全并尽可能提高递送效率。截至目前，美国食品和药物管理局(FDA)已批准了三种利用不同类型的病毒载体进行的八种基因治疗方法，其采用的病毒载体为：腺相关病毒(AAV)载体、慢病毒载体和单纯疱疹病毒型载体。2022年2月为止，已有25种利用病毒载体进行治疗的方案处于后期开发阶段，另有120种正在进行二期试验。在所有三种类型的病毒载体中，慢病毒载体是利用率排名第二的载体，在30％的基因治疗中被使用，仅次于腺相关病毒载体的利用率。
作为逆转录病毒科的一员，慢病毒具有许多特性使其能够适用于基因治疗：慢病毒外源基因装载能力可以达到10kb；慢病毒可以将基因整合到靶细胞基因组中实现稳定的表达，并且不仅可以感染分裂的细胞，也可以感染不分裂的细胞；慢病毒在靶细胞中引起的免疫应答反应较低。
慢病毒感染宿主细胞时，主要步骤包括与宿主细胞结合，与宿主细胞膜融合后释放结构蛋白、酶蛋白和病毒核心，病毒RNA在逆转录酶的作用下逆转录并与整合酶形成整合前复合物，整合前复合物进入细胞核后，整合酶催化其整合至宿主基因组，外源基因前启动子驱动其在细胞质中表达。其中，利用病毒表面糖蛋白与宿主细胞表面受体蛋白的相互作用可以实现对于特定细胞的感染。第一个也是最广泛使用的糖蛋白是水疱性口膜炎病毒糖蛋白(VSV-G)，因为它们具有广泛的趋向性，产生的慢病毒滴度较高并且具有很高的稳定性。
低密度脂蛋白受体(LDL-R)是VSV-G假型慢病毒在细胞表面的主要进入受体。低密度脂蛋白受体是由839个氨基酸残基组成、包含5个功能结构域的36面体单链糖蛋白。它广泛分布于肝细胞、血管平滑肌细胞、单核细胞、巨噬细胞等全身各组织的细胞膜表面。低密度脂蛋白受体胞外结构域包括一个配体结合结构域、一个表皮生长因子前体同源结构域和一个在C端的富含O-连接寡糖的结构域。其中配体结合结构域由7个富含半胱氨酸的重复序列(CR1-CR7)组成。VSV-G能够独立结合两个不同的CR结构域(CR2和CR3)。尽管LDLR在几乎所有组织中都表达，但其在静息状态的T细胞、B细胞、或造血干细胞(HSC)等表面的表达量极低，导致对这些细胞的感染效率非常低下。不论在科研实验、抑或是临床如CAR-T细胞免疫疗法的使用过程中，通过慢病毒方式对这些细胞的改造和利用都受到了极大限制。
发明内容
为了解决这个问题，本公开提供了一种新技术，使用双特异性分子来介导不依赖于VSV-G糖蛋白受体LDLR的基因传递。
本公开的一个方面，提供了一种双特异性抗原结合分子或其片段，其包含第一抗原结合结构域和第二抗原结合结构域，其中所述第一抗原结合结构域用于特异性结合细胞表面抗原；所述第二抗原结合结构域用于特异性结合水疱性口膜炎病毒糖蛋白(VSV-G)。
在另一个方面，本公开提供了一种双特异性抗原结合分子或其片段，其包括：
包含VH、CH1、CH2、CH3、CR2和CR3的第一多肽；和包含VL和CL的第二多肽；
其中VH为第一抗原结合结构域的重链可变区，VL为第一抗原结合结构域的轻链可变区，所述第一抗原结合结构域用于特异性结合细胞表面抗原；CH1、CH2和CH3分别为IgG分子的第一、第二和第三恒定区，CL为轻链恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
在另一个方面，本公开还提供了一种双特异性抗原结合分子或其片段，其包括：
包含scFv的第一抗原结合结构域，所述scFv包含VH和VL；和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述scFv和所述第二抗原结合结构域的CH2和CH3结构域；
其中，所述第一抗原结合结构域用于特异性结合细胞表面抗原；VH为重链可变区，VL为轻链可变区；CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
在另一个方面，本公开还提供了一种双特异性抗原结合分子或其片段，其包括：
包含VHH的第一抗原结合结构域，和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述VHH和所述第二抗原结合结构域的CH2和CH3结构域；
其中，所述第一抗原结合结构域用于特异性结合细胞表面抗原；CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
另一方面，本公开还提供了一种双特异性抗原结合分子或其片段，其包括：
包含细胞表面蛋白的天然配体的第一抗原结合结构域，和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述天然配体和所述第二抗原结合结构域的CH2和CH3结构域；
其中，所述第一抗原结合结构域用于特异性结合细胞表面抗原；CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
在另一个方面，本公开还提供了一种多核苷酸，其编码本公开所提供的双特异性抗原结合分子或其片段。
在另一个方面，本公开还提供了一种重组载体，其包含本公开所提供的多核苷酸。
在另一个方面，本公开还提供了一种宿主细胞，其包含本公开所提供的多核苷酸或重组载体。
在另一个方面，本公开还提供了生产本公开的双特异性抗原结合分子或其片段的方法。
在另一个方面，本公开还提供了一种采用慢病毒载体向对象转导目标基因的方法，其包括向所述对象施用本公开所提供的双特异性抗原结合分子或其片段。
本公开的有益效果：
(1)本公开设计了一种全新的双特异性分子，可以高效地介导VSV-G慢病毒感染不表达VSV-G受体LDLR的细胞。在本公开中，用于特异性结合细胞表面抗原的抗原结合结构域可以被广泛替换以实现特异性地靶向不同种类的细胞。
(2)发明人还发现，对于一些较难感染(LDLR低表达或不表达)的细胞，如原代细胞等，双特异性分子的使用能大大提高目的基因转导效率，从而使得目的基因整合到宿主细胞基因组的几率大大增加，为构建稳定细胞系、对特定基因进行过表达、敲低或敲降等基因功能探究提供了一个有利的途径。
(3)由于慢病毒载体的诸多优点，使其已成为感染T细胞，尤其是用于CAR-T细胞的生产中的主要载体。然而一方面，病毒生产难以规模化，另一方面，T细胞数量的有限性与活化效率的限制也进一步增加了CAR-T细胞的生产的难度。本公开的双特异性分子增强了慢病毒对T细胞的感染效率，在制备CAR-T细胞时可以降低对T细胞数量、病毒滴度的要求，可进一步减少CAR-T细胞的生产成本。
附图说明
图1A至图1F展示了本公开的双特异性抗原结合分子的结构示意图；其中用于结合细胞的一端可以设计成为Fab形式、scFv、单域抗体VHH或者细胞表面特异性膜蛋白的天然配体；用于结合VSV-G假型慢病毒的一端为人源低密度脂蛋白受体(LDLR)的CR2和CR3结构域，该部分也可以替换成为猪、马、牛种属的低密度脂蛋白受体(LDLR)的CR2和CR3结构域；两个结合结构域可以通过抗体的Fc进行连接以增加价态，或者直接用柔性的短linker比如GSG进行连接。
图2展示了利用双特异性抗原结合分子感染细胞的示意图，首先将特定的双特异性分子以一定浓度与细胞在4℃进行孵育，使得双特异性分子与细胞表面的特异性蛋白相结合，通过离心细胞弃去上清中游离的双特异性分子；用一定滴度的VSV-G假型慢病毒重悬细胞，病毒表面的VSV-G蛋白与LDLR的CR2-CR3结构域结合；双特异性分子不仅拉近了细胞与病毒的物理距离，细胞表面的蛋白在受到双特异性分子的刺激后发生内吞，使得病毒颗粒被包裹进入细胞，并且内吞小体中的低pH值促进VSV-G蛋白发生构象改变，与膜融合，释放遗传物质到胞内，通过逆 转录并进一步整合到细胞的基因组中实现长效的基因转导；从内吞开始发生的过程与传统的VSV-G假型慢病毒通过LDLR受体进行感染是相一致的。
图3展示了流式检测不同种类细胞表面LDLR表达的检测结果，通过利用人源低密度脂蛋白受体LDLR的流式抗体，在不同的细胞系中进行表达量的检测，与常用的细胞系相比，未经CD3/CD28抗体激活的人原代T细胞LDLR表达量较低，在CD3/CD28抗体刺激后，LDLR表达发生上调。
图4展示了等效滴度VSV-G假型GFP慢病毒对不同种类细胞感染效率的比较分析结果，通过利用相同滴度的VSV-G假型GFP慢病毒感染不同种类的细胞，并利用流式检验GFP阳性的细胞比例。
图5展示了SDS-PAGE验证不同双特异性分子的表达结果，通过利用SDS-PAGE对双特异性分子的表达进行验证，不同的双特异性分子在蛋白分子量上均符合理论大小。
图6展示了双特异性分子与不同细胞表面膜蛋白结合比例的柱状统计图。通过用不同的双特异性分子分别与不同种类细胞进行孵育，用结合双特异性分子Fc段的荧光二抗指示结合情况，证明表达的蛋白可通过第一结合结构域与特定的细胞结合。
图7展示了不同双特异性分子与过表达了VSV-G蛋白的293T细胞结合比例的柱状统计图。通过用不同的双特异性分子分别与过表达VSV-G蛋白的293T细胞进行孵育，用结合双特异性分子Fc段的荧光二抗指示结合情况，证明表达的蛋白可以通过人源低密度脂蛋白受体LDLR的CR2-CR3结构域与VSV-G蛋白结合。
图8A-C展示了流式检测双特异性分子(靶向HLA)对慢病毒感染Ramos细胞的感染效率的增强效果。
图9A-C展示了流式检测双特异性分子(靶向HLA)对慢病毒感染Jurkat T细胞的感染效率的增强效果。
图10A-C展示了流式检测双特异性分子(靶向HLA)对慢病毒感染人外周血单核细胞的感染效率的增强效果。
图11A-C展示了流式检测双特异性分子(靶向CD19)对慢病毒感染Ramos细胞的感染效率的增强效果。
图12展示了流式检测双特异性分子(靶向CD20)对慢病毒感染Ramos细胞的感染效率的增强效果。
图13展示了流式检测双特异性分子(靶向CD45)对慢病毒感染Ramos细胞的感染效率的增强效果。
图14展示了流式检测双特异性分子(靶向CD40)对慢病毒感染Ramos细胞的感染效率的增强效果。
图15展示了流式检测双特异性分子(靶向CD3)对慢病毒感染Jurkat T细胞的感染效率的增强效果。
图16展示了流式检测双特异性分子(靶向CD3)对慢病毒感染未经刺激的人原代T细胞的感染效率的增强效果。
图17展示了流式检测双特异性分子(靶向CD3)对慢病毒感染CD3/CD28抗体激活的人原代T细胞的感染效率的增强效果。
图18展示了流式检测双特异性分子(靶向CD4)对慢病毒感染未经刺激的人原代T细胞的感染效率的增强效果。
具体实施方式
为了使本公开可更容易理解，首先定义某些术语。另外的定义贯穿详细说明而阐述。
I.定义
在详细描述本公开之前，应当了解的是，本公开并不限于特定的组合物或方法步骤，因为这些组合物或方法步骤可以改变。如在本说明书和随附权利要求书中所使用，除非上下文另外明确说明，否则单数形式“一个/种”和“该”包括复数参考对象。术语“一个”(或“一种”)，以及术语“一个或多个/一种或多种”和“至少一个”在此可以互换地使用。
此外，在此使用“和/或”应当理解为在有或没有另一者的情况下，两个指定的特征或组分中的每一者的特定披露。因此，措词如“A和/或B”中所使用的术语“和/或”旨在包括“A和B”、“A或B”、“A”(单独)、以及“B”(单独)。同样，如在词组如“A、B和/或C”中使用的术语“和/或”旨在包括以下方面：A、B以及C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；以及C(单独)。
除非另外定义，在此所使用的所有技术和科学术语具有与本公开涉及的领域所属的技术人员通常所理解的相同的意义。例如，简明生物医学与分子生物学词典(the Coneise Dictionary of Biomedicine and Molecular Biology)，卓培秀(Juo，PeiShow)，第2版，2002，CRC出版社；细胞与分子生物学词典(The Dictionary of Cell and Molecular Biology)，第3版，1999，美国学术出版社(Academic Press)；以及牛津生物化学与分子生物学辞典(the Oxford Dictionary Of Biochemistry And Molecular Biology)，修订版，2000，牛津大学出版社(Oxford University Press)为技术人员提供了用 于本公开中的许多术语的通用词典。
单位、前缀以及符号均以它们的国际单位系统(SI)接受形式表示。数值范围包括定义该范围的数字。除非另外指明，否则氨基酸序列以氨基到羧基的方向从左到右书写。在此提供的小标题不是不同方面的限制，可以通过作为一个整体参考本说明书来获得这些方面。因此，通过以其全文参考说明书，更完全地定义了就在以下定义的术语。
应当理解，当用语言“包含”来说明方面时，还提供了关于“由……组成”和/或“主要由……组成”描述的其他类似方面。
氨基酸在此通过它们的通常己知的三字母符号或通过由IUPAC-IUB生物化学命名委员会(IUPAC-IUB Biochemical Nomenclature Commission)推荐的单字母符号来提及。同样地，核苷酸通过它们的普遍公认的单字母代码来提及。
本文所用的术语“抗原结合分子”在其最广泛的含义上指特异性结合抗原决定簇的分子。抗原结合分子的实例是免疫球蛋白及其衍生物，例如片段。
术语“抗体”意指通过位于免疫球蛋白分子的可变区内的至少一个抗原识别位点识别并特异性地结合至靶标诸如蛋白、多肽、肽、碳水化合物、多核苷酸、脂质、或上述物质的组合的免疫球蛋白分子。如在此所使用，术语“抗体”涵盖完整多克隆抗体、完整单克隆抗体、抗体片段(诸如Fab、Fab′、F(ab)2、以及Fv片段)、单链可变片段(scFv)、二硫化物稳定的scFv、多特异性抗体诸如从至少两个完整抗体和/或其抗原结合片段中产生的双特异性抗体、嵌合抗体、人源化抗体、人抗体、包含抗体的抗原决定部分的融合蛋白以及包含抗原识别位点的任何其他修饰的免疫球蛋白分子，只要这些抗体展现希望的生物活性。
常规的免疫球蛋白是四聚体，由两条重链和两条轻链组成，组合分子量约150kDa。典型的抗体包含由二硫键相互连接的至少两个重(H)链和两个轻(L)链。每个重链包含重链可变区(在此缩写为VH)和重链恒定区(在此缩写为CH)。该重链恒定区包含三个结构域(CH1、CH2以及CH3)。每个轻链包含轻链可变区(在此缩写为VL)和轻链恒定区。该轻链恒定区包含一个结构域CL。这些VH区和VL区可被进一步细分为称为互补决定区(CDR)的高变区，这些高变区与称为构架区(FW)的更保守的区相互间置。每个VH和VL包含以下列顺序从氨基末端至羧基末端安排的三个CDR和四个FW：FW1、CDR1、FW2、CDR2、FW3、CDR3、FW4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白与宿主细胞或因子的结合，包括免疫系统的不同细胞(例如，效应子细胞)和经典补体系统的第一组分(C1q)。
术语“抗原结合结构域”指抗原结合分子的部位，即一个或多个氨基酸残基，其提供与抗原的相互作用。例如，抗体的抗原结合结构域包含来自互补决定区(CDR)的氨基酸残基。抗原结合结构域的实例包括但不限于Fab、Fab'、F(ab')2和单链Fv(scFv)片段。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-肽接头-VH或可以包括VH-肽接头-VL。
本领域已知五个主要类别的抗体：IgA，IgD，IgE，IgG和IgM，对应的重链恒定结构域分别被称为α，δ，ε，γ和μ，IgG和IgA可以进一步分为不同的亚类，例如IgG可分为IgG1，IgG2，IgG3，IgG4，IgA可分为IgA1和IgA2。来自任何脊椎动物物种的抗体的轻链基于其恒定结构域的氨基酸序列可以被分配到两种明显相异的类型之一，称为κ和λ。
在IgG、IgA和IgD抗体的情形中，该恒定区包含称为CH1、CH2和CH3的三个结构域(IgM和IgE具有第四结构域CH4)。在IgG、IgA和IgD类别中，CH1和CH2结构域被柔性铰链区分离，该铰链区是可变长度的富含脯氨酸和半胱氨酸的区段。每类抗体进一步包含由配对半胱氨酸残基形成的链间和链内二硫键。
术语“Fc”在本文中用于定义免疫球蛋白重链的C端区域，即包含免疫球蛋白重链中能够稳定自身联合的C端恒定区的形成二聚体的两条多肽链。该术语包括天然序列Fc区和变体Fc区。虽然IgG重链的Fc区的边界可以略微变化，但是人IgG重链Fc区通常定义为自Cys226或Pro230延伸至重链的羧基端，例如，IgG Fc域包含IgG CH2和IgG CH3恒定域。除非本文中另外指定，Fc区或恒定区中氨基酸残基的编号方式依照EU编号系统，也称作EU索引，如记载于Kabat等，Sequences of Proteins of Immunological Interest，第5版Public Health Service，National Institutes of Health，Bethesda，MD，1991。
在骆驼科(Camelidae)成员中，相当比例的血清抗体是同源二聚体IgG，分子量约80kD(Hamers-Casterman等，1993，Nature，363，446-448)。这些重链免疫球蛋白(Ig)包含三个结构域，其可变区被称为VHH(variable domain of heavy chain of heavy-chain antibody)。重组VHH(约12至14kD)构成完整的抗原结合结构域并显示出广阔的抗原结合谱。扩大它们的高变区，并表现出独特的特性，如三至四个(与常规抗体VL相互作用的)疏水框架残基被更多亲水 性氨基酸取代。为了稳定扩大的CDR，除了常规的二硫键以外，在单峰骆驼CDR1和CDR3之间，在美洲驼的CDR2和CDR3之间，VHH可具有额外的二硫键(Harmsen和De Haard,2007，Appl Microbiol Biotechnol.，77，13-22；Muyldermans，2001，J Biotechnol.，74，277-302)。扩大的CDR3环可以采取凸面构象，而常规的互补位被限制在凹的或者平面结构(Muyldermans，2001，J Biotechnol.，74，277-302)。这些特征允许VHH识别对于常规抗体而言免疫原性较差的独特表位(Lafaye，2009，Mol Immuno.，46，695-704；Wernery,2001，J Vet Med B Infect Dis Vet Public Health.，48，561-568)。尽管VHH被定义为单价抗体，默认排除任何亲合力效果，被测量表示为体外IC50的生物活性可类似于常规的二价抗体分子(Thys等，2010，Antiviral Res.，87，257-264)。在本公开中，“VHH抗体”、“重链可变区(VHH)”、“VHH结构域”以及“纳米抗体VHH”可以互换使用。
“单克隆抗体”是指涉及高度特异性识别和结合单个抗原决定簇或表位的均质抗体群体。这与典型地包括针对不同抗原决定簇的不同抗体的多克隆抗体相反。
术语“单克隆抗体”涵盖完整单克隆抗体和全长单克隆抗体两者以及抗体片段(诸如Fab、Fab′、F(ab′)2、Fv、单链可变片段(scFv)、包含抗体部分的融合蛋白、以及包含抗原识别位点的任何其他修饰的免疫球蛋白分子。此外，“单克隆抗体”是指通过任何数量的方式制备的此类抗体，包括但不限于通过杂交瘤、噬菌体选择、重组表达、以及转基因动物(例如，在转基因小鼠中表达人抗体)。
术语“人源化抗体”是指从非人(例如，鼠)免疫球蛋白中衍生的抗体，该抗体被工程化成含有最少的非人(例如，鼠)序列。典型地，人源化抗体是人免疫球蛋白，其中来自CDR的残基被来自具有希望的特异性、亲和力以及能力的非人物种(例如，小鼠、大鼠、兔、或仓鼠)的CDR的残基替换(琼斯(Jones)等人，1986，自然，321：522-525；瑞彻曼(Riechmann)等人，1988，自然，332：323-327；威霍英(Verhoeyen)等人，1988，科学(Science)，239：1534-1536)。在一些例子中，人免疫球蛋白的FW残基被来自具有希望的特异性和/或亲和力和/或能力的非人物种的抗体中的相应残基替换。
该人源化抗体可通过取代FW区中的另外残基和/或在替换的非人残基内精修并优化抗体特异性和/或亲和力和/或能力来进一步修饰。通常，该人源化抗体将基本上包含所有至少一个、并且典型地两个或三个可变结构域，这些可变结构域含有所有或基本上所有的相应于非人免疫球蛋白的CDR区，而所有或基本上所有的FW区是人免疫球蛋白共有序列的那些。该人源化抗体还可以包含免疫球蛋白恒定区或结构域(Fc)的至少一部分，典型地是人免疫球蛋白的至少一部分。用于产生人源化抗体的方法的实例描述于美国专利号5,225,539或5,639,641中。
术语“人抗体”意指由人产生的抗体或具有相应于使用本领域中已知的任何技术(例如，在培养物细胞中重组表达、或在转基因动物中表达)由人制备产生的抗体的氨基酸序列的抗体。因此，术语人抗体还涵盖具有相应于最初由人(或其工程化的变体或衍生物)产生但在非人系统中表达(例如，由化学合成产生；在微生物、哺乳动物或昆虫细胞中重组表达；或在动物受试者中表达)的抗体的氨基酸序列的抗体。因此，从人受试者或从人细胞(例如，表达重组抗体或其片段的杂交瘤或细胞系)中获得并且随后在例如小鼠的动物中表达的抗体被认为是人抗体。人抗体的这种定义包括完整或全长抗体、其片段、和/或包含至少一个人重链和/或轻链多核苷酸的抗体，诸如例如包含鼠轻链和人重链多核苷酸的抗体。
术语“嵌合抗体”是指其中免疫球蛋白分子的氨基酸序列从两种或更多种动物物种中衍生的抗体。典型地，轻链和重链两者的可变区相应于从具有希望的特异性和/或亲和力和/或能力的哺乳动物(例如，小鼠、大鼠、兔等)中的一种物种中衍生的抗体的可变区，而恒定区与从另一种物种(通常人)中衍生的抗体中的序列同源以便避免在这些物种中引发免疫应答。
术语“双特异性的”意指抗原结合分子能够特异性结合两个不同的抗原决定簇。通常，双特异性抗原结合分子包含两个抗原结合部位，两个抗原结合部位中的每一个对不同的抗原决定簇特异。在某些实施方案中，双特异性抗原结合分子能够同时结合两个抗原决定簇，尤其是表达在两种不同细胞上的两个抗原决定簇。
术语“抗原”是指被抗体或抗体结合片段识别并特异性结合的物质，广义上，抗原可以包括所选靶标的任何免疫原性片段或决定簇，包括单表位、多表位、单结构域、多结构域、或完整的胞外结构域(ECD)或蛋白质。肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合均可构成抗原。非限制性示例性抗原包括肿瘤抗原或病原体抗原等。“抗原”也可以指引发免疫反应的分子。任何形式的抗原或含有该抗原的细胞或制剂都可以用于生成对抗原决定簇具有特异性的抗体。抗原可以是分离的全长蛋白质、细胞表面蛋白(例如，用在其表面上表达至少一部分抗原的细胞进行免疫的)、或可溶性蛋白质(例如，仅用该蛋白质的ECD部分进行免疫的)或蛋白质构建体(例如，Fc抗原)。该抗原可以在基因修饰的细胞中产生。前述任何抗原可以单独或与本领域已知的一种或多种免疫原性增强佐剂组合使用。编码该抗原的DNA可以是基因组的或非基因组的(例如，cDNA)，并且可以编码足以引起免疫原性应答的至少一部分ECD。可以使用任何载体来转化其中表达抗原的细胞，所述载体包括但不限于腺病毒载体、慢病毒载体、质粒以及非病毒载体如阳离子脂质。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三 级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象存在并且包括至少3-15个氨基酸。由给定的抗体确定其结合的表位的方法是本领域熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用以指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。
术语“氨基酸修饰”(或“修饰的氨基酸”)包括在多肽序列中的氨基酸取代、插入和/或缺失。本文中的“氨基酸取代”或“取代”或“替代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代S32A指第32位的丝氨酸被丙氨酸替换。
“特异性结合”意指该结合对抗原具有选择性，且可以与不想要或非特异的相互作用区分开。可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术来测定抗体结合特异性抗原决定簇的能力，该其他技术例如表面等离振子共振(SPR)技术(在BIAcore仪器上分析)(Liljeblad等，Glyco J，17，323-329(2000))和传统结合测定(Heeley Endocr,Res,28,217-229(2002))。
“亲和力”指分子(例如受体)的单个结合部位和它的结合配偶体(例如配体)之间非共价相互作用的总和的强度。除非另有说明，本文所用的“结合亲和力”指反映结合对(例如抗原结合部分和抗原，或受体和它的配体)的成员之间1:1相互作用的内在结合亲和力。分子X对它的配偶体Y的亲和力通常可以表示为解离常数(KD)，其是解离和结合速率常数(分别为koff和kon)的比值。因此，等同的亲和力可以包含不同的速率常数，只要速率常数的比值保持相同。亲和力可以通过本领域已知的完善方法来测量，包括本文中描述的那些。用于测量亲和力的具体方法是表面等离振子共振(SPR)。
本文所用的术语“价”指抗原结合分子中指定数目的抗原结合区的存在。因此，示例性的，术语“四价双特异性抗原结合分子”是指双特异性抗原结合分子中具有四个抗原结合区，所述的四个抗原结合区可以结合相同或不同的抗原；在一些示例中，例如“对于某靶标为二价”是指在该双特异性抗原结合分子中包含两个针对某个特定靶标的抗原结合区。在另一些示例中，例如CD3单价双特异性抗原结合分子指在该双特异性抗原结合分子中包含一个针对CD3的抗原结合区。
在本公开的一些示例中，所述抗体对于细胞表面抗原和VSV-G均为二价的。在其他的示例中，所述抗体对于细胞表面抗原和VSV-G也可以不是二价的，例如一价、三价、四价等；抗体中对于细胞表面抗原和VSV-G的“价”可以是相同的或不同的。
需说明的是，本公开的抗体可变区的CDR和FR的划分是根据Kabat定义确定的。而其他命名和编号系统，例如Chothia、IMGT或AHo等，也是本领域技术人员已知的。因此，以本公开的抗体序列为基础，包含任何命名系统衍生的一种或多种CDR的人源化抗体均明确地保持在本公开的范围内。
术语“序列同一性”或“序列相似性”或“序列同源性”，是指在将所述序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守置换视为序列同一性的部分之后，候选序列中的氨基酸残基与参比多肽序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
“抗体依赖性细胞介导的细胞毒性”或“ADCC”是指细胞毒性的一种形式，其中结合至存在于某些细胞毒性细胞(例如，天然杀伤(NK)细胞、嗜中性粒细胞、以及巨噬细胞)上的Fc受体(FcR)上的分泌的免疫球蛋白使得这些细胞毒性效应子细胞能够特异性地结合至带有抗原的靶细胞并且随后用细胞毒素杀伤该靶细胞。针对靶细胞表面的特异性高亲和力IgG抗体“武装”细胞毒性细胞并且是这种杀伤所需要的。靶细胞的溶解是细胞外的，要求直接的细胞与细胞接触，并且不涉及补体。应当想到，除了抗体以外，具有特异性结合至带有抗原的靶细胞的能力的包含Fc区 的其他蛋白质(确切地Fc融合蛋白)将能够实现细胞介导的细胞毒性。为了简明起见，由Fc融合蛋白的活性产生的细胞介导的细胞毒性在此又称为ADCC活性。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语“核酸”或“多核苷酸”还包括含有已知的天然核苷酸的类似物的核酸，其具有与参照核酸相似的结合性质，并且以与天然存在的核苷酸相似的方式被代谢(参见，属于Kariko等人的美国专利No.8278036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、单核苷酸多态性(SNP)和互补序列以及明确指出的序列。
术语“载体”意指能够递送，并且在一些方面中，在宿主细胞中表达感兴趣的一个或多个基因或一个或多个序列的构建体。载体的实例包括，但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子缩合剂相连的DNA或RNA表达载体、包封在脂质体中的DNA或RNA表达载体、以及某些真核细胞诸如生产者细胞。
本公开中，除非另有明确规定，用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体或类似物、突变蛋白以及来源于抗体的结合化合物(如抗体及其结合片段)。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答”或“反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为的改变，例如生物区室(如组织、细胞、细胞器等)内的部分成分浓度、密度、粘附或迁移、基因表达速率或分化状态的改变，其改变可以与活化、刺激或处理相关。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，指已将外源核酸引入其中的细胞，包括这类细胞的后代。宿主细胞包括“转化体”和“转化细胞”，其包括初级转化细胞及从其衍生的后代，而不考虑代数。后代在核酸含量上可以并非与亲本细胞完全相同，但可以包含突变。本文包括具有与在最初转化的细胞中筛选或选择的功能或生物学活性相同的功能或生物学活性的突变体后代。宿主细胞是可以用来产生本公开的双特异性抗原结合分子的任意类型的细胞系统。宿主细胞包括培养细胞，例如哺乳动物培养细胞，如包括Jurkat细胞，PBMC细胞，A375细胞，U251细胞及U87细胞或杂交瘤细胞、酵母细胞、昆虫细胞和植物细胞等，以及包含在转基因动物、转基因植物或培养的植物或动物组织中的细胞。
本文所用的术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“稳定转染”或“稳转”是指将外源核酸、DNA或RNA引入和整合到转染细胞的基因组中。术语“稳定转染体”(stable transfectant)是指将外来DNA稳定地整合到基因组DNA中的细胞。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。发明所述的抗体或其抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站(http://imgt.cines.fr)得到，或者从免疫球蛋白杂志(The Immunoglobulin FactsBook，(2001)ISBN：012441351)上获得。
本公开工程化的抗体或其抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。
II.双特异性抗原结合分子或其片段
本公开的一个方面，提供了一种双特异性抗原结合分子(本公开中也称为双特异性抗体)或其片段，其包含第一抗原结合结构域和第二抗原结合结构域，其中所述第一抗原结合结构域用于特异性结合细胞表面抗原；所述第二抗原结合结构域用于特异性结合水疱性口膜炎病毒糖蛋白(VSV-G)。
本公开中，用于特异性结合细胞表面抗原的第一抗原结合结构域可以被广泛替换以实现特异性地靶向不同种类的细胞。
在一些实施方式中，所述细胞可以是低表达低密度脂蛋白受体(LDL-R)的细胞；如本公开所用，术语“低表达”、“表达水平低”相互可以交换的，并且在应用含义上应当意指与“对照物”或“阈值”相比较，至少有2％、3％、4％、5％、6％、7％、8％、9％或10％、优选的至少15％或20％、更优选25％、30％、50％、80％、100％或更显著的降低。例如，可对至少存在一个表达强度低于阈值的基因重复用Student’s T-test进行检测，以确定显著性。
在一些实施方式中，所述细胞可以是不表达低密度脂蛋白受体(LDL-R)的细胞。
在一些实施方式中，所述细胞包括但不限于T细胞、B细胞、NK细胞、造血干细胞(HSC)。
在一些实施方式中，所述T细胞和B细胞为原代细胞。
在一些实施方式中，所述T细胞和B细胞为静息细胞。相比于激活状态的细胞，静息细胞具有更低的LDL-R表达水平。
在一些实施方式中，所述细胞表面抗原选自B细胞表面抗原、NK细胞表面抗原、造血干细胞表面抗原或T细胞表面抗原；示例性的，所述细胞表面抗原选自CD3、CD19、CD45、CD20、CD34、CD40、CD56、CD16、CD133、CD147、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CD4、CD5、CD8A&CD8B、CD7中任一种。
在一些实施方式中，所述第二抗原结合结构域包含低密度脂蛋白受体(LDL-R)的CR2和CR3结构域。
在一些实施方式中，所述CR2和CR3结构域来源于人、马或猪。
在一些实施方式中，所述第二抗原结合结构域包含与SEQ ID NO.1所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
在一些实施方式中，所述第二抗原结合结构域氨基酸序列为：VTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPV(SEQ ID NO.1)。
在一些实施方式中，所述第二抗原结合结构域的氨基酸序列与SEQ ID NO.1相比具有一个或几个氨基酸的取代、插入和/或缺失，改变后的所述第二抗原结合结构域具有与野生型相当的或提高的特异性结合水疱性口膜炎病毒糖蛋白(VSV-G)的活性。
在一些实施方式中，“几个”意指5个或更少，更优选3个或更少，最优选2个或更少。例如，所述第二抗原结合结构域的氨基酸序列与SEQ ID NO.1相比具有5个、4个、3个、2个或1个氨基酸残基的取代、插入和/或缺失。
在一些实施方式中，所述CR2和CR3结构域作为单域抗体，与水疱性口膜炎病毒糖蛋白特异性结合。
在一些实施方式中，所述双特异性抗体与细胞表面抗原结合，以使所述细胞表面包裹上所述双特异性抗体分子，在进行病毒感染时，病毒通过识别所述双特异性抗体上CR2和CR3结构域，与细胞接触，而细胞表面的特异性分子如CD19、CD3、CD45等在被特异性抗体结合后会发生依赖于网格蛋白的内吞，该内吞过程介导了病毒进入靶细胞，最终实现基因的传递。
在一些实施方式中，所述第一抗原结合结构域包含重链(HC)可变区(VH)和/或轻链(LC)可变区(VL)。
本公开中所说的，用于特异性结合细胞表面抗原的第一抗原结合结构域可以包含已公开的针对所述细胞表面抗原的任何抗体的重链可变区和/或轻链可变区，示例性的，可以是针对上述任一细胞表面抗原，例如但不限于CD3、CD19、CD45、CD20、CD34、CD40、CD56、CD16、CD133、CD147、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CD4、CD5、CD8A&CD8B、CD7、HLA的抗体的重链可变区和/或轻链可变区。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.87～95所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；
和/或
包含与SEQ ID NO.96～104所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.87所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.96所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在 一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD19。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.88所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.97所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD3(OKT3)。在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.89所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.98所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD45。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.90所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.99所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD34。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.91所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.100所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD20。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.92所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.101所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD40。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.93所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.102所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD4。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.94所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.103所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原CD3(UCHT1)。
在一些实施方式中，所述第一抗原结合结构域可以包含与SEQ ID NO.95所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的重链可变区；和与SEQ ID NO.104所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列的轻链可变区。在一些实施方式中，所述第一抗原结合结构域可以特异性结合细胞表面抗原HLA。
在一些实施方式中，所述第一抗原结合结构域还可以包含重链(HC)恒定区(CH)或其片段和/或轻链(LC)恒定区(CL)或其片段。
在一些实施方式中，所述重链恒定区或其片段是IgG恒定区；在一些实施方式中，所述重链恒定区或其片段至少包括IgG的第一恒定区CH1。
在一些实施方式中，所述轻链恒定区是κ恒定区或λ恒定区。
在一些实施方式中，所述第一抗原结合结构域包括Fab、Fab'、F(ab')2、scFv或VHH的至少一种。
在一些实施方式中，所述第一抗原结合结构域是单克隆抗体、人源化抗体、人抗体、嵌合抗体、或亲和力优化的抗体。
在一些实施方式中，所述第一抗原结合结构域还包含Fc结构域。
在一些实施方式中，所述Fc结构域来源于鼠或人。
在一些实施方式中，所述Fc结构域还包含能够降低或增强该双特异性抗体的ADCC活性的至少一个突变。
在一些实施方式中，所述Fc结构域包含促进所述Fc结构域的两条链结合的修饰；在一些实施方式中，所述Fc结构包括Knob链和Hole链。
在一些实施方式中，所述第二抗原结合结构域被共价连接至所述第一抗原结合结构域的重链和/或轻链的羧基末端上；
在一些实施方式中，所述第二抗原结合结构域被共价连接至所述第一抗原结合结构域的重链和/或轻链的氨基末端上；
在一些实施方式中，所述第二抗原结合结构域被共价嵌入在所述第一抗原结合结构域的重链和/或轻链的多肽链中。
在一些实施方式中，当所述所述第一抗原结合结构域的重链和轻链均连接有所述第二抗原结合结构域时，所述第二抗原结合结构域与所述轻链和所述重链的连接位点可以相同或不同，例如，一个第二抗原结合结构域连接于重链的羧基末端，另一个第二抗原结合结构域可以连接于轻链的羧基末端、氨基末端、或嵌入在轻链的多肽链中；或者一个第二抗原结合结构域连接于重链的氨基末端，另一个第二抗原结合结构域可以连接于轻链的羧基末端、氨基末端、或嵌入在轻链的多肽链中等。
在一些实施方式中，所述第二抗原结合结构域与所述第一抗原结合结构域的重链和/或轻链通过肽接头连接；在一些实施方式中，所述肽接头选自L1、L2、L3或L4，其中，L1的序列为(G4S)x，x是1-6中的任意整数(SEQ ID NO.7)，L2的序列为(G4S)xA，x是1-6中的任意整数(SEQ ID NO.8)，L3的序列为EPKSSDKTHTCPPCP(SEQ ID NO.9)，L4的序列为DKTHTCPPCP(SEQ ID NO.10)；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
在一些实施方式中，所述双特异性抗原结合分子为四价或六价的。
在一些实施方式中，所述双特异性抗原结合分子对于两个靶标为二价或四价的。
在一些实施方式中，所述双特异性抗原结合分子的示例性分子结构如图1A至图1F所示。
在一个示例中，如图1A所示，所述抗体的第一抗原结合结构域具有Fab结构，第二抗原结合结构域连接在重链的C末端。
在一个示例中，如图1B所示，所述抗体的第一抗原结合结构域具有Fab结构和Fc结构域，所述第二抗原结合结构域连接在重链的C末端(即重链CH3的C末端)。
图1A和图1B所示抗体是四价抗体，其对于细胞表面抗原和VSV-G均为二价。
在另一些示例中，所述第二抗原结合结构域还可以连接在轻链(如轻链CL)的C末端。
在另一个示例中，如图1C所示，所述抗体的第一抗原结合结构域具有Fab结构和Fc结构域，其中，轻链和重链的C末端均连接有第二抗原结合结构域，在这种情况下，所述抗体对于细胞表面抗原是二价的，对于VSV-G是四价的。发明人发现，此种结构的双特异性抗体与VSV-G的结合能力得到明显提高。
图1C所示的抗体是六价的，其中对细胞表面抗原为二价，对VSV-G为四价。
在一个示例中，如图1D所示，所述抗体的第一抗原结合结构域具有scFv结构和Fc结构域，第二抗原结合结构域连接在Fc结构域(即重链CH3)的C末端。
在一个示例中，如图1E所示，所述抗体的第一抗原结合结构域为VHH抗体，第二抗原结合结构域连接在第一抗原结合结构域的C末端。
图1D所示的抗体为二价的，其对于细胞表面抗原和VSV-G均为单价。
在一个示例中，如图1F所示，所述抗体的第一抗原结合结构域具有ligand结构和Fc结构域，第二抗原结合结构域连接在Fc结构域的N末端。
图1F所示的抗体是四价的，其对于细胞表面抗原和VSV-G均为二价。
在上述示例中，所述的第二抗原结合结构域可以通过肽接头连接在第一抗原结合结构域的末端。
在在一些实施方式中，所述双特异性抗原结合分子或其片段包括：
包含VH、CH1、CH2、CH3、CR2和CR3的第一多肽；和包含VL和CL的第二多肽；
其中VH为重链可变区，VL为轻链可变区；CH1、CH2和CH3分别为IgG分子的第一、第二和第三恒定区，CL为轻链恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
在一些实施方式中，如图1B所示，所述第一多肽在N端至C端顺序中包含VH、CH1、CH2、CH3、CR2和 CR3；所述第二多肽在N端至C端顺序中包含VL和CL；
在一些实施方式中，所述的两个多肽缔合以形成靶向细胞表面抗原的抗原结合位点；其一端通过抗体的可变区识别特定的细胞表面抗原(例如T细胞表面的CD3，B细胞表面的CD19等)，在Fc的另一端，通过偶联LDLR的CR2和CR3结构域，与病毒的VSV-G糖蛋白结合。
在一些实施方式中，所述双特异性抗原结合分子为四价的。例如，其包含能够结合至两个不同抗原(细胞表面抗原和VSV-G)的四个抗原结合位点的抗体。
在一些实施方式中，所述双特异性抗原结合分子对于两个靶标(细胞表面抗原和VSV-G)为二价的。
在一些实施方式中，所述第一多肽包含CH3和CR2之间的肽接头；优选地，所述肽接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
在一些实施方式中，所述第一多肽包含与SEQ ID NO.2所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列；所述第二多肽包含与SEQ ID NO.3所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列；
在一些实施方式中，所述第一多肽包含与SEQ ID NO.4所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列；所述第二多肽包含与SEQ ID NO.5所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
在一些实施方式中，如图1C所示，所述第二多肽在N端至C端顺序中还包含CR2和CR3。在一些实施方式中，所述CR2和CR3连接于CL的羧基末端。
在一些实施方式中，所述第二多肽还包含的CL与CR2之间的肽接头；优选地，所述肽接头选自L1、L2、L3或L4；其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
在一些实施方式中，所述双特异性抗原结合分子或其片段包括：
包含scFv的第一抗原结合结构域，所述scFv包含VH和VL；和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述scFv和所述第二抗原结合结构域的CH2和CH3结构域；
其中VH为重链可变区，VL为轻链可变区；CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
在一些实施方式中，如图1D所示，所述的双特异性抗原结合分子或其片段，其在N端至C端顺序中包含scFv、CH2、CH3、CR2和CR3；所述scFv包含VH和VL；
在一些实施方式中，所述双特异性抗原结合分子为四价的。
在一些实施方式中，所述双特异性抗原结合分子对于两个靶标为二价的。
在一些实施方式中，所述的双特异性抗原结合分子或其片段包含scFv和CH2之间的肽接头；优选地，所述接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
在一些实施方式中，所述的双特异性抗原结合分子或其片段包含CH3和CR2之间的肽接头；优选地，所述肽接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
在一些实施方式中，所述的双特异性抗原结合分子或其片段包含与SEQ ID NO.6所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
在一些实施方式中，所述双特异性抗原结合分子或其片段包括：
包含VHH抗体的第一抗原结合结构域；和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述VHH和所述第二抗原结合结构域的CH2和CH3结构域；
其中，CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
在一些实施方式中，如图1E所示，所述的双特异性抗原结合分子或其片段，其在N端至C端顺序中包含VHH、CR2和CR3。
在一些实施方式中，所述双特异性抗原结合分子为二价的。
在一些实施方式中，所述双特异性抗原结合分子对于两个靶标为单价的。
在一些实施方式中，所述的双特异性抗原结合分子或其片段包含VHH和CR2之间的肽接头；优选地，所述接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
在一些实施方式中，所述双特异性抗原结合分子或其片段包括：
包含细胞表面蛋白的天然配体的第一抗原结合结构域，和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述天然配体和所述第二抗原结合结构域的CH2和CH3结构域；
其中，所述第一抗原结合结构域用于特异性结合细胞表面抗原；CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
在一些实施方式中，如图1F所示，所述的双特异性抗原结合分子或其片段，其在N端至C端顺序中包含CR2、CR3、CH2、CH3和天然配体。
在一些实施方式中，所述天然配体为CD40配体(CD40ligand)。在一些实施方式中，所述CD40配体的氨基酸序列如SEQ ID NO.106所示。
在一些实施方式中，所述双特异性抗原结合分子为四价的。
在一些实施方式中，所述双特异性抗原结合分子对于两个靶标为二价的。
在一些实施方式中，所述双特异性抗原结合分子或其片段，其包含CR3和CH2之间的肽接头；优选地，所述接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
在一些实施方式中，在知晓第一抗原结合结构域的重链可变区(VH)和/或轻链可变区(VL)的序列信息、第二抗原结合结构域(CR2和CR3)的序列信息，以及第一抗原结合结构域与第二抗原结合结构域的连接关系的情况下，本领域技术人员能够根据现有技术中CH1、CH2、CH3、CL等的常规序列获得抗体的全长序列(例如以结合其他目标细胞表面抗原的抗原结合结构域的重链和/或轻链可变区(VH和/或VL)直接替换本公开中所提供的抗体全长序列中的VH和VL，以获得结合其他目标细胞表面抗原的双特异性抗体)，以及编码所述抗体的核酸序列。
在另一个方面，本公开提供了本公开的双特异性抗原结合分子或其片段用于介导的受体非依赖性慢病毒载体基因转导的应用；优选地，所述基因转导的对象为低表达或不表达低密度脂蛋白受体的细胞；优选地，所述基因转导的对象选自T细胞、B细胞、NK细胞或造血干细胞；优选地，所述T细胞和所述B细胞为原代细胞；优选地，所述T细胞和所述B细胞为静息细胞(resting cell)。
III.双特异性抗原结合分子或其片段的制备
可以根据本领域中已知的方法来制备本本公开的双特异性抗体。例如，使用诸如由科勒(Kohler)和米尔斯坦(Milstein)(1975)自然256：495所描述的那些杂交瘤方法来产生本公开的双特异性抗体。
使用该杂交瘤方法，小鼠、仓鼠、或其他适当的宿主动物被如上所述免疫成诱导通过抗体的淋巴细胞进行的产生，这些淋巴细胞将特异性结合至免疫抗原。淋巴细胞还可以被体外免疫。免疫之后，分离这些淋巴细胞并且使用例如聚乙二醇与适合的骨髓瘤细胞系融合，以便形成随后可以从未融合的淋巴细胞和骨髓瘤细胞中选择离开的杂交瘤细胞。如通过免疫沉淀、免疫印迹、或通过体外结合测定(例如，放射免疫测定(RIA)；酶联免疫吸附测定(ELISA))所确定的特异性针对所选择抗原的产生单克隆抗体的杂交瘤随后可以使用标准方法(高定(Goding)，单克隆抗体：原理和实践(Monoclonal Antibodies：Principles and Practice)，美国学术出版社，1986)在体外培养物中或在体内作为动物中的腹水肿瘤来繁殖。这些单克隆抗体随后可以对于上述的多克隆抗体从所述的培养基或腹水中纯化出来。
还可以使用如在美国专利号4,816,567中所述的重组DNA方法来制备本公开的双特异性抗体。诸如通过使用特异性扩增编码抗体重链和轻链的基因的寡核苷酸引物的RT-PCR，将编码单克隆抗体的多核苷酸从成熟B细胞或杂交瘤细胞中分离出来，并且使用常规程序确定它们的序列。然后将编码重链和轻链的分离的多核苷酸克隆到适合的表达载体中，当转染时这些表达载体进入不另外产生免疫球蛋白的蛋白质的宿主细胞诸如大肠杆菌细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞或骨髓瘤细胞中，通过这些宿主细胞来产生单克隆抗体。同样，如所述的可以将希望物种的重组抗体或其包含抗原结合片段的分子从表达希望物种的CDR的噬菌体展示文库中分离出来(麦卡弗蒂(McCafferty)等人，自然348：552-554(1990)；克莱森(Clarkson)等人，自然352：624-628(1991)；以及马克斯(Marks)等人，分子生物学杂志(J.Mol.Biol.)222：581-597(1991))。
可以使用重组DNA技术以多种不同方式进一步修饰编码本公开的双特异性抗体的一种或多种多核苷酸，以便产生替代的本公开的双特异性抗体。在一些方面中，例如小鼠单克隆抗体的轻链和重链的恒定结构域可以被取代成(1)例如人抗体的那些区以便产生嵌合抗体或(2)非免疫球蛋白多肽以便产生融合抗体。在一些方面中，恒定区被截断或去除以产生单克隆抗体的希望的抗体片段。可变区的定点或高密度诱变可以被用来优化单克隆抗体的特异性、亲和力等。
在某些方面中，本公开的双特异性抗体是人抗体或其抗原结合片段。人抗体可以使用本领域中已知的不同技术 来直接制备。可以产生体外免疫或从产生针对靶抗原的抗体的免疫个体中分离的永生化人B淋巴细胞(参见，例如科尔(Cole)等人，单克隆抗体和癌症治疗(Monoclonal Antibodies and Cancer Therapy)，阿兰R.丽丝(Alan R.Liss)，第77页(1985)；波伊默(Boemer)等人，免疫学杂志(J.Immunol.)47：86-95(1991)；以及美国专利号5,750,373)。然后可以制备在永生化人B淋巴细胞中编码抗体的一个或多个cDNA并且插入表达载体和/或异源宿主细胞中以便用于表达抗体的非天然存在的重组形式。
同样，本公开的双特异性抗体或其抗原结合片段可以选自噬菌体文库，其中该噬菌体文库将人抗体或其片段表达为具有异源噬菌体蛋白质的融合蛋白，如例如在沃恩(Vaughan)等人，自然-生物技术(Nat.Biotech.)14：309-314(1996)；希茨(Sheets)等人，美国科学院院刊95：6157-6162(1998)；霍金布姆(Hoogenboom)和温特(Winter)，分子生物学杂志227：381(1991)以及马克斯等人，分子生物学杂志222：581(1991))中所述。用于产生和使用抗体噬菌体文库的技术还被描述于美国专利号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；以及7,264,963中，这些美国专利中的每一个通过引用以其全文结合。
亲和力成熟策略和链改组策略(马克思等人，生物技术(BioTechnology)10：779-783(1992)，该参考文献通过引用以其全文结合)是本领域中已知的，并且可以被用来产生高亲和力人抗体或其抗原结合片段。
在一些方面中，本公开的双特异性抗体可以是人源化抗体。也可以使用用于工程化、人源化或表面重修非人抗体或人抗体的方法并且这些方法是本领域中熟知的。人源化、表面重修或类似工程化的抗体可以具有来自非人来源例如但不限于小鼠、大鼠、非人灵长类动物或其他哺乳动物的一个或多个氨基酸残基。这些非人氨基酸残基被经常称为“输入”残基的残基替换，这些输入残基典型地取自已知人序列的“输入”可变、恒定或其他结构域。此类输入的序列可以被用来降低免疫原性或降低、增强或改进结合、亲和力、开(on)速率、关(off)速率、亲合力、特异性、半衰期、或本领域中所知的任何其他适合的特征。通常，CDR残基是直接和最主要地参与影响抗原(如细胞表面抗原)结合的。因此，非人或人CDR序列的部分或全部被维持而可变区和恒定区的非人序列可以用人或其他氨基酸替换。在某些方面中，人CDR被插入非人抗体支架中以便在动物模型系统中制备具有降低免疫原性的抗体，例如“鼠源化”抗体。
本公开的双特异性抗体可以任选地被人源化、表面重修、或工程化，其中保留对细胞表面抗原的高亲和力和其他有利的生物特性。为了实现这个目标，人源化(或人)或工程化的抗体和表面重修的抗体可以任选地通过使用亲本、工程化和人源化序列的三维模型对亲本序列和不同概念人源化和工程化产物进行分析的方法来制备。三维免疫球蛋白模型是普遍可得到的并且是本领域技术人员熟知的。
例证和展示所选择的候选免疫球蛋白序列的可能的三维构象结构的计算机程序是可用的。对这些展示的检查容许分析残基在候选免疫球蛋白序列功能中可能的作用，即，分析影响候选免疫球蛋白结合第一靶标(诸如CD3、CD19、CD45等)和/或第二靶标(VSV-G)能力的残基。以这种方式，可以从共有序列和输入序列中选择并合并构架残基以使得实现希望的抗体特征，诸如对一个或多个靶抗原的亲和力增加。
对在本公开的双特异性抗体的人源化、表面重修或工程化可以使用任何已知的方法来进行，诸如但不限于描述于以下参考文献中的那些：琼斯等人，自然321：522(1986)；瑞彻曼等人，自然332：323(1988)；威霍英等人，科学239：1534(1988))、斯姆斯(Sims)等人，免疫学杂志151：2296(1993)；乔西亚和莱斯克，分子生物学杂志196：901(1987)、卡特等人，美国科学院院刊89：4285(1992)；普雷斯塔(Presta)等人，免疫学杂志151：2623(1993)、美国专利号5,639,641；5,723,323；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；以及7,342,110；WO90/14443；WO90/14424；WO90/14430；以及EP229246，这些参考文献中的每一个通过引用整体结合在此，包括其中引用的参考文献。
在某些方面中，提供本公开的双特异性抗体的片段。已知不同技术用于产生抗体片段。在传统上，这些片段是通过完整抗体的蛋白水解消化而衍生的(例如森本晃司(Morimoto)等人，生物化学与生物物理杂志方法(J.Biochem.Biophy.Methods)24：107-117(1993)；布伦南(Brennan)等人，科学，229：81(1985))。在某些方面中，重组产生本公开的双特异性抗体片段。所有Fab、Fv和scFv抗体片段都可以表达在大肠杆菌或其他宿主细胞中并且从其分泌，因而允许产生大量的这些片段。还可以从以上讨论的抗体噬菌体文库中分离此类抗体片段。这些抗体片段还可以是如在美国专利号5,641,870中所述的线性抗体。用于产生抗体片段的其他技术对于熟练的从业人员将是清楚的。
适用于产生单链抗体scFv的技术可以参见例如美国专利号4,946,778。另外，适用于构建Fab表达文库的方法可以参见例如休斯(Huse)等人，科学246：1275-1281(1989)，以便允许快速和有效的鉴定对细胞表面抗原(如CD3、CD19、CD45等)、或其衍生物、片段、类似物或同源物具有希望的特异性的单克隆Fab片段。抗体片段可以通过本领域的技术来产生，这些抗体片段包括但不限于：(a)由抗体分子的胃蛋白酶消化产生的F(ab′)2片段；(b)由还原F(ab′)2片段的二硫化物桥键产生的Fab片段；(c)由使用木瓜蛋白酶和还原剂处理抗体分子产生的Fab片段以及(d)Fv片段。
在一些方面中，尤其在抗体片段的情况下，抗体或其抗原结合片段可以被修饰以便增加它的血清半衰期。这可 以例如通过以下方式来实现：通过突变抗体或抗体片段中的适当的区域将补救受体结合表位掺入抗体或抗体片段中或通过将表位掺入随后融合至任一端或在中间处的抗体或抗体片段的肽标签中(例如，通过DNA或肽合成)、或通过YTE突变。增加抗体或其抗原结合片段的血清半衰期的其他方法(例如，轭合至异源分子诸如PEG)是本领域中熟知的。
应当指出的是在某些方面中，双特异性抗体分子可以被工程化成直接将CH3结构域融合至对应修饰的抗体或其片段的铰链区。在其他构建体中，肽间隔物可以被插入在铰链区与修饰的CH2和/或CH3结构域之间。例如，可以表达相容的构建体，其中CH2结构域已缺失并且剩余的CH3结构域(修饰或未修饰的)使用5-20个氨基酸的间隔物而连接至铰链区。可以例如添加这样一种间隔物以便确保恒定结构域的调节元件保持游离并且可接近或确保铰链区保持柔性。然而，应当指出氨基酸间隔物可在一些情况下被证明是免疫原性的并且针对构建体引发不希望的免疫应答。因此，在某些方面中，添加至构建体的任何间隔物将是相对非免疫原性的，或甚至总体上省略的，以便维持修饰抗体的希望的生物化学品质。
本公开的双特异性抗体可以从一种或多种载体表达。例如，在一些实施方案中，第一多肽由一种载体表达并且第二多肽由第二种载体表达。在一些实施方案中，第一多肽和第二多肽由一种载体表达。在一些实施方案中，通过使用仅一种载体来增强和/或增加多肽的表达效率。在一些实施方案中，通过表达仅两个多肽来增强和/或增加双特异性抗体的产生效率。在一些实施方案中，与表达三个或更多个多肽相比，通过表达仅两个多肽来增强和/或增加双特异性抗体的产生效率。在一些实施方案中，通过表达仅两个多肽来增强和/或增加活性抗原结合位点的形成。在一些实施方案中，与表达三个或更多个多肽相比，通过表达两个多肽来增强和/或增加活性抗原结合位点的形成。在一些实施方案中，与形成异二聚体分子相比，通过形成同二聚体分子来增强和/或增加双特异性抗体的产生效率。在一些实施方案中，与形成异二聚体分子相比，通过形成同二聚体分子来增强和/或增加双特异性抗体的稳定性。
在另一个方面，本公开提供了一种多核苷酸，其编码本公开的双特异性抗原结合分子或其片段。
本公开的多核苷酸可以呈RNA形式或呈DNA形式。DNA包括cDNA、基因组DNA、以及合成DNA；并且可以是双链或单链的，并且如果是单链的，可以是编码链或非编码(反义)链。在某些方面中，该DNA是用来产生非天然存在的重组抗体的cDNA。
在某些方面中，这些多核苷酸是分离的。在某些方面中，这些多核苷酸是基本上纯的。在某些方面中，这些多核苷酸包含被融合在与例如辅助从宿主细胞表达和分泌多肽的多核苷酸(天然或异源的)相同阅读构架中的成熟多肽的编码序列(例如，用作控制多肽从细胞运输的分泌序列的前导序列)。具有前导序列的多肽是前蛋白并且可以具有由宿主细胞裂解的前导序列以便形成多肽的成熟形式。这些多核苷酸还可以编码作为成熟蛋白加上另外的5′氨基酸残基的前蛋白。在某些方面中，这些多核苷酸被改变成针对某一宿主细胞优化密码子使用。
在某些方面中，这些多核苷酸包含被融合在与异源标记序列相同阅读构架中的成熟抗抗体分子的编码序列，该异源标记序列允许例如纯化编码的多肽。例如，该标记序列可以是例如由pQE-9载体供应的以便提供在细菌宿主的情况下融合至标记物的成熟多肽的纯化的六组氨酸(His6)标签。在其他方面中，该标记序列可以是在使用哺乳动物宿主(例如，COS-7细胞)时，例如从流感血凝素蛋白中衍生的血凝素(HA)标签。
本公开进一步涉及例如编码本公开的双特异性抗体片段、类似物、以及衍生物的所述多核苷酸的变体。
这些多核苷酸变体可以在编码区、非编码区、或两者中含有改变。在一些方面中，这些多核苷酸变体含有产生沉默取代、添加、或缺失的改变，但不改变编码的多肽的特性或活性。在一些方面中，通过由于遗传密码的简并性引起的沉默取代来产生核苷酸变体。可以出于各种原因来产生多核苷酸变体，例如为了优化特定宿主的密码子表达(将人mRNA中的密码子改变成细菌宿主诸如大肠杆菌优选的那些)。
在一些方面中，编码本公开的双特异性抗体的DNA序列可以通过化学合成，例如使用寡核苷酸合成仪来构建。可以基于希望多肽的氨基酸序列并且选择有利于其中将产生感兴趣重组多肽的宿主细胞的那些密码子来设计此类寡核苷酸。标准方法可以适用于合成编码感兴趣的分离多肽的分离多核苷酸序列。例如，可以使用完整的氨基酸序列来构建反向翻译的基因。此外，可以合成含有编码特定分离多肽的核苷酸序列的DNA低聚物。例如，可以合成编码希望多肽的部分的若干小寡核苷酸并且然后进行连接。各个寡核苷酸典型地含有用于互补组装的5′或3′突出端。
一旦组装(通过合成、定点诱变或另一种方法)，编码感兴趣的特定分离多肽的多核苷酸序列将被插入表达载体中并且可操作地连接至适用于在希望的宿主中表达蛋白质的表达控制序列上。合适的组装可以例如通过核苷酸测序、限制性图谱以及在适合宿主中表达生物活性多肽来证实。正如本领域中熟知的，为了在宿主中获得转染基因的高表达水平，该基因必须可操作地连接至在选择的表达宿主中有功能性的转录和翻译表达控制序列上。
在另一个方面，本公开提供了一种重组载体，其包含本公开的多核苷酸。
在某些方面中，重组载体是可复制的DNA构建体，这些DNA构建体具有编码本公开的抗体或/和其抗原结合片段的多肽链、可操作地连接至从哺乳动物、微生物、病毒或昆虫基因中衍生的适合转录或翻译调节元件上的合成或cDNA衍生的DNA片段。转录单元通常包括对以下各项的组装：(1)在基因表达方面具有调节作用的基因元件或多个元件，例如转录启动子或增强子，(2)被转录到mRNA中并且翻译成蛋白质的结构或编码序列，以及(3)适当转录和翻译起始序列和终止序列。此类调节元件可以包括控制转录的操纵子序列。
可以另外地掺入通常由复制起点赋予的在宿主中复制的能力、以及促进识别转化体的选择基因。当DNA区是 功能上彼此相关的时候，它们可操作地连接。例如，如果信号肽(分泌前导)的DNA被表达为参与多肽分泌的前体，则它可操作地连接至多肽的DNA上；如果启动子控制序列的转录，则它可操作地连接至编码序列上；或如果核糖体结合位点为了容许翻译而定位，则它可操作地连接至编码序列上。意图用于酵母表达系统中的结构元件包括能实现通过宿主细胞细胞外分泌翻译的蛋白质的前导序列。可替代地，在不使用前导或运输序列表达重组蛋白的情况下，它可以包括N-末端的甲硫氨酸残基。这个残基可以任选地随后从表达的重组蛋白中裂解，以便提供最终产物。
表达控制序列和表达载体的选择将取决于宿主的选择。可以采用各种各样的表达宿主/载体组合。对于真核宿主的有用表达载体包括例如，包含来自SV40、牛乳头瘤病毒、腺病毒以及巨细胞病毒的表达控制序列的载体。对于细菌宿主的有用表达载体包括已知的细菌质粒，诸如来自大肠杆菌的质粒，包括pCR 1、pBR322、pMB9以及它们的衍生物、更宽宿主范围的质粒，诸如M13和丝状单链DNA噬菌体。
在另一个方面，本公开提供了一种宿主细胞，其包含本公开的多核苷酸或重组载体。包括在适当启动子控制下的原核细胞、酵母、昆虫或更高等真核细胞。原核细胞包括革兰阴性或革兰阳性生物体，例如大肠杆菌或细菌。更高等真核细胞包括如下所述已确立的哺乳动物来源的细胞系。还可以采用无细胞的翻译系统。用于与细菌、真菌、酵母以及哺乳动物细胞宿主一起使用的适当克隆和表达载体由泊威斯(Pouwels)等人(克隆载体：实验室手册(Cloning Vectors：A Laboratory Manual)，纽约，爱思唯尔(Elsevier，N.Y.)，1985)所述，该参考文献的相关披露内容特此通过引用结合。
关于蛋白质产生(包括抗体产生)的方法的另外信息可见于例如关国公开号2008/0187954、美国专利号6,413,746和6,660,501、以及国际专利公开号WO 04009823中，这些专利中的每一个特此通过引用以其全文结合。
还可以有利地采用不同哺乳动物或昆虫细胞培养物系统来表达本公开的双特异性抗体分子或其抗原结合片段。可以进行哺乳动物细胞中的重组蛋白的表达，因为此类蛋白质通常是正确折叠、适当修饰并且完全有功能的。适合的哺乳动物宿主细胞系的实例包括HEK-293和HEK-293T，由格祖曼(Gluzman)(细胞(Cell)23：175，1981)所述的COS-7猴肾脏细胞系；以及其他细胞系，包括例如L细胞、C127、3T3、中国仓鼠卵巢(CHO)、NSO、HeLa、以及BHK细胞系。哺乳动物表达载体可以包含非转录元件，诸如复制起点、连接至有待表达的基因上的适合启动子和增强子、及其他5′或3′侧翼非转录序列、以及5′或3′非翻译序列，诸如必要的核糖体结合位点、聚腺苷酸化位点、剪接供体和受体位点、以及转录终止序列。用于在昆虫细胞中产生异源蛋白的杆状病毒系统由卢科(LuckoW)和萨默斯(Summers)，生物技术6：47(1988)进行了综述。
可以根据任何适合的方法来纯化由转化的宿主产生的抗体或其抗原结合片段。此类标准方法包括，例如色谱法(例如，离子交换、亲和力以及定大小柱色谱法)、离心、差别溶解性、或通过蛋白质纯化的任何其他标准技术。亲和力标签诸如六组氨酸、麦芽糖结合结构域、流感外壳序列、谷胱甘肽S-转移酶等可以被附接至蛋白质上，以便允许通过在适当亲和力柱上穿过而简单纯化。还可以使用例如蛋白水解、核磁共振或x-射线结晶学来物理表征分离蛋白质。
例如，可以首先使用商业上可获得的蛋白质浓缩过滤器，例如Millipore超滤单元来浓缩来自将重组蛋白分泌到培养基中的系统的上清液。在浓缩步骤之后，可以将浓缩物施加至适合的纯化基质中。可替代地，可以采用阴离子交换树脂，例如具有侧链二乙基氨基乙基(DEAE)基团的基质或基底。这些基质可以是丙烯酰胺、琼脂糖、葡聚糖、纤维素或常用于蛋白质纯化的其他类型。可替代地，可以采用阳离子交换步骤。
适合的阳离子交换剂包括包含磺基丙基或羧甲基基团的不同不溶性基质。最后，可以采用使用疏水性RP-HPLC介质(例如，具有侧链甲基或其他脂肪族基团的硅胶)的一个或多个反相高效液相色谱法(RP-HPLC)步骤来进一步纯化抗体分子。还可以不同的组合采用前述纯化步骤中的一些或全部来提供均质重组蛋白。
可以例如通过初始从细胞沉淀物中提取、接着进行一个或多个浓缩、盐析、水性离子交换或大小排阻色谱法步骤来分离在细菌培养物中产生的本公开的双特异性抗体或其抗原结合片段。可以采用高效液相色谱法(HPLC)用于最终的纯化步骤。在重组蛋白的表达中采用的微生物细胞可以通过任何合宜方法来破坏，包括冻融循环、超声、机械破坏、或使用细胞裂解剂。
本领域中已知的用于纯化抗体和其他蛋白质的方法还包括例如描述于美国专利公开号US20080312425、US20080177048以及US20090187005中的那些，这些专利中的每一个特此通过引用以其全文结合。
在另一个方面，本公开提供了一种采用慢病毒载体向对象转导目标基因的方法，其包括向所述对象施用本公开的双特异性抗原结合分子或其片段。
本公开中的慢病毒载体能在其基因组RNA中编码外源基因。含有外源基因的重组病毒载体可通过在所述慢病毒载体基因组中插入外源基因来获得。本公开中所说的“外源基因”可以是需要在细胞中表达的任何目标基因，并可以是编码天然蛋白、或在天然蛋白中删除、取代或插入氨基酸残基而修饰的蛋白的基因。外源基因可插入病毒基因组中蛋白质非编码区的任何目的位置。所述目标基因通过慢病毒载体转到入对象细胞中，并在对象的细胞中表达；进一步的，所述目标基因在对象细胞中表达可以起到预防和或治疗疾病的作用。本领域技术人员可根据需要选自慢病毒载体携带的目标基因，本公开在此不做限定。
实施例
质粒载体构建
(1)对于CD19scfv-VSV-G双特异性抗体，载体构建包括将编码抗人源CD19的scfv片段的DNA插入到编码人抗体Fc段的DNA前，将编码人源LDL-R的CR2-CR3的DNA片段插入到抗体Fc段后，并且将三段连接完整的序列插入可在哺乳动物细胞中表达的表达载体中。其中，该哺乳动物细胞表达载体MG-HSP-v1包含CMV启动子，可以在哺乳动物细胞中启动下游基因表达。
抗人源CD19的scfv片段根据文章(DOI:10.1182/blood-2010-04-281931)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成，其核苷酸序列如SEQ ID NO.56所示；编码人抗体Fc段的DNA根据蛋白质数据库UniProt(P01857·IGHG1_HUMAN)经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成，其核苷酸序列如SEQ ID NO.57所示，编码人源LDL-R CR2-CR3的DNA根据蛋白质数据库UniProt(P01130·LDLR_HUMAN)经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成，其核苷酸序列如SEQ ID NO.58所示。
用重叠延伸聚合酶链式反应(overlap extension polymerase chain reaction)将三种片段连接在一起。所用引物如下表1所示。用BsaI限制性内切酶(NEB,Cat#R3733L)处理表达载体MG-HSP-v1(其核苷酸序列如SEQ ID NO.55所示)使其线性化。
表1
按照下述比例将各成分加入1.5mL Ep管中：
将上述成分混匀后在冰上静置5分钟。加入1μl Exonuclease III(Takara,Cat#2170A)，用移液枪轻吹混匀后在冰上静置60分钟。加入1μl 0.5M EDTA(Invitrogen，Cat#11568896)，混匀后65℃加热5分钟，在冰上静置5分钟。离心后将混合物全部加入到100μl DH5α中，在冰上孵育20分钟，42℃热激45秒，在冰上孵育2分钟，加入无抗LB培养基预摇60分钟后，涂布到带有氨苄抗生素的平板。16小时候挑取单克隆扩增培养，测序正确后提取质粒。
(2)对于CD3(OKT3)-VSV-G、CD3(UCHT1)-VSV-G、CD4-VSV-G、CD20-VSV-G、CD40-VSV-G、CD45-VSV-G、HLA-VSV-G双特异性抗体，需构建两种载体，其一，将编码抗人源CD3(OKT3)、CD3(UCHT1)、CD4、CD20、CD40、CD45或者HLA的VH片段的DNA插入到编码人抗体CH1-Fc段的DNA前，将编码人源LDL-R的CR2-CR3的DNA片段插入到抗体Fc段后，并且将三段连接完整的序列插入可在哺乳动物细胞中表达的表达载体中。其中，该哺乳动物细胞表达载体MG-HSP.v2(合成)包含CMV启动子，可以在哺乳动物细胞中启动下游基因表达。其二，将编码抗人源CD3(OKT3)、CD3(UCHT1)、CD4、CD20、CD40、CD45或者HLA的VL片段的DNA插入到编码人抗体CL段的DNA前，并且将两段连接完整的序列插入可在哺乳动物细胞中表达的表达载体中。其中，该哺乳动物细胞表达载体MG-LSP.V1(合成)包含CMV启动子，可以在哺乳动物细胞中启动下游基因表达。
表2




抗人源CD3(OKT3)的VH和VL片段根据文章(DOI:10.1073/pnas.0402295101)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其中，VH DNA序列如SEQ ID NO.60所示，VL DNA序列如SEQ ID NO.67所示)；抗人源CD3(UCHT1)的VH和VL片段根据文章(DOI:10.1073/pnas.0407359101)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其中，VH DNA序列如SEQ ID NO.61所示，VL DNA序列如SEQ ID NO.68所示)；抗人源CD4的VH和VL片段根据专利(https://patents.google.com/patent/CN101245108B/en)，经金斯瑞公司密码子优化工具 (https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其中，VH DNA序列如SEQ ID NO.62所示，VL DNA序列如SEQ ID NO.69所示)；抗人源CD20的VH和VL片段根据专利(https://patents.google.com/patent/CN101245108B/en)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其中，VH DNA序列如SEQ ID NO.63所示，VL DNA序列如SEQ ID NO.70所示)；抗人源CD40的VH和VL片段根据专利(https://patents.google.com/patent/EP3925977A1/en)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其中，VH DNA序列如SEQ ID NO.64所示，VL DNA序列如SEQ ID NO.71所示)；抗人源CD45的VH和VL片段根据数据库(GenBank:QES69311.1)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其中，VH DNA序列如SEQ ID NO.65所示，VL DNA序列如SEQ ID NO.72所示)；抗人源HLA的VH和VL片段根据文章(DOI:DOI:10.1016/0092-8674(78)90296-9)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其中，VH DNA序列如SEQ ID NO.66所示，VL DNA序列如SEQ ID NO.73所示)；编码人抗体CH1-Fc段的DNA根据蛋白质数据库UniProt(P01857·IGHG1_HUMAN)经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其核苷酸序列如SEQ ID NO.74所示)，编码人抗体CL段的DNA根据蛋白质数据库UniProt(P01834·IGKC_HUMAN)经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成(其核苷酸序列如SEQ ID NO.75所示)，编码人源LDL-R CR2-CR3的DNA与(1)相同。
用重叠延伸聚合酶链式反应(overlap extension polymerase chain reaction)将所需片段连接在一起。所用引物如下表3所示。用BsaI限制性内切酶(NEB,Cat#R3733L)处理表达载体MG-HSP-v2(其核苷酸序列如SEQ ID NO.59所示)或者MG-LSP-v1(其核苷酸序列如SEQ ID NO.76所示)，使其线性化。
表3

对于CD3(OKT3)(或CD3(UCHT1)、CD4、CD20、CD40、CD45、HLA)-VH-IgG1-CH1-Fc-LDLR-CR2/3质粒，按照下述比例将各成分加入1.5mL Ep管中：
对于CD3(OKT3)(或CD3(UCHT1)、CD4、CD20、CD40、CD45、HLA)-VL-CL质粒，按照下述比例将各成分加入1.5mL Ep管中：
将上述成分混匀后在冰上静置5分钟。加入1μl Exonuclease III(Takara,Cat#2170A)，用移液枪轻吹混匀后在冰上静置60分钟。加入1μl 0.5M EDTA(Invitrogen，Cat#11568896)，混匀后65℃加热5分钟，在冰上静置5分钟。离心后将混合物全部加入到100μl DH5α中，在冰上孵育20分钟，42℃热激45秒，在冰上孵育2分钟，加入无抗LB培养基预摇60分钟后，涂布到带有氨苄抗生素的平板。16小时候挑取单克隆扩增培养，测序正确后提取质粒。
(3)对于CD40L-VSV-G双特异性抗体，载体构建包括将编码人源LDL-R的CR2-CR3的DNA片段插入到抗体Fc段前，将编码人源CD40配体的片段的DNA插入到编码人抗体Fc段的DNA后，并且将三段连接完整的序列插入可在哺乳动物细胞中表达的表达载体中。其中，该哺乳动物细胞表达载体MG-HSP-v1包含CMV启动子，可以在哺乳动物细胞中启动下游基因表达。
人源CD40配体的片段根据蛋白质数据库UniProt(P29965·CD40L_HUMAN)，经金斯瑞公司密码子优化工具(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)优化后得到DNA序列并合成，其核苷酸序列如SEQ ID NO.所示；编码人抗体Fc段的DNA与(1)相同，编码人源LDL-R CR2-CR3的DNA与(1)相同。
用重叠延伸聚合酶链式反应(overlap extension polymerase chain reaction)将三种片段连接在一起。所用引物如下表1所示。用BsaI限制性内切酶(NEB,Cat#R3733L)处理表达载体MG-HSP-v1(其核苷酸序列如SEQ ID NO.55所示)使其线性化。
表4
按照下述比例将各成分加入1.5mL Ep管中：
将上述成分混匀后在冰上静置5分钟。加入1μl Exonuclease III(Takara,Cat#2170A)，用移液枪轻吹混匀后在冰上静置60分钟。加入1μl 0.5M EDTA(Invitrogen，Cat#11568896)，混匀后65℃加热5分钟，在冰上静置5分钟。离心后将混合物全部加入到100μl DH5α中，在冰上孵育20分钟，42℃热激45秒，在冰上孵育2分钟，加入无抗LB培养基预摇60分钟后，涂布到带有氨苄抗生素的平板。16小时候挑取单克隆扩增培养，测序正确后提取质粒。
实施例1双特异性抗体分子制备
(1)第-2天：Expi-293F细胞在Union293细胞培养基中悬浮培养(额外添加1％GlutaMax)培养，含5％CO2的37℃培养箱，转速设置为180rpm。确保刚复苏的细胞已培养三代或更多代且活力≥90％。培养并扩增Epi293F细胞，直到转染前一天(第-1天)细胞密度应达到大约3-5×106/mL。
(2)第-1天：按照最终密度2.5-3×106/mL接种细胞，让细胞过夜生长。
(3)第0天：测定活细胞密度和活率。活细胞密度和活率应分别达到4.5-5.5×106/mL和≥95％，才可进行转染。用提前预热的Union293细胞培养基(添加1％GlutaMax)将细胞稀释至3×106活细胞/mL，轻晃培养瓶以混匀细胞。在无菌的15mL离心管中配制用于转染的混合液(以30mL表达体系为例)：
Opti-MEM减血清培养基               3mL
总表达质粒(轻链：重链＝2.5：1)     24μg
polyethylenimine(PEI)转染试剂      96μg
用移液枪将混合物轻柔地吹匀，在室温静置10分钟。在一次性无菌通气摇瓶中加入27mL浓度为3×106/mL的Expi-293F细胞，后将混合物逐滴添加到培养皿的细胞中，轻轻摇晃混匀，将细胞放置在含5％CO2的37℃培养箱中培养24小时。
(4)第1天：在细胞中加入300μL 300mM丙戊酸(VPA，Sigma cat#P4543；用水配制，0.22μM过滤器过滤)和270μL 45％葡萄糖(Sigma cat#G8769)，轻轻摇晃混匀，将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第5天：收蛋白。将细胞转移到50mL离心管中，在4℃以4000rpm离心10分钟，将上清转移到新的50mL离心管。
(6)将Protein G纯化介质(Genscript cat#L00664)混匀后取400μL，加1mL 1×PBS，在4℃以600g离心10分钟，弃上清。重复清洗的操作三遍以去除存储液中的乙醇。将清洗后的Protein G纯化介质加入步骤(5)得到的上清中，在4℃旋转孵育2-4小时。
(7)准备用于纯化的重力柱，将大小合适的亲水性筛板垫入3mL亲和层析柱空柱管中，并用平衡/洗杂液(25mM Tris,150mM NaCl；pH 7.2)提前润湿筛板。将孵育过后的上清转移到柱管中，利用重力使未结合的蛋白与杂质随培养基上清流出，结合有目的蛋白的Protein G纯化介质在筛板的作用下堆积在柱管中。向柱管中缓慢加入10倍柱体积的平衡/洗杂液清洗Protein G纯化介质。缓慢加入1mL洗脱液(0.1M glycine,pH 2-3)，在流出口收集洗脱液(提前加入1/10体积的中和液(1M Tris，pH 7.5-9))，测量蛋白浓度，可重复洗脱步骤直至流出液的蛋白浓度无法检 测到为止。
(8)用50KD的超滤管将蛋白溶剂从洗脱液替换为1×PBS，并浓缩至0.5mg/mL，用0.22μM过滤器过滤，分装后放4℃冰箱短期保存，或-20℃以延长保存时间。
其中，CD3-VSV-G双特异性抗体重链序列如SEQ ID NO.2所示，轻链序列如SEQ ID NO.3所示；CD45-VSV-G双特异性抗体重链序列如SEQ ID NO.4所示，轻链序列如SEQ ID NO.5所示；CD19scfv-VSV-G双特异性抗体序列如SEQ ID NO.6所示。
实施例2 VSV-G假型慢病毒包装
(1)第0天：利用胰蛋白酶消化HEK293T细胞，收集一定量的细胞铺在六孔板中，应控制细胞量在第二天转染时达到约70-80％的汇合度，以增加转染效率。将细胞放置在含5％CO2的37℃培养箱中培养8-24小时。
(2)第1天：细胞完全附着且达到合适密度时即可开始转染。转染前2小时用真空泵吸走HEK293T细胞培养基并更换成1.5mL新鲜的DMEM培养基(培养基中添加10％FBS(56℃加热30分钟灭活)，1％青霉素-链霉素，下述以DMEM完全培养基代指)。在无菌的1.5mL Eppendorf管中配制转染用的质粒混合物，按照下述配方配制每个反应：
用移液枪将混合物轻柔地吹匀，在室温静置10分钟。静置后将混合物逐滴添加到培养皿的细胞中中，呈“十”或“8”字形轻轻摇晃混匀。将细胞放置在含5％CO2的37℃培养箱中培养8小时后，更换2mL新鲜的DMEM完全培养基。
(3)第3天：转染后48小时，在无菌的Eppendorf管中收集HEK293T细胞的培养液。将培养液在4℃以500g离心10分钟去除细胞碎片及杂质，随后使用注射器和滤器进行过滤，获得携带GFP荧光蛋白mRNA的VSV-G假型慢病毒。将过滤后的病毒液分装并保存在-80℃以备使用。
实施例3利用双特异性分子(靶向HLA)增强慢病毒对Ramos细胞的感染
(1)第1天：靶细胞Ramos细胞在IMDM培养基中培养(培养基中添加10％FBS(56℃加热30分钟灭活)，1％青霉素-链霉素，1％GlutaMAX，下述以IMDM完全培养基代指)。感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下300g离心3分钟，弃去培养基。
(2)用提前预冷的IMDM培养基(添加2％FBS)梯度稀释HLA-VSV-G双特异性分子，使终浓度分别为5、2.5、1.25、0.63、0.31、0.16、0.08、0.04、0.00μg/mL，(同时使用只靶向HLA的抗体(不携带LDLR-CR2-CR3结构域)2.5μg/mL作为对照)，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的IMDM培养基(添加2％FBS)以稀释体系中的双特异性分子的浓度，在4℃下300g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的IMDM培养基(添加2％FBS)重悬细胞，在4℃下300g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的IMDM完全培养基将病毒稀释到MOI约0.7，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下300g 离心3分钟，弃去培养基。用1mL提前预热到37℃的IMDM完全培养基重悬细胞，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积，确保细胞密度不超过3×106个/mL。
(7)第4天：感染60小时后，利用流式细胞仪检测感染效率，同时使用未感染的细胞作为对照，结果如图8A至8C所示。
实施例4利用双特异性分子(靶向HLA)增强慢病毒对Jurkat T细胞的感染
(1)第1天：靶细胞Jurkat T细胞在RPMI-1640培养基中培养(培养基中添加10％FBS(56℃加热30分钟灭活)，1％青霉素-链霉素，1％GlutaMAX,0.1％2-巯基乙醇(55mM))，下述以RPMI-1640完全培养基代指)。感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下300g离心3分钟，弃去培养基。
(2)用提前预冷的RPMI-1640培养基(添加2％FBS)梯度稀释HLA-VSV-G双特异性分子，使终浓度分别为5、2.5、1.25、0.63、0.31、0.16、0.08、0.04、0.00μg/mL，(同时使用只靶向HLA的抗体(不携带LDLR-CR2-CR3结构域)2.5μg/mL作为对照)，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的RPMI-1640培养基(添加2％FBS)以稀释体系中的双特异性分子的浓度，在4℃下300g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的RPMI-1640培养基(添加2％FBS)重悬细胞，在4℃下300g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的RPMI-1640完全培养基将病毒稀释到MOI约1，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下300g离心3分钟，弃去培养基。用1mL提前预热到37℃的RPMI-1640完全培养基重悬细胞，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积，确保细胞密度不超过3×106个/mL。
(6)第4天：感染60小时后，利用流式细胞仪检测感染效率，同时使用未感染的细胞作为对照，结果如图9A至9C所示。
实施例5利用双特异性分子(靶向HLA)增强慢病毒对人外周血单核细胞的感染
(1)第1天：复苏冻存的人外周血单核细胞PBMC，重悬计数。在非组织培养物处理的24孔板中每孔中加入1×106个PBMC细胞，并补充最终体积为2mL的完全培养基(X-VIVO 15无血清培养基，2％FBS(56℃加热30分钟灭活)，下述以完全培养基代指)，培养基中加入IL-15(5ng/mL)和IL-7(10ng/mL)。
(2)第2天：感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下500g离心3分钟，弃去培养基。
(2)用提前预冷的完全培养基稀释HLA-VSV-G双特异性分子，使终浓度分别为1.25，0.00μg/mL，(同时使用只靶向HLA的抗体(不携带LDLR-CR2-CR3结构域)2.5μg/mL作为对照)，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的完全培养基以稀释体系中的双特异性分子的浓度，在4℃下500g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的完全培养基重悬细胞，在4℃下500g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的完全培养基将病毒稀释到MOI约1，用0.3mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，加入IL-15(5ng/mL)和IL-7(10ng/mL)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下500g 离心3分钟，弃去培养基。用1mL提前预热到37℃的完全培养基重悬细胞，加入IL-15(5ng/mL)和IL-7(10ng/mL)，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积。
(6)第4天：感染72小时后，利用流式细胞仪检测感染效率，同时使用未感染的细胞作为对照，结果如图10所示。
实施例6利用双特异性分子(靶向CD19)增强慢病毒对Ramos细胞的感染
(1)第1天：靶细胞Ramos细胞在IMDM培养基中培养(培养基中添加10％FBS(56℃加热30分钟灭活)，1％青霉素-链霉素，1％GlutaMAX，下述以IMDM完全培养基代指)。感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下300g离心3分钟，弃去培养基。
(2)用提前预冷的IMDM培养基(添加2％FBS)稀释双特异性分子CD19scfv-VSV-G，使终浓度为5或者0μg/mL，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的IMDM培养基(添加2％FBS)以稀释体系中的双特异性分子的浓度，在4℃下300g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的IMDM培养基(添加2％FBS)重悬细胞，在4℃下300g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的IMDM完全培养基将病毒稀释到MOI约1，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下300g离心3分钟，弃去培养基。用1mL提前预热到37℃的IMDM完全培养基重悬细胞，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积，确保细胞密度不超过3×106个/mL。
(6)第4天：感染72小时后，利用流式细胞仪检测感染效率，同时使用未感染的细胞作为对照，结果如图11所示。
实施例7利用双特异性分子(靶向CD20)增强慢病毒对Ramos细胞的感染
(1)第1天：靶细胞Ramos细胞在IMDM培养基中培养(培养基中添加10％FBS(56℃加热30分钟灭活)，1％青霉素-链霉素，1％GlutaMAX，下述以IMDM完全培养基代指)。感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下300g离心3分钟，弃去培养基。
(2)用提前预冷的IMDM培养基(添加2％FBS)稀释双特异性分子CD20-VSV-G，使终浓度为5或者0μg/mL，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的IMDM培养基(添加2％FBS)以稀释体系中的双特异性分子的浓度，在4℃下300g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的IMDM培养基(添加2％FBS)重悬细胞，在4℃下300g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的IMDM完全培养基将病毒稀释到MOI约1，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下300g离心3分钟，弃去培养基。用1mL提前预热到37℃的IMDM完全培养基重悬细胞，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积，确保细胞密度不超过3×106个/mL。
(6)第4天：感染60小时后，利用流式细胞仪检测感染效率，结果如图12所示。
实施例8利用双特异性分子(靶向CD45)增强慢病毒对Ramos细胞的感染
(1)第1天：靶细胞Ramos细胞在IMDM培养基中培养(培养基中添加10％FBS(56℃加热30分钟灭活)，1％青霉素-链霉素，1％GlutaMAX，下述以IMDM完全培养基代指)。感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下300g离心3分钟，弃去培养基。
(2)用提前预冷的IMDM培养基(添加2％FBS)稀释双特异性分子CD40-VSV-G，使终浓度为5或者0μg/mL，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的IMDM培养基(添加2％FBS)以稀释体系中的双特异性分子的浓度，在4℃下300g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的IMDM培养基(添加2％FBS)重悬细胞，在4℃下300g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的IMDM完全培养基将病毒稀释到MOI约1，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下300g离心3分钟，弃去培养基。用1mL提前预热到37℃的IMDM完全培养基重悬细胞，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积，确保细胞密度不超过3×106个/mL。
(6)第4天：感染72小时后，利用流式细胞仪检测感染效率，结果如图13所示。
实施例9利用双特异性分子(靶向CD40)增强慢病毒对Ramos细胞的感染
(1)第1天：靶细胞Ramos细胞在IMDM培养基中培养(培养基中添加10％FBS(56℃加热30分钟灭活)，1％青霉素-链霉素，1％GlutaMAX，下述以IMDM完全培养基代指)。感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下300g离心3分钟，弃去培养基。
(2)用提前预冷的IMDM培养基(添加2％FBS)稀释双特异性分子CD40-VSV-G或者CD40L-VSV-G，使终浓度为5μg/mL，(同时使用无抗体处理的作为对照)，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的IMDM培养基(添加2％FBS)以稀释体系中的双特异性分子的浓度，在4℃下300g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的IMDM培养基(添加2％FBS)重悬细胞，在4℃下300g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的IMDM完全培养基将病毒稀释到MOI约1，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下300g离心3分钟，弃去培养基。用1mL提前预热到37℃的IMDM完全培养基重悬细胞，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积，确保细胞密度不超过3×106个/mL。
(6)第4天：感染60小时后，利用流式细胞仪检测感染效率，结果如图14所示。
实施例10利用双特异性分子(靶向CD3)增强慢病毒对Jurkat T细胞的感染
(1)第1天：靶细胞Jurkat T细胞在RPMI-1640培养基中培养(培养基中添加10％FBS(56℃加热30分钟灭 活)，1％青霉素-链霉素，1％GlutaMAX,0.1％2-巯基乙醇(55mM))，下述以RPMI-1640完全培养基代指)。感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下300g离心3分钟，弃去培养基。
(2)用提前预冷的RPMI-1640培养基(添加2％FBS)稀释双特异性分子CD3(OKT3)-VSV-G，使终浓度为5或者0μg/mL，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的RPMI-1640培养基(添加2％FBS)以稀释体系中的双特异性分子的浓度，在4℃下300g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的RPMI-1640培养基(添加2％FBS)重悬细胞，在4℃下300g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的RPMI-1640完全培养基将病毒稀释到MOI约2，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下300g离心3分钟，弃去培养基。用1mL提前预热到37℃的RPMI-1640完全培养基重悬细胞，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积，确保细胞密度不超过3×106个/mL。
(6)第4天：感染60小时后，利用流式细胞仪检测感染效率，结果如图15所示。
实施例11利用双特异性分子(靶向CD3)增强慢病毒对未经刺激的人原代T细胞的感染
(1)第1天：复苏冻存的人外周血单核细胞PBMC，重悬计数。在非组织培养物处理的24孔板中每孔中加入1×106个PBMC细胞，并补充最终体积为2mL的完全培养基(X-VIVO 15无血清培养基，2％FBS(56℃加热30分钟灭活)，下述以完全培养基代指)，培养基中加入IL-15(5ng/mL)和IL-7(10ng/mL)。
(2)第2天：感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下500g离心3分钟，弃去培养基。
(2)用提前预冷的完全培养基稀释CD3(OKT3)-VSV-G或者CD3(UCHT1)-VSV-G双特异性分子，使终浓度为5μg/mL，(同时使用无抗体处理的作为对照)，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的完全培养基以稀释体系中的双特异性分子的浓度，在4℃下500g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的完全培养基重悬细胞，在4℃下500g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的完全培养基将病毒稀释到MOI约5，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，加入IL-15(5ng/mL)和IL-7(10ng/mL)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下500g离心3分钟，弃去培养基。用1mL提前预热到37℃的完全培养基重悬细胞，加入IL-15(5ng/mL)和IL-7(10ng/mL)，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积。
(6)第4天：感染72小时后，利用流式细胞仪检测感染效率，结果如图16所示。
实施例12利用双特异性分子(靶向CD3)增强慢病毒对CD3/CD28抗体激活的人原代T细胞的感染
(1)第1天：在无菌水中稀释CD3和CD28抗体最终浓度为1μg/mL。用移液器在非组织培养处理的24孔板中加入0.5mL(＝0.5μg抗体)抗体溶液/孔，在培养箱中37℃孵育4小时。吸去抗体溶液，每孔加入1mL完全培养基(X-VIVO 15无血清培养基，2％FBS(56℃加热30分钟灭活)，下述以完全培养基代指)，在培养箱中37℃孵育15～30分钟。期间完成冻存PBMC的复苏，重悬计数。吸弃培养基，在每孔中加入1×106个PBMC细胞，并补 充最终体积为2mL的完全培养基，空孔可加PBS以防止培养基蒸发。
(2)第2天：加入IL-15(5ng/mL)和IL-7(10ng/mL)。
(3)第4天：感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下500g离心3分钟，弃去培养基。
(2)用提前预冷的完全培养基稀释CD3(OKT3)-VSV-G或者CD3(UCHT1)-VSV-G双特异性分子，使终浓度为5μg/mL，(同时使用无抗体处理的作为对照)，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的完全培养基以稀释体系中的双特异性分子的浓度，在4℃下500g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的完全培养基重悬细胞，在4℃下500g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的完全培养基将病毒稀释到MOI约5，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，加入IL-15(5ng/mL)和IL-7(10ng/mL)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下500g离心3分钟，弃去培养基。用1mL提前预热到37℃的完全培养基重悬细胞，加入IL-15(5ng/mL)和IL-7(10ng/mL)，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积。
(6)第4天：感染72小时后，利用流式细胞仪检测感染效率，结果如图17所示。
实施例13利用双特异性分子(靶向CD4)增强慢病毒对未经刺激的人原代T细胞的感染
(1)第1天：复苏冻存的人外周血单核细胞PBMC，重悬计数。在非组织培养物处理的24孔板中每孔中加入1×106个PBMC细胞，并补充最终体积为2mL的完全培养基(X-VIVO 15无血清培养基，2％FBS(56℃加热30分钟灭活)，下述以完全培养基代指)，培养基中加入IL-15(5ng/mL)和IL-7(10ng/mL)。
(2)第2天：感染前，收集靶细胞并进行计数，将特定数目的细胞收集到无菌的1.5mL Eppendorf管中(每个感染反应约5×105个细胞)，在4℃下500g离心3分钟，弃去培养基。
(2)用提前预冷的完全培养基稀释CD4双特异性分子，使终浓度为5μg/mL，(同时使用无抗体处理的作为对照)，用50μL的混合液重悬步骤1中离心后的细胞，在4℃冰箱中孵育15分钟。孵育结束后在细胞悬液中加入1mL提前预冷的完全培养基以稀释体系中的双特异性分子的浓度，在4℃下500g离心3分钟，弃去培养基。
(3)再次用1mL提前预冷的完全培养基重悬细胞，在4℃下500g离心3分钟，弃去培养基。再一次重复该步骤。
(4)准备用于感染的VSV-G假型慢病毒混合液，用提前预冷的完全培养基将病毒稀释到MOI约5，用0.5mL病毒混合液重悬步骤(3)中离心收集的细胞(此时细胞表面已经结合双特异性分子)，加入IL-15(5ng/mL)和IL-7(10ng/mL)，轻吹混匀后接种到24孔板中，在37℃下1800g离心45分钟(加速9，减速5)。离心后将细胞放置在含5％CO2的37℃培养箱中培养。
(5)第2天：感染24小时后，用移液枪轻吹细胞并全部转移到无菌的1.5mL Eppendorf管中，在室温下500g离心3分钟，弃去培养基。用1mL提前预热到37℃的完全培养基重悬细胞，加入IL-15(5ng/mL)和IL-7(10ng/mL)，并接种到24孔板中。每天检查细胞状态与密度，必要时更换新鲜培养基或扩大细胞培养体积。
(6)第4天：感染72小时后，利用流式细胞仪检测感染效率，结果如图18所示。
从图8至图18中可以看出，在使用等量VSV-G假型慢病毒对Jurkat T细胞、Ramos细胞和人原代T细胞(未刺激或者经CD3/CD28抗体刺激)进行感染，使用相应的双特异性抗体均可提高VSV-G假型慢病毒的感染效率，其中，使用HLA-VSV-G双特异性抗体使Ramos细胞感染效率由4.5％最高提高至53.8％(图8)；使用HLA-VSV-G双特异性抗体使Jurkat T细胞感染效率由38.0％最高提高至75.7％(图9)；使用HLA-VSV-G双特异性抗体使人外周血单核细胞感染效率由0.052％提高至62.7％(图10)；使用CD29scFv-VSV-G双特异性抗体使Ramos细胞感 染效率由21.4％提高至59.4％(图11)；使用CD20-VSV-G双特异性抗体使Ramos细胞感染效率由34.6％提高至68.2％(图12)；使用CD45-VSV-G双特异性抗体使Ramos细胞感染效率由34.6％提高至58.1％(图13)；使用CD40-VSV-G或CD40L-VSV-G双特异性抗体使Ramos细胞感染效率由34.6％提高至42.9％或51.7％(图14)；使用CD3-VSV-G双特异性抗体使Jurkat T细胞感染效率由66.7％最高提高至81.9％(图15)；使用CD3(OKT3)-VSV-G或者CD3(UCHT1)-VSV-G双特异性抗体使未经刺激的人原代T细胞细胞感染效率由9.19％提高至33.46或者30.61％(图16)；使用CD3(OKT3)-VSV-G或者CD3(UCHT1)-VSV-G双特异性抗体使CD3/CD28抗体激活的人原代T细胞感染效率由16.0％提高至或者40.7％或23.0％(图17)；用CD4-VSV-G双特异性抗体使未经刺激的人原代T细胞感染效率由9.19％提高至或者43.05％(图18)；同时比较图18与图16对T细胞表面特异性蛋白的染色可以看出，通过靶向CD3的双特异性分子介导慢病毒感染后，细胞表面CD3表达量下调；通过靶向CD4的双特异性分子介导慢病毒感染使得细胞表面CD4表达量下调，这进一步说明慢病毒通过双特异性分子的作用进入细胞。
另外，通过对图8至图18中平均荧光强度(MFI)结果的分析发现，本公开的双特异性抗体在提高病毒感染效率的同时，还提高了目标基因(GFP)在细胞中的表达水平，其中，使用HLA-VSV-G双特异性抗体使Ramos细胞目标蛋白表达水平由49038最高提高至165640(图8)；使用HLA-VSV-G双特异性抗体使Jurkat T细胞目标蛋白表达水平由8689最高提高至17143(图9)；使用HLA-VSV-G双特异性抗体使人外周血单核细胞目标蛋白表达水平由227提高至4489(图10)；使用CD29scFv-VSV-G双特异性抗体使Ramos细胞目标蛋白表达水平由5238提高至11043(图11)；使用CD20-VSV-G双特异性抗体使Ramos细胞目标蛋白表达水平由127289提高至163324(图12)；使用CD3-VSV-G双特异性抗体使Jurkat T细胞目标蛋白表达水平由18338提高至32697(图15)；使用CD3(OKT3)-VSV-G或者CD3(UCHT1)-VSV-G双特异性抗体使未经刺激的人原代T细胞目标蛋白表达水平由10264提高至58730或34160(图16)；使用CD3(OKT3)-VSV-G或者CD3(UCHT1)-VSV-G双特异性抗体使CD3/CD28抗体激活的人原代T细胞目标蛋白表达水平由225907提高至453000或337512(图17)；使用CD4-VSV-G双特异性抗体使未经刺激的人原代T细胞目标蛋白表达水平由10264提高至22693(图18)。
说明本公开的双特异性抗体能够提高VSV-G假型慢病毒对不同细胞的感染效率和表达效率。

Claims (40)

  1. 一种双特异性抗原结合分子或其片段，其包含第一抗原结合结构域和第二抗原结合结构域，其中所述第一抗原结合结构域用于特异性结合细胞表面抗原；所述第二抗原结合结构域用于特异性结合水疱性口膜炎病毒糖蛋白(VSV-G)。
  2. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述第一抗原结合结构域包含重链(HC)可变区(VH)和/或轻链(LC)可变区(VL)。
  3. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述第一抗原结合结构域还包含重链(HC)恒定区(CH)或其片段和/或轻链(LC)恒定区(CL)或其片段；优选地，所述重链恒定区或其片段是IgG恒定区；和/或所述轻链恒定区是κ恒定区或λ恒定区。
  4. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述第一抗原结合结构域包括Fab、Fab'、F(ab')2、scFv、细胞表面蛋白的天然配体或VHH的至少一种。
  5. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述第一抗原结合结构域是单克隆抗体、人源化抗体、人抗体、嵌合抗体、或亲和力优化的抗体。
  6. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述第一抗原结合结构域还包含Fc结构域；优选地，所述Fc结构域包含能够降低或增强该双特异性抗体的ADCC活性的至少一个突变。
  7. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述细胞表面抗原选自B细胞表面抗原、NK细胞表面抗原、造血干细胞表面抗原或T细胞表面抗原；优选地，所述细胞表面抗原选自CD3、CD19、CD45、CD20、CD34、CD40、CD56、CD16、CD133、CD147、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CD4、CD5、CD8A&CD8B、CD7、HLA中任一种。
  8. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述第二抗原结合结构域包含低密度脂蛋白受体(LDL-R)的CR2和CR3结构域。
  9. 根据权利要求8所述的双特异性抗原结合分子或其片段，其中，所述第二抗原结合结构域包含与SEQ ID NO.1所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
  10. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，
    (a)所述第二抗原结合结构域被共价连接至所述第一抗原结合结构域的重链的羧基末端上；
    (b)所述第二抗原结合结构域被共价连接至所述第一抗原结合结构域的重链的氨基末端上；或，
    (c)所述第二抗原结合结构域被共价嵌入在所述第一抗原结合结构域的重链的多肽链中；
    和/或
    (d)所述第二抗原结合结构域被共价连接至所述第一抗原结合结构域的轻链的羧基末端上；
    (e)所述第二抗原结合结构域被共价连接至所述第一抗原结合结构域的轻链的氨基末端上；或，
    (f)所述第二抗原结合结构域被共价嵌入在所述第一抗原结合结构域的轻链的多肽链中。
  11. 根据权利要求1所述的双特异性抗原结合分子或其片段，其中，所述第二抗原结合结构域与所述第一抗原结合结构域的重链和/或轻链通过肽接头连接；优选地，所述肽接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
  12. 一种双特异性抗原结合分子或其片段，其包括：
    包含VH、CH1、CH2、CH3、CR2和CR3的第一多肽；和包含VL和CL的第二多肽；
    其中VH为第一抗原结合结构域的重链可变区，VL为第一抗原结合结构域的轻链可变区，所述第一抗原结合结构域用于特异性结合细胞表面抗原；CH1、CH2和CH3分别为IgG分子的第一、第二和第三恒定区，CL为轻链恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
  13. 根据权利要求12所述的双特异性抗原结合分子或其片段，其中所述第一多肽在N端至C端顺序中包含VH、CH1、CH2、CH3、CR2和CR3；所述第二多肽在N端至C端顺序中包含VL和CL；
    优选地，所述CR2和CR3结构域包含与SEQ ID NO.1所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
  14. 根据权利要求12所述的双特异性抗原结合分子或其片段，其中所述两个多肽缔合以形成靶向细胞表面抗原的抗原结合位点；优选地，所述细胞表面抗原选自B细胞表面抗原、NK细胞表面抗原、造血干细胞表面抗原或T细胞表面抗原；优选地，所述细胞表面抗原选自CD3、CD19、CD45、CD20、CD34、CD40、CD56、CD16、CD133、CD147、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CD4、CD5、CD8A&CD8B、CD7、HLA中任一种。
  15. 根据权利要求12所述的双特异性抗原结合分子或其片段，其中所述双特异性抗原结合分子为四价或六价的；优选地，所述双特异性抗原结合分子对于两个靶标为二价或四价的。
  16. 根据权利要求13所述的双特异性抗原结合分子或其片段，其中所述第一多肽包含CH3和CR2之间的肽接头；优选地，所述肽接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
  17. 根据权利要求12所述的双特异性抗原结合分子或其片段，其中所述第一多肽包含与SEQ ID NO.2所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列；所述第二多肽包含与SEQ ID NO.3所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列；或者
    所述第一多肽包含与SEQ ID NO.4所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列；所述第二多肽包含与SEQ ID NO.5所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
  18. 根据权利要求13所述的双特异性抗原结合分子或其片段，所述第二多肽在N端至C端顺序中还包含CR2和CR3结构域；优选地，所述第二多肽还包含的CL与CR2之间的肽接头；优选地，所述肽接头选自L1、L2、L3或L4；其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
  19. 一种双特异性抗原结合分子或其片段，其包括：
    包含scFv的第一抗原结合结构域，所述scFv包含VH和VL；和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述scFv和所述第二抗原结合结构域的CH2和CH3结构域；
    其中，所述第一抗原结合结构域用于特异性结合细胞表面抗原；VH为重链可变区，VL为轻链可变区；CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
  20. 根据权利要求19所述的双特异性抗原结合分子或其片段，其在N端至C端顺序中包含scFv、CH2、CH3、CR2和CR3；所述scFv包含VH和VL；
    优选地，所述CR2和CR3结构域包含与SEQ ID NO.1所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
  21. 根据权利要求20所述的双特异性抗原结合分子或其片段，其中所述scFv靶向细胞表面抗原；优选地，所述细胞表面抗原选自B细胞表面抗原、NK细胞表面抗原、造血干细胞表面抗原或T细胞表面抗原；优选地，所述细胞表面抗原选自CD3、CD19、CD45、CD20、CD34、CD40、CD56、CD16、CD133、CD147、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CD4、CD5、CD8A&CD8B、CD7、HLA中任一种。
  22. 根据权利要求19至21中任一项所述的双特异性抗原结合分子或其片段，其中所述双特异性抗原结合分子为四价的。
  23. 根据权利要求19至21中任一项所述的双特异性抗原结合分子或其片段，其中所述双特异性抗原结合分子对于两个靶标为二价的。
  24. 根据权利要求20所述的双特异性抗原结合分子或其片段，其包含scFv和CH2之间的肽接头；优选地，所述肽接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
  25. 根据权利要求20所述的双特异性抗原结合分子或其片段，其包含CH3和CR2之间的肽接头；优选地，所述肽接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
  26. 根据权利要求19所述的双特异性抗原结合分子或其片段，其包含与SEQ ID NO.6所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
  27. 一种双特异性抗原结合分子或其片段，其包括：
    包含VHH的第一抗原结合结构域，和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述VHH和所述第二抗原结合结构域的CH2和CH3结构域；
    其中，所述第一抗原结合结构域用于特异性结合细胞表面抗原；所述CH2、CH3分别为IgG分子的第二和第三 恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
  28. 根据权利要求27所述的双特异性抗原结合分子或其片段，其在N端至C端顺序中包含VHH、CR2和CR3；优选地，所述CR2和CR3结构域包含与SEQ ID NO.1所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
  29. 根据权利要求27所述的双特异性抗原结合分子或其片段，其中，所述细胞表面抗原选自B细胞表面抗原、NK细胞表面抗原、造血干细胞表面抗原或T细胞表面抗原；优选地，所述细胞表面抗原选自CD3、CD19、CD45、CD20、CD34、CD40、CD56、CD16、CD133、CD147、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CD4、CD5、CD8A&CD8B、CD7、HLA中任一种。
  30. 根据权利要求27所述的双特异性抗原结合分子或其片段，所述双特异性抗原结合分子为二价的；优选地，所述双特异性抗原结合分子对于两个靶标为单价的。
  31. 根据权利要求28所述的双特异性抗原结合分子或其片段，其包含VHH和CR2之间的肽接头；优选地，所述接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
  32. 一种双特异性抗原结合分子或其片段，其包括：
    包含细胞表面蛋白的天然配体的第一抗原结合结构域，和包含CR2和CR3的第二抗原结合结构域；以及任选地，用于连接所述天然配体和所述第二抗原结合结构域的CH2和CH3结构域；
    其中，所述第一抗原结合结构域用于特异性结合细胞表面抗原；CH2、CH3分别为IgG分子的第二和第三恒定区；CR2和CR3为低密度脂蛋白受体(LDL-R)的第二和第三CR结构域。
  33. 根据权利要求32所述的双特异性抗原结合分子或其片段，其在N端至C端顺序中包含CR2、CR3、CH2、CH3和天然配体；优选地，所述CR2和CR3结构域包含与SEQ ID NO.1所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列。
  34. 根据权利要求32所述的双特异性抗原结合分子或其片段，其中，所述细胞表面抗原选自B细胞表面抗原、NK细胞表面抗原、造血干细胞表面抗原或T细胞表面抗原；优选地，所述细胞表面抗原选自CD3、CD19、CD45、CD20、CD34、CD40、CD56、CD16、CD133、CD147、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CD4、CD5、CD8A&CD8B、CD7、HLA中任一种。
  35. 根据权利要求32所述的双特异性抗原结合分子或其片段，其中所述双特异性抗原结合分子为四价的；优选地，所述双特异性抗原结合分子对于两个靶标为二价的。
  36. 根据权利要求33所述的双特异性抗原结合分子或其片段，其包含CR3和CH2之间的肽接头；优选地，所述接头选自L1、L2、L3或L4，其中，L1的序列如SEQ ID NO.7所示，L2的序列如SEQ ID NO.8所示，L3的序列如SEQ ID NO.9所示，L4的序列如SEQ ID NO.10所示；优选的，所述L1肽接头为G4S(SEQ ID NO.11)或(G4S)3(SEQ ID NO.12)；更优选的，所述L2肽接头为(G4S)3A(SEQ ID NO.13)或(G4S)4A(SEQ ID NO.14)。
  37. 权利要求1-36中任一项所述的双特异性抗原结合分子或其片段用于介导的受体非依赖性慢病毒载体基因转导的应用；优选地，所述基因转导的对象为低表达或不表达低密度脂蛋白受体的细胞；优选地，所述基因转导的对象选自T细胞、B细胞、NK细胞或造血干细胞；优选地，所述T细胞和所述B细胞为原代细胞。
  38. 一种多核苷酸，其编码权利要求1-36中任一项所述的双特异性抗原结合分子或其片段；或，一种重组载体，其包含所述的多核苷酸；或，一种宿主细胞，其包含所述的多核苷酸或重组载体。
  39. 生产权利要求1-36中任一项所述的双特异性抗原结合分子或其片段的方法。
  40. 一种采用慢病毒载体向对象转导目标基因的方法，其包括向所述对象施用权利要求1-36中任一项所述的双特异性抗原结合分子或其片段。
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