Antibody delivery by dual viral vector system

Abstract

The present invention provides a dual recombinant viral vector system for intracellularly expressing a polypeptide molecule composed of two different peptide chains and having antigen-binding activity, wherein a first chain is expressed by a first viral vector and a second chain is expressed by a second viral vector, and the first chain and the second chain can be assembled into a polypeptide molecule having antigen-binding activity after expression in a cell. The present invention also provides a dual plasmid system for producing the dual recombinant viral vector system, a host cell comprising the dual recombinant viral vector system, a pharmaceutical composition, and uses and methods of delivering the polypeptide molecule to a subject with the dual recombinant viral vector system.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to a Chinese patent application having a filing date of March 8, 2021 and application number 202110252231.1, which is hereby incorporated by reference in its entirety.

[0003] The present invention relates to the field of gene therapy, and in particular to a system and method for expressing antibodies via a dual viral vector system. [Background technology]

[0004] In recent years, it has been demonstrated that recombinant adeno-associated virus (rAAV) can be used to deliver gene components to different tissues and various types of cells in various species, and this new therapeutic method shows good prospects. The rAAV drug Alipogene tiparvovec (Glybera, uniQure), approved by the European Medicines Agency (EMA) in 2012, is composed of the rAAV1 vector and human lipoprotein lipase (LPL) gene, and is intended to treat lipoprotein lipase deficiency (LPLD). So far, two other rAAV drugs have been approved by the FDA and EMA, namely voretigene neparvovec-rzyl (Luxturna, Spark) and onasemnogene abeparvovec (ZOLGENSMA, AveXis / Novartis), the latter of which has been conditionally approved, both of which are rAAV-mediated complete gene replacement therapies. Several clinical gene therapy trials delivering different genetic components by rAAV vectors use mechanisms of action that have proven effective in animal models, including truncated genes such as the B-domain deleted Factor VIII gene for treating hemophilia and micro-dystrophin for treating DMD, small RNAs such as small hairpin RNAs for treating Hepatitis B, microRNAs for treating Huntington's disease, and small nuclear RNAs that induce exon skipping for treating DMD, and expressed antibodies for treating AMD.

[0005] Antibody drugs have already proven to be safe and effective in a wide range of indications. rAAV is an ideal vector for delivering antibody genes, and the length of monoclonal antibody genes is within the packaging capacity of rAAV. The rAAV-based antibody delivery strategy is to express antibody light and heavy chain polypeptides in cells transduced with a single rAAV vector and allow the realization of IgG self-assembly by self-cleaving peptides (e.g., different 2A). However, such strategies have low antibody expression efficiency and may cause toxic effects by resulting in non-functional antibodies or antibody protein aggregates due to inappropriate assembly of antibodies, limiting their clinical application. To date, the only rAAV-based IgG drug that has been in clinical trials is AAV8-VRC07, which expresses HIV-1 neutralizing antibodies and is used for antiviral therapy. Other similar strategies include the expression of soluble antibodies without Fc fragments (RGX-314) or recombinant proteins fused with Fc fragments (AAV2-sFLT01 and ADVM-022).

[0006] Other viral vectors can be used to deliver genetic material, but similarly suffer from low antibody expression and assembly efficiency. There are currently no universal methods for efficient antibody expression in vivo. Summary of the Invention

[0007] The present invention provides a method for efficiently expressing a polypeptide molecule having antigen-binding activity in vivo using a recombinant viral vector, in which the coding nucleic acid sequences for the two polypeptide chains constituting the polypeptide molecule are placed in two expression cassettes, respectively, and inserted between the 5' and 3' inverted repeat sequences of two recombinant viral vectors, respectively. The expression cassettes express the two polypeptide chains constituting the polypeptide molecule antibody, respectively, in a host cell, and are assembled into a polypeptide molecule having antigen-binding activity.

[0008] A first aspect of the present invention provides a dual recombinant viral vector system for expressing in a cell a polypeptide molecule which is composed of two different peptide chains and has antigen-binding activity, a first recombinant viral vector, the genome of which includes a first expression cassette for expressing a first chain of said polypeptide molecule; a second recombinant viral vector whose genome includes a second expression cassette for expressing a second chain of said polypeptide molecule; wherein the first chain and the second chain are distinct, the first recombinant virus and the second recombinant virus are capable of expressing the first chain and the second chain, respectively, after being introduced into a cell, and the first chain and the second chain are capable of assembling into a polypeptide molecule having antigen-binding activity.

[0009] In some embodiments, the first recombinant viral vector and the second recombinant viral vector are independently selected from the group consisting of a recombinant adeno-associated viral vector, a recombinant adenoviral vector, a recombinant HSV vector, a recombinant lentiviral vector, and a recombinant retroviral vector.

[0010] In some embodiments, the first recombinant viral vector and the second recombinant viral vector are recombinant adeno-associated viral vectors, or the first recombinant viral vector and the second recombinant viral vector are lentiviral vectors.

[0011] In some embodiments, the first recombinant viral vector and the second recombinant viral vector are recombinant adeno-associated viral vectors, each comprising an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV Rh10 capsid, or a chimeric AAV capsid or modified capsid.

[0012] In some embodiments, the first recombinant viral vector and the second recombinant viral vector comprise ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAV Rh10.

[0013] In some embodiments, the polypeptide molecule comprises an antigen binding portion having a heavy chain variable region and a light chain variable region, and the first chain comprises the heavy chain variable region of the antigen binding portion and the second chain comprises the light chain variable region of the antigen binding portion.

[0014] In some embodiments, the polypeptide molecule comprises an antigen-binding moiety having a heavy chain and a light chain, wherein the first chain comprises the heavy chain of the antigen-binding moiety and the second chain comprises the light chain of the antigen-binding moiety.

[0015] In some embodiments, the ratio of the first recombinant viral vector to the second recombinant viral vector is 4:1 to 1:8, and preferably 1:1 to 1:4.

[0016] In some embodiments, the polypeptide molecule is a monoclonal antibody or antigen-binding fragment thereof, the first chain is a heavy chain of the monoclonal antibody or antigen-binding fragment thereof, and the second chain is a light chain of the monoclonal antibody or antigen-binding fragment thereof.

[0017] In some embodiments, the antigen-binding fragment of the monoclonal antibody is a Fab fragment, a Fab' fragment or a (Fab') 2 It is a fragment.

[0018] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof specifically binds to an angiogenic factor.

[0019] In some embodiments, the angiogenic factor is selected from VEGF, VEGFR, FGF, PDGF, TGFβ, Ang1, Ang2, integrins, CD31, and plasminogen activators.

[0020] In some embodiments, the polypeptide molecule is a bispecific antibody comprising a first antigen binding portion that specifically binds to a first antigen epitope and a second antigen binding portion that specifically binds to a second antigen epitope, which are distinct from each other, wherein the first antigen binding portion comprises a heavy chain and a light chain, wherein the first chain comprises the heavy chain of the first antigen binding portion and the second chain comprises the light chain of the first antigen binding portion.

[0021] In some embodiments, the first antigen-binding portion of the bispecific antibody is a monoclonal antibody or an antigen-binding fragment thereof, preferably the antigen-binding fragment is a Fab fragment, a Fab' fragment or a (Fab') 2 It is a fragment.

[0022] In some embodiments, the second antigen-binding portion of the bispecific antibody is a single chain form of the antibody.

[0023] In some embodiments, the single chain form of the antibody is a single domain antibody or an scFv.

[0024] In some embodiments, the second antigen-binding portion is linked to the N-terminus or C-terminus of the heavy chain of the first antigen-binding portion, and the first chain comprises the heavy chain of the first antigen-binding portion and the second antigen-binding portion.

[0025] In some embodiments, the second antigen-binding portion is linked to the N-terminus or C-terminus of the light chain of the first antigen-binding portion, and the second chain comprises the light chain of the first antigen-binding portion and the second antigen-binding portion.

[0026] In some embodiments, the first chain comprises a heavy chain of a first antigen-binding moiety and a second antigen-binding moiety, and the second chain comprises a light chain of the first antigen-binding moiety.

[0027] In some embodiments, at least one or both of the first antigen-binding moiety and the second antigen-binding moiety specifically binds to an angiogenic factor.

[0028] In some embodiments, the angiogenic factor is selected from VEGF, VEGFR, FGF, PDGF, TGFβ, Ang1, Ang2, integrins, CD31, and plasminogen activators.

[0029] In some embodiments, the first antigen binding moiety specifically binds to VEGF and the second antigen binding moiety specifically binds to Ang2, or the first antigen binding moiety specifically binds to Ang2 and the second antigen binding moiety specifically binds to VEGF.

[0030] In some embodiments, the polypeptide molecule comprises a first antigen binding moiety that specifically binds to a first antigen epitope and a second binding domain that specifically binds to a second target molecule, wherein the second target molecule is a cytokine, the first antigen binding moiety comprises a heavy chain and a light chain, the first chain comprises the heavy chain of the first antigen binding moiety and the second chain comprises the light chain of the first antigen binding moiety.

[0031] In some embodiments, the second binding domain is a cytokine receptor, an extracellular domain thereof, or a variant thereof.

[0032] In some embodiments, the first antigen-binding portion is a monoclonal antibody or an antigen-binding fragment thereof, and preferably, the antigen-binding fragment is a Fab fragment, a Fab' fragment or a (Fab') 2 It is a fragment.

[0033] In some embodiments, the second binding domain is linked to the N-terminus or C-terminus of the heavy chain of the first antigen-binding portion, and the first chain comprises the heavy chain of the first antigen-binding portion and the second binding domain.

[0034] In some embodiments, the second binding domain is linked to the N-terminus or C-terminus of the light chain of the first antigen-binding portion, and the second chain comprises the light chain of the first antigen-binding portion and the second binding domain.

[0035] In some embodiments, the first chain comprises a heavy chain and a second binding domain of the first antigen-binding moiety, and the second chain comprises a light chain of the first antigen-binding moiety.

[0036] In some embodiments, the first antigen-binding portion specifically binds to the target PD-1, PD-L1, or angiogenic factor, and the second binding domain is a receptor or fragment thereof capable of binding to human transforming growth factor beta (TGFβ), Ang2, or VEGF.

[0037] In some embodiments, the angiogenic factor is selected from VEGF, VEGFR, FGF, PDGF, TGFβ, Ang1, Ang2, integrins, CD31, and plasminogen activators.

[0038] In some embodiments, the first antigen binding portion specifically binds to PD-L1 and the second binding domain is human transforming growth factor beta receptor II (TGFβRII) or a fragment thereof.

[0039] In some embodiments, the dual viral vector system is a composition comprising the first recombinant viral vector and the second recombinant viral vector.

[0040] A second aspect of the present invention provides a dual plasmid system for producing any one of the dual recombinant viral vector systems described above, a first plasmid containing the first expression cassette and viral genomic elements necessary to produce the first recombinant viral vector; and a second plasmid comprising said second expression cassette and the viral genomic elements necessary to produce said second recombinant viral vector.

[0041] A third aspect of the invention provides a host cell transduced with any one of the above-mentioned dual recombinant viral vector systems.

[0042] A fourth aspect of the present invention provides a pharmaceutical composition comprising any one of the dual recombinant viral vector systems described above or a host cell as described above, and a pharma- ceutically acceptable vector.

[0043] A fifth aspect of the present invention provides an application of any one of the aforementioned dual recombinant viral vector systems, the aforementioned dual plasmid system or the aforementioned host cell for producing a polypeptide molecule which is composed of two different peptide chains and has antigen-binding activity.

[0044] A sixth aspect of the present invention provides a method for producing a polypeptide molecule which is composed of two different peptide chains and has antigen-binding activity, comprising the steps of: transducing a host cell with any one of the dual recombinant viral vector systems described above; and obtaining said polypeptide molecule by culturing said transduced host cells under conditions to cause said host cells to express the first and second chains of said polypeptide molecule, which assemble to produce a polypeptide molecule having antigen-binding activity.

[0045] A seventh aspect of the present invention provides an application of any one of the aforementioned dual recombinant viral vector systems in the manufacture of a medicament for delivering a polypeptide molecule, which is composed of two different peptide chains and has antigen-binding activity, to the body of a subject.

[0046] An eighth aspect of the present invention provides a method for delivering a polypeptide molecule composed of two different peptide chains and having antigen-binding activity to a subject, the method comprising administering to a subject in need thereof any one of the dual recombinant viral vector systems described above or the pharmaceutical composition described above.

[0047] A ninth aspect of the present invention provides any one of the aforementioned dual recombinant viral vector systems or the aforementioned pharmaceutical composition for delivering a polypeptide molecule which is composed of two different peptide chains and has antigen-binding activity to the body of a subject. [Brief description of the drawings]

[0048] The present invention will become more fully understood in connection with the following detailed description and the accompanying drawings, in which like elements are numbered in like manner and in which: [Figure 1] Figure 1 shows Avastin expression by dual AAV vectors. (a) Schematic diagram of a vector expressing the complete antibody sequence (AAV-Avastin single vector), a vector expressing only the heavy chain (AAV-Avastin heavy chain, i.e., AAV-Avastin-H), and a vector expressing only the light chain (AAV-Avastin light chain, i.e., AAV-Avastin-L). (b) and (c) Western blot results of Avastin antibody produced by HEK293FT cells transduced with single vector (AAV2-Avastin) and dual vector (AAV2-Avastin-L and AAV2-Avastin-H), where (b) is under non-reducing conditions and (c) is under reducing conditions, the positive control is purified antibody, and the negative control is cell culture supernatant not transduced with recombinant viral vector. d shows Western blot results of Avastin antibody produced by hARPE-19 cells transduced with single vector (AAV2-Avastin) and dual vector (AAV2-Avastin-L and AAV2-Avastin-H), where the positive control was purified antibody. [Diagram 2]Western blot results of Avastin antibody expression by HEK293FT or hARPE-19 cells transduced with single or dual vectors carrying AAV8 capsid, AAV9 capsid and AAV5 capsid (a,b,c), where the positive control is purified antibody. [Diagram 3] Western blot results of Avastin antibody expression by HEK293FT or hARPE-19 cells transduced or transfected with dual AAV viral vectors and dual plasmid vectors, where the positive control is purified antibody. [Figure 4] Western blot results of Avastin antibody expression by hiPSC-RPE transduced with dual AAV viral vectors, where the positive control is purified antibody. [Diagram 5] Figure 1 shows ABTAA expression by dual AAV vectors. (a) Schematic diagram of transfer plasmid expressing complete antibody sequence (pAAV2.CMV.ABTAA) and transfer plasmid expressing only light or heavy chain (pAAV2.CMV.ABTAA-L and pAAV2.CMV.ABTAA-H). (b and c) Western blot results of ABTAA antibody expression by HEK293FT cells transduced with single or dual AAV viral vectors, where (b) is non-reducing condition and (c) is reducing condition, positive control is purified antibody, and negative control is cell culture supernatant not transduced with recombinant viral vector. [Figure 6]Figure 1 shows anti-Ang2 and anti-VEGF bispecific antibody expression by dual AAV vectors. a and b are schematic diagrams of dual AAV vectors expressing bispecific antibodies, where the VEGF antibody variable region shown in figure a is linked to the ABTAA heavy chain, and the VEGF antibody variable region shown in figure b is linked to the ABTAA light chain. c and d are Western blot results of ABTAA-anti-VEGF bispecific antibody expression by HEK293FT cells transduced with the viral dual vectors shown in figures a and b, respectively, where the positive control is purified antibody. [Figure 7] FIG. 1 shows expression of dual AAV vector-delivered Avastin genes in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present invention provides a dual recombinant viral vector system for delivering an antibody component and its application, in which two recombinant viral vectors are used to deliver the two chains of an antibody component, respectively, thereby improving the expression level of the antibody component; in particular, when two recombinant viral vectors are used to deliver the heavy chain and light chain of an antibody component, respectively, it can promote effective assembly of the heavy chain and light chain of the antibody component, improve the pairing efficiency of the heavy chain and light chain, and reduce inaccurate pairing.

[0050] Taking recombinant AAV vectors as an example, the dual recombinant virus vector system of the present invention has shown advantageous application effects. In the conventional design of antibody delivery using rAAV vectors, heavy and light chain ORFs are packaged in the same rAAV vector. Due to the size limitation of gene packaging of rAAV, it is difficult for rAAV to deliver antibodies (e.g., bispecific antibodies) that exceed the rAAV packaging capacity. In addition, to obtain the expression of single light and heavy chains, a self-cleaving peptide (e.g., P2A) or a furin protease cleavage site is inserted between the light and heavy chains, or two promoters are used to drive the expression of the light and heavy chains, respectively. However, it has been found that such single vector designs cause low-level expression of antibodies in cells and generate large amounts of mispairing products. Low-level expression means that high doses of rAAV need to be administered to obtain effective antibody concentrations in practical applications, and the mispairing products are prone to forming undesirable aggregates. In the drug field, the two issues are related to safety and toxicity and are of great concern.

[0051] The present invention provides a strategy for delivering antibodies that are effectively expressed in vivo by dual rAAV vector-mediated gene delivery, where one rAAV vector contains a complete expression cassette including an antibody light chain coding sequence, and the other rAAV vector contains a complete expression cassette including an antibody heavy chain coding sequence, and when cells are simultaneously transduced with the two vectors, the light and heavy chains expressed by the transduced cells pair and assemble to form a full-length and functional antibody, with enhanced antibody expression levels. By adjusting the ratio of light chain vector (LC vector) to heavy chain vector (HC vector), the antibody expression level can be further improved, more effective antibody assembly can be achieved, and there is a higher accuracy rate of heavy and light chain pairing. When expressing antibodies, especially bispecific and even multispecific antibodies, with the dual rAAV vector system of the present invention, there are excellent effects such as effective antibody expression, stable presence in host cells, sustained antibody expression, high cell transformation efficiency, high antibody expression level, and / or high accuracy rate of antibody heavy and light chain assembly. The dual rAAV vector system of the present invention is particularly suitable for delivering antibodies to a subject in vivo, and can achieve the purpose of gene therapy, which can effectively express antibodies in vivo (e.g., in the body of a subject, including in cells, tissues, or organs in the body of the subject), can stably exist in vivo, can persistently express antibodies, does not require the coding sequence of the antibody to be integrated into the genome of cells in the body of the subject, and has high safety. The dual rAAV vector system of the present invention is particularly suitable for delivering antibodies to non-dividing cells, for example, delivering antibodies to the liver, eye, muscle, or cerebrospinal fluid to effectively express functional antibodies in the cells of these tissues or body fluids.

[0052] Similarly, when a recombinant viral vector, such as a recombinant adenoviral vector, a recombinant herpes simplex viral vector, a recombinant lentiviral vector, or a recombinant retroviral vector, is used to deliver each of the two chains of an antibody component using two recombinant viral vectors, it also has the excellent effects of high cell transformation efficiency, high antibody expression level, and / or high accuracy of assembly of the antibody heavy and light chains.

[0053] Unless otherwise indicated, the terms used in the present invention have the meanings generally understood in the art and can be understood by reference to standard textbooks, references and literature known to those skilled in the art. The definitions and interpretations given in this specification are intended to clarify their specific application within the scope of the present invention. All references cited in this specification are incorporated herein by reference in their entirety.

[0054] Without wishing to be bound by any theory, the explanations of the relevant theories or mechanisms in the present invention are merely intended to aid in the understanding of the invention and should not be considered as limiting the aspects protected by the present invention.

[0055] As used herein, the term "comprise", or other forms of terms having similar meanings such as "comprise", "contain", or "have", should be understood to include the recited elements but not exclude the presence of other elements, including cases where the term consists only of the recited elements. The term "consisting of" means consisting only of the recited elements. The term "consisting essentially of" means not excluding elements that do not significantly affect the relevant aspect.

[0056] Unless otherwise specified, the terms "a," "an," or "the" herein include the singular and the plural. The terms "at least one" or "at least one" or other similar expressions are equivalent to "one or more" or "one or more."

[0057] Any numerical range described herein, such as a temperature range, time range, composition or concentration range, or other numerical range, includes the end values, all intermediate ranges, subranges (e.g., a range between an intermediate value and an end value), and all single numerical values, particularly intermediate ranges, subranges, and single integer numerical values, with integer numerical values ​​as end values, and any intermediate ranges, subranges, and all single numerical values ​​described in the numerical range may be excluded from the numerical range.

[0058] As used herein, "about" and "approximately" mean a range of ±20%, ±18%, ±15%, ±12%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5% of the numerical value, including the endpoints of the range and any single numerical value within the range.

[0059] The term "and / or" as used herein should be understood as referring to any one element or any combination of several elements connected by the term.

[0060] In the present invention, the terms "nucleic acid sequence" and "nucleotide sequence" are used interchangeably and refer to a sequence made up of a linkage of bases, sugars and a phosphate backbone. The nucleic acid sequence of the present invention may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), may contain natural or non-natural bases, it may be single-stranded or double-stranded, and may be a coding or non-coding sequence.

[0061] In the present invention, unless otherwise specified, a nucleic acid sequence is usually described from the 5'-terminus to the 3'-terminus, and an amino acid sequence is usually described from the N-terminus to the C-terminus.

[0062] The present invention uses two recombinant viral vectors to express two different peptide chains constituting a polypeptide molecule, the polypeptide molecule being composed of two different peptide chains and having antigen-binding activity, and the two different peptide chains can be assembled into a polypeptide molecule having antigen-binding activity after expression in a cell. Therefore, the present invention provides a dual recombinant viral vector system for expressing the polypeptide molecule, a dual plasmid system for producing the dual recombinant viral vector system, a host cell comprising the dual recombinant viral vector system, a pharmaceutical composition, and a use and method of the dual recombinant viral vector system for delivering the polypeptide molecule.

[0063] Polypeptide molecules The dual recombinant viral vector system of the present invention is suitable for expressing a polypeptide molecule that is composed of two different peptide chains and has antigen-binding activity.

[0064] The term "antigen-binding activity" means that the polypeptide molecule can specifically bind to an antigen epitope via an antigen-binding site (e.g., a heavy chain variable region VH and / or a light chain variable region VL) contained therein, and can induce an immune response. The "polypeptide molecule composed of two different peptide chains and having antigen-binding activity" according to the present invention may be an antibody, or an antibody fusion protein comprising an antibody and a second binding domain. The two different peptide chains constituting the polypeptide molecule have different amino acid sequences, and are referred to as a first chain and a second chain, respectively, in the present invention. The polypeptide molecule being composed of two different peptide chains means that the polypeptide molecule is composed of one or more first chains and one or more second chains; for example, the polypeptide molecule may be composed of one first chain and one second chain, or may be a tetramer composed of two first chains and two second chains, where the first chain is expressed by one recombinant viral vector and the second chain is expressed by another recombinant vector, and the first chain and the second chain can be assembled into a polypeptide molecule having antigen-binding activity after expression in a cell. The "assembly" includes the interaction of the first chain and the second chain to form a polypeptide molecule having antigen-binding activity, the interaction including covalent (e.g., disulfide) mutual linkage, e.g., the linkage between the CH1, CH2 and / or CH3 of the two heavy chain constant regions via a disulfide bond, and / or non-covalent (e.g., hydrogen bond, van der Waals forces, charge effects or hydrophobic effects) interaction, e.g., pairing between the heavy chain variable region and the light chain variable region.

[0065] In the present invention, the terms "first chain" and "second chain" are used simply to distinguish between two different peptide chains constituting the polypeptide molecule, and do not imply that the first chain or the second chain comprises a specific amino acid sequence or has a specific meaning. In the present invention, when describing specific embodiments, for the sake of clarity, the first chain is described as comprising a first sequence and the second chain is described as comprising a second sequence, but as will be understood by those skilled in the art, the two are interchangeable, i.e., the first chain may comprise the second sequence and the second chain may comprise the first sequence.

[0066] The term "antibody", which may also be referred to as "antigen-binding portion" in the present invention, refers to a polypeptide that comprises an antigen-binding site and is capable of specifically binding to and immunoreacting with an antigen epitope. The term "antibody" includes polypeptides having a heavy chain variable region (VH) and a light chain variable region (VL), and further includes polypeptides comprising only a heavy chain variable region VH, e.g., heavy chain antibodies or single domain antibodies (sdAbs). The term "antibody" refers to a monoclonal antibody or an antigen-binding fragment thereof (e.g., Fab, Fab', F(ab')). 2The term "antibody" includes, but is not limited to, monospecific antibodies, i.e., antibodies that specifically bind to a single antigen epitope, and multispecific antibodies, i.e., antibodies that specifically bind to multiple different antigen epitopes (e.g., bispecific, trispecific, etc.). In some embodiments, the "antibody" of the present invention is composed of two different chains, and the two chains are linked to each other and organized as an antibody having antigen-binding activity. The term "single-chain antibody" refers to an antibody composed of a single chain, and the single chain contained in the single-chain antibody contains at least a heavy chain variable region and a light chain variable region derived from an IgG immunoglobulin, and may be, for example, an scFv, and further contains at least a heavy chain variable region, and may be, for example, a single domain antibody or a heavy chain antibody (i.e., containing a heavy chain variable region and a heavy chain constant region). In the present invention, the term "specifically bind" or "targeting" refers to the selective recognition of a specific epitope of an antigen by an antigen-binding polypeptide, i.e., an antibody or antigen-binding portion binds to the antigen or to the specific epitope with greater affinity or longer duration than to other epitopes of a different antigen or the same antigen. The binding ability of an antibody to an antigen epitope can be known, for example, by measuring the dissociation constant by radioimmunoassay (RIA).

[0067] The term "antigen" has the meaning commonly understood by those skilled in the art and includes polypeptides, polysaccharides, glycoproteins, polynucleotides, etc. The term "antigenic epitope", which may also be referred to as an "antigenic determinant cluster", refers to a chemical functional group on the surface of an antigen that determines antigen specificity. An antigenic epitope can be recognized and bound by an antigen-binding site in an antibody.

[0068] The term "monoclonal antibody" refers to a homogeneous antibody directed only to a certain antigen epitope. Compared to typical and common polyclonal antibody preparations that contain different antibodies directed to different antigenic determinant clusters (epitopes), each monoclonal antibody is directed to a single antigenic determinant cluster on an antigen. Although monoclonal antibodies can be obtained by hybridoma culture, in the present invention, the modifier "monoclonal" should not be understood as referring to the homogeneous character of the antibody and the antibody produced by any particular method. The monoclonal antibodies of the present invention can be produced by recombinant DNA methods or obtained by other screening methods.

[0069] A "monoclonal antibody" according to the present invention may be referred to as a full length antibody or complete antibody, i.e. an immunoglobulin molecule (e.g., IgG) or multimer thereof (e.g., IgA or IgM) composed of four polypeptide chains. The four polypeptide chains comprise two identical heavy chains (H) and two identical light chains (L), which are linked together via disulfide bonds to form a tetramer. Each heavy chain is composed of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (composed of domains CH1, CH2 and CH3). Each light chain is composed of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions may be further subdivided into hypervariable regions called complementarity determining regions (CDRs), between which more conservative regions called framework regions (FRs) are inserted. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the hydroxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Depending on the amino acid sequence of the heavy chain constant region, immunoglobulin molecules can be classified into different classes, and there are five major types of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further classified into subclasses, for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The monoclonal antibodies of the present invention include immunoglobulin molecules of any of the above classes or subclasses.

[0070] An "antigen-binding fragment" according to the present invention means one or more portions of a complete antibody, which retain the ability to specifically interact with an antigen epitope. Examples of antigen-binding fragments include: (1) Fab fragment: an antigen-binding fragment produced by digesting a complete antibody with papain, which is composed of the complete light chain (including the VL domain and the CL domain) of a full-length antibody and the heavy chain variable region VH and heavy chain constant region domain CH1 of a full-length antibody, without the hinge chain region; (2) F(ab') 2 Fragment: An antigen-binding fragment produced by digesting a complete antibody with pepsin, which includes the complete light chains (including the VL and CL domains) of two full-length antibodies, the heavy chain variable regions VH and the heavy chain constant region domain CH1 and hinge chain region of two full-length antibodies, and may be considered as a fragment formed by pairing two Fab' fragments having a disulfide bond therebetween, (3) a Fab' fragment, (4) an Fd fragment consisting of a VH domain and a CH1 domain, (5) an Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody, and (6) a single domain antibody sdAb consisting of a VHH domain. Note that the term "antigen-binding fragment" of the present invention further includes antibodies in the form of a single chain, for example, scFv (single-chain Fv formed by linking the VL domain and the VH domain via a linker) and dsFv (single-chain antibody formed by introducing an interchain disulfide bond between the VH and VL of scFv). An "antigen-binding fragment" typically contains at least one or both of the heavy chain variable region and the light chain variable region of a complete antibody, and may further contain a portion of the light chain constant region and / or the heavy chain constant region of the complete antibody, for example, one or two or more of the CL domain, CH1 domain, CH2 domain, and CH3 domain.

[0071] "Single domain antibody (sdAb)" according to the present invention refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). These single domain antibodies can bind to antigens alone without pairing with a corresponding polypeptide that contains CDRs. Common single domain antibodies contain heavy chains but lack the light chains normally found in antibodies, e.g., camelid sdAbs (see, e.g., Hamers-Casterman et al., Nature 363:446-8(1993); Greenberg et al., Nature 374:168-73(1995); Hassanzadeh-Ghassabeh et al., Nanomedicine(Lond),8:1013-26(2013)). A single domain antibody can be obtained from camel heavy chain antibody by artificial engineering and is called VHH, which has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0072] According to the present invention, a "single chain antibody (scFv)" refers to a fusion protein of the heavy and light chain variable regions of an immunoglobulin linked via a peptide linker. The N-terminus of the heavy chain variable region may be linked to the C-terminus of the light chain variable region via a peptide linker, or the N-terminus of the light chain variable region may be linked to the C-terminus of the heavy chain variable region via a peptide linker.

[0073] A "multispecific antibody" according to the present invention means an antibody capable of specifically binding to multiple antigen epitopes. The multiple antigen epitopes may be different epitopes in the same antigen or different epitopes in different antigens. A multispecific antibody comprises multiple antigen-binding moieties, each of which targets one antigen epitope, and the antigen epitopes targeted by the multiple antigen-binding moieties are different from each other. Each antigen-binding moiety may be a monoclonal antibody (e.g., an IgG immunoglobulin), a Fab fragment, a Fab' fragment, a F(ab') fragment, a Fab' ... 2The multispecific antibody may be independently selected from a heavy chain variable region, a light ...

[0074] In some preferred embodiments, the multispecific antibody is a bispecific antibody comprising a first antigen-binding portion that specifically binds to a first antigen epitope and a second antigen-binding portion that specifically binds to a second antigen epitope, the first and second antigen epitopes being different. Bispecific antibodies have various forms and may or may not comprise an Fc region, and in bispecific antibodies that comprise an Fc region, the structure may be symmetric or asymmetric. In some preferred embodiments, the bispecific antibody of the present invention comprises an Fc region. In some preferred embodiments, the bispecific antibody of the present invention is symmetric and comprises two identical first chains and two identical second chains. In some embodiments, the bispecific antibody of the present invention may further comprise one first chain and one second chain. Depending on the difference in the bispecific antibody structure, the first and second chains of the bispecific antibody may each comprise a heavy chain variable region and / or a light chain variable region that targets one antigen epitope or two different antigen epitopes. In some embodiments, the bispecific antibody of the invention is symmetric, the first chain of which comprises a heavy chain that targets a first antigenic epitope and the second chain of which comprises a light chain that targets a second antigenic epitope, hi some embodiments, the bispecific antibody may be an anti-VEGF and Ang2 bispecific antibody.

[0075] An "antibody fusion protein" according to the present invention refers to a fusion protein having various biological functions, obtained by fusing an antibody (which may be referred to as the first antigen-binding portion in the antibody fusion protein) with another polypeptide having biological functions (a second binding domain). The "other polypeptide having biological functions" according to the present invention may be linked to the heavy chain variable region VH, the light chain variable region VL, the heavy chain constant region CH1, CH2, CH3, and / or the light chain constant region CL of the antibody. The "other polypeptide having biological functions" according to the present invention may be another antibody, a toxin, an enzyme, a cytokine, a receptor or a fragment thereof that targets a different antigen epitope, etc. When the antibody is fused to another antibody that targets a different antigen epitope, the antibody fusion protein is a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the second binding domain may be a receptor or a fragment thereof (e.g., an extracellular domain thereof) that specifically binds to its ligand, or a variant thereof. In some embodiments, the ligand of the receptor and the antigen targeted by the first antigen-binding portion are different proteins. In some embodiments, the ligand is a cytokine or a variant thereof, and the cytokine may be, for example, transforming growth factor beta (TGFβ), vascular endothelial growth factor receptor (VEGF), or angiotensin 2 (Ang2). In some embodiments, the second binding domain may be a receptor that specifically binds to these cytokines or variants thereof, such as a transforming growth factor beta (TGFβ) receptor (e.g., TGF-βRII), a vascular endothelial growth factor receptor (VEGF) receptor, or angiotensin 2 (Ang2) receptor, or may even be a fragment of the receptor, e.g., a fragment capable of binding to the cytokine, e.g., the extracellular domain of the receptor or a variant thereof. The cytokine and cytokine receptor may be humanized.In some embodiments, the other polypeptide is the extracellular domain of TGFβRII or a variant thereof, such as the extracellular domain of TGFβRII (see, for example, SEQ ID NO:13 of patent application WO2018 / 205985), the soluble extracellular domain of TGFβRII (see, for example, SEQ ID NO:10 of patent application WO2018 / 129331), or an N-terminal truncation of the extracellular domain of TGF-βRII (see, for example, SEQ ID NO:14 or SEQ ID NO:15 of patent application WO2018 / 205985). The antibody fusion protein formed by fusing an antibody to a cytokine receptor or a fragment thereof can achieve a synergistic therapeutic effect by blocking the interaction between the cytokine receptor and the cytokine while the antibody exerts a therapeutic effect (e.g., an antitumor effect). For example, a receptor of TGFβ, such as TGFβRII or its extracellular domain, can block the neutralizing effect of TGFβ in the tumor microenvironment and achieve a synergistic anti-tumor effect with an antibody, and a receptor or extracellular domain of VEGF or Ang2 can block angiogenesis by blocking the binding of VEGF or Ang2 to its receptor in the environment and achieve a synergistic anti-tumor effect with an antibody. In a preferred embodiment, the first antigen-binding moiety is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-VEGF antibody, or an anti-Ang2 antibody.

[0076] In some embodiments, the polypeptide molecule of the invention comprises an antibody or antigen-binding portion having a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region of the antibody or antigen-binding portion being located on two peptide chains, respectively, in which the first chain and the second chain of the polypeptide molecule comprise the heavy chain variable region and the light chain variable region of the antibody or antigen-binding portion, respectively, e.g., the first chain of the polypeptide molecule comprises the heavy chain variable region of the antibody or antigen-binding portion, and the second chain comprises the light chain variable region of the antibody or antigen-binding portion. The dual recombinant viral vector system of the invention can facilitate the correct pairing of the heavy chain variable region and the light chain variable region to form an active antigen-binding portion. Examples of such antibodies or antigen-binding portions include monoclonal antibodies or their Fab fragments, Fab' fragments, F(ab') fragments, and the like. 2 Examples of the polypeptide molecule expressed by the dual recombinant viral vector system of the present invention include an antibody or an antigen-binding portion having a heavy chain variable region and a light chain variable region, which are located on two peptide chains, a multispecific antibody composed of such an antibody or antigen-binding portion and another antibody or antigen-binding portion, or an antibody fusion protein composed of such an antibody or antigen-binding portion and another protein having a biological function. Furthermore, when another polypeptide having a biological function is further fused to the first chain containing the heavy chain variable region and / or the second chain containing the light chain variable region and expressed by the dual recombinant viral vector system of the present invention, the exact pairing of the heavy chain variable region and the light chain variable region can still be promoted to form an antigen-binding portion having an activity and generate another polypeptide having a biological activity. Therefore, the polypeptide molecule expressed by the dual recombinant viral vector system of the present invention may be an antibody or an antigen-binding portion having a heavy chain variable region and a light chain variable region located on two peptide chains, respectively, or may be a multispecific antibody composed of such an antibody or antigen-binding portion and another antibody or antigen-binding portion, or may be an antibody fusion protein composed of such an antibody or antigen-binding portion and another protein having a biological function.

[0077] Furthermore, the first chain of the polypeptide molecule, other than comprising the heavy chain variable region of the antibody or antigen-binding portion, may further comprise a full length or a part thereof of a heavy chain constant region CH, for example, any one, any two or all of the CH1 domain, CH2 domain and CH3 domain, and the second chain, other than comprising the light chain variable region of the antibody or antigen-binding portion, may further comprise a light chain constant region CL, whereby the first chain and the second chain form an antigen-binding portion having activity due to the heavy chain variable region, heavy chain constant region, light chain variable region and light chain constant region after expression in a cell. Thus, in a preferred embodiment, the polypeptide molecule of the present invention comprises an antibody or antigen-binding portion having a heavy chain and a light chain, in which the first chain and the second chain of the polypeptide molecule comprise the heavy chain and the light chain of the antibody or antigen-binding portion, respectively, e.g., the first chain of the polypeptide molecule comprises the heavy chain of the antibody or antigen-binding portion, and the second chain comprises the light chain of the antibody or antigen-binding portion. Examples of such antibodies or antigen-binding portions include monoclonal antibodies or antigen-binding fragments thereof comprising a heavy chain and a light chain, such as a Fab fragment, a Fab' fragment, a F(ab') 2 The present inventors have discovered that by expressing the heavy and light chains of an antibody in a dual recombinant viral vector system, respectively, it is possible to increase the expression level of the antibody and the assembly efficiency of the heavy and light chains, and that when the proportion of the expressed light chain is high, the expression level of the antibody and the assembly efficiency of the heavy and light chains are also high.

[0078] In some embodiments, the antibody or antigen binding portion comprised in the polypeptide molecule of the invention is an antibody that specifically binds VEGF or an antibody that specifically binds Ang2. In some embodiments, the antibody that specifically binds VEGF is Avastin. In some embodiments, the heavy chain of the antibody that specifically binds VEGF has an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the light chain of the antibody that specifically binds VEGF has an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the antibody that specifically binds Ang2 is ABTAA. The heavy chain of the antibody that specifically binds ABTAA has an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the light chain of the antibody that specifically binds ABTAA has an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:10.

[0079] In the present invention, the term "heavy chain" refers to a chain comprising a heavy chain variable region in a full-length antibody or an antigen-binding fragment thereof comprising at least two chains, and the term "light chain" refers to a chain comprising a light chain variable region in a full-length antibody or an antigen-binding fragment thereof comprising at least two chains. Thus, the term "heavy chain" according to the present invention refers to a chain comprising a heavy chain variable region and at least a part of a heavy chain constant region, for example, a chain comprising a heavy chain variable region VH and all three heavy chain constant regions CH1, CH2 and CH3, or a chain comprising a heavy chain variable region VH and a part of a heavy chain constant region (for example, any one or any two of CH1, CH2 and CH3). The term "light chain" according to the present invention refers to a chain comprising a light chain variable region and at least a part of a light chain constant region, for example, a chain comprising a light chain variable region VL and a light chain constant region CL.

[0080] Furthermore, the first chain comprising the heavy chain and / or the second chain comprising the light chain can be further fused with another polypeptide having biological function (i.e., a second binding domain) to generate another polypeptide having biological activity while still having high expression and high assembly efficiency of the heavy and light chains. Thus, the polypeptide molecule expressed by the dual recombinant viral vector system of the present invention may be composed of an antibody or antigen-binding portion having a heavy chain and a light chain, or may be a multispecific antibody composed of such an antibody or antigen-binding portion and another antibody or antigen-binding portion, or may be an antibody fusion protein composed of such an antibody or antigen-binding portion and another protein having biological function.

[0081] In some preferred embodiments, the ratio of the first chain comprising the antibody heavy chain to the second chain comprising the antibody light chain expressed by the dual recombinant viral vector system of the present invention may be 4:1 to 1:8, for example, 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4 or 1:2 to 1:4, for example, 1:1, 1:2, 1:3 or 1:4, most preferably 1:2 to 1:4. The ratio of the first chain to the second chain may be determined by the amount of the viral genome of the recombinant viral vector comprising the coding sequences of the first chain and the second chain, and the copy number of the coding sequences of the first chain and the second chain contained in the first recombinant viral vector and the second recombinant viral vector. In some embodiments, the coding sequence of the first chain in the first recombinant viral vector system is a single copy. In some embodiments, the coding sequence of the second chain in the second recombinant viral vector system is a single copy. In some embodiments, the coding sequences of functional protein domains (e.g., antibody heavy chain variable region, light chain variable region, heavy chain, light chain, and / or other polypeptides having biological functions) contained in the coding sequences of the first chain and the second chain are also single copies. In some embodiments, the ratio of the first chain to the second chain can be represented by the ratio of the first recombinant viral vector to the second recombinant viral vector. In some embodiments, the ratio of the first recombinant viral vector for expressing the first chain comprising the antibody heavy chain to the second recombinant viral vector for expressing the second chain comprising the antibody light chain can be 4:1 to 1:8, for example, 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4, or 1:2 to 1:4, for example, 1:1, 1:2, 1:3, or 1:4, and is most preferably 1:2 to 1:4. The ratio of the recombinant viral vector can be calculated based on the amount of the viral genome, for example, the viral genome titer. Measurement of the amount of viral genome or viral genome titer is well known to those skilled in the art and can be measured, for example, by PCR such as droplet digital PCR (ddPCR).

[0082] In some preferred embodiments, the polypeptide molecules expressed by the dual recombinant viral vector systems of the present invention are monoclonal antibodies or antigen-binding fragments thereof comprising a heavy chain and a light chain, such as a Fab fragment, a Fab' fragment, a F(ab') fragment, or a Fab' antibody. 2 and the like. The first chain comprises a heavy chain of the monoclonal antibody or antigen-binding fragment, and the second chain comprises a light chain of the monoclonal antibody or antigen-binding fragment. More preferably, the ratio of the first chain to the second chain may be 4:1 to 1:8, for example, 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4 or 1:2 to 1:4, for example, 1:1, 1:2, 1:3 or 1:4, and most preferably 1:2 to 1:4. In some embodiments, the ratio of the first recombinant viral vector for expressing the first strand to the second recombinant viral vector for expressing the second strand may be 4:1 to 1:8, such as 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4 or 1:2 to 1:4, such as 1:1, 1:2, 1:3 or 1:4, and most preferably 1:2 to 1:4.

[0083] In another preferred embodiment, the polypeptide molecule expressed by the dual recombinant viral vector system of the present invention is a bispecific antibody, which comprises a first antigen-binding moiety that specifically binds to a first antigenic epitope and a second antigen-binding moiety that specifically binds to a second antigenic epitope, which are distinct from each other, wherein the first antigen-binding moiety comprises an antibody heavy chain and an antibody light chain, e.g., a full-length antibody (e.g., an IgG immunoglobulin) or an antigen-binding fragment thereof comprising a heavy chain and a light chain, e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fab' ... 2The first chain comprises the heavy chain of the first antigen-binding portion, and the second chain comprises the light chain of the first antigen-binding portion. Any bispecific antibody form having different structures formed thereon can be applied to the present invention, for example, the second antigen-binding portion may be in any single-chain or two-chain form, including, but not limited to, Fab fragments, Fv fragments, scFv or single domain antibodies that specifically bind to the second antigen epitope. When the second antigen-binding portion is an antibody in a single-chain form, the second antigen-binding portion may be linked to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain or the C-terminus of the light chain of the first antigen-binding portion, or may be linked between the heavy chain variable region VH and the heavy chain constant region CH1 of the first antigen-binding portion. When the second antigen-binding portion is an antibody in a two-chain form, the two chains of the second antigen-binding portion may be linked to the N-terminus or C-terminus of the two chains of the first antigen-binding portion, respectively. When the second antigen-binding moiety is a Fab, the "light chain variable region-light chain constant region (VL-CL)" in the second antigen-binding moiety can be linked to the heavy chain N-terminus of the first antigen-binding moiety, and the "heavy chain variable region-heavy chain constant region CH1 (VH-CH1)" in the second antigen-binding moiety can be linked to the light chain N-terminus of the first antigen-binding moiety. When the second antigen-binding moiety is an Fv fragment, the heavy chain variable region of the second antigen-binding moiety can be linked to the heavy chain N-terminus of the first antigen-binding moiety, and the light chain variable region of the second antigen-binding moiety can be linked to the light chain N-terminus of the first antigen-binding moiety.

[0084] In some embodiments, a bispecific antibody is composed of a Fab fragment that specifically binds to a first antigen epitope and two identical scFvs that specifically bind to a second antigen epitope, and the two scFvs are linked to the N-terminus of the heavy and light chains of the Fab fragment, respectively. In such embodiments, the first chain comprises the heavy chain of the Fab fragment that specifically binds to the first antigen epitope and the scFv that specifically binds to the second antigen epitope, and the second chain comprises the light chain of the Fab fragment that specifically binds to the first antigen epitope and the scFv that specifically binds to the second antigen epitope.

[0085] In some other embodiments, the bispecific antibody is composed of an IgG monoclonal antibody that specifically binds to a first antigen epitope and a Fab fragment that specifically binds to a second antigen epitope, the heavy chain of the Fab fragment is linked to the N-terminus of the light chain of the IgG monoclonal antibody, and the light chain of the Fab fragment is linked to the N-terminus of the heavy chain of the IgG monoclonal antibody. In such embodiments, the first chain comprises the heavy chain of the IgG monoclonal antibody that specifically binds to the first antigen epitope and the light chain of the Fab fragment that specifically binds to the second antigen epitope, and the second chain comprises the light chain of the IgG monoclonal antibody that specifically binds to the first antigen epitope and the heavy chain of the Fab fragment that specifically binds to the second antigen epitope.

[0086] In some alternative embodiments, the bispecific antibody is composed of an IgG monoclonal antibody that specifically binds to a first antigenic epitope and a single domain antibody that specifically binds to a second antigenic epitope, where the single domain antibody is linked to the N-terminus or C-terminus of the light chain of the IgG monoclonal antibody or to the N-terminus or C-terminus of the heavy chain of the IgG monoclonal antibody, preferably the single domain antibody is linked on the heavy chain of the IgG monoclonal antibody (e.g., at the N-terminus or C-terminus of the heavy chain). In this embodiment, the first chain comprises a heavy chain of an IgG monoclonal antibody that specifically binds to a first antigen epitope and a single domain antibody that specifically binds to a second antigen epitope, and the second chain comprises a light chain of an IgG monoclonal antibody that specifically binds to the first antigen epitope, or the first chain comprises a heavy chain of an IgG monoclonal antibody that specifically binds to the first antigen epitope and the second chain comprises a light chain of an IgG monoclonal antibody that specifically binds to the first antigen epitope and a single domain antibody that specifically binds to the second antigen epitope.

[0087] In some alternative embodiments, the bispecific antibody is composed of an IgG monoclonal antibody that specifically binds to a first antigenic epitope and an scFv that specifically binds to a second antigenic epitope, where the scFv is linked to the N-terminus or C-terminus of the light chain of the IgG monoclonal antibody or to the N-terminus or C-terminus of the heavy chain of the IgG monoclonal antibody, preferably the scFv is linked to the C-terminus of the heavy chain of the IgG monoclonal antibody. In this embodiment, the first chain comprises a heavy chain of an IgG monoclonal antibody that specifically binds to a first antigen epitope and an scFv that specifically binds to a second antigen epitope, and the second chain comprises a light chain of an IgG monoclonal antibody that specifically binds to the first antigen epitope, or the first chain comprises a heavy chain of an IgG monoclonal antibody that specifically binds to the first antigen epitope, and the second chain comprises a light chain of an IgG monoclonal antibody that specifically binds to the first antigen epitope and an scFv that specifically binds to the second antigen epitope.

[0088] In some other embodiments, the bispecific antibody is composed of a Fab fragment that specifically binds to a first antigen epitope, an scFv that specifically binds to a second antigen epitope, and an Fc fragment, where the N-terminus of the scFv is linked to the C-terminus of the heavy chain of the Fab fragment, and the C-terminus of the scFv is linked to the N-terminus of the Fc fragment, and such a type of bispecific antibody may be considered to be obtained by inserting an scFv that specifically binds to a second antigen epitope between the heavy chain constant regions CH1 and CH2 of an IgG monoclonal antibody that specifically binds to the first antigen epitope. In such embodiments, the first chain comprises the heavy chain of the Fab fragment that specifically binds to the first antigen epitope, the scFv that specifically binds to the second antigen epitope, and the Fc fragment, and the second chain comprises the light chain of the Fab fragment that specifically binds to the first antigen epitope.

[0089] More preferably, in a dual recombinant viral vector system for expressing a bispecific antibody, the ratio of the first chain to the second chain may be 4:1 to 1:8, for example, 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4 or 1:2 to 1:4, for example, 1:1, 1:2, 1:3 or 1:4, and most preferably 1:2 to 1:4. In some embodiments, the ratio of the first recombinant viral vector for expressing the first chain to the second recombinant viral vector for expressing the second chain may be 4:1 to 1:8, for example, 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4 or 1:2 to 1:4, for example, 1:1, 1:2, 1:3 or 1:4, and most preferably 1:2 to 1:4.

[0090] In another preferred embodiment, the polypeptide molecule expressed by the dual recombinant viral vector system of the present invention may further be an antibody fusion protein comprising a first antigen-binding moiety that specifically binds to a first target molecule and a second binding domain that specifically binds to a second target molecule, wherein the first target molecule is an antigen or antigen epitope, and the first antigen-binding moiety is a full-length antibody (e.g., an IgG immunoglobulin) or an antigen-binding fragment thereof comprising a heavy chain and a light chain, e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a Fab' fragment. 2 The first chain comprises a heavy chain of the first antigen-binding moiety and the second chain comprises a light chain of the first antigen-binding moiety. The second target molecule is a cytokine or a variant thereof and the second binding domain is a cytokine receptor or an extracellular binding domain thereof, or a truncated or variant thereof. The second target molecule and the first target molecule are different. The second binding domain can be linked on the heavy chain (e.g., at its N-terminus and / or C-terminus) and / or on the light chain (e.g., at its N-terminus and / or C-terminus) of the first antigen-binding moiety.

[0091] The term "variant" refers to a polynucleotide (or polypeptide) that has sequence similarity to a parent nucleic acid (or polypeptide) and can retain the activity of the polynucleotide (or polypeptide). For example, a nucleic acid variant may have one or more nucleotide insertions, substitutions, and / or deletions at the 5'-terminus, 3'-terminus, and / or one or more internal sites compared to the parent nucleic acid. A variant may further have at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity with the parent nucleic acid. A polypeptide variant may have one or more amino acid insertions, substitutions, and / or deletions at the 5'-terminus, 3'-terminus, and / or one or more internal sites compared to the parent polypeptide. A variant may further have at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the parent polypeptide. Sequence identity may be determined by sequence alignment programs known to those of skill in the art, including, but not limited to, BLAST, FASTA, or Needleman.

[0092] In some preferred embodiments, the second target molecule is a cytokine, such as transforming growth factor β, vascular endothelial growth factor (VEGF), or angiotensin (Ang2). In some embodiments, the second binding domain is a cytokine receptor, such as a transforming growth factor β (TGFβ) receptor (e.g., TGF-βRII), a vascular endothelial growth factor receptor (VEGF) receptor, or angiotensin (Ang2) receptor, or a fragment thereof (e.g., an extracellular domain ECD) or a variant thereof. The cytokine and cytokine receptor may be humanized. In some embodiments, the second binding domain is the extracellular domain of TGFβRII or a variant thereof, such as the extracellular domain of TGFβRII (see, for example, SEQ ID NO:13 of patent application WO2018 / 205985), the soluble extracellular domain of TGFβRII (see, for example, SEQ ID NO:10 of patent application WO2018 / 129331), or an N-terminal truncation of the extracellular domain of TGF-βRII (see, for example, SEQ ID NO:14 or SEQ ID NO:15 of patent application WO2018 / 205985). In a preferred embodiment, the first antigen-binding moiety is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-VEGF antibody, or an anti-Ang2 antibody.

[0093] More preferably, in the dual recombinant viral vector system for expressing the antibody fusion protein, the ratio of the first chain to the second chain may be 4:1 to 1:8, for example, 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4 or 1:2 to 1:4, for example, 1:1, 1:2, 1:3 or 1:4, and most preferably 1:2 to 1:4. In some embodiments, the ratio of the first recombinant viral vector for expressing the first chain to the second recombinant viral vector for expressing the second chain may be 4:1 to 1:8, for example, 4:1 to 1:6, 2:1 to 1:6, 2:1 to 1:4, 1:1 to 1:4 or 1:2 to 1:4, for example, 1:1, 1:2, 1:3 or 1:4, and most preferably 1:2 to 1:4.

[0094] The antibodies or antigen-binding portions (e.g., monoclonal antibodies or antigen-binding fragments thereof, first and / or second antigen-binding portions in bispecific antibodies, and first antigen-binding portions in antibody fusion proteins) expressed by the dual recombinant viral vector system of the present invention may be antibodies or antigen-binding portions that specifically bind to any antigen, including, but not limited to, microbial antigens, tumor-specific antigens, cytokines, angiogenesis-related target points, immune checkpoints, and the like. In some particularly preferred embodiments, the antibodies or antigen-binding portions specifically bind to angiogenesis-related target points. The angiogenesis-related target points are angiogenic factors, i.e., factors that stimulate blood vessel development, e.g., promoting blood vessel generation, endothelial cell proliferation, and / or vascular stability. The angiogenic factors can be treated, e.g., as therapeutic target points for diseases associated with angiogenesis, using inhibitors (e.g., antibodies) against these angiogenic factors. The angiogenic factors include, but are not limited to, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGFβ), angiotensin 1 (Ang1), angiotensin 2 (Ang2), integrin, CD31, and plasminogen activators.In some embodiments, the antibody or antigen-binding portion of the invention is an antibody against an angiogenic factor, the angiogenic factor being one, two or more selected from vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGFβ), angiotensin 1 (Ang1), angiotensin 2 (Ang2), integrin, CD31, and plasminogen activators. As one of skill in the art would know, when it is referred to as an antibody or antigen-binding portion specifically binding to a particular antigen, it is meant that the antibody or antigen-binding portion specifically binds to any epitope on that antigen.

[0095] In some embodiments, the antibody or antigen-binding portion may be an antibody against vascular endothelial growth factor (VEGF) and / or angiotensin 2 (Ang2). In some embodiments, the anti-VEGF antibody is Avastin. In some embodiments, the anti-VEGF antibody has a heavy chain CDR sequence identical to the three heavy chain CDR sequences contained in the heavy chain sequence shown in SEQ ID NO:19 (e.g., which may be encoded by the nucleotide sequence shown in SEQ ID NO:2). In some embodiments, the anti-VEGF antibody has a heavy chain variable region sequence identical to the heavy chain variable region sequence contained in the heavy chain sequence shown in SEQ ID NO:19 (e.g., which may be encoded by the nucleotide sequence shown in SEQ ID NO:2). In some embodiments, the anti-VEGF antibody has a heavy chain sequence shown in SEQ ID NO:19 (e.g., which may be encoded by the nucleotide sequence shown in SEQ ID NO:2). In some embodiments, the anti-VEGF antibody has a light chain CDR sequence identical to the three light chain CDR sequences contained in the light chain sequence shown in SEQ ID NO:20 (e.g., which may be encoded by the nucleotide sequence shown in SEQ ID NO:5). In some embodiments, the anti-VEGF antibody has a light chain variable region sequence identical to the light chain variable region sequence contained in the light chain sequence shown in SEQ ID NO:20 (e.g., which may be encoded by the nucleotide sequence shown in SEQ ID NO:5). In some embodiments, the anti-VEGF antibody has a light chain sequence shown in SEQ ID NO:20 (e.g., which may be encoded by the nucleotide sequence shown in SEQ ID NO:5). In some embodiments, the anti-Ang2 antibody is ABTAA. In some embodiments, the anti-Ang2 antibody has a heavy chain CDR sequence identical to the three heavy chain CDR sequences contained in the heavy chain variable region sequence shown in SEQ ID NO:21, or has a heavy chain CDR sequence identical to the three heavy chain CDR sequences contained in the heavy chain sequence encoded by the nucleotide sequence shown in SEQ ID NO:7.In some embodiments, the anti-Ang2 antibody has a heavy chain variable region sequence set forth in SEQ ID NO:21 or has a heavy chain variable region sequence identical to the heavy chain variable region sequence contained in the heavy chain sequence encoded by the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the anti-Ang2 antibody has a light chain CDR sequence identical to the three light chain CDR sequences contained in the light chain variable region sequence set forth in SEQ ID NO:22 or has a light chain CDR sequence identical to the three light chain CDR sequences contained in the light chain sequence encoded by the nucleotide sequence set forth in SEQ ID NO:10. In some embodiments, the anti-Ang2 antibody has a light chain variable region sequence set forth in SEQ ID NO:22 or has a light chain variable region sequence identical to the light chain variable region sequence contained in the light chain sequence encoded by the nucleotide sequence set forth in SEQ ID NO:10. In some embodiments, the heavy chain of the anti-Ang2 antibody has an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the light chain of the anti-Ang2 antibody has an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:10.

[0096] It should be understood that in the present invention, when referring to an amino acid sequence being linked to the N-terminus of another amino acid sequence, it means that the C-terminus of the former is linked to the N-terminus of the latter, and when referring to an amino acid sequence being linked to the C-terminus of another amino acid sequence, it means that the N-terminus of the former is linked to the C-terminus of the latter. In the present invention, when referring to two peptide chains or two amino acid sequences being linked or connected, it should be understood that they may be directly linked or linked via a peptide linker (also called a linking peptide). The peptide linker may comprise or consist of a flexible glycine and a soluble serine or threonine. The peptide linker may be, for example, a GS linker, and may be, for example, 5 to about 25 amino acids in length. The term "GS linker" refers to the GS combination of glycine (G) and serine (S) for linking multiple proteins together to form a fusion protein. A common GS combination is (GGGGS)n, and the length of the linker sequence can be changed by changing the size of n, for example, (GGGGS) 3 (i.e., GGGGSGGGSGGGGS, SEQ ID NO:16), which may be encoded, for example, by the sequence shown in SEQ ID NO:11, with glycine and serine being further combined to generate different linker sequences, for example, GGGGSGGGS (SEQ ID NO:17). Peptide linkers may also be other sequences, for example, ESKYGPPSPPSP (SEQ ID NO:23), which may be encoded, for example, by the nucleic acid sequence shown in SEQ ID NO:14. Suitable peptide linkers can be determined by one of skill in the art.

[0097] As will be appreciated by those skilled in the art, the first chain and / or the second chain may further comprise a leader sequence (i.e., signal peptide) that allows for secretion of the encoded polypeptide, so that the first chain and / or the second chain are produced in a secreted form from cells transduced with the recombinant viral vector. The signal peptide may be located at the N-terminus of the first chain and / or the second chain. In some embodiments, the polypeptide molecule is a monoclonal antibody or antigen-binding fragment thereof, and the first chain comprises, in order from N-terminus to C-terminus, a leader sequence, a heavy chain of the monoclonal antibody or antigen-binding fragment thereof, and the second chain comprises, in order from N-terminus to C-terminus, a leader sequence, a light chain of the monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the polypeptide molecule is a bispecific antibody, where the first chain comprises, in order from N-terminus to C-terminus, a leader sequence, a heavy chain of the first antigen binding moiety, a connecting peptide, a second antigen moiety in single chain form, and the second chain comprises, in order from N-terminus to C-terminus, a leader sequence, a light chain of the first antigen binding moiety. In some embodiments, the polypeptide molecule is an antibody fusion protein, where the first chain comprises, in order from N-terminus to C-terminus, a leader sequence, a heavy chain of the first antigen binding moiety, a connecting peptide, a second binding domain, and the second chain comprises, in order from N-terminus to C-terminus, a leader sequence, a light chain of the first antigen binding moiety. In some embodiments, the leader sequence may be MGWSCIILFLVATATGVHS (SEQ ID NO:18). In some embodiments, the coding nucleotide sequence of the leader sequence is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:9. Recombinant viral vectors

[0098] The term "vector" according to the present invention means any molecule, such as a nucleic acid, a plasmid or a virus, capable of transporting a heterologous nucleic acid contained therein into a host cell (either in a free form or integrated into the host cell genome).

[0099] "Recombinant" according to the present invention means that the vector contains a heterologous sequence, which may be a nucleic acid of interest, for expressing a target protein, e.g., a polypeptide molecule of the present invention.

[0100] According to the present invention, the terms "heterologous nucleic acid", "heterologous sequence" or "nucleic acid of interest" are used interchangeably and refer to a nucleic acid sequence that is not naturally occurring in the host cell, the vector containing it. The heterologous nucleic acid may be a nucleic acid sequence that encodes a target protein, e.g., encodes a polypeptide molecule of the present invention.

[0101] As used herein, the terms "recombinant viral vector", "recombinant virus" or "recombinant viral particle" are used interchangeably and refer to a recombinant virus or recombinant viral particle that can mediate the transfer of a nucleic acid of interest (e.g., a coding nucleic acid sequence for the first and second strands contained in a polypeptide molecule of the invention) into a cell, thereby allowing expression of a polypeptide product, and typically includes a recombinant viral genome (e.g., packaged within a viral capsid, some viruses further comprising an envelope glycoprotein) packaged within the viral particle, the recombinant viral genome including the nucleic acid of interest. Recombinant viral vectors that can be used to express the polypeptide molecules of the invention can be viral vectors derived from various types of viruses, including, but not limited to, adeno-associated viral (AAV) vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, herpes simplex viral (HSV) vectors, and the like.

[0102] The method of producing a recombinant viral vector varies depending on the type of viral vector used, but all of them are well known to those skilled in the art. In general, the recombinant viral vector can be obtained by co-transfecting a packaging cell line with an expression plasmid containing a recombinant viral genome (usually containing at least a heterologous nucleic acid and cis nucleic acid elements necessary for virus production, such as a terminal repeat sequence, a packaging signal sequence, etc.), a packaging plasmid, and optionally other helper plasmids. The co-transfected packaging cell line can be cultured under appropriate conditions to collect viral particles from the cell lysate and / or culture supernatant. When the packaging cell line contains the viral nucleic acid elements contained in the packaging plasmid, the packaging cell line can be transfected only with the expression plasmid without using the packaging plasmid. The medium for producing the recombinant viral vector is known to those skilled in the art, and may be a commercially available or custom-made medium, or may be further supplemented with one or more cell culture components known in the art to increase the titer of recombinant viral particles in the production culture, including but not limited to glucose, vitamins, amino acids, and / or growth factors. Recombinant viral vector production cultures can be grown under conditions appropriate for the particular host cells (i.e., packaging cells) used. After recombinant viral vectors are produced, viral particles can be purified from cell lysates or viral supernatants, if necessary, by a variety of common methods, including column chromatography, CsCl gradients, and the like. For example, multiple column purification steps can be used, such as purification on anion exchange columns, affinity rham and / or cation exchange columns.

[0103] "Transfect" according to the present invention means transferring a polynucleotide such as a nucleic acid molecule, a plasmid, etc. from outside a cell into a cell to make the polynucleotide function within the cell. Transfection methods are well known to those skilled in the art, such as calcium phosphate or liposome-mediated transfection.

[0104] Various types of cells can be used as packaging cell lines. For example, available packaging cell lines include, but are not limited to, mammalian (including human) cells, insect cells, plant cells, microorganisms or yeast, such as insect-derived cell lines such as HEK293 series cells (e.g., HEK293A, HEK293T or HEK293FT), HeLa cells, A549 cells, Vero cells, hARPE-19 cells and SF-9. As known to those skilled in the art, appropriate packaging cell lines can be selected according to the type of recombinant viral vector.

[0105] The first and second recombinant viral vectors of the present invention may be viral vectors derived from the same or different types of viruses, for example, both may be adeno-associated viral (AAV) vectors, adenoviral vectors, herpes simplex viral (HSV) vectors, lentiviral vectors, or retroviral vectors. In some embodiments, the viral elements (including nucleic acid elements, capsid, and optional envelope glycoproteins, etc.) contained in the first and second recombinant viral vectors may be the same, and only the heterologous nucleic acid contained therein may be different. In some embodiments, the first and second recombinant viral vectors are derived from the same type of virus, but the viral elements contained therein may be the same or different, for example, each may be derived from a different serotype of the same type of virus.

[0106] In the dual recombinant viral vector system of the present invention, the first recombinant viral vector and the second recombinant viral vector of the present invention may be provided in the form of a composition, for example, mixed with each other, or each may be provided in a single form, i.e., not mixed with each other.

[0107] "Recombinant viral vector genome (vg)" or "recombinant viral genome (vg)" as used herein interchangeably refers to a polynucleotide sequence that can be packaged into a viral capsid, which at least includes an expression cassette containing a nucleic acid of interest and cis nucleic acid elements necessary for viral production, and may further include other viral nucleic acid elements. Depending on the type of recombinant viral vector, the recombinant viral vector genome may include single-stranded DNA, double-stranded DNA, or single-stranded or double-stranded RNA. In some embodiments, the amount or titer of the recombinant viral vector genome can be measured in vg / mL.

[0108] The term "expression cassette" refers to a segment of linear nucleic acid, which comprises a nucleic acid of interest operably linked to an expression regulatory sequence. The nucleic acid of interest encodes an expression product, for example, the first or second strand of a polypeptide molecule of the present invention. The expression regulatory sequence refers to a sequence having a regulatory effect on the process of expressing a target polypeptide by the nucleic acid of interest, and includes, but is not limited to, a promoter, an enhancer, a terminator, a selection marker gene, a post-transcriptional regulatory element, an internal ribosome entry site (IRES) and / or an adenylation signal. In the present invention, the first expression cassette for expressing the first strand and the second expression cassette for expressing the second strand may comprise the same or different expression regulatory sequences, for example, the same or different promoters, the same or different enhancers, the same or different terminators, the same or different selection marker genes, the same or different post-transcriptional regulatory elements, the same or different internal ribosome entry sites (IRES) and / or the same or different adenylation signals.

[0109] Promoters that can be used in the present invention include, but are not limited to, promoters from viruses, mammals (including humans), bacteria, yeast, or plants. Promoters may be constitutive, inducible, or cell- or tissue-specific. Examples of viral promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the RSV promoter, the chicken β-actin (CBA) promoter, the CMV early enhancer / chicken β-actin (CAG) promoter, the simian virus 40 (SV40) promoter, the 35S and 19S RNA promoters of the cauliflower mosaic virus (CaMV), the coat protein promoter of the tobacco mosaic virus (TMV), and the like. Examples of plant promoters include, but are not limited to, heat shock promoters. Examples of mammalian promoters include, but are not limited to, the human elongation factor 1a (EF1a) promoter, the human ubiquitin C (UCB) promoter, the mouse phosphoglycerate kinase (PGK) promoter, the RNA polymerase III type promoters (e.g., U6 and H1), and the like. Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the mouse phosphoglycerate kinase (PGK) promoter, and the EF1a promoter. Examples of inducible promoters include, but are not limited to, the zinc-induced sheep metallothionein (MT) promoter, the dexamethasone (Dex)-induced mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, and the like.Cell or tissue specific promoters can initiate expression of genes in specific tissues or cell types and may be used to deliver the polypeptide molecules of the present invention to specific cells, tissues or organs, examples of which include, but are not limited to, the rhodopsin kinase (RK) promoter, which drives expression of the nucleic acid of interest in rod and cone photoreceptor cells and retinal cell lines, the liver-specific TTR promoter, the human alpha 1-antitrypsin (hAAT) promoter and the TBG promoter, the mature neuron-specific hSyn promoter, the projection neuron-specific CaMKIIa promoter, the astrocyte-specific GfaABC1D promoter, and the cardiac muscle-specific cTNT promoter. When using the dual recombinant viral vector system of the present invention to deliver a polypeptide molecule for gene therapy, it is expected that the polypeptide molecule will be expressed in cells in the subject's body, so promoters from viruses, mammals (including humans) are preferred. When using host cells to produce the polypeptide molecules of the present invention, promoters from any origin that have promoter activity in the host cells may be used.

[0110] The post-transcriptional regulatory element can increase the expression level of a polypeptide of interest in a host cell. The post-transcriptional regulatory element that can be used in the present invention may be, for example, a viral post-transcriptional regulatory element, such as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a hepatitis B virus post-transcriptional regulatory element (HBVPRE) or an RNA transport element (RTE).

[0111] The expression cassette may further include a gene for a selectable marker that is effective in a cell (e.g., a packaging cell line for producing a recombinant viral vector, or a cell transduced with a recombinant viral vector), such as a drug resistance selectable marker. Such a selectable marker gene may encode a factor that is necessary for the survival or growth of cells grown in a selective medium. Host cells that are not transformed with a vector containing a selectable gene will not survive in the medium. The selectable marker may be a protein that confers resistance to an antibiotic or other toxin, complements an auxotrophic defect, or supplies a vital nutrient that is retained from the medium; examples of antibiotics or other toxins include, but are not limited to, ampicillin, hygromycin, neomycin, methotrexate, kanamycin, gentamicin, zeocin, or tetracycline.

[0112] The adenylation signal sequence may be located in a transcription terminator region, e.g., in the 3' untranslated region, examples of which include, but are not limited to, bovine growth hormone (BGH) poly(A), SV40 late poly(A), thymidine kinase (TK) poly(A) sequences, and the like.

[0113] The present invention further provides a dual plasmid system for producing said dual recombinant viral vector system, comprising a first plasmid for producing a first recombinant viral vector and a second plasmid for producing a second recombinant viral vector, respectively. The first plasmid comprises a recombinant viral genome comprising a first expression cassette, and the second plasmid comprises a recombinant viral genome comprising a second expression cassette. The first and second plasmids are respectively co-transfected with an optional packaging plasmid and an optional other helper plasmid into a packaging cell line to obtain a first recombinant viral vector and a second recombinant viral vector. For different types of recombinant viral vectors, those skilled in the art already know how to construct the plasmids for producing recombinant viral vectors, comprising recombinant viral genomes, packaging plasmids and other helper plasmids, and also know how to select a packaging cell line and co-transfect the packaging cell line with these plasmids to obtain a recombinant viral vector.

[0114] Recombinant AAV vectors (rAAV vectors) The recombinant viral vector of the present invention (e.g., the first recombinant viral vector and / or the second recombinant viral vector) may be a recombinant adeno-associated viral vector. Adeno-associated virus (AAV) is a non-enveloped naturally replication-deficient DNA virus belonging to the parvoviruses, whose single-stranded DNA genome is about 4.7 kb long and contains genes encoding replication proteins (Rep) and viral capsid (Cap), and two 145 bp inverted terminal repeats (ITRs) flanking the genes. Since the ITRs are the minimal self-sequences essential for AAV replication and packaging, and are also cis-acting elements essential for AAV integration, replication and packaging, the other parts of the AAV viral genome can be replaced with an expression cassette containing a nucleic acid of interest, and the genes encoding Rep and Cap can be provided in trans on another plasmid to produce a recombinant AAV vector. If the viral genome does not have the rep and cap genes, the AAV vector may exist in the form of an episome in the cell nucleus of the transduced cell without integrating into the genome of the transduced cell. In natural systems, AAV infection requires the participation of a helper virus (e.g., adenovirus or herpesvirus), which allows AAV to replicate in infected cells and provides the resulting genes, e.g., E1A, E1B, E2A, E4, VA, etc. Recombinant AAV vectors are produced without the use of helper viruses, using the corresponding helper functions provided by plasmids containing the necessary genes contained in the helper virus.

[0115] A "recombinant AAV vector" according to the present invention refers to an AAV particle that contains structural and / or functional nucleic acid elements derived from AAV and is capable of transferring a nucleic acid of interest contained in its viral genome into a cell, and typically comprises an AAV capsid and a recombinant viral genome packaged within the capsid, said recombinant viral genome comprising an expression cassette for expressing the first or second strand of a polypeptide molecule of the present invention. As known to those skilled in the art, said viral genome further comprises AAV terminal inverted repeats (ITRs) flanking the expression cassette.

[0116] AAV has different serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and AAV Rh10, etc. The capsid protein of AAV of each serotype can bind to a different receptor, for example, AAV2 binds to heparan sulfate proteoglycan (HSPG), AAV1, AAV5 and AAV6 bind to N-linked sialic acid, AAV3 binds to heparan, and AAV9 binds to N-terminal galactose residues, thereby AAV of different serotypes have different targeting properties to different cells, tissues or organs, etc. For example, AAV1-9 can target various types of cells in the retina, such as pigment epithelium and photoreceptor cells, AAV8 has hepatocyte tropism in mice, AAV8 and AAV9 can target various muscle types throughout the body, with AAV9 having a higher tropism for muscle, and AAV9 can penetrate the blood-brain barrier (BBB) ​​in mice. Those skilled in the art can know the tropism of AAV vectors with different AAV serotypes and different AAV capsids for different cells, tissues and organs.

[0117] The first and / or second recombinant AAV vectors of the present invention can be constructed using nucleic acid elements derived from various different AAV serotypes, and in particular, the capsid serotype can be selected based on the cells, tissues and / or organs that are desired to be targeted. In particular, the first and second recombinant AAV vectors of the present invention can independently use capsids from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV Rh10, chimeric AAV capsids or modified AAV capsids. In a preferred embodiment, the first and second recombinant AAV vectors contain capsids with the same tropism, e.g., capsids that can target the same cells, tissues and / or organs, and preferably capsids from the same AAV serotype. In some embodiments, the first recombinant AAV vector and / or the second recombinant AAV vector comprise a capsid from AAV2, AAV5, AAV8 or AAV9.

[0118] Chimeric and modified AAV capsids may be used to alter the tropism of AAV vectors and to alter or increase the targeting of AAV vectors to specific cells, tissues and / or organs. The term "tropism" refers to the targeting of a recombinant viral vector to a specific cell, tissue and / or organ. The term "chimeric AAV capsid" refers to an AAV capsid protein that contains amino acid sequences (e.g., domains) from two or more different AAV serotypes and can form an AAV capsid, examples of which include, but are not limited to, the capsid of AAV DJ. The term "modified AAV capsid" refers to an AAV capsid protein that is formed by inserting, substituting or deleting one or more amino acids, or by inserting a polypeptide derived from another binding domain, examples of which include, but are not limited to, AAV2-7m8. In some embodiments, the first recombinant AAV vector and / or the second recombinant AAV vector include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAV Rh10. The ITRs included in the first recombinant AAV vector and the second recombinant AAV vector may be ITRs from the same AAV serotype or different AAV serotypes. In some embodiments, the ITRs of the first recombinant AAV vector and / or the second recombinant AAV vector are from AAV2, AAV5, AAV8, or AAV9. In some embodiments, the ITRs of the first recombinant AAV vector and the second recombinant AAV vector are from the same AAV serotype, e.g., both are from AAV2, AAV5, AAV8, or AAV9.

[0119] In the first and / or second recombinant AAV vectors of the present invention, the capsid and ITRs may be from the same or different AAV serotypes. In some embodiments, in the first and / or second recombinant AAV vectors, the ITRs and capsids may be from different AAV serotypes, for example, AAV2 ITRs may be selected for use with non-AAV2 capsids, for example, AAV5, AAV8 or AAV9 capsids. In some embodiments, in the first and / or second recombinant AAV vectors, the ITRs and capsids are from the same AAV serotype, for example, both from AAV2, AAV5, AAV8 or AAV9. In a preferred embodiment, the ITRs of the first recombinant AAV vector and the second recombinant AAV vector are from the same serotype, e.g., AAV2, and both capsids are also from the same AAV serotype, e.g., AAV2, AAV5, AAV8, or AAV9.

[0120] Methods for generating recombinant AAV vectors are known to those skilled in the art, and recombinant AAV can be produced, for example, by providing a packaging cell line with a nucleic acid molecule comprising a recombinant AAV genome, a nucleic acid molecule comprising nucleotides encoding AAV rep and cap proteins, and a nucleic acid molecule comprising a helper virus (e.g., adenovirus or herpes simplex virus) gene. The cap and rep genes of AAV may be pre-integrated into the packaging cell, and thus, during production, the packaging cell line may be transfected with the nucleic acid molecule comprising the recombinant AAV genome and the nucleic acid molecule comprising the helper virus gene. The nucleic acid molecule may be provided, for example, by a plasmid. Recombinant AAV production methods that can be used include the tri-plasmid co-transfection method, i.e., co-transfecting a transfer plasmid comprising a recombinant AAV genome, a pRC plasmid comprising the AAV rep and cap genes, and a pHelper plasmid comprising helper genes (e.g., E2A, E4 and VA RNA genes) into a packaging cell line, culturing the cells under appropriate conditions, and isolating and purifying the recombinant AAV vector from the cell lysate and / or culture supernatant. Here, a transfer plasmid for producing a recombinant AAV expression vector may contain an expression cassette and AAV terminal inverted repeat sequences flanking the expression cassette. Recombinant adenovirus vectors

[0121] The recombinant viral vector of the present invention (e.g., the first recombinant viral vector and / or the second recombinant viral vector) may be a recombinant adenoviral vector. Adenovirus is a non-enveloped double-stranded linear DNA virus with a genome of about 36 kb. The adenoviral genome includes inverted terminal repeats (ITRs) at both ends, a packaging signal sequence, early genes (encoding regulatory genes E1, E2, E3 and E4) and late genes (encoding structural genes L1-L5). Adenoviruses include more than 50 serotypes and are divided into six different subgroups, A to F. The recombinant viral vector of the present invention may be derived from any one of the serotypes of adenovirus, preferably subgroup C adenovirus or its mutants, such as Ad2, Ad5 and Ad55. E3 is a region not required for viral replication, and deletion of the E3 region does not affect the production of recombinant adenoviral vectors. The deletion of E1A and / or E1B may render the recombinant virus unable to replicate, resulting in the generation of a replication-deficient adenoviral vector. The adenoviral vector of the present invention may be a defective adenoviral vector, for example, a replication-deficient adenoviral vector, which is deleted of E1A and / or E1B. In the production of a recombinant adenoviral vector, the late genes, E1, E2, E3 and / or E4, may be provided in trans by another nucleic acid molecule (e.g., a plasmid), so that all or part of these genes in the lentiviral genome may be deleted and replaced with an expression cassette containing a nucleic acid of interest, and the late genes, E1, E2, E3 and / or E4, may be provided in trans on another nucleic acid molecule or molecules (e.g., a plasmid) to produce a recombinant lentiviral vector. In some embodiments, E3 may not be provided. In some embodiments, the recombinant adenoviral vector is replication-deficient and has at least a portion of E1 deleted, in which case E2 and E4 are provided in trans on another nucleic acid molecule (e.g., a plasmid) and E1 may be provided by a packaging cell line, such as the HEK293-derived cell line and the PER.C6 cell line, which contain the E1 region of adenovirus.

[0122] A "recombinant adenoviral vector" according to the present invention refers to an adenoviral particle that contains structural and / or functional nucleic acid elements derived from an adenovirus and is capable of transferring a nucleic acid of interest contained in its viral genome into a cell, and typically comprises an adenoviral capsid and a recombinant adenoviral genome packaged within the capsid. The recombinant adenoviral genome comprises an expression cassette for expressing the first or second strand of a polypeptide molecule of the present invention. As known to those skilled in the art, the recombinant adenoviral genome further comprises at least terminal inverted repeats (5'ITR and 3'ITR) located at both ends of the genome and a Ψ packaging signal sequence. The recombinant adenoviral vector of the present invention may be derived from Ad2, Ad5 and Ad55.

[0123] The recombinant adenovirus vector containing a nucleic acid of interest can be produced by any technique known to those skilled in the art, for example, by the AdEasy method or AdMAX method known in the art. For example, a shuttle plasmid containing a nucleic acid of interest and an adenovirus base plasmid containing a part of an adenovirus gene can be used to transfect a packaging cell line to obtain a recombinant adenovirus vector. The shuttle plasmid contains a recombinant adenovirus genome, which contains an expression cassette (which contains a nucleic acid of interest, for example, the first expression cassette or the second expression cassette of the present invention) and cis nucleic acid elements essential for the production of a recombinant adenovirus, such as a packaging signal sequence, such as a terminal inverted repeat sequence. The adenovirus base plasmid may contain late genes, E1, E2, E3 and / or E4, but does not contain a packaging signal sequence. A recombinant adenovirus can be produced by transfecting a packaging cell line with the shuttle plasmid and the adenovirus base plasmid and allowing them to undergo homologous recombination or site-specific recombination in the cells. The site-specific recombination can utilize, for example, the Cre / loxP recombinase system.

[0124] In the present invention, the first recombinant viral vector and the second recombinant viral vector may be derived from the same or different types of adenovirus, and if both are derived from the same type of adenovirus, it may contain the same or different viral elements, such as the same or different 5'ITR, 3'ITR, Ψ packaging signal sequence, late genes, E1, E2, E3 and / or E4. Recombinant retroviral vectors

[0125] The recombinant viral vector (e.g., the first recombinant viral vector and / or the second recombinant viral vector) of the present invention may be a recombinant reverse transcription vector. Retroviruses are RNA viruses, and the viral genome is RNA. When a host cell is infected with a retrovirus, the viral genome RNA is reverse transcribed into a DNA intermediate and integrated into the chromosomal DNA of the infected cell. The retroviral genome and proviral DNA have three genes, gag, pol, and env, flanked by two long terminal repeat (LTR) sequences, and a Ψ packaging signal sequence is present between the 5'LTR and the gag gene. The gag gene encodes internal structural (matrix, capsid, and core-shell body) proteins, and the env gene encodes viral envelope glycoproteins. The pol gene encodes multiple enzymes, including an RNA-guided DNA polymerase reverse transcriptase that transcribes viral RNA as double-stranded DNA, an integrase that integrates the DNA generated by the reverse transcriptase into the host chromosomal DNA, and a protein enzyme that processes the encoded gag and pol genes. The 5' and 3' LTRs facilitate transcription and polyadenylation of virion RNA. In producing a recombinant retroviral vector, the gag, pol and env genes can be provided in trans by another nucleic acid molecule (e.g., a plasmid), so that these genes in the retroviral genome can be deleted and replaced with an expression cassette containing the nucleic acid of interest, and the gag, pol and env genes provided in trans on another nucleic acid molecule or molecules (e.g., a plasmid) to produce a recombinant reverse transcription viral vector.

[0126] A "recombinant retroviral vector" according to the present invention refers to a retroviral particle that contains structural and / or functional nucleic acid elements derived from a retrovirus and is capable of transferring a nucleic acid of interest contained in its viral genome into a cell, and typically comprises a retroviral capsid, a recombinant retroviral genome packaged within the capsid, and viral envelope glycoproteins, said recombinant retroviral genome comprising an expression cassette for expressing the first or second strand of a polypeptide molecule of the present invention. As known to those skilled in the art, a recombinant retroviral genome comprises at least long terminal repeats (5'LTR and 3'LTR) located at both ends of the genome, and further comprises a Ψ packaging signal sequence, which may further optionally comprise a polypurine tract PPT. A recombinant retroviral vector of the present invention may be derived, for example, from Moloney murine leukemia virus (MMLV) or murine stem cell virus (MSCV). A recombinant retroviral vector may be replication-competent or replication-deficient. Typically, retroviral vectors are replication-defective, in that the gene coding regions essential for replication and packaging of viral particles are deleted or replaced by other genes, such that upon infection of the initial target cell, the virus is unable to continue its typical cell lysis pathway. Such retroviral vectors and the materials necessary for the production of such viruses (e.g., packaging cell lines) are commercially available (e.g., from Clontech).

[0127] A recombinant retroviral vector can have its tropism altered using heterologous envelope glycoproteins (e.g., envelope glycoproteins from other viruses), i.e., using heterologous env genes (e.g., env genes from other viruses) in the production of the recombinant retroviral vector. For example, VSV-G envelope glycoprotein or Sindbis virus E2 glycoprotein may be used. Recombinant retroviral vectors packaged with heterologous envelope glycoproteins may also be referred to as pseudotyped recombinant retroviral vectors.

[0128] Methods for producing recombinant retroviral vectors are known to those skilled in the art. For example, recombinant retroviral vectors can be produced by providing a packaging cell line with a nucleic acid molecule comprising a recombinant retroviral genome, one or more (e.g., two) nucleic acid molecules comprising the gag, pol and env genes. The nucleic acid molecule may be a plasmid. The gag, pol and env genes may be provided in two separate plasmids, one plasmid comprising the gag and pol genes alone and another plasmid comprising the env gene, such that recombination does not occur between the plasmids. Here, the recombinant retroviral genome comprises an expression cassette (which comprises a nucleic acid of interest, e.g., the first expression cassette or the second expression cassette of the present invention) and cis nucleic acid elements essential for the production of a recombinant retrovirus, such as long terminal repeats (5'LTR and 3'LTR) and the Ψ packaging signal sequence.

[0129] In the present invention, the first and second recombinant viral vectors may be derived from the same or different types of retroviruses, and if both are derived from the same type of retrovirus, they may contain the same or different viral elements, such as the same or different 5'LTR, 3'LTR, Ψ packaging signal sequence, gag, pol and / or env genes. Recombinant lentiviral vectors

[0130] The recombinant virus vector of the present invention (e.g., the first recombinant virus vector and / or the second recombinant virus vector) may be a recombinant lentivirus vector. Lentivirus belongs to retroviruses, and unlike other retroviruses, lentiviruses can integrate into the genome of non-dividing cells, and typical lentiviruses include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Similar to other retroviruses, the lentivirus genome is flanked by two long terminal repeat (LTR) sequences and contains gag, pol, and env genes, which are the main genes encoding viral proteins, and there is a Ψ packaging signal sequence between the 5'LTR and the gag gene. It should be noted that lentiviruses further contain other helper genes involved in synthesis regulation, viral RNA processing, and other replication functions, including tat, rev, nef, vif, vpr, and vpu). In producing a recombinant lentiviral vector, nef, vif, vpr and vpu are not essential, the tat gene may be unnecessary by modification of the 5'LTR, and the gag, pol, env and rev genes can be provided in trans by another nucleic acid molecule (e.g., a plasmid), so that these genes in the lentiviral genome can be deleted and replaced with an expression cassette comprising the nucleic acid of interest, and the gag, pol, env and rev genes are provided in trans on another nucleic acid molecule or molecules (e.g., a plasmid) to produce a recombinant lentiviral vector.

[0131] A "recombinant lentiviral vector" according to the present invention means a lentiviral particle that contains structural and / or functional nucleic acid elements derived from a lentivirus and is capable of transferring a nucleic acid of interest contained in its viral genome into a cell, and typically comprises a lentiviral capsid, a recombinant lentiviral genome packaged in the capsid, and viral envelope glycoproteins, said recombinant lentiviral genome comprising an expression cassette for expressing the first or second strand of a polypeptide molecule of the present invention. As known to those skilled in the art, a recombinant lentiviral genome further comprises at least long terminal repeat sequences (5'LTR and 3'LTR) located at both ends of the genome, and a Ψ packaging signal sequence, which may further optionally contain Rev-response elements (RRE) and / or DNA flap elements (cPPT / CTS), etc. The recombinant lentiviral vector of the present invention may be derived from HIV-1, HIV-2, SIV or FIV. The recombinant lentiviral vector may be replication-competent or replication-deficient. Typically, recombinant lentiviral vectors are replication-defective, in that the gene coding regions essential for replication and packaging of viral particles are deleted or replaced with other genes, such that upon infection of the initial target cell, the virus is unable to proceed through its typical cytolytic pathway.

[0132] The 3'LTR in the recombinant lentiviral vector of the present invention may be self-inactivating (SIN 3'LTR), for example, the U3 region is deleted, so that the lentiviral vector cannot replicate autonomously. The U3 region of the 5'LTR in the recombinant lentiviral vector of the present invention may be replaced with a heterologous promoter that is not dependent on Tat, for example, a promoter from another virus, such as the RSV promoter, thus eliminating the dependency of U3 on Tat protein during transcription, and the Tat sequence may be removed during the production of the recombinant lentiviral vector.

[0133] A recombinant lentiviral vector can be modified in its tropism by using a heterologous envelope glycoprotein (e.g., an envelope glycoprotein from another virus), i.e., by using a heterologous env gene (e.g., an env gene from another virus) in the production of the recombinant lentiviral vector. For example, VSV-G envelope glycoprotein or Sindbis virus E2 glycoprotein can be used. A recombinant lentiviral vector packaged with a heterologous envelope glycoprotein can be referred to as a pseudotyped recombinant lentiviral vector.

[0134] The production of recombinant lentiviral vectors is well known to those skilled in the art, and can be produced, for example, by providing a packaging cell line with a nucleic acid molecule comprising a recombinant lentiviral genome, one or more (e.g., two) nucleic acid molecules comprising gag, pol, rev genes, and a nucleic acid molecule comprising an env gene. Recombinant lentiviral vectors can be produced using a tri- or tetra-plasmid system. In one tetra-plasmid system, the first plasmid is deleted for helper proteins (e.g., tat, vif, vpu, vpr, and nef) and contains the gag and pol genes, the second plasmid contains the rev gene, the third plasmid contains the env gene of the vesicular stomatitis virus envelope protein (VSV-G), and the fourth plasmid contains the recombinant lentiviral genome. Recombinant lentiviral vectors and systems for producing viruses are commercially available (e.g., from Cell Biolabs, Inc.). Here, the recombinant lentivirus genome comprises an expression cassette (wherein the expression cassette comprises a nucleic acid of interest, e.g., the first expression cassette or the second expression cassette of the present invention) and cis nucleic acid elements essential for the production of a recombinant lentivirus, such as long terminal repeats (5'LTR and 3'LTR) and the Ψ packaging signal sequence, and may further optionally comprise a Rev-response element (RRE) and / or a DNA flap element (cPPT / CTS), etc.

[0135] In the present invention, the first recombinant viral vector and the second recombinant viral vector may be derived from the same or different types of lentivirus, and if both are derived from the same type of lentivirus, it may contain the same or different viral elements, for example the same or different 5'LTR, 3'LTR, Ψ packaging signal sequence, gag, pol, rev and / or env genes. Recombinant HSV vectors

[0136] The recombinant viral vector of the invention (e.g., the first recombinant viral vector and / or the second recombinant viral vector) may be a recombinant HSV vector. Herpes simplex virus (HSV) is an enveloped, capsid-containing double-stranded DNA virus, whose genome includes immediate early genes, early genes, and late genes. HSV has two serotypes, HSV-1 and HSV-2. Because HSV virus replication in host cells is a lytic infection and may cause host cell death, recombinant HSV usually has some immediate early genes (e.g., ICP4 and / or ICP27) knocked out, making the virus replication-deficient, and the virus regains replication ability only after the cell complements ICP4 and / or ICP27. Therefore, the recombinant HSV vector may be replication-deficient.

[0137] A "recombinant HSV vector" according to the present invention means an HSV particle that contains structural and / or functional nucleic acid elements derived from HSV and is capable of transferring a nucleic acid of interest contained in its viral genome into a cell, typically comprising an HSV capsid, a recombinant HSV genome packaged within the capsid, and viral envelope glycoproteins, said recombinant HSV viral genome comprising an expression cassette for expressing the first or second strand of a polypeptide molecule of the invention.

[0138] Recombinant HSV vectors can be produced by homologous recombination between an HSV base plasmid and a plasmid carrying a nucleic acid of interest. For example, usually, the HSV base plasmid contains the main functional genes of HSV, but some immediate early genes (e.g., ICP4 and / or ICP27) are deleted, and then homologous recombination occurs after co-transfecting the HSV base plasmid and a plasmid carrying a nucleic acid of interest (e.g., an expression cassette containing a nucleic acid of interest, e.g., the first expression cassette or the second expression cassette of the present invention) into a producer cell line expressing ICP4 and / or ICP27. Examples of producer cell lines include, but are not limited to, Vero cells.

[0139] In the present invention, the first recombinant viral vector and the second recombinant viral vector may be derived from the same or different types of HSV virus, and if both are derived from the same type of HSV virus, they may contain the same or different viral elements. Production of Polypeptide Molecules

[0140] The dual recombinant viral vector system of the present invention may be used to produce the polypeptide molecule in vitro, for example by transducing a host cell with a first recombinant viral vector and a second recombinant viral vector contained in the dual recombinant viral vector system of the present invention, and expressing the polypeptide molecule in the host cell by culturing the host cell under suitable conditions. When the first recombinant viral vector and the second recombinant viral vector are provided in the form of a composition, the host cell may be transduced with a composition comprising the first recombinant viral vector and the second recombinant viral vector. When the first recombinant viral vector and the second recombinant viral vector are provided separately, the host cell may be transduced with one recombinant viral vector and then with the other recombinant viral vector, and the order of transduction may be arbitrary, with the first recombinant viral vector transducing first, or the second recombinant viral vector transducing first. The suitable conditions are available to a person skilled in the art and are usually suitable for the growth of the host cell. The first and second recombinant viral vectors express the first and second chains of the polypeptide molecule in the host cell, and the first and second chains can spontaneously assemble into the polypeptide molecule. The host cell is in vitro. The polypeptide molecule can be collected and purified from the host cell culture for further application, or the host cell expressing the polypeptide molecule can be used for further application, such as injection into the body of a subject, to achieve a therapeutic purpose.

[0141] The produced polypeptide molecule may be secreted extracellularly, or the secreted polypeptide molecule may be collected from the host cell culture supernatant and, if necessary, purified by methods well known in the art.

[0142] As used herein, the term "transduction" means that a viral particle infects a cell and has a function within the cell.

[0143] A "host cell" according to the present invention means a cell that contains a nucleic acid of interest and expresses a polypeptide encoded thereby, including a cell into which a foreign nucleic acid (e.g., a recombinant viral vector of the present invention) has been introduced and its progeny. A host cell may be isolated or in vitro. Examples of host cells include bacteria, yeast, mammalian cells, etc. Suitable host cells include, but are not limited to, insect cells, such as Spodoptera frugiperda cells, microorganisms, including bacteria, yeast cells, such as Saccharomyces cerevisiae or Pichia pastoris, fungi, such as Trichoderma reesei, Aspergillus, Aureobasidum, and Penicillium species, and mammalian cells, such as human cell lines, including Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, COS cells, African green monkey kidney cells (Vero cells), canine kidney cells (e.g., MDCK cells), rat pituitary cells (GH1 cells), human embryonic kidney cells (e.g., HEK 293, HEK293A, HEK293T, HEK293FT, etc.), Hela cells, or hARPE-19 cells, etc.

[0144] Those skilled in the art know how to select an appropriate host cell depending on the type of recombinant viral vector used.

[0145] The dual recombinant viral vector system of the present invention may be for in vivo production of said polypeptide molecule, for example, for producing said polypeptide molecule in the body of a subject, i.e., for delivering said polypeptide molecule to the body of a subject. After being delivered to the body of a subject, the first and second recombinant viral vectors can express the first and second chains of said polypeptide molecule in cells, tissues and / or organs of the body of the subject by transducing the cells of said cells, tissues and / or organs. After being expressed, the first and second chains of said polypeptide molecule can spontaneously assemble into said polypeptide molecule to provide a desired effect, for example, a therapeutic effect, in the subject.

[0146] The dual recombinant viral vector system of the present invention can be specifically expressed in a specific cell, tissue and / or organ by the tropism and / or tissue-specific promoter of the recombinant viral vector, thereby achieving the purpose of targeted therapy. The produced polypeptide molecule is secreted outside the cell and can be detected, for example, in serum.

[0147] The recombinant viruses of the invention can express target antibodies in an amount of at least about 9 μg / ml, at least about 10 μg / ml, at least about 50 μg / ml, at least about 100 μg / ml, at least about 200 μg / ml, at least about 300 μg / ml, at least about 400 μg / ml, at least about 500 μg / ml, at least about 600 μg / ml, at least about 700 μg / ml, at least about 800 μg / ml, at least about 900 μg / ml, or at least about 1000 μg / ml in the serum of a subject.

[0148] "Subject" according to the present invention refers to an animal (including a human) in need of alleviating, preventing and / or treating a disease. In some embodiments, "subject" refers to a mammal, including but not limited to a human, mouse, rat, rabbit, guinea pig, cow, horse, cat, dog, rodent or primate. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. Drug Compositions

[0149] The first and second recombinant viral vectors in the dual recombinant viral vector system of the present invention may be provided separately or in the form of a composition. The first and / or second recombinant viral vector may be provided in the form of a pharmaceutical composition, which comprises an effective amount of the first and / or second recombinant viral vector, and an optional pharmaceutically acceptable vector. As is well known in the art, a "pharmaceutically acceptable vector" is an inactive substance that facilitates administration of an active ingredient, and the pharmaceutical composition can be provided in different dosage forms, for example, liquid solutions, suspensions, emulsions, or as solid forms suitable for dissolving or suspending in liquid before use.

[0150] "Pharmaceutically acceptable vectors" include, but are not limited to, diluents, fillers, antioxidants, preservatives, stabilizers, osmotic pressure regulators, buffers and / or emulsifiers, etc. Depending on the route of administration, pharma- ceutically acceptable vectors include, but are not limited to, sugars, starches, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyalcohols, alginic acid, phosphate buffer, emulsifiers, isotonic saline and / or athermal water, etc. The pharmaceutical composition according to the present invention may be prepared into a clinically acceptable dosage form, such as capsules, tablets, lozenges, suppositories, ointments, lotions, pastes, aerosols, sprays, gels, patches, injections, etc. delivery

[0151] The dual recombinant viral vector system or drug composition of the present invention may be delivered to a subject by any suitable route, e.g., systemically or locally, for example, administered orally, rectally, transmucosally, intranasally, by inhalation, intravaginally, intrathecally, intracerebroventricularly, intraocularly, extragastrointestinal (e.g., intravenously, subcutaneously, intradermally, intramuscularly, intraperitoneally), etc.

[0152] The dual recombinant viral vector system or drug composition of the present invention may be delivered into any type of cell, including, but not limited to, neural cells (including cells of the peripheral and central nervous system, e.g., neuronal cells and dendritic cells), lung cells, ocular cells (including retinal cells, retinal pigment epithelium and corneal cells), epithelial cells (e.g., intestinal and airway epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells and diaphragm cells), liver cells, kidney cells, cancer cells and tumor cells.

[0153] The dual recombinant viral vector system or drug composition of the present invention may be delivered to any tissue or organ, including, but not limited to, blood vessels, lungs, skin, heart, eye (including the vitreous and retina, particularly the retinal pigment epithelium RPE), muscle (including skeletal muscle, cardiac muscle, smooth muscle and diaphragm), central nervous system (including the spinal cord, brain parenchyma and cerebrospinal fluid) and liver, and may also be delivered to tumors (including tumors or lymph nodes).

[0154] The most appropriate route in any given case will depend on the nature and severity of the condition being treated and the nature of the particular viral vector being used. Injection may be performed using any suitable device, such as, for example, a syringe, needle, cannula or catheter, or the recombinant viral vector may be delivered to the brain by stereotactic injection.

[0155] To achieve the desired level of expression of the polypeptide molecules, the dual recombinant viral vector system of the present invention may be administered to the subject one or more times (e.g., 2, 3, 4 or more times). In some embodiments, the first and second recombinant viral vectors may be administered to the subject once a year. In some embodiments, the first and second recombinant viral vectors are administered to the subject up to once every 5 years. In some embodiments, the first and second recombinant viral vectors are administered to the subject up to once every 10 years.

[0156] The dosage of the first and second recombinant viral vectors can be adjusted by those skilled in the art depending on, for example, the disease state, the subject, and the treatment schedule. The first and second recombinant viral vectors are usually administered in a "therapeutically effective amount", and "treatment" according to the present invention means preventing or alleviating (e.g., reducing, alleviating, or curing) at least one symptom associated with a disease state. A "therapeutically effective amount" according to the present invention is an amount sufficient to prevent or alleviate (e.g., reducing, alleviating, or curing) at least one symptom associated with a disease state. The total dose and fractional doses of the first and second recombinant viral vectors administered to a subject depend on the mode of administration, the disease being treated, the pathology of the individual subject, the specific viral vector and the polypeptide molecule being delivered, and can be determined in a general manner. The unit of dosage can be, for example, vg / kg body weight. Disease to be treated

[0157] The dual recombinant viral vector system and methods of the present invention may be used to prevent or treat one or more diseases in a subject by either producing the polypeptide molecules in vitro and applying the produced polypeptide molecules to treat the disease, or by delivering the dual recombinant viral vector system into the subject's body thereby expressing the polypeptide molecules in cells, tissues or organs within the subject's body.

[0158] The disease to be prevented or treated depends on the specific polypeptide molecule expressed and the specific site of delivery. Such diseases include, but are not limited to, viral infections, tumors, and diseases associated with angiogenesis. Examples of viral infections include, but are not limited to, infections by viruses selected from the following: coronavirus, filovirus, flavivirus, norovirus, orthomyxovirus, paramyxoviridae, rubulavirus, measles virus, parvovirus, small RNA virus, foot and mouth disease virus, enterovirus, coxsackievirus, poliovirus, rhinoviridae, poxviridae, reoviridae, rotavirus, HIV virus, leukemia virus, rubella virus, hepatitis C virus (HCV), hepatitis B virus, Epstein Barr virus, influenza virus, human immunodeficiency virus (HIV), respiratory syncytial virus, and any combination thereof.

[0159] Examples of tumors include, but are not limited to, hematological tumors and solid tumors, where hematological tumors include, but are not limited to, myeloma, leukemia, and lymphoma, and solid tumors include, but are not limited to, colorectal cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, Hodgkin's lymphoma, hepatocellular carcinoma, end stage renal cancer, prostate cancer, thyroid cancer, and ovarian cancer.

[0160] Examples of diseases associated with angiogenesis include, but are not limited to, macular degeneration (including age-related macular degeneration), arthritis (including rheumatoid arthritis), arteriosclerosis, diabetic retinopathy, hemangiomas, inflammatory diseases, etc. The dual recombinant viral vector system of the present invention may further be used to treat autoimmune diseases such as multiple sclerosis.

[0161] Unless otherwise specified, all of the methods and materials in the examples described below are common products that are commercially available. As will be appreciated by those skilled in the art, the methods and materials described below are merely illustrative and should not be construed as limiting the scope of the invention.

[0162] Example 1 Dual rAAV Vector-Mediated Avastin (Bevacizumab) Expression in HEK 293 and hARPE-19 Cells Compared to Single rAAV Vectors A. Materials and Methods 1. AAV Vector Design and Generation Schematics of the vector expressing the complete antibody sequence (AAV-Avastin single vector), the vector expressing only the light chain (AAV-Avastin light chain, i.e., AAV-Avastin-L), and the vector expressing only the heavy chain (AAV-Avastin heavy chain, i.e., AAV-Avastin-H) are shown in Figure 1a, and the transfer plasmid pAAV2.CMV.Avastin expressing the complete antibody sequence, and the transfer plasmids pAAV2.CMV.Avastin-L and pAAV2.CMV.Avastin-H expressing only the light chain or the heavy chain each contain the two ITRs and the elements between them shown in Figure 1a. In designing a single vector for AAV-mediated antibody expression, an F2A self-cleaving peptide motif was inserted between the heavy and light chain coding sequences to achieve simultaneous expression of two polypeptides under the control of one promoter (see, e.g., US20110065779A1, US9527904B2, US10093947B2, US20180155412A1).

[0163] Recombinant AAV was produced by transfecting 293FT cells with three plasmids: pAAV.R2C2 plasmid (i.e., pRC plasmid) expressing the rep and cap genes of AAV2, pAdDelta6 plasmid (i.e., pHelper plasmid) expressing helper genes from adenovirus, and a transfer plasmid; see Xiao and Samulski, 1998, J Virol. Here, the pAdDelta6 plasmid was constructed by synthesizing DNA by hand with reference to the Addgene plasmid #112867 sequence. The pAAV.R2C2 plasmid was constructed by removing the AAV8 cap gene from Addgene plasmid #112864 pAAV.R2C8 and inserting the AAV2 cap gene; the AAV2 genome sequence can be found in GenBank: AF043303.

[0164] Construction of single vector transfer plasmid: The target antibody (Avastin) gene is designed and synthesized in series in the order of heavy chain leader sequence, heavy chain, F2A, light chain leader sequence, and light chain, and Not I and Hind III enzyme cleavage sites are added upstream and downstream, respectively, and then it is constructed between the promoter and poly A of the AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH (constructed by synthesizing DNA by yourself with reference to Addgene plasmid #105530) by enzyme cleavage and ligation, transformed, and clone-amplified to obtain the recombinant AAV antibody transfer plasmid pAAV2.CMV.Avastin, and the target sequence is aligned by Sanger sequencing. If it is consistent, it indicates that the inserted sequence is completely accurate. Here, the sequence used is as follows, where the Avastin sequence is referred to https: / / go.drugbank.com / drugs / DB00112:

[0165] [Table 1-1] [Table 1-2]

[0166] Construction of dual vector transfer plasmid (containing heavy chain transfer plasmid and light chain transfer plasmid): Heavy chain transfer plasmid: The heavy chain gene of the target antibody (Avastin) is designed and synthesized in the order of heavy chain leader sequence and heavy chain, and Not I and Hind III enzyme cleavage sites are added upstream and downstream, respectively. It is constructed between the promoter and poly A of the AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH by enzyme cleavage and ligation, transformed, and clone-amplified to obtain the recombinant AAV antibody heavy chain transfer plasmid pAAV2.CMV.Avastin-H, and the target sequence is aligned by Sanger sequencing. If it is consistent, it indicates that the inserted sequence is completely correct.

[0167] The heavy chain leader sequence and the heavy chain sequence in the heavy chain transfer plasmid are the same as the heavy chain leader sequence and the heavy chain sequence, respectively, in the single vector transfer plasmid.

[0168] Light chain transfer plasmid: The light chain gene of the target antibody (Avastin) is designed and synthesized in the order of light chain leader sequence and light chain, and Not I and Hind III enzyme cleavage sites are added upstream and downstream, respectively. It is constructed between the AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH promoter and poly A by enzyme cleavage and ligation, transformed, and clone-amplified to obtain the recombinant AAV antibody light chain transfer plasmid pAAV2.CMV.Avastin-L, and the target sequence is aligned by Sanger sequencing. If it is consistent, it indicates that the inserted sequence is completely correct.

[0169] The light chain leader sequence and the light chain sequence in the light chain transfer plasmid are the same as the light chain leader sequence and the light chain sequence, respectively, in the single vector transfer plasmid.

[0170] pAAV.R2C2, pAdDelta6 and each of the above three transfer plasmids were co-transfected into 2E+08 HEK293FT cells at a molar ratio of 1:1:1 with a total amount of 1040 μg DNA. Recombinant AAV vectors were obtained 48 hours after transfection. Cells were detached from the attachment plate with 0.25% Trypsin-EDTA and lysed by freezing and thawing three times. After centrifugation at 12,000 rpm for 8 minutes, the supernatants were collected to obtain the recombinant AAV vectors AAV2-Avastin (single vector) or AAV2-Avastin-L (light chain vector in dual vector) and AAV2-Avastin-H (heavy chain vector in dual vector), respectively, and the viral genome titer of each recombinant AAV vector crude cell lysate was determined by droplet digital PCR (ddPCR).

[0171] 2. Vector transduction and antibody detection HEK293FT cells were transduced with AAV2-Avastin or with both AAV2-Avastin-L and AAV2-Avastin-H in combination, where the ratios of AAV2-Avastin-L to AAV2Avastin-H were 1:4, 1:2, 1:1, 2:1, 4:1, and 6:1, respectively (based on viral genome titer). Vectors containing only the light chain or only the heavy chain were also transduced and used as controls. 1.2E+05 293FT cells were seeded into each well of a 24-well plate and incubated at 37°C with DMEM / 10% FBS complete medium in 5% CO. 2 The medium was removed, and the viral vector diluted in the above complete medium to a total amount of 5.0E+10 vg was added. The cells were incubated at 37°C in 5% CO 2 After incubation in a culture box for 2.5 hours with 0.1 mL of fresh complete medium, the cells were continued to be cultured. Cell culture supernatants were collected 48 hours after transduction. Each sample was diluted with sampling buffer under non-reducing (Fig. 1b) and reducing conditions (Fig. 1c), respectively, and Western blot was performed with goat anti-human IgG antibody (Invitrogen, Cat No: A18811) to detect Avastin expression.

[0172] hARPE-19 cells were seeded into 24-well plates at 8.0E+04 cells per well and incubated in F12 / 10% FBS medium at 37°C with 5% CO 2 The medium was removed, and viral vectors diluted in F12 / 10% FBS medium to a total amount of 5.0E+10vg were added. AAV2-Avastin, AAV2-Avastin-L, AAV2-Avastin-H, and combinations of AAV2-Avastin-L and AAV2Avastin-H at viral genome titer ratios of 1:4, 1:2, 1:1, 2:1, and 4:1, respectively, were cultured at 37°C, 5% CO 2 After incubation in a culture box for 2.5 hours with 0.1 mL of fresh complete medium, the cells were continued to be cultured. Cell culture supernatants were collected 48 hours after transduction. Each sample was diluted with sampling buffer under non-reducing conditions (Fig. 1d), and Western blot was performed with goat anti-human IgG antibody (Invitrogen, Cat No: A18811) to detect Avastin expression.

[0173] The Western blot positive control was purified Avastin antibody and the negative control was non-transduced virus cell supernatant.

[0174] B. Results As shown in Figure 1, both single vector transduction and dual vector transduction were successful in producing secreted Avastin antibody from HEK293FT cells (Figure 1b and c) and hARPE-19 cells (Figure 1d). As the ratio of light chain vector increased, the antibody concentration also increased. Figures b and d are non-reducing buffer sampling electrophoresis, in which the heavy and light chains of Avastin antibody exist in the form of tetramers, and Figure c is reducing buffer sampling electrophoresis, in which Avastin antibody is dissociated and the heavy and light chains exist in the form of single chains, respectively.

[0175] Example 2. Expression of dual rAAV vectors carrying other serotype capsids mediated Avastin in cell lines A. Materials and Methods In another embodiment, the AAV vector capsid is of serotype AA8, AAV9, or AAV5. The pAAV.R2C9 plasmid was constructed by removing the AAV8 cap gene from Addgene plasmid #112864 pAAV.R2C8 and inserting the AAV9 cap gene, the AAV9 cap gene sequence can be found in GenBank: AY530579.1. The pAAV.R2C5 plasmid was constructed by removing the AAV8 cap gene from Addgene plasmid #112864 pAAV.R2C8 and inserting the AAV5 cap gene, the AAV5 genome sequence can be found in NCBI sequence: NC_006152. The packaging step was the same as that described in Materials and Methods 1 of Example 1, except that the AAV genome rep and cap genes pAAV.R2C2 were replaced with pAAV.R2C8, pAAV.R2C9 and pAAV.R2C5 plasmids containing the above serotype cap genes, respectively, and pAdDelta6 and plasmids containing the complete antibody expression cassette pAAV2.CMV.Avastin or only the antibody heavy chain pAAV2.CMV.Avastin-H or light chain pAAV2.CMV.Avastin-L expression cassettes were used. The plasmids were co-transfected with HEK293FT cells, and 48 hours later, the cell culture supernatants and intracellular viruses were harvested to obtain AAV8-Avastin, AAV8-Avastin-L, and AAV8-Avastin-H, AAV9-Avastin, AAV9-Avastin-L, and AAV9-Avastin-H, AAV9 capsids, AAV5-Avastin, AAV5-Avastin-L, and AAV5-Avastin-H, respectively, and the viral genome titers were measured using the ddPCR method.

[0176] HEK293FT cells were seeded in a 24-well plate at a density of 1.2E+05 cells per well, and hARPE-19 cells were seeded at a density of 8.0E+04 cells per well, and 500 μL DMEM / 10% FBS or F12 / 10% FBS medium was added and cultured overnight. After 24 hours, the medium was removed, and recombinant AAV or recombinant AAV combination with a total amount of about 1.3E+10vg and a titer of 4.0E+10vg / mL or a total amount of about 3.8E+10vg and a titer of 1.2E+11vg / mL was added (the ratio was based on the viral genome titer). AAV8-Avastin, AAV8-Avastin-L: AAV8-Avastin-H 4:1, AAV8-Avastin-L: AAV8-Avastin-H 2:1, AAV8-Avastin-L: AAV8-Avastin-H 1:1, AAV8-Avastin-L: AAV8-Avastin-H 1:2, AAV9-Avastin, AAV9-Avastin-L: AAV9-Avastin-H 4:1, AAV9-Avastin-L: AAV9-Avastin-H 2:1, AAV9-Avastin-L: AAV9-Avastin-H 1:1, AAV9-Avastin-L: AAV9-Avastin-H 1:2, AAV5-Avastin, AAV5-Avastin-L: AAV5-Avastin-H 4:1, AAV5-Avastin-L: AAV5-Avastin-H 2:1, AAV5-Avastin-L: AAV5-Avastin-H 1:1 or AAV5-Avastin-L: AAV5-Avastin-H 1:2.

[0177] 37°C, 5% CO 2After incubation in a culture box for 2.5 hours with 1 mL of fresh medium, the cells were incubated for 67 hours. The cell culture supernatant was collected and subjected to Western blotting with anti-human IgG antibody to detect Avastin antibody expression.

[0178] B. Results As shown in Figure 2, single vector or dual vector transduced HEK293FT or hARPE-19 cells with AAV8 capsid, AAV9 capsid and AAV5 capsid (a, b, c) could express Avastin antibody, and dual vector with AAV8 capsid and AAV9 capsid showed higher expression level in 293FT cell-mediated antibody than single vector, and the expression level increased with the increase of light chain vector ratio. Dual vector with AAV5 capsid showed no obvious relationship with light heavy chain ratio, but the expression amount was higher than that of single vector, and had high tropism to hAPRE-19 cells.

[0179] Avastin expressed by a single vector had bands with molecular weights significantly larger or smaller than the antibody itself, which may be an antibody single chain that is not correctly paired, and the impure expression limited the in vivo application of AAV-delivered antibodies. The antibodies expressed by the dual vector system with AAV8 capsid, AAV9 capsid, and AAV5 capsid showed a significantly reduced content of mispairing bands.

[0180] Example 3: Comparison of expression of plasmid-transfected and viral vector-transduced antibodies in vitro In another example, the efficiency of antibodies expressed by AAV viral vectors and plasmid vectors was compared.

[0181] A. Materials and Methods AAV2-Avastin, AAV2-Avastin-L and AAV2-Avastin-H were packaged and produced as in the packaging step described in Materials and Methods 1 of Example 1, and the viral genome titer was measured using the ddPCR method. HEK293FT cells were seeded into a 24-well plate at a density of 2.0E+05 cells per well, and 500 μL of DMEM / 10% FBS medium was added, and hARPE-19 cells were seeded at a density of 8.0E+04 cells per well, and F12 / 10% FBS medium was added, and the cells were cultured overnight. After 24 hours, HEK293FT cells were transduced with AAV viral vectors at a total amount of 1.0E+10vg, titer of 3.3E+10vg / mL, and volume of 300μL per well, and transfer plasmids were co-transfected at a total mass of 0.1μg or 1.0μg (corresponding to 1.0E+10 or 1.0E+11 copies) with twice the mass of PEI into HEK293FT and hARPE-19 cells, respectively. The recombinant AAV or recombinant AAV combination and plasmid combination were, respectively, AAV2-Avastin, AAV2-Avastin-H, AAV2-Avastin-L, AAV2-Avastin-L: AAV2-Avastin-H 4:1, AAV2-Avastin-L: AAV2-Avastin-H 2:1, AAV2-Avastin-L: AAV2-Avastin-H 1:1, pAAV2.CMV.Avastin, pAAV2.CMV.Avastin-H, pAAV2.CMV.Avastin-L, pAAV2.CMV.Avastin-L: pAAV2.CMV.Avastin-H 4:1, pAAV2.CMV.Avastin-L: pAAV2.CMV.Avastin-H 2:1, pAAV2.CMV.Avastin-L: pAAV2.CMV.Avastin-H 1:1, pAAV2.CMV.Avastin-L: pAAV2.CMV.Avastin-H 1:2.

[0182] 37°C, 5% CO 2 After incubation in a culture box for 2.5 hours with 1 mL of fresh medium, the cells were incubated for 67 hours. The cell culture supernatant was collected and subjected to Western blotting with anti-human IgG antibody to detect Avastin antibody expression.

[0183] B. Results As shown in Figure 3, the positive control is purified Avastin antibody, and the sampling amount is 20ng. Figure a: Plasmid-transfected cells expressing antibodies (lanes 1-4), whose gene copy number is 10 times that of the viral vector, still have lower expression levels than those of viral vector-transduced cells (lanes 8-10). As for the viral vector, the dual vector-expressing antibodies have higher expression levels than the single vector, and the viral dual vector supernatant antibody concentrations are above 1ng / μL, and the antibody expression level increases with the increase in the light chain ratio. Figure b: When the genome copy numbers of the plasmid vector and the viral vector are similar, whether HEK293FT (lanes 1-7) cells or hARPE-19 cells (lanes 8 and 9) are transfected, the supernatant antibody concentrations are both much lower than those of the viral vector (lane 12), i.e., much lower than 1ng / μL.

[0184] Example 4 Dual rAAV Vector-Mediated Avastin Expression in hiPSC-RPE Cells In this example, recombinant AAV dual vectors were transduced into human induced pluripotent stem cell-differentiated retinal pigment epithelium (hiPSC-RPE) expressing antibodies.

[0185] A. Materials and Methods Retinal pigment epithelial precursor cells (RPE precursor, Zhongsheng Suyuan) were continuously cultured in Matrigel-coated 6-well plates after resuscitation, and the medium contained RPE maintenance (Nuwacell, RP0106-H), Maintenance supplement (Nuwacell, RP01016-H), Blebbistatin, penicillin, and streptomycin, and the medium was changed every 2 days. After 12 days, the cells were passaged once and seeded into 12-well plates at a density of 1.0E+06 cells per well and continued to be cultured in the above medium, and the medium was changed every 2 days. On the 13th day after seeding, a total amount of 1.5E+10vg of virus-transduced cells was added according to the method described in Example 1 and Example 2, and recombinant AAV or recombinant AAV combination was added, respectively. AAV2-Avastin, AAV2-Avastin-H, AAV2-Avastin-L, AAV2-Avastin-L: AAV2-Avastin-H 4:1, AAV2-Avastin-L: AAV2-Avastin-H 2:1, AAV2-Avastin-L:AAV2-Avastin-H 1:1.

[0186] Culture supernatants were collected on days 2, 6, 12, 15, and 18 after transduction, and new medium was added. Avastin antibodies in the supernatants were detected by the Western blot method described in Examples 1 to 3. The sampling amount of the Avastin positive control was 40 ng, the sampling amount of the detection target supernatant was 20 μL, and the sampling buffer was a non-reducing buffer.

[0187] B. Results As shown in Figure 4, Avastin antibody expressed and secreted by hiPSC-RPE cells significantly increased from day 2 after AAV transduction, and the secretion level remained constant when detected on day 18. The expression level of the antibody from dual AAV vector-transduced cells was significantly superior to that of the single AAV vector.

[0188] Example 5. Dual rAAV Vector-Mediated ABTAA Expression in 293 Cells In some examples, dual AAV vectors were used to deliver the anti-Ang2 antibody ABTAA.

[0189] A. Materials and Methods Similar to the construction method of the Avastin transfer plasmid in Example 1, a schematic diagram of the transfer plasmid pAAV2.CMV.ABTAA expressing the complete ABTAA antibody sequence, and the transfer plasmids pAAV2.CMV.ABTAA-L and pAAV2.CMV.ABTAA-H expressing only the light chain or the heavy chain is shown in Figure 5a.

[0190] The target antibody (ABTAA) gene was designed and synthesized in the order of heavy chain leader sequence, heavy chain, F2A, light chain leader sequence, and light chain, and Not I and Hind III enzyme cleavage sites were added upstream and downstream, respectively, and it was constructed between the promoter and poly A of the AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH by enzyme cleavage and ligation, transformed, and clone-amplified to obtain the transfer plasmid pAAV2.CMV.ABTAA of the AAV-ABTAA single vector. ABTAA was obtained by reverse translation and codon optimization with reference to the heavy chain variable region (US 9,902,767 B2 SEQ ID NO: 7) and light chain variable region (US 9,902,767 B2 SEQ ID NO: 9) of SAIT-ANG2-AB-m10D6 in US 9,902,767 B2, and ligated onto the human IgG4 heavy chain constant region and light chain constant region, respectively. Target sequence alignment was performed by Sanger sequencing, and a match indicated that the inserted sequence was completely correct. Here, the sequences used were:

[0191] [Table 2-1] [Table 2-2]

[0192] The construction of the dual vector transfer plasmid (containing the heavy chain transfer plasmid and the light chain transfer plasmid) is similar to the method described in Example 1: Heavy chain transfer plasmid: The heavy chain gene of the target antibody (ABTAA) is designed and synthesized in the order of heavy chain leader sequence, heavy chain, and Not I and Hind III enzyme cleavage sites are added upstream and downstream, respectively. It is constructed between the promoter and poly A of the AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH by enzyme cleavage and ligation, transformed, and clone-amplified to obtain the AAV-ABTAA heavy chain transfer plasmid pAAV2.CMV.ABTAA-H, and the target sequence is aligned by Sanger sequencing. If it is consistent, it indicates that the inserted sequence is completely correct.

[0193] The heavy chain leader sequence and the heavy chain sequence in the heavy chain transfer plasmid are the same as the heavy chain leader sequence and the heavy chain sequence, respectively, in the single vector transfer plasmid.

[0194] Light chain transfer plasmid: The light chain gene of the target antibody (ABTAA) is designed and synthesized in the order of light chain leader sequence and light chain, and Not I and Hind III enzyme cleavage sites are added upstream and downstream, respectively. It is constructed between the promoter and poly A of the AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH by enzyme cleavage and ligation, transformed, and clone-amplified to obtain the AAV-ABTAA light chain transfer plasmid pAAV2.CMV.ABTAA-L, and the target sequence is aligned by Sanger sequencing. If it is consistent, it indicates that the inserted sequence is completely correct.

[0195] The light chain leader sequence and the light chain sequence in the light chain transfer plasmid are the same as the light chain leader sequence and the light chain sequence, respectively, in the single vector transfer plasmid.

[0196] Recombinant AAV packaging and harvesting were performed in accordance with the method of Example 1, with pAAV.R2C2, pAdDelta6, and each of the three transfer plasmids above co-transfected into 2E+08 HEK293FT cells at a molar ratio of 1:1:1 with a total amount of 1040 μg DNA. Recombinant AAV vectors AAV2-ABTAA (single vector) or AAV2-ABTAA-L (light chain vector in dual vector) and AAV2-ABTAA-H (heavy chain vector in dual vector) were harvested 48 hours after transfection, and the viral genome titer of each recombinant AAV vector crude cell lysate was determined by droplet digital PCR (ddPCR).

[0197] 2. Vector transduction and antibody detection HEK293FT cells were transduced with AAV2-ABTAA or with both AAV2-ABTAA-L and AAV2-ABTAA-H, where the ratios of AAV2-ABTAA-L to AAV2-ABTAA-H were 1:4, 1:2, 1:1, 2:1, and 4:1, respectively (based on viral genome titer). Vectors containing only the light chain or only the heavy chain were also transduced and used as controls. 1.2E+05 293FT cells were seeded into each well of a 24-well plate and incubated at 37°C with DMEM / 10% FBS complete medium in 5% CO. 2 The medium was removed, and the viral vector diluted with the above complete medium to a total amount of 7.0E+09 vg was added. The cells were incubated at 37°C in 5% CO 2 After incubation in a culture chamber for 2.5 hours with 0.1 mL of fresh complete medium, the cells were continued to be cultured. Cell culture supernatants were collected 48 hours after transduction. Each sample was diluted with sampling buffer under non-reducing (Fig. 5b) and reducing conditions (Fig. 5c), respectively, and Western blot was performed with goat anti-human IgG antibody (Invitrogen, Cat No: A18811) to detect ABTAA expression.

[0198] B. Results As shown in Figure 5, both single vector transduced and dual vector transduced HEK293FT cells successfully produced ABTAA antibody. Figure b is non-reduced buffer sampling electrophoresis, in which the ABTAA antibody heavy and light chains exist in the form of tetramers, and Figure c is reduced buffer sampling electrophoresis, in which the ABTAA antibody is dissociated and the heavy and light chains exist in the form of single chains. The expression level of dual vector transduced (lanes 2-6) was much higher than that of single vector (lane 1). As the ratio of light chain vector increased, not only the concentration of expressed antibody increased, but also the content of hetero bands due to mispairing decreased.

[0199] Example 6 Dual rAAV Vector-Mediated Bispecific Antibody Expression In this example, dual AAV vectors are used to deliver bispecific antibodies, where the first antibody is ABTAA, the sequence of which is the same as ABTAA described in Example 5, and the second antibody is an anti-VEGF single chain antibody (VEGF scFv). The variable region of the second antibody is linked onto the heavy or light chain of the first antibody and delivered by one AAV vector, and correspondingly, the light or heavy chain of the first antibody is delivered by another AAV vector.

[0200] A. Materials and Methods 1. Vector construction and virus packaging: The first combination scheme: ABTAA-H-G15-VEGF-LH and ABTAA-L dual vector, as shown in Figure 6a. ABTAA-H-G15-VEGF-LH vector, anti-VEGF antibody light chain and heavy chain variable region are linked to ABTAA heavy chain via a linking peptide segment, the gene is designed and synthesized in series in the order of ABTAA heavy chain leader sequence, ABTAA heavy chain, linking peptide, VEGF-scFv-VL-VH, and Not I and Hind III enzyme cleavage sites are added upstream and downstream, respectively, and it is constructed between the promoter and poly A of AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH by enzyme cleavage and ligation, transformed and cloned to obtain the transfer plasmid of ABTAA-H-G15-VEGF-LH, the ABTAA sequence is consistent with that in Example 5, and the heavy chain and light chain variable region of VEGF antibody are constructed by splicing with reference to patent US 6,884,879 B1 SEQ ID NO:7 and NO:8, respectively, and the target sequence alignment is performed by Sanger sequencing, and if it is consistent, it indicates that the inserted sequence is completely correct. where the sequences used are:

[0201] [Table 3-1] [Table 3-2]

[0202] The transfer plasmid for ABTAA-L is the same as the transfer plasmid for the AAV-ABTAA light chain in Example 5, where the sequence used is:

[0203] [Table 4]

[0204] The second combination method: ABTAA-L-E4-VEGF-LH and ABTAA-H dual vector, as shown in Figure 6b. In the ABTAA-L-E4-VEGF-LH vector, the light chain and heavy chain variable region of the anti-VEGF antibody are linked to the light chain of ABTAA through a segment of a linking peptide, and the gene is designed and synthesized in series in the order of ABTAA light chain leader sequence, ABTAA light chain, linking peptide, and VEGF-scFv-VL-VH, and Not I and Hind III enzyme cleavage sites are added upstream and downstream, respectively, and it is constructed between the promoter and poly A of the AAV expression vector pAAV.CMV.PI.EGFP.WPRE.bGH by enzyme cleavage and ligation, and transformed and clone-amplified to obtain the transfer plasmid of ABTAA-L-E4-VEGF-LH, and the target sequence alignment is performed by Sanger sequencing, and if it is consistent, it indicates that the inserted sequence is completely correct. Here, the sequence used is as follows:

[0205] [Table 5]

[0206] The transfer plasmid for ABTAA-H is the same as the transfer plasmid for AAV-ABTAA heavy chain in Example 5, where the sequence used is:

[0207] [Table 6]

[0208] Recombinant AAV packaging and harvesting were consistent with the method of Example 5, where pAAV.R2C2, pAdDelta6 and each of the transfer plasmids listed above were co-transfected into 2E+08 HEK293FT cells at a molar ratio of 1:1:1 with a total amount of 1040 μg DNA. Recombinant AAV vectors ABTAA-H-G15-VEGF-LH, ABTAA-L, ABTAA-L-E4-VEGF-LH, and ABTAA-H were harvested 48 hours post-transfection, and the viral genome titers of each recombinant AAV vector crude cell lysate were determined by droplet digital PCR (ddPCR).

[0209] 2. Vector transduction and antibody detection HEK293FT cells were transduced with ABTAA-H-G15-VEGF-LH in combination with ABTAA-L, or ABTAA-L-E4-VEGF-LH in combination with ABTAA-H, where the ratios of ABTAA light chain (ABTAA-L) to heavy chain (ABTAA-H) vectors were 6:1, 4:1, 2:1, 1:1, 1:2, and 1:4, respectively (based on viral genome titers), and each AAV vector was also used to transduce cells alone. 1.2E+05 293FT cells were seeded into each well of a 24-well plate and incubated at 37°C in DMEM / 10% FBS complete medium in 5% CO. 2 The medium was removed, and the viral vector diluted with the above complete medium to a total amount of 7.0E+09 vg was added. The cells were incubated at 37°C in 5% CO 2 After incubation in a culture box with 0.1% NaCl for 2.5 hours, 1 mL of fresh complete medium was added and continued to culture. Cell culture supernatant was collected 48 hours after transduction. Each sample was diluted with non-reducing sampling buffer, and Western blot was performed with goat anti-human IgG antibody (Invitrogen, Cat No: A18811) to detect ABTAA expression, where the positive control was purified ABTAA antibody.

[0210] B. Results As shown in Figure 6, both AAV vector combination methods can generate ABTAA-VEGF bispecific antibodies. When the second antibody is linked to the first antibody heavy chain, the higher the light chain vector ratio, the higher the antibody expression level, and no obvious mispairing bands were observed, as shown in Figure 6c. When the second antibody is linked to the first antibody light chain, many heterogeneous bands due to mispairing appeared, as shown in Figure 6d, and no obvious heterogeneous bands were observed until the light-heavy chain ratio reached 6:1 (Figure 7d, lane 1). The expression of ABTAA monospecific antibodies (lanes 7 and 8) by dual AAV vectors was more efficient than the expression of bispecific antibodies by dual vectors.

[0211] Example 7: Expression of dual AAV vector-delivered Avastin gene in mice In this example, the dual AAV vector was used to deliver the Avastin antibody gene into mice, and the delivery efficiency was compared with that of a single AAV vector. The AAV serotype was type 8, which is the same as the vector described in Example 2.

[0212] A. Materials and Methods Animal study design: For the study, 18 SPF grade C57BL / 6 mice were selected and randomly divided into 6 groups of 3 mice each, and were injected once via the tail vein with the corresponding total amount of the following vectors or vector combinations at a volume of 100 μL / mouse: AAV8-Avastin 2E+11vg AAV8-Avastin 6E+11vg AAV8-Avastin-L:AAV8-Avastin-H 1:1 2E+11vg AAV8-Avastin-L:AAV8-Avastin-H 1:1 6E+11vg AAV8-Avastin-L:AAV8-Avastin-H 2:1 2E+11vg AAV8-Avastin-L:AAV8-Avastin-H 2:1 6E+11vg

[0213] Mouse sera were collected and prepared before and after administration. The serum collection times were as follows: 0 d before administration of the test animals, and 1 d, 3 d, 7 d, 10 d, 14 d, 21 d, 28 d, 35 d, 49 d, 63 d, and 84 d after the start of administration. Avastin protein concentrations in serum samples were measured using ELISA, and the mean and SD of blood concentrations at each blood sampling point were calculated. Curves at the time of administration were created using GraphPad Prism 6.

[0214] B. Results As shown in Figure 7, Avastin expression could be detected 3 days after single AAV or dual vector was administered to mice, and its blood concentration increased rapidly, then decreased, and finally stabilized, with the concentration peaking around 35 days after injection and reaching a plateau around 45 days. The peak blood concentrations of the high- and low-titer single vector groups were 122.42±33.23μg / mL and 44.21±13.50μg / mL, respectively, and the final stable concentrations were 121.61±6.96μg / mL and 30.54±6.66μg / mL, respectively. When the ratio of light chain to heavy chain of the dual vector is 1:1, the peak blood concentrations of the high and low titer groups are 339.53 ± 37.19 μg / mL and 148.69 ± 16.63 μg / mL, respectively, and the final stable concentrations are 272.69 ± 26.53 μg / mL and 111.84 ± 19.56 μg / mL, respectively.When the ratio of light chain to heavy chain of the dual vector is 2:1, the peak blood concentrations of the high and low titer groups are 417.52 ± 20.35 μg / mL and 166.64 ± 14.08 μg / mL, respectively, and the final stable concentrations are 345.68 ± 26.38 μg / mL and 140.73 ± 34.34 μg / mL, respectively. The blood concentration in the high titer group was significantly higher than that in the low titer group, indicating that there is a good correspondence between the injected vector titer and the antibody expression level. In the same titer group, the dual vectors all showed higher expression levels than the single vectors, and when the total titer was the same but the proportion of light chains was higher than that of heavy chains, the antibody expression level was further improved.

[0215] The embodiments of the present invention are not limited to the above embodiments, and those skilled in the art may make various changes and improvements to the present invention in form and detail without departing from the spirit or scope of the present invention, all of which are deemed to fall within the protection scope of the present invention.

Claims

1. A dual recombinant viral vector system for expressing in a cell a polypeptide molecule that is composed of two different peptide chains and has antigen-binding activity, comprising: a first recombinant viral vector, the genome of which includes a first expression cassette for expressing a first chain of said polypeptide molecule; a second recombinant viral vector whose genome includes a second expression cassette for expressing a second chain of said polypeptide molecule; wherein the first chain and the second chain are distinct from each other, the first recombinant viral vector and the second recombinant viral vector are capable of expressing the first chain and the second chain, respectively, after being introduced into a cell, and the first chain and the second chain are capable of assembling into a polypeptide molecule having antigen-binding activity.

2. 2. The dual recombinant viral vector system of claim 1, wherein the first recombinant viral vector and the second recombinant viral vector are independently selected from the group consisting of a recombinant adeno-associated viral vector, a recombinant adenoviral vector, a recombinant HSV vector, a recombinant lentiviral vector, and a recombinant retroviral vector.

3. the first recombinant viral vector and the second recombinant viral vector are recombinant adeno-associated viral vectors, or the first recombinant viral vector and the second recombinant viral vector are lentiviral vectors; The recombinant viral vector is a recombinant adeno-associated viral vector and comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV Rh10 capsid, or a chimeric AAV capsid or modified capsid; and / or The recombinant adeno-associated virus vector comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAV Rh10; A dual recombinant viral vector system according to claim 1 or 2.

4. 4. The dual recombinant viral vector system of any one of claims 1 to 3, wherein the polypeptide molecule comprises an antigen binding moiety having a heavy chain variable region and a light chain variable region, the first chain comprising the heavy chain variable region of the antigen binding moiety and the second chain comprising the light chain variable region of the antigen binding moiety.

5. 5. The dual recombinant viral vector system of claim 4, wherein the polypeptide molecule comprises an antigen binding moiety having a heavy chain and a light chain, the first chain comprising the heavy chain of the antigen binding moiety and the second chain comprising the light chain of the antigen binding moiety.

6. The dual recombinant viral vector system of claim 4 or 5, wherein the ratio of the first recombinant viral vector to the second recombinant viral vector is 4:1 to 1:8, or 1:1 to 1:

4.

7. 7. The dual recombinant viral vector system of any one of claims 4 to 6, wherein the polypeptide molecule is a monoclonal antibody or antigen-binding fragment thereof, the first chain is a heavy chain of the monoclonal antibody or antigen-binding fragment thereof, and the second chain is a light chain of the monoclonal antibody or antigen-binding fragment thereof.

8. the monoclonal antibody or antigen-binding fragment thereof specifically binds to an angiogenic factor; 8. The dual recombinant viral vector system of claim 7, wherein the angiogenic factor is selected from VEGF, VEGFR, FGF, PDGF, TGFβ, Ang1, Ang2, integrins, CD31 and plasminogen activators.

9. 7. The dual recombinant viral vector system of any one of claims 4 to 6, wherein the polypeptide molecule is a bispecific antibody comprising a first antigen binding moiety that specifically binds to a first antigen epitope and a second antigen binding moiety that specifically binds to a second antigen epitope, the first and second antigen epitopes being distinct, the first antigen binding moiety comprising a heavy chain and a light chain, the first chain comprising the heavy chain of the first antigen binding moiety and the second chain comprising the light chain of the first antigen binding moiety.

10. 10. The dual recombinant viral vector system of claim 9, wherein the second antigen-binding portion is an antibody in a single chain form, the antibody in a single chain form being a single domain antibody or an scFv.

11. the second antigen-binding portion is linked to the N-terminus or C-terminus of the heavy chain of the first antigen-binding portion, the first chain comprising the heavy chain of the first antigen-binding portion and the second antigen-binding portion; Or, 11. The dual recombinant viral vector system of claim 10, wherein the second antigen-binding portion is linked to the N-terminus or C-terminus of the light chain of the first antigen-binding portion, and the second chain comprises the light chain of the first antigen-binding portion and the second antigen-binding portion.

12. 12. The dual recombinant viral vector system of claim 10 or 11, wherein the first chain comprises a heavy chain of a first antigen binding moiety and a second antigen binding moiety, and the second chain comprises a light chain of the first antigen binding moiety.

13. at least one or both of the first and second antigen-binding moieties specifically bind to an angiogenic factor; The dual recombinant viral vector system according to any one of claims 10 to 12, wherein the angiogenic factor is selected from VEGF, VEGFR, FGF, PDGF, TGFβ, Ang1, Ang2, integrins, CD31 and plasminogen activators.

14. 14. The dual recombinant viral vector system of claim 13, wherein the first antigen-binding moiety specifically binds VEGF and the second antigen-binding moiety specifically binds Ang2, or the first antigen-binding moiety specifically binds Ang2 and the second antigen-binding moiety specifically binds VEGF.

15. 7. The dual recombinant viral vector system of claim 4, wherein the polypeptide molecule comprises a first antigen binding moiety that specifically binds to a first antigen epitope and a second binding domain that specifically binds to a second target molecule, the second target molecule being a cytokine, the first antigen binding moiety comprises a heavy chain and a light chain, the first chain comprising the heavy chain of the first antigen binding moiety and the second chain comprising the light chain of the first antigen binding moiety.

16. the second binding domain is a cytokine receptor, an extracellular domain thereof or a variant thereof, the first antigen-binding portion specifically binds to a target PD-1, PD-L1, or an angiogenic factor, and the second binding domain is a receptor or a fragment thereof capable of binding to human transforming growth factor β (TGFβ), Ang2, or VEGF; 16. The dual recombinant viral vector system of claim 15, wherein the angiogenic factor is selected from VEGF, VEGFR, FGF, PDGF, TGFβ, Ang1, Ang2, integrins, CD31 and plasminogen activators.

17. the second binding domain is linked to the N-terminus or C-terminus of the heavy chain of the first antigen-binding moiety, the first chain comprising the heavy chain of the first antigen-binding moiety and the second binding domain; or 17. The dual recombinant viral vector system of claim 15 or 16, wherein the second binding domain is linked to the N-terminus or C-terminus of the light chain of the first antigen binding moiety, and the second chain comprises the light chain of the first antigen binding moiety and the second binding domain.

18. 18. The dual recombinant viral vector system of any one of claims 15 to 17, wherein the first chain comprises a heavy chain and a second binding domain of a first antigen binding moiety and the second chain comprises a light chain of the first antigen binding moiety.

19. A host cell transduced with a dual recombinant viral vector system according to any one of claims 1 to 18.

20. A pharmaceutical composition comprising a dual recombinant viral vector system according to any one of claims 1 to 18 or a cell according to claim 34 and a pharma- ceutically acceptable vector.

21. A method for producing a polypeptide molecule that is composed of two different peptide chains and has antigen-binding activity, comprising the steps of: transducing a host cell with a dual recombinant viral vector system according to any one of claims 1 to 18; and obtaining said polypeptide molecule by culturing said transduced host cells under conditions to cause said host cells to express the first and second chains of said polypeptide molecule, which assemble to produce a polypeptide molecule having antigen-binding activity.

22. A dual recombinant viral vector system described in any one of claims 1 to 18 or a pharmaceutical composition described in claim 20 for delivering a polypeptide molecule which is composed of two different peptide chains and has antigen-binding activity to the body of a subject.

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