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WO2023279803A1 - Rbv的蛋白结合分子及其用途 - Google Patents

Rbv的蛋白结合分子及其用途 Download PDF

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WO2023279803A1
WO2023279803A1 PCT/CN2022/087557 CN2022087557W WO2023279803A1 WO 2023279803 A1 WO2023279803 A1 WO 2023279803A1 CN 2022087557 W CN2022087557 W CN 2022087557W WO 2023279803 A1 WO2023279803 A1 WO 2023279803A1
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seq
protein
rbv
antibody
binding molecule
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PCT/CN2022/087557
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French (fr)
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高光
崔院平
陈宣洪
程远政
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高光
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]

Definitions

  • the present invention relates to the technical field of antibodies, more specifically, it relates to protein-binding molecules of RBV and uses thereof.
  • Rabies virus (“RBV” for short) belongs to the genus Lyssavirus of the family Rhabdoviridae. The shape is elastic, the nucleocapsid is helical and symmetrical, with an envelope on the surface and contains single-stranded RNA. It is the pathogen that causes rabies. Rabies virus has two main antigens: one is the glycoprotein antigen on the outer membrane of the virus, which can combine with acetylcholine receptors to make the virus neurotoxic and produce neutralizing antibodies and hemagglutination inhibitory antibodies in the body.
  • the neutralizing antibody has a protective effect; the other is the inner layer of the nucleoprotein antigen, which can cause the body to produce complement-fixing antibodies and precipitins, and has no protective effect.
  • Rabies is a zoonotic infectious disease caused by the rabies virus.
  • RBV contains 5 major proteins (L, N, G, M1 and M2) and 2 minor proteins (P40 and P43).
  • L protein presents transcription function;
  • N protein is the main nucleoprotein that composes the virion and is the main component of inducing rabies cell immunity, and is often used in the diagnosis, classification and epidemiological research of rabies virus;
  • G protein is the glycoprotein that constitutes the surface filaments of the virus , with the characteristics of agglutinating red blood cells is the structure of the combination of rabies virus and cell receptors, which plays a key role in the pathogenesis and immunity of rabies virus;
  • M1 protein is a specific antigen, and forms a cell surface antigen with M2.
  • Antibodies against the G protein can protect humans from RBV infection by inhibiting the initial stages of the infection cycle mediated by it and neutralizing viral infectivity.
  • RBV enters the human body and reaches the central nervous system along the peripheral afferent nerves. Therefore, bites on the head, neck, upper limbs, etc. and those with large and deep wounds have more chances of developing the disease.
  • RBV is mainly present in the medulla, cerebral cortex, cerebellum and spinal cord of affected animals. Salivary glands and saliva often contain a large number of viruses. Rabies can be caused by biting, scratching or mucous membrane infection by a rabid animal. Under certain conditions, it can also be transmitted through respiratory aerosols.
  • rabies vaccine or anti-rabies immunoglobulin can be used to prevent related diseases caused by RBV infection in the world.
  • Rmab monoclonal antibody
  • This type of antibody has good neutralizing effect, It has the advantages of high safety, low cost, and mass production, but the clinical effect is not particularly good.
  • Single domain antibody Single domain antibody, sdAb, also known as nanobody (nanobody), has only one heavy chain variable region domain (VHH). This domain was originally discovered in a heavy chain antibody (hcAb) isolated from the serum of camelids and sharks, and the VHH fragment was amplified by genetic means. VHH is currently known as the smallest unit that can bind target antigens.
  • Single-domain antibodies have a series of advantages such as simple structure, high affinity and stability, strong tissue penetration and low immunogenicity, and are the latest technology in the field of antibody drugs. At present, the application of single domain antibody technology to solve viral infectious diseases has become the consensus of scientists at home and abroad.
  • Invent a low-cost, high-yield protein molecule that can specifically prevent or treat RBV infection-related diseases, so as to make up for or replace the insufficient production capacity of anti-rabies immunoglobulin in the market and the state of short supply.
  • the present invention provides the following technical solutions:
  • the present invention provides protein binding molecule of RBV, it comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1 is selected from SEQ ID NO:1,4,7,10,13,16,19, CDR2 Selected from SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, CDR3 selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21.
  • the protein binding molecule of RBV comprises:
  • CDR1 as shown in SEQ ID NO: 19
  • CDR2 as shown in SEQ ID NO: 20
  • CDR3 as shown in SEQ ID NO: 21.
  • the protein binding molecules of the RBV include four framework regions FR1, FR2, FR3 and FR4, wherein FR1 is selected from SEQ ID NO: 22, 26, 30, 34, 38, 42, 46, and FR2 is selected from From SEQ ID NO: 23, 27, 31, 35, 39, 43, 47, FR3 is selected from SEQ ID NO: 24, 28, 32, 36, 40, 44, 48, FR4 is selected from SEQ ID NO: 25, 29 , 33, 37, 41, 45, 49.
  • the protein binding molecule of RBV comprises:
  • FR1 as shown in SEQ ID NO: 30, FR2 as shown in SEQ ID NO: 31, FR3 as shown in SEQ ID NO: 32, FR4 as shown in SEQ ID NO: 33; or,
  • FR1 as shown in SEQ ID NO: 34 FR2 as shown in SEQ ID NO: 35, FR3 as shown in SEQ ID NO: 36, FR4 as shown in SEQ ID NO: 37; or,
  • the protein binding molecule of the RBV has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence selected from SEQ ID NO: 50-56. %, 98%, 99%, 100% sequence identity, and can specifically bind G protein.
  • the amino acid sequence of the RBV protein binding molecule is shown in SEQ ID NO: 50-56.
  • the RBV protein binding molecule is a humanized antibody.
  • the protein binding molecule of RBV is a humanized antibody, which includes three complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1 is selected from SEQ ID NO: 64, 67, 70, 73, 76, 79 , CDR2 is selected from SEQ ID NO:65,68,71,74,77,80, CDR3 is selected from SEQ ID NO:66,69,72,75,78,81.
  • the RBV protein binding molecule is a humanized antibody comprising:
  • the protein binding molecule of the RBV is a humanized antibody comprising four framework regions FR1, FR2, FR3 and FR4, wherein FR1 is selected from SEQ ID NO: 82, 86, 90, 94, 98, 102, FR2 is selected from SEQ ID NO:83,87,91,95,99,103, FR3 is selected from SEQ ID NO:84,88,92,96,100,104, FR4 is selected from SEQ ID NO:85,89 , 93, 97, 101, 105.
  • the RBV protein binding molecule is a humanized antibody comprising:
  • the protein-binding molecule of RBV is a humanized antibody, which has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence selected from SEQ ID NO: 106-111. %, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the protein-binding molecule of RBV is a humanized antibody whose amino acid sequence is shown in SEQ ID NO: 106-111.
  • the RBV protein binding molecule is an antibody comprising an immunoglobulin single variable domain.
  • the immunoglobulin single variable domain is a heavy chain variable region domain.
  • the protein binding molecule of RBV includes an immunoglobulin Fc region.
  • the RBV protein-binding molecule binds RBV protein less than 1 ⁇ 10 -7 M.
  • the present invention also provides nucleic acid molecules encoding the above-mentioned protein-binding molecules of RBV.
  • the nucleic acid molecule encoding the protein binding molecule of RBV has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the nucleic acid molecules encoding the protein-binding molecules of RBV are shown in SEQ ID NO: 57-63 and 112-117.
  • the present invention also provides a host cell expressing the above-mentioned RBV protein-binding molecules.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned RBV protein-binding molecule and one or more pharmaceutically acceptable excipients.
  • the RBV protein-binding molecules, detection kits, and pharmaceutical compositions of the present invention are used in the preparation of medicines for treating and/or preventing and/or diagnosing diseases related to RBV infection.
  • the invention uses the phage display library technology to screen the protein-binding molecule against rabies virus with high specificity, high affinity and high stability.
  • the protein-binding molecule is aimed at the G protein of RBV, and the RBV protein-binding molecule is an antibody comprising an immunoglobulin single variable domain, which has better anti-RBV virus compared with the amino acid sequence and antibody of the prior art neutralizing inhibitory function.
  • Figure 1 is a diagram of the amplification of VHH fragments of a single domain antibody
  • Fig. 2 is the diversity analysis of phage display library
  • Figures 3, 4, and 5 are for the selection of phage monoclonals for Phage ELISA identification
  • Figures 6, 7, and 8 are the phage ELISA of the fourth round of panning products of the phage display library cell panning;
  • Figures 9, 10, and 11 show flow cytometric detection after expression of the constructed G protein single domain antibody:
  • Figure 12 shows pDonor-CMV-G protein-puro plasmid structure
  • Figure 13 is a schematic diagram of the expression vector Lenti-hIgG1-Fc2;
  • Figure 14 is flow cytometry detection of antibody binding to CHO cells after humanization
  • Figure 15 is the affinity flow detection after expression of the humanized G protein single domain antibody
  • Figure 16 Flow cytometric detection of G protein recombinant cell lines
  • Figure 17 Flow cytometry detection of binding of humanized antibody to CHO-GM004 cells
  • Figure 18 Flow cytometry detection of the binding of antibodies to CHO cells after humanization
  • the abscissa values in Figure 9 are 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 along the positive direction; Figure 10, Figure 11, Figure 14, Figure 15, Figure 16, Figure 10 17.
  • the values of the abscissa in Figure 18 are the same as those in Figure 9.
  • antibody or “immunoglobulin” used interchangeably herein, whether referring to heavy chain antibodies or conventional 4 chain antibodies, are used as generic terms to include full length antibodies, individual antibodies thereof, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments such as VHH domains or VH/VL domains, respectively).
  • sequence as used herein (e.g.
  • immunoglobulin sequence in terms of “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) is to be understood generally Unless a more restrictive interpretation is required herein, both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding said sequence are included.
  • domain refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, domains are responsible for individual functional properties of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or domain.
  • immunoglobulin domain refers to a globular region of an antibody chain (eg, a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or to a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by their maintenance of the immunoglobulin fold characteristic of antibody molecules, consisting of a 2-layer sandwich of approximately seven antiparallel beta-sheet strands, optionally stabilized by conserved disulfide bonds, arranged in two beta-sheets .
  • immunoglobulin variable domain refers to a domain substantially defined in the art and hereinafter referred to as "framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3”, respectively.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to an antibody for an antigen by having an antigen-binding site.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding an antigenic epitope without pairing with another immunoglobulin variable domain.
  • An example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody”, eg an immunoglobulin single variable domain VH and VL (VH domain and VL domain).
  • Another example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") of Camelidae as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments, and VHH antibodies, are antigen-binding immunoantibodies known as "heavy chain antibodies” (ie, “antibodies lacking light chains")
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments, and VHH antibodies, are antigen-binding immunoantibodies known as “heavy chain antibodies” (ie, "antibodies lacking light chains")
  • Variable domains of globulins Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of lightchains"; Nature 363, 446-448 (1993) ).
  • VHH domain is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as the "VH domain”) and the variable domain present in conventional 4-chain antibodies.
  • the variable domain of the light chain (which is referred to herein as the "VL domain”) is distinguished.
  • a VHH domain specifically binds an epitope without the need for another antigen binding domain (in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • - FR1 comprises amino acid residues at positions 1-30
  • - CDR1 comprises amino acid residues at positions 31-35
  • - FR2 comprises amino acids at positions 36-49,
  • - CDR2 comprises amino acid residues at positions 50-65
  • - FR3 comprises amino acid residues at positions 66-94
  • - CDR3 comprises amino acid residues at positions 95-102, and
  • - FR4 comprises amino acid residues at positions 103-113.
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e. according to One or more positions of the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows).
  • numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • the total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domains and polypeptides containing them can be summarized as follows:
  • VHH domains (which have been naturally "designed” to bind antigen functionally in the absence and interaction of light chain variable domains) can serve a single and relatively small function Antigen-binding structural units, domains or polypeptides. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suitable for practical use as a single antigen-binding protein or as a single variable domain of an immunoglobulin, but require some kind of form or another combination to provide a functional antigen binding unit (eg in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain).
  • a functional antigen binding unit eg in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain.
  • VHH domains - alone or as part of a larger polypeptide - offers many advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments)
  • VHH domain can be expressed from a single gene and does not require post-translational folding or modification
  • VHH domains can be easily engineered into multivalent and multispecific formats (formatting);
  • -VHH domains are highly soluble and have no tendency to aggregate
  • VHH domains are highly stable to heat, pH, proteases and other denaturing agents or conditions, and thus can be prepared, stored or transported without the use of refrigeration equipment, thereby achieving cost, time and environmental savings;
  • VHH domain is relatively small compared to conventional 4-chain antibodies and antigen-binding fragments thereof (approximately 15 kDa or 1/10 the size of conventional IgG), thus exhibiting a higher Tissue penetration and can be administered in higher doses;
  • VHH domains can display so-called cavity binding properties (especially due to their extended CDR3 loop compared to conventional VH domains), allowing access to targets and epitopes inaccessible to conventional 4-chain antibodies and antigen-binding fragments thereof.
  • VHH domains derived from Camelidae can be modified by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a human conventional 4-chain antibody.
  • Humanization also referred to herein as “sequence optimization", in addition to humanization, “sequence optimization” may also cover other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as the removal of potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences, and in a specific embodiment, may contain the human framework region sequences of IGHV3.
  • domain antibody also known as “Dab” and “dAb”
  • Dab domain antibody
  • dAb dAb
  • the antigen-binding properties need to be assessed, e.g. by using a library of human single VH or VL domain sequences. Specific options.
  • domain antibodies have a molecular weight of about 13 kDa to about 16 kDa and, if derived from fully human sequences, do not need to be humanized for, eg, human therapeutic use. As in the case of VHH domains, domain antibodies are also well expressed in prokaryotic expression systems, thereby significantly reducing overall manufacturing costs.
  • Domain antibodies have been disclosed, for example, in: Ward, E.S., et al.: "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli"; Nature 341:544-546 (1989); Holt, L.J. et al. : “Domain antibodies: proteins for therapy”; TRENDS in Biotechnology 21(11):484-490 (2003).
  • epitope or the term “antigenic determinant” used interchangeably refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Antigenic determinants usually comprise chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be a "linear " epitope or "conformational” epitope. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed.
  • Epitopes for a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • linear epitopes can be determined, for example, by simultaneously synthesizing a large number of peptides on a solid support, wherein these peptides correspond to parts of the protein molecule, and allowing these peptides, while still attached to the support, to antibody response.
  • These techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci.
  • conformational epitopes can be identified by determining the spatial configuration of amino acids such as by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See eg Epitope Mapping Protocols (ibid).
  • Antibodies can be competitively screened for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for antigen binding. A high-throughput method to obtain antibodies binding to the same epitope based on their cross-competition is described in International Patent Application WO 03/48731. Therefore, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present invention for binding to the same epitope on the RBV protein can be obtained using routine techniques known to those skilled in the art.
  • the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (eg, immunoglobulin single variable domain of the invention) molecule can bind.
  • the specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity.
  • the affinity represented by the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the greater the distance between the epitope and the antigen-binding molecule.
  • affinity can also be expressed as an association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner, depending on the particular antigen of interest.
  • Avidity is a measure of the strength of binding between an antigen-binding molecule (eg, an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing the same) and the associated antigen.
  • Avidity is related to both the affinity between its antigen binding site on the antigen binding molecule and the number of associated binding sites present on the antigen binding molecule.
  • RBV protein binding molecule means any molecule capable of specifically binding an RBV protein.
  • a RBV protein binding molecule may comprise an antibody as defined herein against a RBV protein or a conjugate thereof.
  • RBV protein-binding molecules also encompass so-called “SMIPs” ("Small Modular Immunopharmaceuticals”), or immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.
  • SIPs Small Modular Immunopharmaceuticals
  • IgSF immunoglobulin superfamily antibodies
  • RBV protein binding molecule may alternatively refer to a monovalent molecule that binds the F protein of RBV (i.e., a molecule that binds to one epitope of the F protein of RBV), as well as a bivalent or multivalent binding molecule (i.e., a molecule that binds to more than one epitope). molecular).
  • the "RBV protein binding molecule” of the present invention may comprise at least one immunoglobulin single variable domain, such as VHH, that binds an RBV protein.
  • an "RBV protein binding molecule” of the invention may comprise two immunoglobulin single variable domains, such as VHH, that bind an RBV protein.
  • RBV protein binding molecules that contain more than one immunoglobulin single variable domain are also referred to as "formatted” RBV protein binding molecules.
  • Formatted RBV protein binding molecules may also comprise linkers and/or moieties with effector functions, such as half-life extending moieties (such as immunoglobulin single variable domains that bind serum albumin), in addition to RBV protein-binding immunoglobulin single variable domains. variable domain), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region.
  • the "RBV protein binding molecules" of the invention also encompass bispecific antibodies, which contain immunoglobulin single variable domains that bind different antigens.
  • the RBV protein-binding molecules of the invention will be expressed at preferably 10 ⁇ 7 to 10 ⁇ 11 moles/liter (M), more preferably 10 ⁇ 8 to 10 ⁇ 11 moles/liter, or even more, as measured in a Biacore or KinExA assay.
  • M moles/liter
  • KD dissociation constant
  • KA association constant
  • Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., as described herein). Radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay.
  • SPR surface plasmon resonance
  • RIA Radioimmunoassay
  • EIA enzyme immunoassay
  • amino acid residues will be referred to according to the standard three-letter or one-letter amino acid codes as known and agreed upon in the art.
  • amino acid difference refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence as compared to another sequence.
  • substitution will preferably be a conservative amino acid substitution, which means that an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and its effect on the function, activity or other biological properties of the polypeptide Little or no effect.
  • a conservative amino acid substitution is preferably that an amino acid in the following groups (i)-(v) is replaced by another amino acid residue in the same group: (i) smaller Aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) Polar positively charged residues: His, Arg, and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
  • sequence identity For determination of sequence identity see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 Sequence Analysis Primer, and Devereux, J., eds., M Stockton Press, New York, 1991.
  • a polypeptide or nucleic acid molecule is considered “substantially isolated” when it is separated from another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component).
  • a polypeptide or nucleic acid molecule is considered “substantially isolated” when it has been purified by at least 2-fold, especially at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more.
  • a "substantially isolated” polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique (eg, a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).
  • Affinity matured anti-RBV protein antibodies particularly VHH or domain antibodies, have one or more changes in one or more CDRs that result in an affinity for the RBV protein compared to their respective parental anti-RBV Protein antibodies increased.
  • Affinity matured anti-RBV protein antibodies can be prepared, for example, by methods known in the art as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad.
  • subject means a mammal, especially a primate, especially a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the active compound ie, antibody molecule, immunoconjugate
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, to the extent they are incompatible with the active compounds, are possible in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be incorporated into the compositions.
  • compositions generally must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.
  • Sterile injectable solutions can be prepared by mixing the active compound in the required amount in an appropriate solvent and, if necessary, adding one or a combination of ingredients enumerated above, followed by sterile microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with carrier materials to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, and pharmaceutically acceptable combination of carriers.
  • Dosage regimens can be adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce in association with the required pharmaceutical carrier. desired therapeutic effect.
  • dosages range from about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of recipient body weight.
  • dosages may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or within the range of 1-20 mg/kg.
  • Exemplary treatment regimens entail weekly dosing, once every two weeks, once every three weeks, once every four weeks, once monthly, once every 3 months, once every 3-6 months, or an initial dosing interval Slightly shorter (eg once a week to once every three weeks) followed by longer dosing intervals (eg monthly to once every 3-6 months).
  • the antibody molecule can also be administered as a sustained release formulation, in which case less frequent dosing is required. Dosage and frequency vary according to the half-life of the antibody molecule in the patient. In general, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. Dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively lower doses are administered at infrequent intervals over a prolonged period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient exhibits partial or complete amelioration of disease symptoms. Thereafter, the patient can be dosed in a prophylactic regimen.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the therapeutic Duration, other drugs, compounds and/or materials used in conjunction with the particular composition employed, age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.
  • a "therapeutically effective amount" of an RBV protein binding molecule of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of injury or disability resulting from disease affliction.
  • a "therapeutically effective amount” preferably inhibits viral growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 99%.
  • the ability to inhibit viral growth can be assessed in animal model systems predictive of efficacy against RBV inhibition.
  • a therapeutically effective amount therapeutically relieves symptoms in a subject can be determined by those skilled in the art depending on factors such as the size of the subject, the severity of the subject's symptoms and the particular composition or route of administration chosen.
  • compositions of the present invention may be administered by one or more routes of administration using one or more methods known in the art.
  • routes of administration include nebulized inhalation, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or nebulized inhalation.
  • the present invention provides the use and method of the RBV protein binding molecules, nucleic acid molecules, host cells, immunoconjugates and pharmaceutical compositions of the present invention in the prevention and/or treatment of diseases related to RBV infection.
  • One aspect of the present invention provides a method for preventing and/or treating an RBV infectious disease in a subject, comprising administering the RBV protein-binding molecule of the present invention to the subject, so that the RBV infectious disease of the subject is prevented and/or treat.
  • the RBV protein used for immunization was expressed by CHO cells (expression vector Lenti-CMV-puro, prepared according to conventional methods, and purified by nickel column affinity chromatography to obtain RBV protein.
  • An American llama (alpaca) was selected for immunization. 6 times After the immunization, 100 mL of peripheral blood was extracted from American llamas for the construction of phage display library. 5 mL of peripheral blood was collected, and the centrifuge tubes with collected blood samples were placed in a 37°C incubator for 1 hour; then the blood samples were Transfer to 4°C overnight; 2. Transfer the serum to a new sterile centrifuge tube, centrifuge at 5000rpm for 20min; use ELISA to detect the immune titer, and the results are shown in the table below.
  • sequence serum dilution ratio OD value 1 1:1k 2.748 2 1:2k 2.57 3 1:4k 2.583 4 1:8k 2.421 5 1:16k 2.287 6 1:32k 2.00
  • an antigen expression vector is constructed.
  • the constructed G protein-Fc protein expression vector was subjected to plasmid extraction, and after transiently transfecting CHO cells, cultured continuously for 10 days, centrifuged to collect the culture supernatant, filtered with a 0.45 ⁇ m filter membrane, and the filtrate was transferred to a sterile centrifuge tube , the antibody was purified using a Protein A column.
  • the full-length G protein was synthesized by gene and subcloned into the lentiviral expression vector Lenti-CMV-puro to construct the antigen overexpression vector.
  • the constructed Lenti-CMV-G protein (Full)-puro vector was packaged with lentivirus, infected with CHO-S cells, and screened with puromycin 48 hours later to construct CHO-GM004 recombinant cell lines.
  • the constructed CHO-G protein recombinant cell line and CHO cells were incubated with Rafivirumab positive antibody (1 ⁇ g/10 6 cell) respectively. After incubation for 1 hour, PBS was washed 3 times; PE-anti-Human IgG (1:200) was added and protected from light.
  • a recombinant cell line that overexpresses the target protein incubate the phage library with empty cells and cells that overexpress the target protein in sequence, and after several times of washing to remove non-specifically bound phage, use glycine or TEA to bind to the cell surface
  • the recombinant phages were eluted and amplified; after 3-4 rounds of panning, single clones were selected for ELISA detection.
  • M13KO7 volume 10 x volume x OD600 x 5x 10 8 /M13KO7 titer; put the infected bacterial solution on a shaker, 37°C, 225rpm for 45min; put the bacterial solution in a centrifuge, centrifuge at 4000rpm for 10min, discard Remove the supernatant, resuspend with 2YT-AK medium, 800 ⁇ l per well, reset in the shaker, 30°C, 210rpm overnight culture; at the same time, the ELISA plate was shaken off the antigen, washed three times with PBST washing solution, washed with 3% MPBS was blocked with 250 ⁇ l/well, overnight at 4°C; and an additional blank plate was blocked as BLANK; the next day, the 96-well deep-well plate was placed in a centrifuge and centrifuged at 4000rpm for 10min; the milk in the ELISA plate was discarded and used Wash 4 times with 200 ⁇ L PBST; first add 50
  • VHH antibody sequences obtained from the analysis were separately gene-synthesized and subcloned in tandem with human IgG1Fc into the expression vector Lenti-hIgG1-Fc2 (vector schematic diagram 13). After the vector was verified to be correct by sequencing, the endotoxin-free plasmid was prepared using the Qiagen plasmid extraction kit.
  • LVTransm transfection reagent and the antibody expression vector Lenti-hIgG1-Fc2 Take out the LVTransm transfection reagent and the antibody expression vector Lenti-hIgG1-Fc2 from the refrigerator, thaw at room temperature, and mix completely by blowing up and down with a pipette gun. Remove the PBS buffer and warm to room temperature. Take 500 ⁇ L LVTransm to one well of a 24-well plate, add 4 ⁇ g Lenti-hIgG1-Fc2, pipette up and down to mix well, add 12 ⁇ L LVTransm, immediately pipette up and down to mix, and let stand at room temperature for 10 minutes. The mixture here is called DNA/LVTransm complex.
  • virus suspension Take CVS-11 virus seeds (usually freeze-dried virus seeds) for appropriate dilution, inoculate well-growing BSR cells with 0.1 MOI infection dose, and culture them at 37°C and 5% carbon dioxide for 1 day. Transfer to 34°C to continue culturing. After 2 days, collect the culture supernatant, centrifuge at 4,000 rpm for 10 minutes at 4°C to remove cell debris, take the supernatant and add 10% newborn bovine serum, mix well and divide into small tubes, -70 Store below °C for later use.
  • Pre-titration of the virus solution Take 1 tube of the frozen virus suspension, thaw it quickly under running water, and make a 5-fold serial dilution starting from 1:5 on a 24-well culture plate, take 100 ⁇ L of the virus solution and add 400 ⁇ L containing 10% In the DMEM culture solution of infested newborn bovine blood, mix thoroughly, transfer 50 ⁇ L of each dilution to a 96-well culture plate, make 2 parallel copies of each dilution, and add 5x10 6 /ml BSR cells to each well 50 ⁇ L of the suspension was incubated at 37°C and 5% carbon dioxide for 24 hours.
  • Virus titer 20X (average value of fluorescence focus in the highest dilution factor well x5 + mean value of fluorescence focus in adjacent wells with lower dilution factor)/2x virus dilution factor in the lower well
  • Preparation of virus liquid for neutralization take 1 tube of virus suspension, and operate in the same way as the pre-titration of virus liquid. The proportion of fluorescent foci in each well was counted under a fluorescent microscope, and the virus dilution at which 80% to 95% of the cells were infected by the virus was used as the virus dilution for the neutralization test.
  • Samples to be tested were serially diluted 3-fold with DMEM culture medium containing 10% inactivated neonatal bovine serum, that is, pre-added 100 ⁇ L to each well of a 96-well culture plate for culture Take 50 ⁇ L of the sample to be tested and add it, which is a 1:3 dilution. After mixing well, draw 50 ⁇ L and add it to the 100 ⁇ L culture medium in the next well to become a 1:9 dilution. Serially dilute several wells to an appropriate dilution. , 50 ⁇ L in the last well was discarded.
  • the D10 sequence of the single domain antibody was screened for humanization.
  • the homology model of the antibody was obtained through modeling, and the distance between CDRs was analyzed by using abysis software.
  • a range of framework amino acids as well as rare amino acids, these amino acid positions often affect the conformation or antigen-binding activity of the CDR.
  • the human germline is obtained by IMGT analysis, and after splicing the selected human germline framework with the CDRs of the antibody, compare the framework region sequences of the designed humanized antibody and the original antibody to find out the framework region sequences of the two
  • IMGT analysis the framework region sequences of the designed humanized antibody and the original antibody to find out the framework region sequences of the two
  • the homology modeling results of the parental antibody determine whether these amino acid positions with differences will affect the conformation or antigen-binding activity of the CDR.
  • amino acids similar to those on the surface of human antibodies were selected for replacement, and humanized antibodies (HM-D10-1 to HM-D10-6) were designed.
  • the humanized antibodies designed above were gene-synthesized and subcloned in tandem with human IgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc2 ( Figure 13). After the vector was verified to be correct by sequencing, the endotoxin-free plasmid was prepared using the Qiagen plasmid extraction kit.
  • the humanized antibody expression vector was transiently transfected into 293F cells for small-scale expression, the culture supernatant was collected, and flow cytometry was used to detect the binding of the humanized antibody to the antigen on the cell membrane surface of recombinant cells CHO and CHO-GM004. See Figure 18.
  • the humanized antibodies HM1 and HM4 both specifically bind to the recombinant CHO-native G protein cells, and the binding force is equivalent to that of the D10 antibody.
  • the GM004 recombinant protein was immobilized on the CM5 chip with 10mM Acetate buffer, and the HM1 and HM4 positive humanized antibodies and the original antibody D10 were used as the mobile phase respectively to detect the binding ability of the antibody to the target protein GM004 before and after humanization, as shown in Figure 19. Shown, affinity test results:
  • the single-domain antibodies HM1 and HM4 obtained in the previous project were connected in series with human IgG1Fc to construct a bivalent antibody expression vector (HM1-ggggsggggsggggs-HM4-IgG1Fc); the humanized antibody HM4 antibody sequence and human IgG1FC region were used to construct a bivalent antibody expression vector (HM4-ggggsggggsggggs-HM4-IgG1Fc); gene synthesis was performed separately, and subcloned into the Lenti-hIgG1-Fc2-Puro vector to construct a bivalent antibody expression vector. After the vector was verified to be correct by sequencing, the endotoxin-free plasmid was prepared using the Qiagen plasmid extraction kit.
  • the amino acid sequence of the HM1 antibody is shown in SEQ ID NO.106
  • the amino acid sequence of the HM4 antibody is shown in SEQ ID NO.109;
  • the experimental method is as 2.4
  • Embodiment 6 animal pharmacodynamics verification
  • Virus CVS10 strain, which was inoculated into the brain of mice for infection.
  • mice (15-16 g) were adaptively cultured in the animal room for 1 day in advance.
  • the experimental results showed that: the survival rate of the test product and immunoglobulin in the experimental group was 100%, while the survival rate of the Virus group was 0.

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Abstract

提供一种预防或治疗RBV感染相关疾病的蛋白分子，包括利用噬菌体展示库技术筛选得到的针对狂犬病毒的蛋白结合分子。所述蛋白结合分子针对RBV的G蛋白，该分子包含一免疫球蛋白单一可变结构域的抗体。

Description

RBV的蛋白结合分子及其用途 技术领域
本发明涉及抗体技术领域，更具体地说，它涉及RBV的蛋白结合分子及其用途。
背景技术
狂犬病毒(简称“RBV”)属于弹状病毒科(Rhabdoviridae)狂犬病毒属(Lyssavirus)。外形呈弹状，核衣壳呈螺旋对称，表面具有包膜，内含有单链RNA。是引起狂犬病的病原体，狂犬病毒具有两种主要抗原：一种是病毒外膜上的糖蛋白抗原，能与乙酰胆碱受体结合使病毒具有神经毒性，并使体内产生中和抗体及血凝抑制抗体，中和抗体具有保护作用；另一种为内层的核蛋白抗原，可使体内产生补体结合抗体和沉淀素，无保护作用。狂犬病是由狂犬病毒引起的人畜共患的传染病。
RBV含有5种主要蛋白(L、N、G、M1和M2)和2种微小蛋白(P40和P43)。L蛋白呈现转录作用；N蛋白是组成病毒粒子的主要核蛋白是诱导狂犬病细胞免疫的主要成分，常用于狂犬病病毒的诊断、分类和流行病学研究；G蛋白是构成病毒表面纤突的糖蛋白，具有凝集红细胞的特性是狂犬病病毒与细胞受体结合的结构，在狂犬病病毒致病与免疫中起着关键作用；M1蛋白为特异性抗原，并与M2构成细胞表面抗原。针对G蛋白的抗体可以抑制其介导的感染周期的起始阶段并中和病毒的感染性从而保护人体免受RBV感染。
RBV进入人体，沿周围传入神经而到达中枢神经系统，因此头、颈部、上肢等处咬伤和创口面积大而深者发病机会多。RBV主要存在于患病动物的延脑、大脑皮层、小脑和脊髓中。唾液腺和唾液中也常含有大量病毒，人被患狂犬病的动物咬伤、抓伤或经粘膜感染均可引起狂犬病，在特定条件下也可以通过呼吸道气溶胶传染。
目前世界范围内才采用注射狂犬疫苗或抗狂犬病免疫球蛋白可用于预防RBV感染引起的相关疾病，只有印度2016年上市了一个名为Rmab治疗狂犬病的单抗，这类抗体具有中和效果好、安全性高、成本低、可大量生产优点，但在临床上效果并不是特好。由于抗狂犬病免疫球蛋白产能不足，且价格昂贵，国内持续处于供不应求状态，因而发展一种成本低、产能高且可以特异性地预防或治疗RBV感染相关疾病的抗体药物是当务之急。
单域抗体(single domain antibody，sdAb)，又称纳米抗体(nanobody)，仅有一个重链可变区结构域(VHH)。该结构域最初发现于骆驼科动物和鲨鱼的血清中分离出的一种重链抗体(heavy chain antibody，hcAb)，通过基因手段，扩增出其中的VHH片段。VHH是目前己知的可结合目标抗原的最小单位。单域抗体具有结构简单，亲和力及稳定性高，组织渗透力强和免疫原性低等一系列优点，是抗体药物领域里的最新技术。目前，应用单域抗体技术来解决病毒感染类疾病已成为国内外科学家的共识。
发明内容
发明一种成本低、产能高且可以特异性地预防或治疗RBV感染相关疾病的蛋白分子，弥补或者取代市场上抗狂犬病免疫球蛋白产能不足，供不应求的状态。
为实现上述目的，本发明提供了如下技术方案：
在一个方面，本发明提供了RBV的蛋白结合分子，其包括三个互补决定区CDR1、CDR2和CDR3，其中CDR1选自SEQ ID NO：1、4、7、10、13、16、19，CDR2选自SEQ ID NO：2、5、8、11、14、17、20，CDR3选自SEQ ID NO：3、6、9、12、15、18、21。
在一些实施方案中，所述RBV的蛋白结合分子包括：
1)如SEQ ID NO：1所示的CDR1、如SEQ ID NO：2所示的CDR2、如SEQ ID NO：3所示的CDR3；或者，
2)如SEQ ID NO：4所示的CDR1、如SEQ ID NO：5所示的CDR2、如SEQ ID NO：6所示的CDR3；或者，
3)如SEQ ID NO：7所示的CDR1、如SEQ ID NO：8所示的CDR2、如SEQ ID NO：9所示的CDR3；或者，
4)如SEQ ID NO：10所示的CDR1、如SEQ ID NO：11所示的CDR2、如SEQ ID NO：12所示的CDR3；或者，
5)如SEQ ID NO：13所示的CDR1、如SEQ ID NO：14所示的CDR2、如SEQ ID NO：15所示的 CDR3；或者，
6)如SEQ ID NO：16所示的CDR1、如SEQ ID NO：17所示的CDR2、如SEQ ID NO：18所示的CDR3；或者，
7)如SEQ ID NO：19所示的CDR1、如SEQ ID NO：20所示的CDR2、如SEQ ID NO：21所示的CDR3。
在一些实施方案中，所述RBV的蛋白结合分子包括四个框架区FR1、FR2、FR3和FR4，其中FR1选自SEQ ID NO：22、26、30、34、38、42、46，FR2选自SEQ ID NO：23、27、31、35、39、43、47，FR3选自SEQ ID NO：24、28、32、36、40、44、48，FR4选自SEQ ID NO：25、29、33、37、41、45、49。
在一些实施方案中，所述RBV的蛋白结合分子包括：
1)如SEQ ID NO：22所示的FR1、如SEQ ID NO：23所示的FR2、如SEQ ID NO：24所示的FR3、如SEQ ID NO：25所示的FR4；或者、
2)如SEQ ID NO：26所示的FR1、如SEQ ID NO：27所示的FR2、如SEQ ID NO：28所示的FR3、如SEQ ID NO：29所示的FR4；或者、
3)如SEQ ID NO：30所示的FR1、如SEQ ID NO：31所示的FR2、如SEQ ID NO：32所示的FR3、如SEQ ID NO：33所示的FR4；或者、
4)如SEQ ID NO：34所示的FR1、如SEQ ID NO：35所示的FR2、如SEQ ID NO：36所示的FR3、如SEQ ID NO：37所示的FR4；或者、
5)如SEQ ID NO：38所示的FR1、如SEQ ID NO：39所示的FR2、如SEQ ID NO：40所示的FR3、如SEQ ID NO：41所示的FR4；或者、
6)如SEQ ID NO：42所示的FR1、如SEQ ID NO：43所示的FR2、如SEQ ID NO：44所示的FR3、如SEQ ID NO：45所示的FR4；或者、
7)如SEQ ID NO：46所示的FR1、如SEQ ID NO：47所示的FR2、如SEQ ID NO：48所示的FR3、如SEQ ID NO：49所示的FR4。
在一些实施方案中，所述RBV的蛋白结合分子与选自SEQ ID NO：50～56的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同一性，且能够特异性结合G蛋白。优选的，所述RBV的蛋白结合分子的氨基酸序列如SEQ ID NO：50～56所示。
在一些实施方案中，所述RBV的蛋白结合分子为人源化抗体。
在一些实施方案中，所述RBV的蛋白结合分子为人源化抗体，其包括三个互补决定区CDR1、CDR2和CDR3，其中CDR1选自SEQ ID NO：64、67、70、73、76、79，CDR2选自SEQ ID NO：65、68、71、74、77、80，CDR3选自SEQ ID NO：66、69、72、75、78、81。
在一些实施方案中，所述RBV的蛋白结合分子为人源化抗体，其包括：
1)如SEQ ID NO：64所示的CDR1、如SEQ ID NO：65所示的CDR2、如SEQ ID NO：66所示的CDR3；或者，
2)如SEQ ID NO：67所示的CDR1、如SEQ ID NO：68所示的CDR2、如SEQ ID NO：69所示的CDR3；或者，
3)如SEQ ID NO：70所示的CDR1、如SEQ ID NO：71所示的CDR2、如SEQ ID NO：72所示的CDR3；或者，
4)如SEQ ID NO：73所示的CDR1、如SEQ ID NO：74所示的CDR2、如SEQ ID NO：75所示的CDR3；或者，
5)如SEQ ID NO：76所示的CDR1、如SEQ ID NO：77所示的CDR2、如SEQ ID NO：78所示的CDR3；或者，
6)如SEQ ID NO：79所示的CDR1、如SEQ ID NO：80所示的CDR2、如SEQ ID NO：81所示的CDR3。
在一些实施方案中，所述RBV的蛋白结合分子为人源化抗体，其包括四个框架区FR1、FR2、FR3和 FR4，其中FR1选自SEQ ID NO：82、86、90、94、98、102，FR2选自SEQ ID NO：83、87、91、95、99、103，FR3选自SEQ ID NO：84、88、92、96、100、104，FR4选自SEQ ID NO：85、89、93、97、101、105。
在一些实施方案中，所述RBV的蛋白结合分子为人源化抗体，其包括：
1)如SEQ ID NO：82所示的FR1、如SEQ ID NO：83所示的FR2、如SEQ ID NO：84所示的FR3、如SEQ ID NO：85所示的FR4；或者、
2)如SEQ ID NO：86所示的FR1、如SEQ ID NO：87所示的FR2、如SEQ ID NO：88所示的FR3、如SEQ ID NO：89所示的FR4；或者、
3)如SEQ ID NO：90所示的FR1、如SEQ ID NO：91所示的FR2、如SEQ ID NO：92所示的FR3、如SEQ ID NO：93所示的FR4；或者、
4)如SEQ ID NO：94所示的FR1、如SEQ ID NO：95所示的FR2、如SEQ ID NO：96所示的FR3、如SEQ ID NO：97所示的FR4；或者、
5)如SEQ ID NO：98所示的FR1、如SEQ ID NO：99所示的FR2、如SEQ ID NO：100所示的FR3、如SEQ ID NO：101所示的FR4；或者、
6)如SEQ ID NO：102所示的FR1、如SEQ ID NO：103所示的FR2、如SEQ ID NO：104所示的FR3、如SEQ ID NO：105所示的FR4。
在一些实施方案中，所述RBV的蛋白结合分子为人源化抗体，其与选自SEQ ID NO：106～111的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同一性。优选的，所述RBV的蛋白结合分子为人源化抗体的氨基酸序列如SEQ ID NO：106～111所示。
在一些实施方案中，所述的RBV的蛋白结合分子为包含一免疫球蛋白单一可变结构域的抗体。
在一些实施方案中，免疫球蛋白单一可变结构域为一重链可变区结构域。
在一些实施方案中，所述的RBV的蛋白结合分子，其包括免疫球蛋白Fc区。
在一些实施方案中，所述的RBV的蛋白结合分子，其结合RBV蛋白小于1×10 -7M。
在一些实施方案中，其中所述RBV蛋白分子形成多价链接。
本发明还提供了编码上述RBV的蛋白结合分子的核酸分子。
在一些实施方案中，编码所述RBV的蛋白结合分子的核酸分子与选自SEQ ID NO：57～63和112～117的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同一性。优选的，编码所述RBV的蛋白结合分子的核酸分子如SEQ ID NO：57～63和112～117所示。
在另一个方面，本发明还提供了一种宿主细胞，其表达上述所述的RBV的蛋白结合分子。
在另一个方面，本发明还提供了一种药物组合物，其包括上述所述的RBV的蛋白结合分子和一种或多种药学上可接受的赋形剂。
在另一个方面，本发明的RBV的蛋白结合分子、检测试剂盒、药物组合物在制备用于治疗和/或预防和/或诊断与RBV感染相关疾病的药物中的用途。
本发明利用噬菌体展示库技术筛选得到的具有高特异性、高亲和力和高稳定性的针对狂犬病毒的蛋白结合分子。所述蛋白结合分子针对RBV的G蛋白，所述RBV蛋白结合分子为包含一免疫球蛋白单一可变结构域的抗体，与现有技术的氨基酸序列和抗体相比，具有更好的针对RBV病毒的中和抑制功能。
附图说明
图1为单域抗体VHH片段扩增图；
图2为噬菌体展示库多样性分析；
图3、4、5为挑选噬菌体单克隆，进行Phage ELISA鉴定；
图6、7、8为噬菌体展示库cell panning第四轮淘选产物phage ELISA；
图9、10、11为构建的G蛋白单域抗体表达后流式检测：
图12显示pDonor-CMV-G protein-puro质粒结构；
图13为表达载体Lenti-hIgG1-Fc2示意图；
图14为流式细胞仪检测人源化后抗体与CHO细胞的结合情况；
图15为人源化G蛋白单域抗体表达后亲和力流式检测；
图16G蛋白重组细胞株流式检测；
图17流式细胞仪检测人源化后抗体与CHO-GM004细胞的结合情况；
图18流式细胞仪检测人源化后抗体与CHO细胞的结合情况；
图19人源化抗体HM1和HM4和原始抗体D10亲和力检测；
图20双价抗体的表达纯化SDS-PAGE检测；
Lane M:蛋白marker
Lane 1:Lenti-HM4-HM4-hIgG1-Fc2MW～54kd
Lane 2:Lenti-HM1-HM4-hIgG1-Fc2MW～54kd
Lane 3:Lenti-HM4-HM4-HisMW～30kd
Lane 4:Lenti-HM1-HM4-HisMW～30kd
图21生存率曲线图；
其中，图9的横坐标数值沿正方向依次为10 0、10 1、10 2、10 3、10 4、10 5、10 6；图10、图11、图14、图15、图16、图17、图18的横坐标数值与图9相同。
具体实施方式
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人,“MolecularCloning:A Laboratory Manual”(第2版),第1-3卷,Cold Spring Harbor LaboratoryPress(1989)；Lewin,“Genes IV”，Oxford University Press,New York,(1990)；及Roitt等人,“Immunology”(第2版),Gower Medical Publishing,London,New York(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用，术语(多肽或蛋白的)“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或结构域的功能。
如本文所用的术语“免疫球蛋白结构域”是指抗体链(例如常规4链抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征，其由排列在两个β折叠中任选由保守二硫键稳定的约7个反平行β折叠股的2层夹层组成。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
如本文所用的术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情 况下特异性结合抗原表位的免疫球蛋白可变结构域。本发明含义中的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH及VL(VH结构域及VL结构域)。免疫球蛋白单一可变结构域的另一实例为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH结构域、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,SongaEB,Bendahman N,Hamers R.:“Naturally occurring antibodies devoid of lightchains”；Nature 363,446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本发明的上下文中，术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”以及“Nanobody○R”及“Nanobody○R结构域”(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。
例如Riechmann及Muyldermans,J.Immunol.Methods 231,25-38(1999)中所示，对于骆驼科的VHH结构域所应用的氨基酸残基，根据Kabat等人给出的VH结构域的一般编号法来编号(“Sequence of proteins of immunological interest”,US PublicHealth Services,NIH Bethesda,MD,公开案第91号)。根据该编号法，
-FR1包含在位置1-30处的氨基酸残基，
-CDR1包含在位置31-35处的氨基酸残基，
-FR2包含在位置36-49处的氨基酸，
-CDR2包含在位置50-65处的氨基酸残基，
-FR3包含在位置66-94处的氨基酸残基，
-CDR3包含在位置95-102处的氨基酸残基，且
-FR4包含在位置103-113处的氨基酸残基。
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述替代方法还可以类似地应用于VHH结构域。然而，除非另有说明，否则在本说明书、权利要求书及附图中，将遵循如上所述的根据Kabat且适于VHH结构域的编号。
VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本文所述的目的。
VHH结构域及含有其的多肽的其他结构特性及功能性质可总结如下：
VHH结构域(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。此区分VHH结构域与常规4链抗体的VH及VL结构域，这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于这些独特性质，使用VHH结构域—单独或作为较大多肽的一部分—提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：
-仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；
-VHH结构域可自单一基因表达且不需要翻译后折叠或修饰；
-VHH结构域可容易地改造成多价及多特异性格式(格式化)；
-VHH结构域高度可溶且无聚集趋势；
-VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；
-VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；
-VHH结构域与常规4链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；
-VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.vander Linden et al.,Journal of Immunological Methods,240(2000)185–195；Lietal.,J Biol Chem.,287(2012)13713–13721；Deffar et al.,African Journal ofBiotechnology Vol.8(12),pp.2645-2652,17June,2009和WO94/04678。
源自骆驼科的VHH结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而经“人源化”(本文中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其他修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列，且在一具体实施方案中，可含IGHV3的人框架区序列。
如本文所用，术语“结构域抗体”(亦称为“Dab”及“dAb”)特别用于指代非骆驼科哺乳动物的抗体(特别是人4链抗体)的VH或VL结构域。为了以单一抗原结合结构域的形式(即在不与VL域或VH域分别配对的情况下)结合表位，需要例如通过使用人单一VH或VL结构域序列的文库对所述抗原结合性质进行具体选择。
与VHH一样，结构域抗体的分子量为约13kDa至约16kDa，且若源自完全人序列，则不需要进行人源化以供例如人治疗使用。正如在VHH结构域的情况下，结构域抗体在原核表达系统中也很好地得以表达，从而显著降低总制造成本。
“结构域抗体”已公开于例如以下文献中：Ward,E.S.,等人:“Bindingactivities of a repertoire of single immunoglobulin variable domains secretedfrom Escherichia coli”；Nature 341:544-546(1989)；Holt,L.J.等人:“Domainantibodies:proteins for therapy”；TRENDS in Biotechnology 21(11):484-490(2003)。
此外，本领域技术人员还将了解，有可能将一个或多个上述CDR“移植”于其他“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。
如本文所用，术语“表位”或可互换使用的术语“抗原决定簇”指抗体的互补位所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。参见，例如，Epitope Mapping Protocols in Methodsin Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位。参见例如Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。举例而言，线性表位可通过例如以下方法来确定：在固体支持物上同时合成大量肽，其中这些肽对应于蛋白质分子的各部分，且使这些肽在仍然与支持物连接的情况下与抗体反应。这些技术在本领域中为已知的且描述于例如美国专利第4,708,871号；Geysen等人(1984)Proc.Natl.Acad.Sci.USA81:3998-4002；Geysen等人(1986)Molec.Immunol.23:709-715中。类似地，构象表位可通过诸如通过例如x射线结晶学及2维核磁共振确定氨基酸的空间构形加以鉴别。参见例如Epitope Mapping Protocols(同上)。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位 的抗体的高通量方法描述于国际专利申请WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本发明的抗体分子竞争结合RBV蛋白上的相同表位的抗体及其抗原结合片段。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)分子可结合的不同类型抗原或表位的数目。可基于抗原结合分子的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合分子之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合分子(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合分子上的抗原结合位点之间的亲和力，以及存在于抗原结合分子上的相关结合位点的数目。
如本文所用，术语“RBV蛋白结合分子”意指任何能够特异性结合RBV蛋白的分子。RBV蛋白结合分子可以包括针对RBV蛋白的如本文定义的抗体或其缀合物。RBV蛋白结合分子还涵盖所谓的“SMIP”(“小模块免疫药物”)，或者免疫球蛋白超家族抗体(IgSF)或CDR移植分子。
“RBV蛋白结合分子”或者可以指结合RBV的F蛋白的单价分子(即与RBV的F蛋白的一个表位结合的分子)，以及二价或多价结合分子(即结合一个以上表位的结合分子)。本发明的“RBV蛋白结合分子”可以包含至少一个结合RBV蛋白的免疫球蛋白单一可变结构域如VHH。在一些实施方案中，本发明的“RBV蛋白结合分子”可以包含两个结合RBV蛋白的免疫球蛋白单一可变结构域如VHH。含有一个以上的免疫球蛋白单一可变结构域的RBV蛋白结合分子亦称为“格式化的”RBV蛋白结合分子。格式化的RBV蛋白结合分子除结合RBV蛋白的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中，本发明的“RBV蛋白结合分子”还涵盖双特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。
通常，本发明的RBV蛋白结合分子将以如于Biacore或KinExA测定中测量的优选10 -7至10 -11摩尔/升(M)、更优选10 -8至10 -11摩尔/升、甚至更优选10 -9至10 -11、甚至更优选10 -10至10 -11或更低的解离常数(KD)，和/或以至少10 7M-1、优选至少10 8M-1、更优选至少10 9M-1，更优选至少10 10M -1、例如至少10 11M -1的缔合常数(KA)结合所要结合的抗原(即RBV蛋白)。任何大于10 -4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。“序列相似性”指示相同或代表保守氨基酸取代的氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST 程序来确定相同性。对于序列相同性的确定，可以参见例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics andGenome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysisof Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,NewJersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,AcademicPress,1987Sequence Analysis Primer,Gribskov,M.andDevereux,J.,eds.,MStockton Press,New York,1991。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“基本上分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“基本上分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“基本上分离的”多肽或核酸分子优选基本上为均质的。
“亲和力成熟”的抗RBV蛋白抗体，特别是VHH或结构域抗体，在一个或多个CDR中具有一个或多个变化，所述变化导致对RBV蛋白的亲和力相比于其各自的亲本抗RBV蛋白抗体有所增加。亲和力成熟的抗RBV蛋白抗体可通过例如由以下所述的本领域中已知的方法来制备：Marks等人,1992,Biotechnology 10:779-783或Barbas等人,1994,Proc.Nat.Acad.Sci,USA 91:3809-3813.；Shier等人,1995,Gene 169:147-155；Yelton等人,1995,Immunol.155:1994-2004；Jackson等人,1995,J.Immunol.154(7):3310-9；及Hawkins等人,1992,J.MoI.Biol.226(3):889896；KS Johnson及RE Hawkins,“Affinitymaturation of antibodies using phage display”,Oxford University Press 1996。
如本文所用的术语“对象”意指哺乳动物，尤其灵长类动物，尤其是人。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即抗体分子、免疫缀合物包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用。
这些组合物还可含有佐剂，如防腐剂、润湿剂、乳化剂和分散剂。
药学上可接受的载体包括无菌水溶液或分散液和用于临时制备无菌注射液或分散液的粉末剂。这些用于药学活性物质的介质和试剂的使用是本领域公知的。常规介质或试剂，除了任何与活性化合物不相容的范围外，都可能在本发明的药物组合物中。还可以向组合物中掺入补充的活性化合物。
治疗性组合物一般必须是无菌的并且在制备和贮存条件下稳定的。可以将组合物配制成溶液、微乳状液、脂质体或其他适合高药物浓度的有序结构。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液态聚乙二醇等)及其合适的混合物的溶剂或分散剂。例如，通过使用包衣，例如卵磷脂，在分散液的情况下通过保持所需的颗粒大小，以及通过使用表面活性剂，可以保持适当的流动性。
通过将活性化合物以需要的量混入合适的溶剂中，并且根据需要加入以上列举的成分中的一种或其组合，接着无菌微过滤，可制备无菌注射液。通常，通过将活性化合物掺入到含有基本分散介质和上面所列其他所需成分的无菌载体中制备分散剂。对于用于制备无菌注射液的无菌粉末剂，优选的制备方法是真空干燥和冷冻干燥(冻干)，由其预先无菌过滤的溶液得到活性成分加任何额外所需成分的粉末。
可以与载体材料组合制备单一剂量形式的活性成分的量根据所治疗的对象和特定给药方式而不同。可以与载体材料组合制备单一剂量形式的活性成分的量一般是产生治疗效果的组合物的量。通常，以100％计，这个量的范围是大约0.01％至大约99％的活性成分，优选大约0.1％至大约70％，最优选大约1％至大约30％的活性成分，与药学上可接受的载体相组合。
可以调节剂量方案以提供最佳的期望的反应(例如，治疗反应)。例如，可以施用单一推注，可以随时间施用几次分开的剂量，或者根据治疗状况的紧急情况所需，可以按比例减小或增加剂量。特别有利的是将肠胃外组合物配制成容易给药并且剂量均匀的剂量单位形式。此处使用的剂量单位形式是指适合作为单位剂量用于所治疗的对象的物理不连续单位；每个单位含有预定量的活性化合物，经计算该预定量的活性化合物与需要的药物载体组合产生所需的治疗效果。
对于抗体分子的给药而言，剂量范围为约0.0001至100mg/kg，更通常为0.01至20mg/kg受者体重。例如，剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重，10mg/kg体重或20mg/kg体重，或在1-20mg/kg范围内。示例性的治疗方案需要每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。
或者，抗体分子也可以作为持续释放制剂来给药，在此情况中需要频率较低的给药。剂量和频率根据抗体分子在患者中的半衰期而不同。通常，人抗体表现出最长的半衰期，之后是人源化抗体、嵌合抗体和非人类抗体。给药剂量和频率根据处理是预防性的还是治疗性的而不同。在预防性应用中，在长时间内以较不频繁的间隔给予相对较低的剂量。有些患者在余生中持续接受处理。在治疗性应用中，有时需要以较短的间隔给予较高的剂量，直到疾病的进展减轻或停止，优选直到患者表现为疾病症状部分或完全改善。之后，可以以预防性方案给患者给药。
本发明药物组合物中活性成分的实际剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物或其酯、盐或酰胺的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
本发明的RBV蛋白结合分子的“治疗有效量”优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于RBV相关疾病的治疗，相对于未接受治疗的对象，“治疗有效量”优选地将病毒生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约99％。抑制病毒生长的能力可以在预测对抑制RBV的疗效的动物模型系统中评价。或者，也可以通过检查抑制RBV生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。治疗有效量的治疗性缓解对象的症状。本领域技术人员可以根据如下因素确定这种量，如对象的大小、对象症状的严重性和选择的特定组合物或给药途径。
本发明的组合物可以利用本领域公知的一种或多种方法通过一种或多种给药途径给药。本领域技术人员应当理解，给药途径和/或方式根据期望的结果而不同。本发明RBV蛋白结合分子的优选给药途径包括雾化吸入、静脉内、肌肉内、皮内、腹膜内、皮下、脊柱或其他肠胃外给药途径，例如注射或雾化吸入。
疾病预防和治疗：本发明提供了本发明所述RBV蛋白结合分子、核酸分子、宿主细胞、免疫缀合物及药物组合物在预防和/或治疗RBV感染的相关的疾病中用途和方法。本发明的一方面提供一种预防和/或治疗对象中的RBV感染性疾病的方法，包括给该对象施用本发明的RBV蛋白结合分子，使得所述对象的RBV感染性疾病得到预防和/或治疗。
具体实施例：
实施例1：针对RBV蛋白的单域抗体的筛选
1.1免疫和免疫后血清效价测定
免疫用的RBV蛋白由CHO细胞表达(表达载体Lenti-CMV-puro，按照常规方法制备，经镍柱亲和层析纯化得到RBV蛋白。选取一只美洲无峰驼(alpaca)进行免疫。6次免疫结束后，采取美洲无峰驼提取100mL外周血，用于噬菌体展示库的构建。采集5ml外周血，将收集有血液样本的离心管置于37℃培养箱内放置1小时；然后将血液样本转移至4℃过夜；2.将血清转移至一个新的无菌离心管中，5000rpm离心20min；采用ELISA检测免疫效价，结果见下表所示。
序列 血清稀释比列 OD值
1 1:1k 2.748
2 1:2k 2.57
3 1:4k 2.583
4 1:8k 2.421
5 1:16k 2.287
6 1:32k 2.00
7 1:64k 1.655
8 PBS 0.11
注：稀释比例越高，OD值越高，代表效价越高，可以用于后续的筛选实验。
1.2 RBV G蛋白-FC抗原制备
通过基因合成G蛋白的胞外段(20AA-458AA)序列，在C端添加human IgG1Fc标签，且G蛋白和Fc之间添加肠激酶酶切位点，用于去除FC标签；亚克隆至真核表达载体中，构建抗原表达载体。将构建好的G蛋白-Fc蛋白表达载体进行质粒大抽，瞬时转染CHO细胞后，连续培养10天，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，使用Protein A柱子纯化抗体。
1.3 G蛋白重组细胞株筛选
通过基因合成G蛋白的全长，亚克隆至慢病毒表达载体Lenti-CMV-puro，构建抗原过表达载体。将构建好的Lenti-CMV-G蛋白(Full)-puro载体包装慢病毒，感染CHO-S细胞，48小时后使用嘌呤霉素进行筛选，构建CHO-GM004重组细胞株。构建好的CHO-G蛋白重组细胞株和CHO细胞分别孵育Rafivirumab阳性抗体(1μg/10 6cell)，孵育1h后，PBS清洗3次；加入PE-anti-Human IgG(1:200)，避光孵育45min，PBS清洗3次后，使用500ul PBS重悬细胞，进行流式检测，结果见图16所示，可以看出G蛋白重组细胞株可以用于筛选阳性抗体。
1.4 PBMC分离及VHH抗体片段的获得
采集50ml外周血，使用淋巴细胞分离液分选PBMC。提取RNA，使用PrimeScript TM II 1st Strand cDNA Synthesis Kit进行反转录，制备cDNA。再进行VHH片段的扩增，扩增PCR后采用琼脂糖电泳回收750bp产物，在进行第二轮扩增，其产物采用琼脂糖电泳检测并回收产物，大小为400bp，结果见图1所示。
1.5噬菌体展示库的构建
1)菌体展示载体的构建
SfiI分别酶切pCom F载体和上述获得的VHH PCR胶回收产物，50℃酶切过夜。使用1％的琼脂糖凝胶分离pCom F载体片段，切取5000bp的载体片段进行胶回收；同时使用DNA片段回收试剂盒纯化PCR酶切产物，并用NanoDrop测定浓度。酶切好的pCom F载体和VHH片段使用T4ligase连接，16℃连接过夜。
2)噬菌体连接产物电转化大肠杆菌
准备电转杯、连接产物和电转感受态置于冰上预冷；取预冷的建库连接产物加入到电转感受态中，置于冰上1min，向每个电转杯中加入70μL DNA/感受态混合物，将电转杯放于冰上；按照2500V，5ms进行电转；电击结束后，立刻加入平衡至室温的SOC培养基重悬菌体，37℃摇床培养1小时。取菌液15mL直接进行噬菌体拯救，其余5mL电转产物加入等体积的50％甘油，均匀混合后保存在-80℃。另外取菌液20μL，加入980μL 2YT培养基进行稀释后，取100μL稀释后产物，加入900μL 2YT培养基进行第二步稀释，取50μL均匀涂布在含有氨苄青霉素的LB平板上，37℃培养过夜。第二天，取出平板，计算每个连接能够产生的克隆数，计算库容。同时挑取平板上的单克隆20个到含氨苄青霉素的2YT培养基中，37℃震荡培养约6-8小时，送菌液测序(测序通用引物M13R)，看库的多样性，见图2所示，序列差异代表多样性，可以看出多样性好。
3)噬菌体展示库的复苏和拯救，噬菌体沉淀
将电转后的转化产物用2YT稀释调整至OD600为0.2左右，加入终浓度为100ug/mL的氨苄青霉素置于恒温摇床中，37℃，225rpm培养，至OD600为0.5时停止；投入M13KO7，摇匀后37℃静置30min后，37℃，225rpm培养1h。M13KO7体积＝10 x体积x OD600 x 5 x 108/M13KO7滴。将菌液6000rpm离心10min后用2YT-AK培养基重悬，25℃，200rpm过夜培养；将菌液10000rpm离心15min；弃去沉淀，将上清转至新的离心管，管内加入1/5菌液体积的PEG/NaCl，混匀后放置4℃，静置2h。将沉淀的噬菌体上清，10000rpm，4℃，离心30min；弃去上清，每个50ml离心管的沉淀(噬菌体)用1ml无菌PBS重悬；将重悬的噬菌体转移至1.5mLEP管中，置于离心机中，12000g，4℃，离心5min；将上清转移至新的1.5mlEP管，每管加入250μl的PEG/NaCl，混匀后4℃静置10min。12000g离心10min，弃上清，加入1ml PBS重悬。12000g离心5min，弃沉淀，将上清转移至新的1.5mlEP管。2000g离心5mi，将上清转移 至新的1.5mlEP管，得到噬菌体原始库。取10μl沉淀投入90μl 2YT培养基，记做10 -1，依次往后10倍稀释至10 -9，取10 -7、10 -8、10 -9三个梯度20μl稀释好的样品投入200μl事先准备好的OD600为0.5的ER2738，混匀后放于37℃水浴锅，静置10min，每108μl涂1块LB-AMP固体平板，37℃过夜，第二天数斑以确定Titer。Titer计算：选取斑数在30-300之间的平板，两块平板取平均值，斑数量乘以稀释倍数再乘以100得到titer，见图3所示，说明库容量大。
4)噬菌体展示库的固相panning
使用ELISA板，包被目的蛋白，经历数次洗脱步骤后，使用TEA将结合在固定抗原上的重组噬菌体洗脱下来，并进行扩增；经历3-4轮淘选后，挑选单克隆进行测序。
用PBS稀释抗原至50μg/ml，150μl/well，共包被3well，置于4℃包被过夜；吸去target蛋白，用3％MPBS室温封闭1h；将lib phage或上轮扩增的沉淀用450μl 3％MPBS稀释，将6x10 11Pfu的phage(每孔2x10 11Pfu)投入到对照蛋白包被孔中，每孔投入150μl的稀释好的phage，室温孵育1h；吸去target蛋白的MPBS，并将对照孔中的phage转至包被target蛋白的孔中，室温孵育1-1.5h；将phage吸去，用0.05％PBST洗涤8-10次，每次2-3min，再用PBS洗涤4-5次，每次2-3min，同时用PBS洗涤事先封闭的1.5ml EP管；用1xTEA洗脱6～8min，每孔200μl，收集洗脱下来的产物至预先封闭过的EP管中，每孔再加入100μl Tris-HCl中和；取10μl output产物投入90μl 2YT培养基，记做100，依次往后10倍稀释至10 -2，取10 1、10 0、10 -1、10 -2四个梯度20μl稀释好的样品投入200μl事先准备好的OD600为0.5的ER2738(10 1是直接将未稀释的产物20μl加入ER2738)，混匀后放于37℃水浴锅，静置10min，每108μl涂1块LB-AMP固体平板，37℃过夜，第二天数斑以确定Titer。Titer计算：选取斑数在30-300之间的平板，两块平板取平均值，用斑数量乘以稀释倍数再乘以洗脱体积，见图4所示，说明在扩增。
5)噬菌体展示库的cell-based panning
使用过表达靶蛋白的重组细胞株，将噬菌体库依次与空细胞和过表达靶蛋白的细胞进行孵育后，经数次洗涤洗去非特异性结合的噬菌体后，使用甘氨酸或TEA将结合在细胞表面上的重组噬菌体洗脱下来，并进行扩增；经历3-4轮淘选后，挑选单克隆进行ELISA检测。
提前一天用1％PBSA封闭1.5ml EP管，4℃过夜；Target cell和control cell各取1x10 7，用PBS洗涤三次，用10mL1％PBSA重悬，放置于脱色摇床室温低速封闭1h；向control cell中投入1x10 11的噬菌体，孵育1h，target cell继续封闭；将孵育结束的细胞置于离心机中，1000g离心5min，弃去target cell上清(1％PBSA)，并将control cell的上清小心吸出，加入到target cell管中，重悬后，置于摇床，低速孵育1h；将target cell置于离心机中，1000g离心5min；(同时用PBS洗涤三次昨天封闭的1.5ml EP管)，弃去target cell上清，加入4ml PBS重悬，分装至封闭的1.5ml EP管，用PBS离心洗涤5次，再将细胞转移至另外四个EP管，继续洗涤5次，然后将细胞转移至同一个EP管中；用200μl PBS重悬细胞，然后加入200μl 2xTEA迅速吹吸，直至溶液不再粘稠，再加入200μl Tris-HCl中和，即得到产物；测定Output库的titer，同固相panning方案，见图5所示，可以看出库容量在富集。
6)Phage ELISA
分装2YT-Amp培养基至96孔深孔板，每孔500μl，挑取output平板上的单克隆，37℃，225rpm培养至OD600直至0.5；最后两孔H11和H12不挑克隆，只放培养基，作为空白对照；同时用CBS包被抗原至ELISA板，浓度1μg/ml，100μl/well，37℃，包被2h；另取一块96孔深孔板，分装2YT-A培养基，每孔500μl，用排枪依次吸取OD600为0.5的菌液10μl至新分装的96孔板中，置于37℃，225rpm培养过夜，此为送样测序菌液；向OD600为0.5的菌液中，加入M13KO7，混匀后放于37℃，静置15min；
M13KO7体积＝10 x体积x OD600 x 5x 10 8/M13KO7滴度；将侵染结束的菌液置于摇床上，37℃，225rpm培养45min；将菌液置于离心机中，4000rpm离心10min，弃去上清，用2YT-AK培养基重悬，每孔800μl，重置于摇床中，30℃，210rpm过夜培养；同时ELISA板甩掉抗原，用PBST洗液洗三遍后，用3％MPBS封闭250μl/well，4℃过夜；并额外封闭一块空白板，作为BLANK；第二天，将96孔深孔板置于离心机中，4000rpm离心10min；将ELISA板中的牛奶弃去，用200μL PBST洗涤4次；在每孔先加入50μl PBST，再一一对应加入50μL离心后的噬菌体上清，置4℃孵育1h；弃去上清，并用PBST洗涤5次；用PBST稀释HRP-Anti M13二抗，每孔100μl，4℃孵育45min后，洗去二抗，PBST洗5次，TMB 常温显色10min，盐酸终止，读数，选取S/N比大的值的克隆，用保种的菌液送测；见图6～8所示，S/N值越大越好。
实施例2：针对RBV的单域抗体的初步评价鉴定
2.1单域抗体的表达载体的构建和表达
根据噬菌体单克隆Elisa检测结果，挑选阳性克隆进行测序，获得15个VHH抗体序列。将分析获得的VHH抗体序列分别进行基因合成，与human IgG1Fc串联亚克隆至表达载体Lenti-hIgG1-Fc2中(载体示意图13)。载体经测序验证无误后，使用Qiagen质粒大抽试剂盒制备去内毒素质粒备用。
从冰箱中取出LVTransm转染试剂及抗体表达载体Lenti-hIgG1-Fc2，室温解冻后，用移液枪上下吹打完全混匀。取出PBS缓冲液，温热至室温。取500μLPBS至24孔板的一个孔，加入4μg Lenti-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入12μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。此处的混合物称为DNA/LVTransm复合物。将上述532μL DNA/LVTransm复合物加入到1.5mL CHO细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO2培养箱，130RPM培养6～8小时后，加入1.5mL新鲜的FreeStyle TM 293培养基，将细胞重新放回培养箱中继续培养。连续培养3天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，进行后续的流式和ELISA检测。
2.2流式检测重组抗体与靶蛋白的结合
从液氮中复苏CHO和CHO-GM004细胞株，调整细胞状态至对数生长期；将两种细胞分别分为若干份，每份细胞的数量为5*10 5个细胞；将表达的抗体分别孵育靶细胞，充分混匀后，室温孵育1小时；800xg室温离心5分钟，去掉含有抗体的上清，使用PBS洗涤细胞3次；加入1μL PE标记的Anti-human IgG，充分混匀后，室温避光孵育30分钟；800xg室温离心5分钟，去掉含有二抗的上清，使用PBS洗涤细胞3次；使用500uL PBS重悬细胞，进行流式分析。
2.3单域抗体的表达纯化
从冰箱中取出LVTransm转染试剂及单链抗体表达载体，室温解冻后，用移液枪上下吹打完全混匀。取出PBS或HBSS缓冲液，温热至室温。取2mL PBS至6孔板的一个孔，分别加入130μg Lenti-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入400μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。
将上述DNA/LVTransm复合物加入到50mL CHO细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO2培养箱，130RPM培养6～8小时后，加入50mL新鲜的FreeStyle TM 293培养基，将细胞重新放回培养箱中继续培养。连续培养7天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，使用Protein A柱子纯化抗体。采用流式检测如图9～11所示，获得了7个候选抗体。
Figure PCTCN2022087557-appb-000001
2.4候选抗体的细胞中和抑制实验
RBV抗体中和抑制试验快速荧光灶抑制试验法。
实验步骤：
(一)中和用病毒的制备
1)病毒悬液的制备：取CVS-11毒种(一般为冻干毒种)做适当稀释，以0.1MOI感染量接种生长良好的BSR细胞，于37℃、5％二氧化碳条件下培养1天后转入34℃继续培养，2天后收集培养上清液，于4℃以每分钟4000转离心10分钟去除细胞碎片，取上清液加入10％新生牛血清，混匀后分装小管，-70℃以下冻存备用。
2)病毒液的预滴定：取冻存的病毒悬液1支，经流水速融后，在24孔培养板上从1:5开始做5倍系列稀释，取100μL病毒液加入400μL含10％灭能新生牛血淸的DMEM培养液中，充分混匀后，每个稀释度取50μL转移至96孔培养板，每个稀释度平行做2份，每孔再加入5x10 6/ml的BSR细胞悬液50μL，于37℃、5％二氧化碳条件下培养24小时。培养结束后弃上清液，PBS洗1遍，再加入80％冷丙酮，每孔50μL，4℃固定30分钟，或-30℃固定10分钟，弃丙酮，待挥发干燥后每孔加入50μL工作浓度的FITC标记的狂犬病病毒核蛋白抗体染色，于37℃孵育30分钟，用PBS洗3遍，甩干，每孔加80％甘油50μL,于荧光显微镜下观察；计数每孔中的荧光灶数，取每孔荧光灶数在30以下的孔，记录相邻4孔荧光灶数，取其平均值，计算如下：
病毒滴度(FFU/ml)＝20X(最高稀释倍数孔荧光灶平均值x5+相邻孔稀释倍数较低的荧光灶平均值)/2x稀释倍数较低孔病毒稀释倍数
3)中和用病毒液的制备：取病毒悬液1支，按病毒液的预滴定同法操作。在荧光显微镜下计数每孔中的荧光灶比例，以80％～95％的细胞被病毒感染的病毒稀释度为中和试验用病毒稀释度。
取冻存的病毒悬液1支，经流水融化后，用含5％灭能新生牛血清的DMEM培养液将病毒稀释至中和试验用病毒稀释度，置冰浴备用。
4)狂犬病免疫球蛋白标准品/阳性对照抗体工作液的制备：用含10％灭能新生牛血清的DMEM培养液将狂犬病免疫球蛋白标准品/阳性对照抗体做3倍系列稀释，即在96孔培养板中每孔预先加入100μL培养液，取50μL标准品/阳性对照抗体加入其中，成为1:3稀释度，充分混合后，吸取50μL加入下一孔100μL培养液中，成为1:9稀释度，如此系列稀释若干孔至适宜稀释度。
(二)待测样本工作液的制备
待测样本(抗体样品应预先经56℃、30分钟灭能)用含10％灭能新生牛血清的DMEM培养液做3倍系列稀释，即在96孔培养板中每孔预先加人100μL培养液，取50μL待测样本加入其中，即为1:3稀释度，充分混合后，吸取50μL加人下一孔100μL培养液中，成为1:9稀释度，如此系列稀释若干孔至适宜稀释度，最后一孔中50μL弃去。
(三)测定方法
将稀释后的标准品/阳性对照抗体及待测样本各孔中加入中和用病毒液，50μL/孔，同时设正常细胞对照孔(只加100μL DMEM于孔中)，以及中和用病毒对照孔(含5％灭能新生牛血清的DMEM100μL,加入中和用病毒50μL,)，混匀后置37℃中和1小时，每孔加入Ix10 6个/ml的BSR细胞悬液50μL，于37℃、5％二氧化碳条件下培养24小时。待培养结束吸干培养液，每孔中加入PBS100μL清洗并吸干后，每孔加人预冷至4℃的80％丙酮50μL，4℃固定30分钟，或-30℃固定10分钟，弃丙酮，待挥发干燥后加人工作浓度的荧光标记狂犬病病毒核蛋白抗体，50μL/孔，37℃孵育30分钟，弃去液体，用PBS洗板2～3次，甩干，每孔加人80％甘油50μL,荧光显微镜下观察。
最终获得的结果如下表：
序号 名称 规格 结果
1 SDAB19051501-4-H8纯化抗体 1.36mg/ml,400μl 543.4
2 SDAB19051501-D10纯化抗体 1.55mg/ml,400μl 183250.3
3 SDAB1905150-2-43纯化抗体 1.68mg/ml,300μl 395.2
4 SDAB1905150-2-52纯化抗体 1.49mg.ml,400μl 365.9
5 SDAB1905150-2-59纯化抗体 0.98mg/ml,500μl 329.8
6 SDAB1905150-2-60纯化抗体 0.66mg/ml,400μl 951.5
7 SDAB1905150-2-79纯化抗体 0.96mg/ml,500μl 318.7
对照抗体1 Rafivirumab抗体 2.1mg/mL,200μl 1613
对照抗体2 Foravirumab抗体 2.5mg/mL,200μl 17759.6
注：结果数值越大，代表中和抑制能力越好，从结果可以看出，SDAB19051501-D10抗体是阳性对照抗体Rafivirumab的113.6倍，是阳性对照抗体Foravirumab的10.3倍。
实施例3：人源化抗体
3.1人源化抗体基因合成及表达载体构建
根据细胞中和抑制实验结果筛选单域抗体D10序列做人源化，首先通过建模获得该抗体的同源模型，结合abysis软件分析距离CDRs
Figure PCTCN2022087557-appb-000002
范围的框架氨基酸以及稀有氨基酸，这些氨基酸位点通常影响CDR的构象或抗原结合活性。然后通过IMGT分析获得人源germline，通过将选定的人源germline框架与抗体的CDRs进行拼接后，比对设计获得的人源化抗体与原始抗体的框架区序列，找出两者框架区序列中存在差异的氨基酸位点，通过分析亲本抗体的同源建模结果，确定这些存在差异的氨基酸位点是否会影响CDR的构象或抗原结合活性。在维持抗体活性并兼顾减少异源性基础上选用与人抗体表面残基相似的氨基酸进行替换，设计抗体人源化(HM-D10-1到HM-D10-6)。
Figure PCTCN2022087557-appb-000003
将上述设计的人源化抗体分别进行基因合成，与human IgG1Fc串联亚克隆至表达载体pcDNA3.4-hIgG1-Fc2中(图13)。载体经测序验证无误后，使用Qiagen质粒大抽试剂盒制备去内毒素质粒备用。
3.2人源化抗体瞬时转染表达
1)从冰箱中取出LVTransm转染试剂及抗体表达载体，室温解冻后，用移液枪上下吹打完全混匀。取出PBS缓冲液，温热至室温。取500μl PBS至24孔板的一个孔，加入4μg pcDNA3.4-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入12μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。
2)将上述DNA/LVTransm复合物加入到1.5mL 293F细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO2培养箱，130RPM培养6～8小时后，加入1.5mL新鲜的OPM-293CD05培养基，将细胞重新放回培养箱中继续培养。
3)连续培养3天后，离心收集培养基上清，用0.45μm的滤膜过滤，纯化抗体，进行流式检测。
3.3流式检测人源化抗体与靶蛋白的结合
1)从液氮中复苏CHO和CHO-GM004细胞株，使用CHOGrow CD1培养基调整细胞状态至对数生长期；
2)将细胞分别分为若干份，每份细胞的数量为5*10 5个细胞。
3)将表达的抗体分别孵育靶细胞，充分混匀后，室温孵育1小时。
4)800xg室温离心5分钟，去掉含有抗体的上清，使用PBS洗涤细胞3次。
5)加入1uL PE或者Alexa488标记的Anti-human IgG，充分混匀后，室温避光孵育30分钟。
6)800xg室温离心5分钟，去掉含有二抗的上清，使用PBS洗涤细胞3次。
7)使用500uL PBS重悬细胞，进行流式分析。
3.4人源化抗体的表达纯化
1)从冰箱中取出LVTransm转染试剂及单链抗体表达载体，室温解冻后，用移液枪上下吹打完全混匀。取出PBS，温热至室温。取2mL PBS至6孔板的一个孔，分别加入130μg pcDNA3.4-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入400μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。
2)将上述DNA/LVTransm复合物加入到50mL 293F细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO2培养箱，130RPM培养6～8小时后，加入50mL新鲜的OPM-293CD05培养基，将细胞重新放回培养箱中继续培养。
3)连续培养7天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，使用Protein A柱子纯化抗体。
3.5结果
3.5.1抗体表达载体构建测序结果
所有构建的抗体表达载体均经Sanger测序，抗体表达载体均完全正确。
3.5.2人源化抗体流式细胞检测结果
将人源化的抗体表达载体瞬时转染293F细胞进行小试表达，收集培养基上清，采用流式细胞仪检测人源化后抗体与重组细胞CHO和CHO-GM004细胞膜表面的抗原结合情况，见图18所示。
根据流式检测结果，人源化抗体HM1，HM4均特异性与重组CHO-native G protein细胞结合，且结合力与D10抗体相当。
3.6人源化抗体亲和力检测
将GM004重组蛋白使用10mMAcetate缓冲液固定在CM5芯片上，分别以HM1和HM4阳性人源化抗体及原始抗体D10作为流动相，检测人源化前后抗体与靶蛋白GM004的结合能力，见图19所示，亲和力检测结果：
Figure PCTCN2022087557-appb-000004
结果分析：根据亲和力检测结果，人源化抗体与原始抗体的亲和力一致。
从结果我们可以看出人源化抗体HM1和HM4与原始抗体D10亲和力相当。
实施例4：多价连接的抗体序列设计
4.1载体构建
1.Fc标签双价抗体表达载体构建
将前期项目获得的单域抗体HM1和HM4与human IgG1Fc串联，构建双价抗体表达载体(HM1-ggggsggggsggggs-HM4-IgG1Fc)；将人源化抗体HM4抗体序列和human IgG1FC区构建双价抗体表达载体(HM4-ggggsggggsggggs-HM4-IgG1Fc)；分别进行基因合成，亚克隆至Lenti-hIgG1-Fc2-Puro载体中，构建双价抗体表达载体。载体经测序验证无误后，使用Qiagen质粒大抽试剂盒制备去内毒素质粒。
HM1抗体氨基酸序列如SEQ ID NO.106所示
HM4抗体氨基酸序列如SEQ ID NO.109所示；
2.His标签双价抗体表达载体构建
将步骤1中构建的两个双价抗体表达载体Fc标签更换为His标签，为了防止His标签隐藏，在抗体序列和His标签之间添加G4S，同时添加2个6×His标签，构建His标签双价抗体表达载体，即HM1-ggggsggggsggggs-HM4-His和HM4-ggggsggggsggggs-M4-His。测序验证无误后，制备去内毒素大抽质 粒。
4.2双价抗体的表达纯化
1.从冰箱中取出LVTransm转染试剂及单链抗体表达载体，室温解冻后，用移液枪上下吹打完全混匀。取出PBS缓冲液，温热至室温。分别取2mL PBS至6孔板的两个孔，分别加入130μg抗体表达载体，移液枪上下吹打充分混匀后，加入400μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。
2.将上述DNA/LVTransm复合物加入到50mL 293F细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO2培养箱，130RPM培养6～8小时后，加入50mL新鲜的FreeStyle TM 293培养基，将细胞重新放回培养箱中继续培养。
3.连续培养7天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，分别使用Protein A和镍柱亲和柱纯化抗体。
4.SDS-PAGE检测蛋白纯度。
4.3结果
双价抗体的表达纯化SDS-PAGE检测：(见图20)其中：
Lane M:蛋白marker
Lane 1:Lenti-HM4-HM4-hIgG1-Fc2MW～54kd
Lane 2:Lenti-HM1-HM4-hIgG1-Fc2MW～54kd
Lane 3:Lenti-HM4-HM4-HisMW～30kd
Lane 4:Lenti-HM1-HM4-HisMW～30kd
实施例5：细胞功能验证
5.1通过细胞中和抑制实验考察人源化单域抗体和多价链接的对狂犬病毒的中和抑制效果
实验方法如2.4
5.2实验结果如下表：
Figure PCTCN2022087557-appb-000005
注：结果数值越大，代表中和抑制能力越好，从结果可以看出，人源化抗体无论是双价还是单价都好于对照抗体Rafivirumab和Foravirumab。
实施例6：动物药效学验证
6.1实验方案：
(1)病毒：CVS10毒株，采用小鼠脑内接种感染。
(2)实验动物：小鼠(15-16g)提前在动物房进行适应性培养1天。
(3)实验观察指标：小鼠存活率及其它临床体征观察。
(4)抗体样品数量：3个。
(5)所需动物数量：5个样品(3个实验组+1个空白病毒对照+1个免疫球蛋白对照)，每组10只小鼠(2个实验人员同时平行做)。
Figure PCTCN2022087557-appb-000006
6.2实验结果
Figure PCTCN2022087557-appb-000007
生存率曲线图(如附图21所示)
实验结果表明：实验组供试品与免疫球蛋白的生存率一样，都是100％，而Virus组的生存率为0。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (14)

  1. RBV的蛋白结合分子，其包括三个互补决定区CDR1、CDR2和CDR3，其中CDR1选自SEQ ID NO：1、4、7、10、13、16、19，CDR2选自SEQ ID NO：2、5、8、11、14、17、20，CDR3选自SEQ ID NO：3、6、9、12、15、18、21。
  2. 权利要求1所述的RBV的蛋白结合分子，其中所述RBV的蛋白结合分子包括：
    1)如SEQ ID NO：1所示的CDR1、如SEQ ID NO：2所示的CDR2、如SEQ ID NO：3所示的CDR3；或者，
    2)如SEQ ID NO：4所示的CDR1、如SEQ ID NO：5所示的CDR2、如SEQ ID NO：6所示的CDR3；或者，
    3)如SEQ ID NO：7所示的CDR1、如SEQ ID NO：8所示的CDR2、如SEQ ID NO：9所示的CDR3；或者，
    4)如SEQ ID NO：10所示的CDR1、如SEQ ID NO：11所示的CDR2、如SEQ ID NO：12所示的CDR3；或者，
    5)如SEQ ID NO：13所示的CDR1、如SEQ ID NO：14所示的CDR2、如SEQ ID NO：15所示的CDR3；或者，
    6)如SEQ ID NO：16所示的CDR1、如SEQ ID NO：17所示的CDR2、如SEQ ID NO：18所示的CDR3；或者，
    7)如SEQ ID NO：19所示的CDR1、如SEQ ID NO：20所示的CDR2、如SEQ ID NO：21所示的CDR3。
  3. 权利要求1～2中任一项所述的RBV的蛋白结合分子，其中所述RBV的蛋白结合分子包括四个框架区FR1、FR2、FR3和FR4，其中FR1选自SEQ ID NO：22、26、30、34、38、42、46，FR2选自SEQ ID NO：23、27、31、35、39、43、47，FR3选自SEQ ID NO：24、28、32、36、40、44、48，FR4选自SEQ ID NO：25、29、33、37、41、45、49。
  4. 权利要求1～3中任一项所述的RBV的蛋白结合分子，其中所述RBV的蛋白结合分子为人源化抗体。
  5. 权利要求1～4所述的RBV的蛋白结合分子，其为包含一免疫球蛋白单一可变结构域的抗体。
  6. 权利要求5所述的RBV的蛋白结合分子，免疫球蛋白单一可变结构域为人源化的重链可变区结构域。
  7. 权利要求1～6中任一项所述的RBV的蛋白结合分子，其包括免疫球蛋白Fc区。
  8. 权利要求1～7中任一项所述的RBV的蛋白结合分子，其结合RBV蛋白小于1×10 -7M。
  9. 权利要求1～7中任一项所述的RBV的蛋白结合分子，其中所述RBV蛋白分子形成多价链接。
  10. 一种编码权利要求1～6中任一项所述的RBV的蛋白结合分子的核酸分子。
  11. 一种检测试剂盒，其包括权利要求1～9中任一项所述的RBV的蛋白结合分子。
  12. 一种宿主细胞，其表达权利要求1～9中任一项所述的RBV的蛋白结合分子。
  13. 一种药物组合物，其包括权利要求1～9中任一项所述的RBV的蛋白结合分子和一种或多种药学上可接受的赋形剂。
  14. 权利要求1～9中任一项所述的RBV的蛋白结合分子、权利要求11所述的检测试剂盒、权利要求13所述的药物组合物在制备用于治疗和/或预防和/或诊断与RBV感染相关疾病的药物中的用途。
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