CN114835802B - Protein binding molecules against respiratory syncytial virus - Google Patents

Protein binding molecules against respiratory syncytial virus Download PDF

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CN114835802B
CN114835802B CN202210358214.0A CN202210358214A CN114835802B CN 114835802 B CN114835802 B CN 114835802B CN 202210358214 A CN202210358214 A CN 202210358214A CN 114835802 B CN114835802 B CN 114835802B
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CN114835802A (en
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高光
崔院平
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Gmaxx Biologics Ltd
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Abstract

The present invention relates to a protein binding molecule directed against RSV and to the use of single domain antibodies directed against the RSV F protein for the prevention and treatment of RSV-related diseases. The single domain antibody has better neutralization inhibition effect on RSV than the currently marketed drugs.

Description

Protein binding molecules against respiratory syncytial virus
The present application is a divisional application of the chinese patent application with application number 202010743933.5.
Technical Field
The invention relates to the field of medical biology, in particular to a single domain antibody and a derivative protein thereof.
The invention also relates to the application of the antibody and the protein.
Background
Respiratory syncytial virus (abbreviated as "RSV") belongs to the genus pneumovirus of the Paramyxoviridae family and is an enveloped single-stranded negative-strand RNA virus. The most prominent feature of RSV viral infection leading to cytopathy is the formation of viral syncytia.
RSV has two major membrane glycoproteins, the G protein, which is an attachment protein that mediates binding of the virus to the cell surface, and the F protein, which mediates fusion of the virus and host cell membrane, allowing the nucleocapsid of the virus to enter the cytoplasm and initiate replication of the virus. The F protein is highly conserved and forms trimeric spikes that undergo conformational changes upon activation. Antibodies to protein F can inhibit the initiation of its mediated infectious cycle and neutralize viral infectivity to protect humans from RSV infection.
RSV is the primary pathogen causing severe lower respiratory tract infections including bronchiolitis (bronchiolitis) and pneumonia (pneumaria) in infants and young children and causes annual epidemics during the winter months. The virus also causes substantial disease burden in the elderly, adults with cardiopulmonary disorders and immunosuppression are also at risk of RSV severe infection, and immune responses against RSV virus do not prevent repeat infection.
There is currently no vaccine worldwide available to prevent diseases associated with RSV infection, and the only pharmaceutical product on the market is a humanized monoclonal antibody directed against the F proteinIt prevents the spread of the virus to the lower respiratory tract by binding to the respiratory syncytial virus fusion protein. The drug is currently only used prophylactically in children at extremely high risk of severe RSV infection, this limited use being due at least in part to the high cost of such products. Thus, it is urgent to develop an antibody drug which is low in cost and can specifically prevent or treat diseases associated with RSV infection.
Single domain antibodies (single domain antibody, sdAb), also known as nanobodies (nanobodies), have only one heavy chain variable region domain (VHH). This domain was originally found as a heavy chain antibody (heavy chain antibody, hcAb) isolated from serum of camelids and sharks, from which VHH fragments were amplified by genetic means. VHH is the smallest unit currently known to bind an antigen of interest. The single domain antibody has a series of advantages of simple structure, high affinity and stability, strong tissue penetration, low immunogenicity and the like, and is the latest technology in the field of antibody medicines. Currently, the application of single domain antibody technology to solve virus infection diseases has become common knowledge of domestic and foreign scientists.
Disclosure of Invention
It is an object of the present invention to provide protein binding molecules (RSVF protein binding molecules) directed against Respiratory Syncytial Virus (RSV).
It is a second object of the present invention to provide an isolated nucleotide encoding the above-described RSVF protein binding molecule.
It is a third object of the present invention to provide pharmaceutical compositions containing the protein binding molecules of RSVF as described above.
It is a fourth object of the present invention to provide the use of the above-described RSVF protein binding molecules.
According to one aspect of the invention, the protein binding molecules against respiratory syncytial virus with high specificity, high affinity and high stability are screened using single B cell single domain antibody rapid screening techniques. The protein binding molecule is directed against the F protein of RSV, which is an antibody comprising an immunoglobulin single variable domain, which has better neutralizing and inhibitory function against RSV virus than amino acid sequences and antibodies of the prior art. The specific technical scheme is as follows:
optimizing antigen F protein (SEQ ID NO. 1) by a gene synthesis method, carrying out codon optimization according to human codon preference, synthesizing an optimized F protein gene (SEQ ID NO. 2), subcloning into a eukaryotic expression vector, transiently transfecting 293F cells, and then expressing and purifying to obtain the antigen F protein. Immunizing alpaca with antigen F protein, collecting alpaca peripheral blood, separating Peripheral Blood Mononuclear Cells (PBMC), performing flow separation by using fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) coupled RSV F protein and 647-Anti-Camelid VHH antibody and incubating the PBMC, screening out single B cells expressing single domain antibody against F protein and cloning the antibody sequences of the single B cells, and performing expression and purification verification to finally obtain the Anti-RSV single domain antibody and the sequence thereof.
The present invention provides RSV F protein binding molecules comprising at least one immunoglobulin single variable domain capable of specifically binding to RSV F protein. In some embodiments, the RSV F protein binding molecule comprises only one immunoglobulin single variable domain that specifically binds to RSV F protein. In some embodiments, the RSV F protein binding molecule comprises two or more VHHs that specifically bind to RSV F protein.
In some embodiments, the at least one immunoglobulin single variable domain comprises a CDR1, CDR2, and CDR3 selected from the group consisting of:
(1) CDR1 as shown in SEQ ID No.4, CDR2 as shown in SEQ ID No.5 and CDR3 as shown in SEQ ID No. 6; (HB 10)
(2) CDR1 as shown in SEQ ID No.9, CDR2 as shown in SEQ ID No.10 and CDR3 as shown in SEQ ID No. 11; (HC 12)
(3) CDR1 as shown in SEQ ID No.14, CDR2 as shown in SEQ ID No.15 and CDR3 as shown in SEQ ID No. 16; (HB 1)
(4) CDR1 as shown in SEQ ID No.19, CDR2 as shown in SEQ ID No.20 and CDR3 as shown in SEQ ID No. 21; (HF 8)
(5) CDR1 as shown in SEQ ID No.24, CDR2 as shown in SEQ ID No.25 and CDR3 as shown in SEQ ID No. 26; (HG 3)
(6) CDR1 as shown in SEQ ID No.29, CDR2 as shown in SEQ ID No.30 and CDR3 as shown in SEQ ID No. 31; (HG 10)
(7) CDR1 as shown in SEQ ID No.34, CDR2 as shown in SEQ ID No.35 and CDR3 as shown in SEQ ID No. 36; (HA 6)
(8) CDR1 as shown in SEQ ID NO.39, CDR2 as shown in SEQ ID NO.40 and CDR3 (HH 9) as shown in SEQ ID NO. 41.
According to a preferred embodiment of the invention, the protein binding molecule for RSV F according to the invention comprises an amino acid selected from the group consisting of amino acid sequences having the amino acid sequence:
(a) Amino acid as shown in SEQ ID NO. 3; (HB 10)
(ii) An amino acid as shown in SEQ ID NO. 8; (HC 12)
(iii) An amino acid as shown in SEQ ID NO. 13; (HB 1)
(iv) Amino acid as shown in SEQ ID NO. 18; (HF 8)
(v) An amino acid as shown in SEQ ID NO. 23; (HG 3)
(vi) An amino acid as shown in SEQ ID NO. 28; (HG 10)
(vii) An amino acid as shown in SEQ ID NO. 33; (HA 6)
(viii) An amino acid as shown in SEQ ID NO. 38. (HH 9)
According to a preferred embodiment of the present invention, an isolated nucleotide encoding a protein binding molecule of RSV F as described above has the nucleotide sequence:
(a) A nucleotide as shown in SEQ ID NO. 7; (HB 10)
(b) A nucleotide as shown in SEQ ID NO. 12; (HC 12)
(c) A nucleotide as shown in SEQ ID NO. 17; (HB 1)
(d) A nucleotide as shown in SEQ ID NO. 22; (HF 8)
(e) A nucleotide as shown in SEQ ID NO. 27; (HG 3)
(f) A nucleotide as shown in SEQ ID NO. 32; (HG 10)
(g) A nucleotide as shown in SEQ ID NO. 37; (HA 6) or
(h) A nucleotide as shown in SEQ ID NO. 42. (HH 9)
The invention also relates to expression vectors and host cells containing the above-described nucleotides, which can be used for expression and production of the single domain antibodies of the invention.
The invention provides at least eight single domain antibodies against RSV F protein, the CDR sequences, amino acid sequences and nucleotide sequences of which are listed in table 2, which antibodies specifically bind to the F protein of RSV.
The invention also provides derived proteins of the RSV F protein binding molecules, including: a single domain antibody specific to the F protein can also be obtained by a sequence with high sequence homology with the CDR1-3 of the invention. In some embodiments, sequences having "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" with the sequences in the antibodies (1) - (8) may achieve the objects of the invention, i.e., the scope of the derivative proteins of the invention.
Protein binding molecules of the invention include antibodies, monomers thereof, or combinations thereof with other proteins. Also included are various variants, such as: the resulting deglycosylated antibody retains the same function as the parent non-deglycosylated antibody, comprising one or more mutations at the N-glycosylation site of one or more CDRs of its variable domain.
In some embodiments, at least one immunoglobulin single variable domain in an RSV F protein binding molecule of the invention is a VHH. In other embodiments, these VHHs are humanized VHHs comprising an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to any one of SEQ ID NOs 43-53. Alternatively, the amino acid sequence of the VHH comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any of SEQ ID NOS: 43-53. For example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.
In some embodiments, the RSV F protein binding molecules of the invention are obtained via affinity maturation. The affinity-matured RSV F protein binding molecule can have one or more changes in one or more CDRs that result in an increase in affinity for the RSV F protein as compared to the parent RSV F protein binding molecule.
In some embodiments, the RSV F protein binding molecules of the invention comprise an immunoglobulin Fc region in addition to at least one immunoglobulin single variable domain capable of specifically binding to an RSV F protein. Inclusion of an immunoglobulin Fc region in the RSV F protein binding molecules of the invention can allow the binding molecules to form dimers while extending the in vivo half-life of the molecules. The Fc region useful in the present invention may be derived from immunoglobulins of different subtypes, e.g., igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM.
In some embodiments, mutations may be introduced into the Fc sequence, thereby making the mutant Fc more susceptible to homodimer or heterodimer formation. The knob-hole model, as mentioned in Ridgway, presta et al 1996 and Carter 2001, which uses the steric effect of the amino acid side chain groups of the Fc contact interface, makes heterodimer formation between different Fc mutations easier; as in CN 102558355A or CN103388013a, the ionic interaction force between Fc contact interfaces is changed by changing the charge of the amino acids at the Fc contact interfaces, so that heterodimers (CN 102558355A) are more easily formed between different pairs of Fc mutations, or homodimers (CN 103388013 a) are more easily formed between Fc having the same mutation.
The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, more preferably a human IgG1 Fc region. In some embodiments, the amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO. 65 and the nucleotide sequence is shown in SEQ ID NO. 66.
In some specific embodiments, the RSV F protein binding molecules of the invention are molecules formed by binding specific antibodies shown in tables 2 and 4 to a human immunoglobulin Fc region.
In another aspect, the RSV F protein binding molecules of the invention also encompass anti-RSV F protein antibody molecules that are capable of binding to the same epitope on the RSV F protein as a VHH consisting of the amino acid sequence of any one of the invention.
The KD of the binding molecule of the RSV F protein of the present invention to the RSV F protein may be less than 1X 10 -7 M, preferably less than 1X 10 -8 M, more preferably less than 1X 10 -9 M, more preferably less than 1X 10 -10 M, particularly preferably less than 1X 10 -11 M。
The RSV F protein binding molecules of the invention are capable of inhibiting RSV 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%.
Furthermore, the RSV F protein binding molecules of the invention are resistant to alkaline and oxidative treatments. For example, after about 8 hours, preferably about 16 hours, more preferably about 24 hours, or more preferably about 32 hours of treatment with a strong base (e.g., 500mM ammonium bicarbonate), the activity of the RSV F protein binding molecule of the present invention remains unchanged. Alternatively, the RSV F protein binding molecule activity of the invention remains unchanged after about 2 hours, preferably about 4 hours or more preferably about 8 hours of treatment with an oxidizing agent (1% hydrogen peroxide).
Furthermore, the RSV F protein binding molecules of the invention have stability at high concentrations. For example, at a concentration of about 100mg/ml, more preferably about 150mg/ml, more preferably about 200mg/ml, or more preferably about 250mg/ml, the RSV F-protein binding molecules of the present invention remain stable against aggregation.
Pharmaceutical composition
In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of the RSV F protein binding molecules of the invention formulated with a pharmaceutically acceptable carrier. Such compositions may comprise one or a combination (e.g., two or more different) of the RSV F protein binding molecules or immunoconjugates of the invention. For example, the pharmaceutical compositions of the invention may contain a combination of antibody molecules that bind to different epitopes on a target antigen.
According to a further aspect of the invention, the invention also includes the use of the above-described RSV F protein binding molecules, including pharmaceutical use, i.e. the use of the above-described antibodies or derivatives for the manufacture of a medicament for the prevention and/or treatment of diseases associated with RSV infection. The use may also include the use of the antibodies in detection reagents for detection of diseases associated with RSV infection.
The present invention prepares and screens the single domain antibody against RSV, and the current cytopharmacodynamics has proved that the single domain antibody has the specific drug on RSV virusBetter neutralization inhibition effect.
Drawings
FIG. 1 shows single cells double positive for FITC protein and 647
The abscissa indicates FITC-F protein, the ordinate indicates APC-Anti-Camelid VHH antibody, and cells in the range of P3 refer to single B cells that were double positive for antibody detection.
FIG. 2 is a clone gel electrophoresis pattern of single B cell antibody sequences;
wherein fig. 2A is a plate number S190116; fig. 2B is a plate No. S190117.
FIG. 3 shows the results of the similarity of the plate sequences of S190116 and S190117;
FIG. 4 shows FACS detection after expression of a constructed F protein single domain antibody;
FIG. 5 shows the flow fluorescence values of K562-native F protein stable transgenic cells of the S190116 plate antibody;
FIG. 6 shows the K562 cell line flow fluorescence values of the S190116 plate antibody:
FIG. 7 shows FACS detection of F-protein single domain antibody expression constructed by S190117 plate;
FIG. 8 shows the flow fluorescence values of K562-native F protein stable transgenic cells of the S190117 plate antibody;
FIG. 9 shows the flow fluorescence values of K562 cell lines of the S190117 plate antibody;
FIG. 10 shows the structure of the pDOOR-CMV-F protein-puro plasmid;
FIG. 11 shows the effect of neutralizing anti-RSV F protein single domain antibodies in inhibiting RSV virus;
wherein (a) normal HEP-2 cells (b) RSV infected cells+anti-RSV F protein single domain antibodies
(c) RSV infected cells (d) RSV infected cells + vehicle control
FIG. 12 is a schematic representation of the expression vector Lenti-hIgG1-Fc 2;
FIG. 13 is FACS detection after expression of humanized F protein single domain antibodies;
FIG. 14 shows the detection of the antigen binding of humanized antibodies to the surface of the cell membrane of recombinant K562-native F protein by flow cytometry;
FIG. 15 shows the detection of binding of humanized antibodies to K562 cells by flow cytometry;
FIG. 16 shows SDS-PAGE detection of expression purification of diabodies.
Detailed Description
Definition of the definition
Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to standard handbooks, such as Sambrook et al, "molecular cloning: A Laboratory Manual" (2 nd edition), vol.1-3, cold Spring Harbor LaboratoryPress (1989); lewis, "Genes IV", oxford University Press, new York, (1990); and Roitt et al, "Immunology" (2 nd edition), gower Medical Publishing, london, new York (1989), and the general prior art cited herein; moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically detailed may be, and have been, performed in a manner known per se, which will be appreciated by those skilled in the art. Reference is also made to, for example, standard handbooks, the above-mentioned general prior art and other references cited therein.
The terms "antibody" or "immunoglobulin" are used interchangeably herein to refer to either heavy chain antibodies or conventional 4 chain antibodies, unless otherwise indicated, as general terms to include full length antibodies, individual chains thereof, and all portions, domains, or fragments thereof (including but not limited to antigen binding domains or fragments, e.g., VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" (e.g. in terms of "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) as used herein is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the context requires a more defined interpretation.
As used herein, the term "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independent of the rest of the protein. In general, a domain is responsible for a single functional property of a protein, and in many cases can be added, removed, or transferred to other proteins without losing the function of the remainder of the protein and/or the domain.
The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (e.g., 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. The immunoglobulin domain is characterized in that it maintains the immunoglobulin folding characteristics of the antibody molecule, consisting of a 2-layer sandwich of about 7 antiparallel β -sheet strands, optionally stabilized by a conserved disulfide bond, arranged in two β -sheets.
The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain consisting essentially of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to an antigen to an antibody by having an antigen binding site.
The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that is capable of specifically binding an epitope without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain within the meaning of the invention is a "domain antibody", e.g. an immunoglobulin single variable domain VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is the "VHH domain" (or simply "VHH") of the family Camelidae, as defined below.
"VHH domains", also known as heavy chain single domain antibodies, VHH domains, VHH antibody fragments and VHH antibodies, are the variable domains of antigen-binding immunoglobulins known as "heavy chain antibodies" (i.e. "antibodies lacking light chains") (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)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (which is referred to herein as a "VH domain") present in conventional 4-chain antibodies, and the light chain variable domain (which is referred to herein as a "VL domain") present in conventional 4-chain antibodies. The VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain). VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain.
In the context of the present invention, the terms "heavy chain single domain antibody", "VHH domain", "VHH antibody fragment", "VHH antibody" and "Nanobody o R domain" ("Nanobody" is a trademark of Ablynx n.v. company, ghent, belgium) are used interchangeably.
For example, amino acid residues used for the VHH domain of the family Camelidae are numbered according to the general numbering of the VH domain given by Kabat et al ("Sequence of proteins of immunological interest", US PublicHealth Services, NIH Bethesda, MD, publication No. 91) as shown in FIG. 2 of Riechmann and Muyledermans, J.Immunol. Methods 231,25-38 (1999). According to this numbering process, the number of the cells is,
FR1 comprises amino acid residues at positions 1-30,
CDR1 comprising amino acid residues at positions 31-35,
FR2 comprises the 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.
It should be noted, however, that the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to 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), as is well known in the art for VH domains and VHH domains. This means that in general, numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
Alternative methods of numbering amino acid residues of VH domains are known in the art, which may also be similarly applied to VHH domains. However, unless otherwise indicated, in the present description, claims and figures, numbering according to Kabat and as appropriate for VHH domains as described above will be followed.
The total number of amino acid residues in the VHH domain will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.
Other structural and functional properties of VHH domains and polypeptides containing them can be summarized as follows:
VHH domains (which have been naturally "designed" to functionally bind to an antigen in the absence of and without interaction with a light chain variable domain) can be used as single and relatively small functional antigen binding building blocks, domains, or polypeptides. This distinguishes VHH domains from the VH and VL domains of conventional 4-chain antibodies, which are not themselves generally suitable for practical use as a single antigen-binding protein or immunoglobulin single variable domain, but need to be combined in some form or another to provide a functional antigen-binding unit (e.g., in the form of a conventional antibody fragment such as a Fab fragment; or in the form of an scFv consisting of a VH domain covalently linked to a VL domain).
Because of these unique properties, the use of VHH domains, alone or as part of a larger polypeptide, provides a number of significant advantages over the use of conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., fab-or F (ab') 2-fragments):
only a single domain is required to bind antigen with high affinity and high selectivity, so that neither the presence of two separate domains nor the assurance that the two domains are present in the appropriate spatial conformation and configuration is required (e.g. scFv typically require the use of specifically designed linkers);
the 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 aggregation tendency;
VHH domains are highly stable to heat, pH, proteases and other denaturants or conditions, and thus can be prepared, stored or transported without the use of refrigeration equipment, thereby achieving cost, time and environment savings;
VHH domains are easy to prepare and relatively inexpensive, even on the scale required for production;
the VHH domain is relatively small compared to conventional 4-chain antibodies and antigen-binding fragments thereof (about 15kDa or 1/10 of the size of conventional IgG), thus exhibiting higher tissue permeability and can be administered at higher doses compared to conventional 4-chain antibodies and antigen-binding fragments thereof;
VHH domains can exhibit so-called cavity binding properties (especially due to their extended CDR3 loops compared to conventional VH domains) so that targets and epitopes not reachable by conventional 4-chain antibodies and antigen-binding fragments thereof can be reached.
Methods for obtaining VHH binding to a specific antigen or epitope have previously been disclosed in the following documents: vander Linden et al Journal of Immunological Methods,240 (2000) 185-195; liet al, J Biol chem.,287 (2012) 13713-13721; deffar et al African Journal ofBiotechnology Vol.8 (12), pp.2645-2652,17June,2009 and WO94/04678.
A camelidae-derived VHH domain may be "humanized" (also referred to herein as "sequence optimisation" which may also encompass, in addition to humanisation, other modifications to the sequence by one or more mutations that provide VHH-modifying properties, such as removal of potential post-translational modification sites) 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 corresponding positions in the human conventional 4-chain antibody VH domain. The humanized VHH domain may contain one or more fully human framework region sequences and in a particular embodiment may contain human framework region sequences of IGHV 3.
As used herein, the term "domain antibody" (also referred to as "Dab" and "Dab") is particularly used to refer to the VH or VL domain of an antibody (particularly a human 4-chain antibody) of a non-camelidae mammal. In order to bind an epitope in the form of a single antigen-binding domain (i.e. without pairing with a VL domain or VH domain, respectively), specific selection of the antigen-binding properties is required, for example, by using a library of human single VH or VL domain sequences.
Like VHH, domain antibodies have a molecular weight of about 13kDa to about 16kDa and, if derived from fully human sequences, do not require humanization for use in, for example, human therapy. 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 in, for example, the following documents: ward, E.S., et al, "Bindingactivities of arepertoire of single immunoglobulin variable domains secretedfrom Escherichia coli"; nature341:544-546 (1989); holt, l.j. Et al, "domainntiibodies: proteins for therapy"; TRENDS in Biotechnology 21 (11): 484-490 (2003).
Furthermore, one skilled in the art will also appreciate that it is possible to "graft" one or more of the above CDRs onto other "scaffolds (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for such CDR grafting are known in the art.
As used herein, the term "epitope" or the term "epitope" is used interchangeably to refer to any epitope on an antigen to which the paratope of an antibody binds. An epitope typically comprises a chemically active surface group of a molecule, such as an amino acid or sugar side chain, and typically has specific three-dimensional structural features as well as specific charge features. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which may be a "linear" epitope or a "conformational" epitope. See, e.g., epitope Mapping Protocols in Methodsin Molecular Biology, volume 66, g.e.Morris, ed. (1996). In linear epitopes, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In conformational epitopes, points of interaction exist across amino acid residues of the protein that are separated from each other.
The epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, e.g., epitope Mapping Protocols in Methods in Molecular Biology, volume 66, g.e.Morris, ed. (1996). For example, a linear epitope may be determined by, for example, the following methods: a large number of peptides are synthesized simultaneously on a solid support, wherein these peptides correspond to portions of the protein molecule, and these peptides are reacted with antibodies while still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al (1984) Proc.Natl. Acad. Sci. USA81:3998-4002; geysen et al (1986) molecular.Immunol.23:709-715. Similarly, conformational epitopes can be identified by determining the spatial conformation of amino acids, such as by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., epitope Mapping Protocols (supra).
Antibodies can be screened for binding to the same epitope competitively using conventional techniques known to those skilled 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 binding to an antigen. High throughput methods for obtaining antibodies binding to the same epitope based on their cross-competition are described in international patent application WO 03/48731. Thus, antibodies and antigen binding fragments thereof that compete with the antibody molecules of the invention for binding to the same epitope on RSV F protein can be obtained using conventional techniques known to those of skill in the art.
In general, the term "specific" refers to the number of different types of antigens or epitopes to which a particular antigen binding molecule or antigen binding protein (e.g., immunoglobulin single variable domain of the invention) molecule can bind. The specificity of an antigen binding molecule may be determined based on its affinity and/or avidity. Affinity, expressed by the dissociation equilibrium constant (KD) of an antigen to an antigen binding protein, is a measure of the strength of binding between an epitope and an antigen binding site on an antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding molecule (alternatively, affinity can also be expressed as association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, depending on the particular antigen of interest, affinity can be determined in a known manner. Avidity is a measure of the strength of binding between an antigen binding molecule (e.g., an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide comprising the same) and the antigen of interest. Affinity is related to both: affinity with the antigen binding sites on its antigen binding molecule, and the number of relevant binding sites present on the antigen binding molecule.
As used herein, the term "RSV F protein binding molecule" means any molecule capable of specifically binding to an RSV F protein. The RSV F protein binding molecule can comprise an antibody or conjugate thereof as defined herein directed against the RSV F protein. RSV F protein binding molecules also encompass so-called "SMIPs" ("small modular immunopharmaceuticals"), or immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.
"RSVF protein binding molecule" may alternatively refer to monovalent molecules that bind to the F protein of RSV (i.e., molecules that bind to one epitope of the F protein of RSV), as well as bivalent or multivalent binding molecules (i.e., binding molecules that bind to more than one epitope). The "RSV F protein binding molecules" of the invention can comprise at least one immunoglobulin single variable domain that binds to an RSV F protein, such as a VHH. In some embodiments, a "RSV F protein binding molecule" of the invention can comprise two immunoglobulin single variable domains that bind to RSV F proteins, such as VHH. RSV F protein binding molecules comprising more than one immunoglobulin single variable domain are also referred to as "formatted" RSV F protein binding molecules. The formatted RSV F protein binding molecules can also comprise a linker and/or moiety having effector functions, such as a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (e.g., serum albumin) and/or conjugated polymer (e.g., PEG) and/or Fc region, in addition to the immunoglobulin single variable domain that binds RSV F protein. In some embodiments, the "RSV F protein binding molecules" of the invention also encompass bispecific antibodies that contain immunoglobulin single variable domains that bind to different antigens.
In general, the RSV F protein binding molecules of the invention will be measured as the preferred 10 as in the Biacore or kinex a assay -7 To 10 -11 Mol/liter (M), more preferably 10 -8 To 10 -11 Molar/liter, even more preferably 10 -9 To 10 -11 Even more preferably 10 -10 To 10 -11 Or lower dissociation constant (KD), and/or at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 More preferably at least 10 10 M -1 For example at least 10 11 M -1 Is associated with the antigen to which it is bound (i.e., RSV F protein). Any of more than 10 -4 KD values for M are 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., radioimmunoassays (RIA), enzyme Immunoassays (EIA), and sandwich competitive assays, as described herein.
Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as known and agreed upon in the art. When comparing two amino acid sequences, the term "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 the other sequence. In the case of a substitution, the substitution will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue of similar chemical structure, with little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g. conservative amino acid substitutions are preferably those in which one amino acid in the following groups (i) - (v) is replaced by another amino acid residue in the same group: (i) smaller aliphatic nonpolar or low polar residues: ala, ser, thr, pro and Gly; (ii) a polar negatively charged residue and (uncharged) amide: asp, asn, glu and Gln; (iii) a polar positively charged residue: his, arg and Lys; (iv) a larger aliphatic nonpolar residue: met, leu, ile, val and Cys; (v) aromatic residues: phe, tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: substitution of Ala with Gly or Ser; arg is replaced by Lys; asn is substituted with Gln or His; asp is substituted with Glu; cys is replaced by Ser; gln is substituted with Asn; glu is substituted with Asp; substitution of Gly with Ala or Pro; his is substituted with Asn or Gln; lie is substituted with Leu or Val; leu is substituted with Ile or Val; lys is substituted with Arg, gin or Glu; met is substituted with Leu, tyr or Ile; phe is substituted with Met, leu or Tyr; ser is substituted by Thr; thr is replaced by Ser; trp is substituted with Tyr; tyr is substituted 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 may be used to determine identity. For a determination of sequence identity, reference can be made, for example, to: 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., academic Press,1987 and Sequence AnalysisPrimer, gribskov, m.and deveeux, j., eds., MStockton Press, new York,1991.
A polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been separated from at least one other component (e.g., another protein/polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or micro-component) with which it is ordinarily associated in that source or medium (medium), as compared to its natural biological source and/or the reaction medium or medium from which the polypeptide or nucleic acid molecule was obtained. In particular, a polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold and up to 1000-fold or more. The "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by suitable techniques, such as suitable chromatographic techniques, e.g., polyacrylamide gel electrophoresis.
An "affinity matured" anti-RSV F protein antibody, particularly a VHH or domain antibody, has one or more changes in one or more CDRs that result in an increase in affinity for the RSV F protein as compared to its respective parent anti-RSV F protein antibody. Affinity-matured anti-RSV F protein antibodies can be prepared, for example, by methods known in the art as described below: marks et al 1992,Biotechnology 10:779-783 or barbes et al 1994,Proc.Nat.Acad.Sci,USA 91:3809-3813; thier et al, 1995,Gene 169:147-155; yelton et al, 1995, immunol.155:1994-2004; jackson et al, 1995, J.Immunol.154 (7): 3310-9; and Hawkins et al, 1992, J.MoI.biol.226 (3): 889896; KS Johnson and RE Hawkins, "Affinitymaturation of antibodies using phage display", oxford University Press 1996.
The term "subject" as used herein means a mammal, particularly a primate, particularly a human.
As used herein, "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. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, immunoconjugate, may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying 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, except insofar as they are incompatible with the active compound, are possible in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.
Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, the dispersants are prepared by incorporating the active compound into a sterile carrier which contains a basic dispersion medium and the other required ingredients enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) from a previously sterile-filtered solution thereof to yield a powder of the active ingredient plus any additional desired ingredient.
The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form is generally the amount of the composition that produces a therapeutic effect. Typically, this amount ranges 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% of the active ingredient, on a 100% basis, in combination with a pharmaceutically acceptable carrier.
The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased as needed for the emergency of the treatment situation. It is particularly 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 suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier.
For administration of antibody molecules, the dosage range is about 0.0001 to 100mg/kg, more typically 0.01 to 20mg/kg of the recipient body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens require weekly, biweekly, tricyclically, weekly, monthly, 3 months, 3-6 months, or a slightly shorter initial dosing interval (e.g., weekly to tricyclically) followed by longer post dosing intervals (e.g., monthly to 3-6 months).
Alternatively, the antibody molecule may be administered as a sustained release formulation, in which case less frequent administration is required. Dosages and frequencies will vary depending on the half-life of the antibody molecule in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over a long period of time. Some patients continue to receive treatment for the remainder 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 a partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.
The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response to the particular patient, composition and mode of administration without toxicity to the patient. The dosage level selected will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the present invention or its esters, salts or amides, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition being used, the age, sex, weight, condition, general health and medical history of the patient undergoing treatment, and like factors well known in the medical arts.
The "therapeutically effective amount" of the RSV F protein binding molecules of the invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease asymptomatic periods, or prevention of damage or disability due to disease distress. For example, for the treatment of RSV-related disease, 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% relative to an untreated subject. The ability to inhibit viral growth can be evaluated in an animal model system that predicts efficacy for inhibiting RSV. Alternatively, it may be assessed by examining the ability to inhibit RSV growth, which inhibition may be determined in vitro by assays well known to those of skill in the art. A therapeutically effective amount of the therapeutic agent relieves symptoms in the subject. Such amounts may be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.
The compositions of the present invention may be administered by one or more routes of administration using one or more methods known in the art. Those skilled in the art will appreciate that the route and/or mode of administration will vary depending upon the desired result. Preferred routes of administration for the RSV F protein binding molecules of the invention include aerosol inhalation, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or aerosol inhalation.
Disease prevention and treatment
The invention provides uses and methods of the RSV F protein binding molecules, nucleic acid molecules, host cells, immunoconjugates and pharmaceutical compositions of the invention in the prevention and/or treatment of diseases associated with RSV infection. In one aspect, the invention provides a method of preventing and/or treating RSV infectious disease in a subject comprising administering to the subject an RSV F protein binding molecule of the invention such that RSV infectious disease in the subject is prevented and/or treated.
Kit for detecting a substance in a sample
Also included within the scope of the invention are kits comprising a RSV F protein binding molecule, immunoconjugate or pharmaceutical composition of the invention, and instructions for use. The kit may further comprise at least one additional agent or one or more additional RSV F protein binding molecules of the invention (e.g., binding molecules that bind different epitopes of the RSV F protein). Kits typically include a label that indicates the intended use of the kit contents. The term label includes any written or recorded material provided on or with or otherwise with the kit.
Examples
The following examples and experimental examples are provided to further illustrate the present invention and should not be construed as limiting the present invention. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., fritsch, e.f. and maniis, t. (1989) Molecular Cloning: a Laboratory Manual,2nd edition,Cold spring Harbor Laboratory Press.
The expression vector pcDNA3.1 used in the following examples was purchased from Invitrogen corporation, and cell lines including 293F cells, 293F-SVP16 cells, K562 cells, HEP-2 cells were purchased from American Type Culture Collection (ATCC) and cultured according to the corresponding instructions.
The positive control antibody used in the following examples was Palivizumab(from Celastomer, specification: 100 mL/count, lot number NDC 60574-4113-1).
[ example 1 ]: screening of Single-Domain antibodies against RSV F protein
1.1 construction of library (single B cell screening against F protein):
The RSV F protein for immunization is expressed by 293F cells (expression vector pcDNA3.1-Neolass, prepared by conventional method, and purified by magnetic bead affinity chromatography of control antibody Palivizumab to obtain RSV F protein, selecting one llama (alpaca) for immunization.4 times, extracting lymphocytes from 50mL peripheral blood of the camel, sorting peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) by using lymphocyte separation solution, dividing the PBMC into two parts, the first part serving as a negative control, adding fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) coupled RSV F protein and 647-Anti-Camelid VHH antibody (gold, cat#A 01994) were incubated on ice for 1 hour in the dark, centrifuged at 4℃for 5 minutes, the supernatant containing the antibody was removed, washed 3 times with pre-chilled PBS, and then the cell pellet was resuspended with 500uL of pre-chilled PBS buffer, and the FITC and 647 fluorescent double positive single B cells were sorted out using a sorting flow cytometer onto 96-well reaction plates to which the cell lysate had been added for cloning of single domain antibodies, the results are shown in FIG. 1, the abscissa represents FITC-F protein, the ordinate represents APC-Anti-Camelid VHH antibody, and cells in the range of P3 refer to reverse transcription of antibody detection double positive single B cells.1.2 single B cell lysate
Preparing reverse transcription reagent (no nucleic acid water, IGEPAL (10%), random primers (300 ng/uL) RNase inhibitor (40U/uL), reverse transcriptase (200U/uL), 5 Xreverse transcription buffer, DL-DTT (100 mM), 25mM DNTP, RNase inhibitor (40U/uL) into the above-mentioned single B cells with double positive FITC and 647 after cleavage, performing PCR to make RNA reverse transcription into cDNA, diluting cDNA sample by adding 10uL sterile ultra-pure water into each well after finishing PCR reaction, cloning of 1.3 single B cell antibody sequence
A VHH screening reaction system (Ex Taq HS,10 XBuffer, dNTP (25 mM), 1st 5'forward primer mix (10. Mu.M), 1st 3'reverse primer mix (10. Mu.M)) was prepared, and appropriate cDNA products were added to perform VHH sequence screening, and after the reaction was completed, the PCR products were analyzed by 1% agarose gel electrophoresis, and the target fragment of about 400bp was isolated and sequenced. The results are shown in FIG. 2.
[ example 2 ]: preliminary evaluation of single domain antibodies against RSV F
2.1 construction and expression of expression vectors for Single-Domain antibodies
The coding sequence of 42 single-domain antibodies obtained by sequencing analysis is subjected to similarity analysis (shown in figure 3), 33 strains are selected for gene synthesis, subcloned into an expression vector Lenti-hIgG1-Fc2 (purchased from adedge corporation) in series with human IgG1Fc, cultured, plasmids are extracted by using Qiagen plasmids, and sequencing verification is carried out. The PBS buffer was removed and warmed to room temperature. One well of a 24-well plate was filled with 500. Mu.l PBS, and the expression vectors Lenti-hIgG1-Fc2 and LVTransm (Lipofectamine 2000 and Lipofectamine 3000) were immediately stirred up and down with a pipette and mixed, and then allowed to stand at room temperature for 10 minutes. Transfer to 1.5mL of 293F-SVP16 cells (ATCC), gently shake and mix well. The cells were exposed to 5% CO at 37 ℃ 2 After culturing for 6-8 hours in an incubator at 130RPM, 1.5mL of fresh FreeStyle is added TM 293 medium, the cells were returned to the incubator for continued culture. After 3 days of continuous culture, the culture supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube.
2.2 expression purification of Single-Domain antibodies
The PBS buffer was removed and warmed to room temperature. Taking 2mL PBS to one well of a 6-well plate, and adding the expression vector Lenti-hIgG1-F respectivelyc2 and LVTransm are immediately blown up and down by a pipette and evenly mixed, and then the mixture is kept stand for 10 minutes at room temperature. Transfer to 50mL293F-SVP16 cells, mix thoroughly with gentle shaking. The cells were exposed to 5% CO at 37 ℃ 2 After culturing for 6-8 hours in an incubator at 130RPM, 50mL of fresh FreeStyle is added TM 293 medium, the cells were returned to the incubator for continued culture. After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube and purified using a Protein A column to obtain antibodies.
2.3 detection of specific binding of anti-RSV F protein candidate single domain antibodies to RSV F protein
RSV F recombinant protein was diluted with sterile PBS to a final concentration of 5ug/mL. Taking a new 96-well plate, adding 100 uL/well, and coating at 4 ℃ overnight;
Washing 3 times with PBST (0.05% Tween 20 in PBS) to wash off the coating solution; blocking was performed for 2 hours at 37℃with 200 uL/well of 3% BSA; after washing the well plate 3 times using PBST to wash off the blocking buffer; 100ul of diluted anti-RSV F protein single domain antibody (1 ug/ml) was added and incubated for 1 hour at room temperature, control wells were PBS; and washed 3 times with PBST to wash off the liquid in the wells; 100uL of HRP-Protein A (1:10000 dilution) was added and incubated for 1 hour at room temperature; washing the well plate 3 times using PBST to wash off the liquid in the well;
adding 100 uL/hole TMB color development solution, and incubating for 15 minutes at room temperature in a dark place; 50 uL/Kong Zhongzhi of liquid was added and the OD in the wells was read using an ELISA reader. Wherein when the ratio of OD value for RSV F protein divided by the blank (PBS) OD value > =4, it is determined that the candidate antibody is capable of binding to RSV F protein; meanwhile, when the OD value of the candidate antibody capable of binding to the RSV F antigen protein divided by the OD value of the positive control antibody Palivizumab is > =0.7, the candidate antibody is considered to specifically bind to the RSV F moiety, and the results are shown in table 1.
Table 1S190116:
table 1S190117:
2.4 investigation of RSV F protein binding of anti-RSV F protein single domain antibodies to cell surfaces by FACS
Recombinant K562 cells (K562-native F protein cell lines) transiently expressing RSV F protein on a membrane were obtained by transfecting K562 cells (ATCC) with a plasmid carrying the RSV F full-length protein gene (pDONOR-CMV-Fprotein-puro, iCarTab preparation, original plasmid structure shown in FIG. 10).
Taking a K562-native F protein cell strain and a K562 cell strain, and regulating the cell state to a logarithmic growth phase by using 1640 complete medium containing 10% FBS; dividing the two cells into several parts, each cell number being 5×10 5 The expressed antibodies were incubated with the target cells, respectively, and after thoroughly mixing, incubated at room temperature for 1 hour. Centrifuging at room temperature for 5 minutes, removing the supernatant containing the antibody, and washing the cells 3 times by using PBS;
adding 1uL of PE marked Anti-human IgG, fully and uniformly mixing, and incubating for 30 minutes at room temperature in a dark place; centrifuging at room temperature for 5 minutes, removing the supernatant containing the secondary antibody, and washing the cells 3 times by using PBS; cells were resuspended using 500uL PBS and flow analyzed. The results are shown in FIG. 4.
The results showed that 10 antibody strains of S190116-HB10, HC12, HE3, HF7 and S190117-HB1, HF8, HG3, HG10, HA6, HH9 had good binding ability. Finally 8 antibodies HB10, HC12, HB1, HF8, HG3, HG10, HA6 and HH9 were selected for cell neutralization inhibition experiments. The CDR sequences, amino acid sequences and nucleotide sequences of the 8 antibodies HB10, HC12, HB1, HF8, HG3, HG10, HA6, HH9 screened are shown in Table 2:
TABLE 2 Single domain antibodies against RSV
2.5 examination of neutralizing inhibitory Effect of anti-RSV F-protein Single-domain antibody by cell neutralizing inhibitory experiment
Dilution of antibodies: and respectively carrying out 8-fold dilution and then 8-fold gradient dilution on the 8 purified antibodies, wherein the obtained dilution multiples are respectively 8, 16, 32, 64, 128, 256, 512 and 1024. The sample size in each well was 50. Mu.L, and 6 wells were prepared in this experiment.
Addition of RSV virus: according to the result of the pre-experiment, the TCID50 of the experimental virus is 3. Viruses were diluted to the desired titer with serum-free DMEM, then 50 μl was added to each well, and the 96-well plates were placed in a 5% incubator at 37 ℃ and incubated for 2h.
Adding cells: prepared HEP-2 cell CO 2 Digestion counts. Based on the counting results, the cells were diluted to 10 5 And each mL. Plates incubated for 2h above were removed and counted HEP-2 cells were added at 100. Mu.L per well. Transfer to 37 ℃ 5% CO 2 Culturing in an incubator for 3-7 days, and observing day by day.
After obvious lesions are found, the liquid is added to the fluorogenic substrate and the luminescence value is read by a chemiluminescent instrument. A fluorescence value below 5000 indicates no lesions, the higher the fluorescence value, the more viral content. The results are shown in Table 3 and FIG. 11.
TABLE 3 Table 3
Humanized antibody sequence design
The single domain antibody HG10 sequences screened according to example 2 were humanized using a design of surface amino acid substitutions.
TABLE 4 humanized anti-RSV antibodies
3.1 humanized antibody Gene Synthesis and expression vector construction
The humanized antibodies designed as described above were individually subjected to gene synthesis and subcloned into the expression vector Lenti-hIgG1-Fc2 (vector schematic as shown in FIG. 12) in tandem with human IgG1Fc (available from addgene). After the vector is verified by sequencing, the Qiagen plasmid megapump kit is used for preparing the endotoxin-removing plasmid for standby.
3.2 expression and purification of humanized antibodies
1. The LVTransm (Lipofectamine 2000 and Lipofectamine 3000) transfection reagent and antibody expression vector were removed from the freezer, thawed at room temperature, and thoroughly mixed by pipetting up and down. The PBS or HBSS buffer was removed and warmed to room temperature. Mu.l of PBS was added to one well of a 24-well plate, 4. Mu.g of Lenti-hIgG1-Fc2 was added, and after sufficiently mixing by pipetting up and down, 12. Mu.l of LVTransm was added, immediately mixing by pipetting up and down, and standing at room temperature for 10 minutes.
2. The above DNA/LVTransm complex was added to 1.5mL of 293F-SVP16 (ATCC) cells, and mixed thoroughly with gentle shaking. The cells were placed in a 37℃5% CO2 incubator at 130RPM for 6-8 hours, and 1.5mL fresh FreeStyle was added TM 293Expression Medium medium, and the cells were returned to the incubator for continued culture.
3. After 3 days of continuous culture, the culture supernatant was collected by centrifugation, filtered through a 0.45 μm filter, and the antibody was purified and subjected to flow assay.
3.3 flow detection of humanized antibody binding to target protein
1. The K562-native F protein cell line was recovered from liquid nitrogen and cell state was adjusted to logarithmic growth phase using 1640, 10% FBS complete medium.
2. The cells are divided into several parts, each cell number is 5 x 10≡5 cells.
3. The expressed antibodies were incubated with the target cells, respectively, and after thoroughly mixing, incubated at room temperature for 1 hour.
4.800Xg were centrifuged at room temperature for 5 minutes, the supernatant containing the antibody was removed, and the cells were washed 3 times with PBS.
5. 1uL PE-labeled Anti-human IgG was added, and after thoroughly mixing, incubated at room temperature for 30 minutes in the dark.
6.800Xg were centrifuged at room temperature for 5 minutes, the supernatant containing the secondary antibody was removed, and the cells were washed 3 times with PBS.
7. Cells were resuspended using 500uL PBS and flow analyzed. .
3.4ELISA detection of binding of recombinant antibodies to target proteins
1. Protein F of RSV was diluted with sterile PBS to a final concentration of 5ug/mL. A new 96-well plate was taken and coated overnight at 4℃with 100 uL/well.
2. The antigen coating was removed and washed 3 times with PBST (0.5% tween).
3. Blocking was performed for 2 hours at 37℃with 200 uL/well of 3% BSA;
4. after removal of the blocking buffer, the well plate was washed 3 times with PBST;
5. 100ul of diluted antibody (1 ug/ml) was added and incubated for 1 hour at room temperature, control wells were PBS;
6. remove the liquid in the wells and wash 3 times with PBST;
7. 100uL of HRP-Protein A (1:10000 dilution) was added and incubated for 1 hour at room temperature;
8. after removing the liquid from the wells, the well plate was washed 3 times with PBST;
9. adding 100 uL/hole TMB color development liquid;
10. incubating for 15 minutes at room temperature in a dark place;
11. adding 50 uL/Kong Zhongzhi liquid;
12. the o.d. values in the wells were read using a microplate reader.
3.5 expression purification of humanized antibodies
1. And taking out the LVTransm transfection reagent and the single-chain antibody expression vector from the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to completely mix uniformly. The PBS or HBSS buffer was removed and warmed to room temperature. 2mL of PBS was taken into one well of a 6-well plate, 130. Mu.g of Lenti-hIgG1-Fc2 was added, and after the mixture was thoroughly mixed by pipetting up and down, 400. Mu.L of LVTransm was added, immediately mixed by pipetting up and down, and left to stand at room temperature for 10 minutes.
2. The above DNA/LVTransm complex was added to 50mL of 293F-SVP16 (ATCC) cells and thoroughly mixed with gentle shaking. The cells were placed in a 37℃5% CO2 incubator at 130RPM for 6-8 hours, and 50mL fresh FreeStyle was added TM 293 medium, the cells were returned to the incubator for continued culture.
3. After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube and the antibody was purified using a Protein A column.
3.6 humanized antibody affinity detection
The F protein of RSV was immobilized on a CM5 chip using 10mM Acetate buffer, and the binding ability of the humanized antibodies to the F protein of the target protein RSV was measured before and after humanization using the positive humanized antibody and the original antibody prepared as described above as mobile phases.
3.7 post-humanization neutralization inhibition assay
Experimental procedure is detailed in 2.5
3.8 results
Construction of 3.8.1 antibody expression vector sequencing results
All constructed antibody expression vectors were sequenced by Sanger and were completely correct (fig. 13).
3.8.2 humanized antibody flow cytometry detection results
The humanized antibody expression vector is transiently transfected into 293F cells for small test expression, culture medium supernatant is collected, the antigen binding condition of the humanized antibody and the surface of a recombinant cell K562-native F protein cell membrane is detected by using a flow cytometer (figure 14), and the binding condition of the humanized antibody and the K562 cells is detected by using the flow cytometer (figure 15).
According to the flow detection result, the humanized antibodies HG10-2, HG10-3, HG10-4 and HG10-5 are specifically combined with recombinant K562-native F protein cells, and the combination force is equivalent to that of the HG10 antibody.
The results of the 3.8.3 humanized antibody ELISA assays are shown in Table 5.
Table 5:
HG10-3 and HG10-5 were selected for expression purification and affinity detection of antibodies based on the results of flow and ELISA detection using HG10 antibody as a positive control
3.8.4 affinity assay results
Determination of binding of HG10, HG10-3 and HG10-5 Single Domain antibodies to RSVF protein
Table 6 affinity assay results:
analysis of results: based on the affinity detection results, the humanized antibody has a consistent affinity with the original antibody.
3.8.5 results of neutralization inhibition test after humanization
Neutralization inhibition assay before and after humanization of HG10 single domain antibodies
Table 7 neutralization test results
Analysis of results: according to the neutralization inhibition test result, the humanized antibody is superior to the non-humanized antibody.
Multivalent ligation antibody sequence design
4.1 vector construction
Fc tag bivalent antibody expression vector construction
The single domain antibodies HG10 and HB1 obtained in the previous project are connected in series with the human IgG1Fc to construct a bivalent antibody expression vector (HG 10-ggggsgggsgggsgggs-HB 1-IgG1 Fc); constructing a bivalent antibody expression vector (HG 10-5-ggggsgggsgggsgggs-HG 10-5-IgG1 Fc) by using a humanized antibody HG10-5 antibody sequence and a human IgG1FC region; respectively carrying out gene synthesis, subcloning into a Lenti-hIgG1-Fc2-Puro vector, and constructing a bivalent antibody expression vector. After the vector was verified by sequencing, the endotoxin-removed plasmid was prepared using the Qiagen plasmid megapump kit.
HG10 antibody amino acid sequence shown in SEQ ID NO.28
The HB1 antibody has an amino acid sequence shown in SEQ ID NO. 13;
the amino acid sequence of the HG10-5 antibody is shown as SEQ ID NO. 51;
his tag diabody expression vector construction
The Fc tags of the two bivalent antibody expression vectors constructed in the step 1 are replaced by His tags, G4S is added between the antibody sequence and the His tags for preventing the His tags from being hidden, and 2 6 XHis tags are added at the same time, so that the His tag bivalent antibody expression vectors, namely HG 10-ggggsgggsgggsgggs-HB 1-His and HG 10-5-ggggggsgggsgggsggs-HG 10-5-His, are constructed. After sequencing verification, preparing the endotoxin-removing large drawing plasmid.
4.2 expression purification of diabodies
1. And taking out the LVTransm transfection reagent and the single-chain antibody expression vector from the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to completely mix uniformly. The PBS buffer was removed and warmed to room temperature. Respectively taking 2mL of PBS to two holes of a 6-hole plate, respectively adding 130 mug antibody expression vector, blowing up and down by a pipette to fully mix, adding 400 mug LVTransm, immediately blowing up and down by the pipette to mix, and standing for 10 minutes at room temperature.
2. The DNA/LVTransm complex was added to 50mL of 293F cells, gently swirled and thoroughly mixed. The cells were placed in a 37℃5% CO2 incubator at 130RPM for 6-8 hours, and 50mL fresh FreeStyle was added TM 293 Medium, to be fineThe cells were returned to the incubator for continued culture.
3. After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube and the antibodies were purified using Protein A and nickel column affinity columns, respectively.
SDS-PAGE detects protein purity.
4.3 results
Expression of diabodies purified SDS-PAGE detection: (see FIG. 16)
SEQUENCE LISTING
<110> Suzhou Gao Hongli Biotech Co., ltd
<120> protein binding molecules against respiratory syncytial virus
<130> JS1981-22P150638-DIV
<150> CN201910711455.7
<151> 2019-08-02
<160> 66
<170> PatentIn version 3.5
<210> 1
<211> 488
<212> PRT
<213> respiratory syncytial virus
<400> 1
Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser
1 5 10 15
Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile
20 25 30
Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp
35 40 45
Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala
50 55 60
Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Ala Asn Ser
65 70 75 80
Lys Ala Lys Lys Glu Ala Pro Arg Gly Met Arg Tyr Thr Met Asn Leu
85 90 95
Gln Arg Asn Val Asn Val Thr Asp Ser Leu Lys Lys Lys Lys Lys Phe
100 105 110
Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Ile Ala
115 120 125
Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser
130 135 140
Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val
145 150 155 160
Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys
165 170 175
Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile
180 185 190
Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile
195 200 205
Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr
210 215 220
Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro
225 230 235 240
Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val
245 250 255
Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu
260 265 270
Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys
275 280 285
Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly
290 295 300
Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn
305 310 315 320
Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln
325 330 335
Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser
340 345 350
Glu Val Asn Leu Cys Asn Ile Asp Ile Phe Asn Pro Lys Tyr Asp Cys
355 360 365
Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser
370 375 380
Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser
385 390 395 400
Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr
405 410 415
Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr
420 425 430
Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro
435 440 445
Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp
450 455 460
Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe
465 470 475 480
Ile Arg Lys Ser Asp Glu Leu Leu
485
<210> 2
<211> 545
<212> PRT
<213> Artificial
<220>
<223> codon optimized F protein
<400> 2
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Gln Asn Ile Thr Glu Glu Phe Tyr
20 25 30
Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu Arg
35 40 45
Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile Lys
50 55 60
Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys Gln
65 70 75 80
Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu Met
85 90 95
Gln Ser Thr Pro Ala Ala Asn Ser Lys Ala Lys Lys Glu Ala Pro Arg
100 105 110
Gly Met Arg Tyr Thr Met Asn Leu Gln Arg Asn Val Asn Val Thr Asp
115 120 125
Ser Leu Lys Lys Lys Lys Lys Phe Leu Gly Phe Leu Leu Gly Val Gly
130 135 140
Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu Glu
145 150 155 160
Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala
165 170 175
Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val Leu
180 185 190
Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn Lys
195 200 205
Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln
210 215 220
Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Ala
225 230 235 240
Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu
245 250 255
Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu
260 265 270
Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met
275 280 285
Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu
290 295 300
Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu
305 310 315 320
Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr
325 330 335
Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro
340 345 350
Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr
355 360 365
Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Ile Asp
370 375 380
Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp
385 390 395 400
Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr
405 410 415
Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys
420 425 430
Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr
435 440 445
Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys
450 455 460
Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu
465 470 475 480
Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu
485 490 495
Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu
500 505 510
Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile
515 520 525
Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu
530 535 540
Arg
545
<210> 3
<211> 122
<212> PRT
<213> Artificial
<220>
<223> HB10 antibodies
<400> 3
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Pro Phe Ser
20 25 30
Asp Tyr Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu
35 40 45
Trp Val Ser Gly Ile Tyr Lys Asp Gly Ser Gly Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Ile Tyr
85 90 95
Tyr Cys Leu Ser Gly Trp Gly Leu Asp Gly Leu Pro Arg Gly Ser Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 4
<211> 9
<212> PRT
<213> Artificial
<220>
<223> CDR1/HB10
<400> 4
Gly Phe Pro Phe Pro Phe Ser Asp Tyr
1 5
<210> 5
<211> 6
<212> PRT
<213> Artificial
<220>
<223> CDR2/HB10
<400> 5
Tyr Lys Asp Gly Ser Gly
1 5
<210> 6
<211> 11
<212> PRT
<213> Artificial
<220>
<223> CDR3/HB10
<400> 6
Gly Trp Gly Leu Asp Gly Leu Pro Arg Gly Ser
1 5 10
<210> 7
<211> 366
<212> DNA
<213> Artificial
<220>
<223> nucleotide encoding HB10
<400> 7
gaggtgcagc tggtggagtc tgggggaggc ttggcgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt ccccttcccc ttcagtgatt atgccatgac ctgggtccgc 120
caggctccag gaaaggagct cgagtgggtg tccggcattt ataaggatgg tagtggcact 180
tactatgcag actccgtgaa ggggcgcttc accatctcca gagacaacgc caagaatatg 240
ctgtatctgc aaatgaacaa cctgaaacct gaggacacgg ggatatatta ctgtttgagt 300
ggatggggct tggacggtct tccccggggt tcctggggcc aggggaccca ggttaccgtc 360
tcctcg 366
<210> 8
<211> 127
<212> PRT
<213> Artificial
<220>
<223> HC12 Single Domain antibody
<400> 8
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Gly Ala Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Asn Gly Gly Ser Thr Val Arg Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Leu
65 70 75 80
Leu Gln Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Lys Glu Phe Phe Phe Gly Ser Trp Cys Leu Ser Ser Gly Lys
100 105 110
Ala Ser Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 7
<212> PRT
<213> Artificial
<220>
<223> CDR1/HC12
<400> 9
Gly Ser Thr Leu Gly Ala Tyr
1 5
<210> 10
<211> 6
<212> PRT
<213> Artificial
<220>
<223> CDR2/HC12
<400> 10
Ser Ser Asn Gly Gly Ser
1 5
<210> 11
<211> 18
<212> PRT
<213> Artificial
<220>
<223> CDR3/HC12
<400> 11
Lys Glu Phe Phe Phe Gly Ser Trp Cys Leu Ser Ser Gly Lys Ala Ser
1 5 10 15
Gln Ser
<210> 12
<211> 381
<212> DNA
<213> Artificial
<220>
<223> nucleotide encoding HC12
<400> 12
gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgacactc 60
tcctgtgcag cctctggatc cactttgggt gcttatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaagg ggtctcatgt attagtagta atggtggtag tacagtgcgg 180
gcagactccg tgaggggccg attcaccatc tccagagaca acgccaagaa cacggtgctg 240
ctgcagatga acaacctgca acctgaggac acagcaattt actactgtgc agcaaaggag 300
ttcttcttcg gtagctggtg ccttagcagt gggaaagcgt ctcagtcctg gggccagggg 360
acccaggtca ccgtctcctc t 381
<210> 13
<211> 126
<212> PRT
<213> Artificial
<220>
<223> HB1 Single Domain antibody
<400> 13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Gly Thr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Gly Gly Ser Ile Val Val Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Leu Leu
65 70 75 80
Gln Met Asn Asn Leu Gln Leu Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Ala Lys Thr Tyr Phe Phe Gly Ser Trp Cys His Ser Asn Gly Trp Thr
100 105 110
Ser Glu Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Thr
115 120 125
<210> 14
<211> 8
<212> PRT
<213> Artificial
<220>
<223> CDR1/HB1
<400> 14
Gly Ser Thr Leu Gly Thr Tyr Ala
1 5
<210> 15
<211> 7
<212> PRT
<213> Artificial
<220>
<223> CDR2/HB1
<400> 15
Ile Ser Ser Gly Gly Ser Ile
1 5
<210> 16
<211> 18
<212> PRT
<213> Artificial
<220>
<223> CDR3/HB1
<400> 16
Lys Thr Tyr Phe Phe Gly Ser Trp Cys His Ser Asn Gly Trp Thr Ser
1 5 10 15
Glu Ser
<210> 17
<211> 378
<212> DNA
<213> Artificial
<220>
<223> nucleotide encoding HB1
<400> 17
gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgcgcag cctctggatc cactttgggt acttatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaagg ggtctcatgt attagtagtg gtggtagtat agtcgttgca 180
gactccgtga agggccgatt caccatttcc agagacaacg ccaagaacac ggtgcttctg 240
caaatgaaca acctacaact tgaggacaca gcaatttact actgtgcagc aaagacgtac 300
ttcttcggta gctggtgtca tagcaatggg tggacgtccg agtcctgggg ccaggggact 360
caggtcaccg tctccacg 378
<210> 18
<211> 128
<212> PRT
<213> Artificial
<220>
<223> HF8 single domain antibody
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Leu Met Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Asp
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Asp Tyr Tyr Cys
85 90 95
Ala Ala Ala Arg Phe Asp Ser Leu Tyr Gly Ser Ser Cys Phe Arg Ala
100 105 110
Ala Leu Tyr Glu Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 19
<211> 8
<212> PRT
<213> Artificial
<220>
<223> CDR1/HF8
<400> 19
Gly Phe Thr Leu Asp Tyr Tyr Ala
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial
<220>
<223> CDR2/HF8
<400> 20
Ile Ser Arg Ser Gly Leu Met Thr
1 5
<210> 21
<211> 19
<212> PRT
<213> Artificial
<220>
<223> CDR3/HF8
<400> 21
Ala Arg Phe Asp Ser Leu Tyr Gly Ser Ser Cys Phe Arg Ala Ala Leu
1 5 10 15
Tyr Glu Asn
<210> 22
<211> 384
<212> DNA
<213> artificial
<220>
<223> nucleotide encoding HF8
<400> 22
gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt cactttggat tattatgccg taggctggtt ccgccaggcc 120
ccagggaggg agcgtgaggg ggtctcatgt attagtagga gtggtcttat gacaaactat 180
gccgactccg tgaagggccg attcaccgtc tccagagaca acgccaagaa cacggtagat 240
ttgcaaatga acagcctgaa acctgacgac acggccgatt attactgtgc agcagcccgg 300
ttcgactccc tgtatggtag tagttgcttc agagcggcgc tatatgagaa ttggggccag 360
gggacccagg tcaccgtctc ctca 384
<210> 23
<211> 125
<212> PRT
<213> Artificial
<220>
<223> HG3 single domain antibodies
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Thr Val Gly Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Glu Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Glu Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Thr
115 120 125
<210> 24
<211> 7
<212> PRT
<213> Artificial
<220>
<223> CDR1/HG3
<400> 24
Gly Phe Thr Leu Asp Ser Tyr
1 5
<210> 25
<211> 5
<212> PRT
<213> Artificial
<220>
<223> CDR2/HG3
<400> 25
Thr Val Gly Gly Arg
1 5
<210> 26
<211> 17
<212> PRT
<213> Artificial
<220>
<223> CDR3/HG3
<400> 26
Glu Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val Glu
1 5 10 15
Ser
<210> 27
<211> 375
<212> DNA
<213> Artificial
<220>
<223> nucleotide encoding HG3
<400> 27
gaggtgcagc tggtggagtc tgggggaggc ttggtgcagt ctggggggtc tctgagactc 60
tcctgtgagg cctctggatt cactttggat agttatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg ggtctcatgt attactgttg gtggtagaac aaactatgca 180
gacccggtga agggccgatt caccatttcg agagacaacg ccaagaacac ggtgtatctg 240
caaatgaata gcctgaaacc tgaggacaca gccatttatt actgttcagc agaagatcga 300
ctgttcggcc tttgtagcct atccccaaaa atcgttgagt cctggggcca ggggacccag 360
gtcaccgtct ccacg 375
<210> 28
<211> 125
<212> PRT
<213> Artificial
<220>
<223> HG10 single domain antibody
<400> 28
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Thr Val Gly Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Thr
115 120 125
<210> 29
<211> 8
<212> PRT
<213> Artificial
<220>
<223> CDR1/HG10
<400> 29
Gly Phe Thr Leu Asp Ser Tyr Ala
1 5
<210> 30
<211> 5
<212> PRT
<213> Artificial
<220>
<223> CDR2/HG10
<400> 30
Thr Val Gly Gly Arg
1 5
<210> 31
<211> 17
<212> PRT
<213> Artificial
<220>
<223> CDR3/HG10
<400> 31
Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val Asp
1 5 10 15
Ser
<210> 32
<211> 375
<212> DNA
<213> Artificial
<220>
<223> nucleotide encoding HG10
<400> 32
gaggtgcagc tggtggagtc tgggggaggc ttggtgcagt ctggggggtc tctgagactc 60
tcctgtgagg cctctggatt cactttggat agttatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg ggtctcatgt attactgttg gtggtagaac aaactatgca 180
gacccggtga agggccgatt caccatttct agagacaacg ccaagaacac ggtgtatctg 240
caaatgaaca gcctgaaacc tgaggacaca gccatttatt actgttcagc agatgatcga 300
ctgttcggcc tttgtagcct atccccaaaa atcgttgact cctggggcca ggggacccag 360
gtcaccgtct ccacg 375
<210> 33
<211> 124
<212> PRT
<213> Artificial
<220>
<223> HA6 Single Domain antibodies
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Asn Ile Glu
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Val Ile Thr Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Ile Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Val Arg Ala Glu Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Ala Asp Leu Arg Tyr Phe Asn Pro Tyr Gly Pro Asp Arg Arg Leu Glu
100 105 110
Val Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 34
<211> 7
<212> PRT
<213> Artificial
<220>
<223> CDR1/HA6
<400> 34
Gly Ser Thr Ser Asn Ile Glu
1 5
<210> 35
<211> 5
<212> PRT
<213> Artificial
<220>
<223> CDR2/HA6
<400> 35
Thr Ser Gly Gly Ser
1 5
<210> 36
<211> 16
<212> PRT
<213> Artificial
<220>
<223> CDR3/HA6
<400> 36
Asp Leu Arg Tyr Phe Asn Pro Tyr Gly Pro Asp Arg Arg Leu Glu Val
1 5 10 15
<210> 37
<211> 372
<212> DNA
<213> Artificial
<220>
<223> nucleotide encoding HA6
<400> 37
gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggactc tctgagactc 60
tcctgtgcag cctctggaag cacctctaat atcgaaacca tgggatggta ccgccaggct 120
ccagggaagc agcgcgagtt ggttgcagta attactagtg gtggcagcac aagctatgca 180
gactccatag agggccgatt caccatctcc agagtcagag ccgagaacac actctatctg 240
caaatgaaca gcctgaaacc tgaggacacg gccgtgtatt actgtaaggc agatcttagg 300
tactttaacc catatggccc cgacaggcgt ctcgaagttt ggggccaggg cacccaggtc 360
accgtctcct ca 372
<210> 38
<211> 123
<212> PRT
<213> Artificial
<220>
<223> HH9 Single Domain antibodies
<400> 38
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Leu Ser Leu Arg Ala Tyr
20 25 30
Gln Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile
35 40 45
Ser Cys Ser Ile Asp Ser Gly Ala Thr Ile Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ile Val Ser Arg Asp Ser Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Ser Asp Phe Cys Ser Arg Asn Pro Ala Gln Tyr Asn Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 39
<211> 8
<212> PRT
<213> Artificial
<220>
<223> CDR1/HH9
<400> 39
Arg Leu Ser Leu Arg Ala Tyr Gln
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial
<220>
<223> CDR2/HH9
<400> 40
Ser Ile Asp Ser Gly Ala Thr Ile
1 5
<210> 41
<211> 14
<212> PRT
<213> Artificial
<220>
<223> CDR3/HH9
<400> 41
Arg Ser Asp Phe Cys Ser Arg Asn Pro Ala Gln Tyr Asn Tyr
1 5 10
<210> 42
<211> 369
<212> DNA
<213> Artificial
<220>
<223> nucleotide encoding HH9
<400> 42
gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ccggggggtc tctgagactc 60
tcctgtgcag cctccagact ctctttgcgt gcctatcaaa taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg gatctcatgt agtatcgata gtggcgcgac cataacttat 180
gcagactccg tgaagggccg attcatcgtc tccagagaca gtgccaagaa cacggtgtat 240
ctgcaaatga acaacctgaa acctgaggac acagccgttt attactgtgc agcacggtcc 300
gacttctgtt cacggaaccc ggcacaatat aactactggg gccaggggac ccaggtaacc 360
gtctcctca 369
<210> 43
<211> 125
<212> PRT
<213> artificial sequence
<400> 43
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Val Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 44
<211> 125
<212> PRT
<213> artificial sequence
<400> 44
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Thr Val Gly Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 45
<211> 125
<212> PRT
<213> artificial sequence
<400> 45
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Thr Val Gly Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 46
<211> 125
<212> PRT
<213> artificial sequence
<400> 46
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Thr Val Gly Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 47
<211> 125
<212> PRT
<213> artificial sequence
<400> 47
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Thr Val Gly Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 48
<211> 125
<212> PRT
<213> artificial sequence
<400> 48
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Val Gly Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 49
<211> 125
<212> PRT
<213> artificial sequence
<400> 49
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Val Gly Gly Arg Thr Tyr Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 50
<211> 125
<212> PRT
<213> artificial sequence
<400> 50
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Val Gly Gly Arg Thr Tyr Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 51
<211> 125
<212> PRT
<213> artificial sequence
<400> 51
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Val Gly Gly Arg Thr Tyr Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 52
<211> 125
<212> PRT
<213> artificial sequence
<400> 52
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Val Gly Gly Arg Thr Tyr Tyr Ala Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 53
<211> 125
<212> PRT
<213> artificial sequence
<400> 53
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val
35 40 45
Ser Ala Ile Thr Val Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
85 90 95
Ala Asp Asp Arg Leu Phe Gly Leu Cys Ser Leu Ser Pro Lys Ile Val
100 105 110
Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 54
<211> 375
<212> DNA
<213> artificial sequence
<400> 54
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat agctatgcca tgagctgggt ccgacaggcc 120
cctggcaaag gacttgaatg ggtgtccgcc atcacagtcg gcggcagaac ctactacgcc 180
gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgagagc cgaggacacc gccgtgtact actgtagcgc cgacgataga 300
ctgttcggcc tgtgcagcct gtctcctaag atcgtggatt cttggggcca gggcaccctg 360
gtcacagtct cttct 375
<210> 55
<211> 375
<212> DNA
<213> artificial sequence
<400> 55
gaggtgcagc tggttgaatc tggcggagga ctggttcagt ctggcggctc tctgagactg 60
agctgtgaag ccagcggctt taccctggac agctatgcca tcggctgggt ccgacaggct 120
cctggcaaag gacttgagtg ggtgtcctgc atcacagtcg gcggcaggac caattacgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaacg ccaagaacac cgtgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 56
<211> 375
<212> DNA
<213> artificial sequence
<400> 56
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
agctgtgaag ccagcggctt taccctggac agctatgcca tcggctgggt ccgacaggct 120
cctggcaaag gacttgagtg ggtgtcctgc atcacagtcg gcggcaggac caattacgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaacg ccaagaacac cgtgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 57
<211> 375
<212> DNA
<213> artificial sequence
<400> 57
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat tcttatgcca tcggctgggt ccgacaggcc 120
cctggaaaag gacttgagtg ggtgtcctgc atcacagtcg gcggcaggac caattacgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaacg ccaagaacac cgtgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 58
<211> 375
<212> DNA
<213> artificial sequence
<400> 58
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat agctatgcca tgagctgggt ccgacaggcc 120
cctggcaaag gacttgaatg ggtgtcctgc atcacagtcg gcggcaggac caattacgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaacg ccaagaacac cgtgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 59
<211> 375
<212> DNA
<213> artificial sequence
<400> 59
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat agctatgcca tgagctgggt ccgacaggcc 120
cctggcaaag gacttgaatg ggtgtccgcc atcacagtcg gcggcaggac caattatgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaacg ccaagaacac cgtgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 60
<211> 375
<212> DNA
<213> artificial sequence
<400> 60
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat agctatgcca tgagctgggt ccgacaggcc 120
cctggcaaag gacttgaatg ggtgtccgcc atcacagtcg gcggcagaac ctactatgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaacg ccaagaacac cgtgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 61
<211> 375
<212> DNA
<213> artificial sequence
<400> 61
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat agctatgcca tgagctgggt ccgacaggcc 120
cctggcaaag gacttgaatg ggtgtccgcc atcacagtcg gcggcagaac ctactatgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cgtgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 62
<211> 375
<212> DNA
<213> artificial sequence
<400> 62
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat agctatgcca tgagctgggt ccgacaggcc 120
cctggcaaag gacttgaatg ggtgtccgcc atcacagtcg gcggcagaac ctactatgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgaagcc tgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 63
<211> 375
<212> DNA
<213> artificial sequence
<400> 63
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
tcttgtgccg ccagcggctt caccctggat agctatgcca tgagctgggt ccgacaggcc 120
cctggcaaag gacttgaatg ggtgtccgcc atcacagtcg gcggcagaac ctactatgcc 180
gatcctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgagagc cgaggacacc gccatctact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 64
<211> 375
<212> DNA
<213> artificial sequence
<400> 64
gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60
agctgtagcg ccagcggctt caccctggat agctatgcca tgcactgggt ccgacaggcc 120
cctggcaaag gcctggaata tgtgtctgcc atcaccgtcg gcggcagaac ctactacgcc 180
gattctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240
cagatgagca gcctgagagc cgaggatacc gccgtgtact actgcagcgc cgacgataga 300
ctgttcggcc tgtgttctct gagccccaag atcgtggatt cttggggcca gggcacactg 360
gtcacagtgt catct 375
<210> 65
<211> 227
<212> PRT
<213> Homo sapiens
<400> 65
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 66
<211> 681
<212> DNA
<213> Homo sapiens
<400> 66
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600
aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 660
ctctccctgt ctccgggtaa a 681

Claims (8)

1. A single domain antibody directed against respiratory syncytial virus, which specifically binds to protein F of respiratory syncytial virus, comprising an immunoglobulin single variable domain comprising three antibody complementarity determining regions CDR1, CDR2 and CDR3, the complementarity determining regions being:
CDR1 as shown in SEQ ID No.29, CDR2 as shown in SEQ ID No.30 and CDR3 as shown in SEQ ID No. 31.
2. The single domain antibody against respiratory syncytial virus according to claim 1, characterized in that the antibody comprises amino acids having the following amino acid sequence: an amino acid as shown in SEQ ID NO. 28.
3. The single domain antibody against respiratory syncytial virus according to claim 1, wherein the immunoglobulin single variable domain is a humanized heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 43-47.
4. The single domain antibody against respiratory syncytial virus according to claim 1, wherein the antibody further binds to an immunoglobulin Fc region.
5. The single domain antibody against respiratory syncytial virus of claim 1, wherein the antibody forms a multivalent linkage.
6. A nucleic acid molecule encoding the single domain antibody of any one of claims 1-5 against respiratory syncytial virus.
7. A pharmaceutical composition comprising the single domain antibody of claim 1 directed against respiratory syncytial virus and a pharmaceutically acceptable carrier.
8. Use of a single domain antibody against respiratory syncytial virus according to claim 1 for the preparation of a medicament for the prevention and/or treatment of diseases which are associated with respiratory syncytial virus infection.
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