CN112513076B - Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof - Google Patents

Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof Download PDF

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CN112513076B
CN112513076B CN202080002271.4A CN202080002271A CN112513076B CN 112513076 B CN112513076 B CN 112513076B CN 202080002271 A CN202080002271 A CN 202080002271A CN 112513076 B CN112513076 B CN 112513076B
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张军方
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Shenzhen Yinnuosai Biology Technology Co ltd
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Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof. The binding molecule is capable of specifically binding to the spike protein of SARS-COV-2 and comprises at least one immunoglobulin single variable domain. The SARS-COV-2-Spike protein binding molecule provided by the invention can specifically bind to SARS-COV-2-Spike protein, and effectively block the binding of SARS-COV-2-Spike protein and human body cell ACE2 receptor, thereby blocking the infection process of SARS-COV-2 to cells, inhibiting the infection and amplification of SARS-COV-2, playing the role of long-acting inhibition of SARS-COV-2 in vivo, and effectively avoiding the repeated occurrence of SARS-COV-2 in vivo.

Description

Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof.
Background
At present, no specific medicine exists in COVID-19, and rapid development of effective medicines is urgently needed. Although the developed broad-spectrum small-molecule antiviral drugs such as Reidcisvir, Favipiravir and the like have certain curative effect on COVID-19, the drug has no specificity to SARS-COV-2 and has limited curative effect, so the drug is difficult to become a specific drug of COVID-19.
Disclosure of Invention
Aiming at the problems that the prior antiviral drug has no specificity to the novel coronavirus of the novel coronavirus, has poor treatment effect and is difficult to become a specific drug for SARS-COV-2, the invention provides a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a SARS-COV-2 spike protein binding molecule capable of specifically binding to SARS-COV-2 spike protein and comprising at least one immunoglobulin single variable domain, wherein the CDR1, CDR2 and CDR3 of said immunoglobulin single variable domain are selected from any one of the group consisting of:
1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3;
2) CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6;
3) CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 9;
4) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 12;
5) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14 and CDR3 shown in SEQ ID NO. 15;
6) CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17 and CDR3 shown in SEQ ID NO. 18;
7) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20 and CDR3 shown in SEQ ID NO. 21;
8) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23 and CDR3 shown in SEQ ID NO. 24;
9) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26 and CDR3 shown in SEQ ID NO. 27;
10) CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29 and CDR3 shown in SEQ ID NO. 30;
11) CDR1 shown in SEQ ID NO. 31, CDR2 shown in SEQ ID NO. 32 and CDR3 shown in SEQ ID NO. 33;
12) CDR1 shown in SEQ ID NO. 34, CDR2 shown in SEQ ID NO. 35 and CDR3 shown in SEQ ID NO. 36;
13) CDR1 shown in SEQ ID NO. 37, CDR2 shown in SEQ ID NO. 38 and CDR3 shown in SEQ ID NO. 39;
14) CDR1 shown in SEQ ID NO. 40, CDR2 shown in SEQ ID NO. 41 and CDR3 shown in SEQ ID NO. 42;
15) CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 44 and CDR3 shown in SEQ ID NO. 45;
16) CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 47 and CDR3 shown in SEQ ID NO. 48;
17) CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 50 and CDR3 shown in SEQ ID NO. 51;
18) CDR1 shown in SEQ ID NO. 52, CDR2 shown in SEQ ID NO. 53 and CDR3 shown in SEQ ID NO. 54;
19) CDR1 shown in SEQ ID NO. 55, CDR2 shown in SEQ ID NO. 56 and CDR3 shown in SEQ ID NO. 57;
20) CDR1 shown in SEQ ID NO. 58, CDR2 shown in SEQ ID NO. 59 and CDR3 shown in SEQ ID NO. 60;
21) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62 and CDR3 shown in SEQ ID NO: 63.
Compared with the prior art, the SARS-COV-2 Spike protein (SARS-COV-2-Spike protein) binding molecule provided by the invention can specifically bind to SARS-COV-2-Spike protein, effectively block the binding of SARS-COV-2-Spike protein and human body cell ACE2 receptor, further block the infection process of SARS-COV-2 to cells, and inhibit the infection and amplification of SARS-COV-2. The SARS-COV-2-Spike protein binding molecule provided by the invention also has the characteristics of good specificity of binding with SARS-COV-2-Spike protein, high biological activity and stability and no toxic or side effect. Meanwhile, the SARS-COV-2-Spike protein binding molecule provided by the invention can play a role of long-acting inhibition on SARS-COV-2 in vivo, and effectively avoids the repeated infection or positive infection of SARS-COV-2 in vivo.
Preferably, the immunoglobulin single variable domain is a single domain antibody.
Preferably, the single domain antibody comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS 64-84.
Preferably, the single domain antibody comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS 64-84.
Preferably, the single domain antibody comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOS 64-84.
Preferably, the single domain antibody comprises any one of the amino acid sequences of SEQ ID NO 64-84.
Preferably, the SARS-COV-2 spike protein binding molecule further comprises an immunoglobulin Fc region.
The inclusion of an immunoglobulin Fc region in the SARS-COV-2 spike protein binding molecule allows the binding molecule to form a dimer while further extending the in vivo half-life of the molecule. The Fc region used in the present invention may be from different subtypes of immunoglobulin, for example, IgG (IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM.
Preferably, the immunoglobulin Fc region is the Fc region of human IgG 1.
Preferably, the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO 85.
The stability and biological activity of the combined molecule fused with the Fc region are further improved, and the KD value of the combined molecule combined with SARS-COV-2 spike protein is further reduced.
Preferably, the SARS-COV-2 spike protein binding molecule comprises at least one amino acid sequence of SEQ ID NO 86-106.
The invention also provides a nucleic acid molecule for coding the SARS-COV-2 spike protein binding molecule, wherein the nucleic acid molecule is RNA, DNA or cDNA, which can be obtained by artificial synthesis or separated from proper natural sources.
The invention also provides an expression vector containing the nucleic acid molecule and an expression control element thereof. The expression vector typically comprises at least one nucleic acid molecule provided herein operably linked to one or more suitable expression regulatory elements (promoters, enhancers, terminators, integration factors, selection markers, leaders, reporters, and the like). The selection of such elements and their sequences for expression in a particular host cell is within the knowledge of one skilled in the art.
The invention also provides host cells comprising and expressing the nucleic acid molecules. The host cell is a cell for expressing a heterologous protein, including a bacterial cell, a fungal cell, or a mammalian cell.
The invention also provides a method for obtaining the SARS-COV-2 spike protein binding molecule, which comprises the following steps:
a. culturing the host cell of claim 16 under conditions that allow expression of the SARS-COV-2 spike protein binding molecule;
b. collecting the SARS-COV-2 spike protein binding molecule expressed by the host cell from the culture of step a.
The recombination of specific nucleic acid molecules into expression vectors and expression into host cells by transformation or transfection methods, selection of markers, methods of inducing protein expression, culture conditions, and the like are known in the art. Techniques for the isolation and purification of protein binding molecules are well known to those skilled in the art.
In addition, the SARS-COV-2 spike protein binding molecule of the invention can also be obtained by other methods known in the art for producing proteins of known sequence, such as chemical synthesis.
The invention also provides an immunoconjugate comprising a SARS-COV-2 spike protein binding molecule of any one of the above conjugated to a therapeutic moiety.
The invention also provides a pharmaceutical composition comprising the SARS-COV-2 spike protein binding molecule and/or the immunoconjugate described above, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the invention may further comprise other adjuvants and auxiliary materials, etc. according to the needs.
The "pharmaceutically acceptable carrier" according to the present invention includes any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, and the like which are physiologically compatible. 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., the binding molecule, immunoconjugate, may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound, as is well known to those skilled in the art.
The invention also provides application of the pharmaceutical composition in preparing a medicine for treating or preventing novel pneumonia caused by the coronavirus disease.
The invention also provides a kit for detecting SARS-COV-2, comprising any one of the SARS-COV-2 spike protein binding molecules.
The invention also provides the using method of the kit for detecting SARS-COV-2, under the condition that the SARS-COV-2 spike protein binding molecule and the SARS-COV-2 spike protein can form a compound, the SARS-COV-2 spike protein binding molecule is contacted with a detection sample and a control sample, and the formation of the compound is detected; the presence of SARS-COV-2 in the sample is determined by the difference in complex formation between the test sample and the control sample.
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FIG. 1 is an agarose gel electrophoresis of total RNA extracted in example 1 of the present invention, wherein M: DNA marker 2000, lane 1: total RNA;
FIG. 2 is an agarose gel electrophoresis image of the PCR amplification product in Step1 of nested PCR amplification single domain antibody gene in example 1 of the present invention, wherein M: DNA marker 2000, lane 1: (ii) amplification products;
FIG. 3 is an agarose gel electrophoresis of PCR amplification products in Step2 of nested PCR amplification of single domain antibody gene in example 1 of the present invention, wherein DNA marker 2000, lane 1: (ii) amplification products;
FIG. 4 is an agarose gel electrophoresis of colony PCR amplification products for calculating library insertion rates in example 1 of the present invention, wherein M: DNA marker 2000; lanes 1-8: 8 colonies were picked;
FIG. 5 is a graph showing the change in viral load in the respiratory tracts of rhesus monkeys in the treatment group and the control group according to the change in days in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Definition of
Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art that will be understood by those skilled in the art. Moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically recited may be and have been performed in a manner known per se to those of skill in the art.
The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin domain consisting essentially of four "framework regions" referred to in the art as "framework region 1" or "FR 1", "framework region 2" or "FR 2", "framework region 3" or "FR 3", and "framework region 4" or "FR 4", wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art as "complementarity determining region 1" or "CDR 1", "complementarity determining region 2" or "CDR 2", and "complementarity determining region 3" or "CDR 3". Thus, the general structure or sequence of an immunoglobulin single variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Immunoglobulin single variable domains confer antigen specificity to antibodies by virtue of having an antigen binding site.
Conventional IgG antibody molecules generally consist of a light chain comprising 1 variable region (VL) and 1 constant region (CL) and a heavy chain comprising 1 variable region (VH) and 3 constant regions (CH1, CH2, CH 3). Single domain antibodies (sdabs), which are antibodies lacking the light chain of the antibody and having only the variable region of the heavy chain, are also called nanobodies (nanobodies) because of their small molecular weight. Single domain antibodies specifically bind epitopes without the need for additional antigen binding domains. Single domain antibodies are small, stable and efficient antigen recognition units formed from immunoglobulin single variable domains.
It is well known in the art that the total number of amino acid residues in each CDR may differ for single domain antibodies.
The total number of amino acid residues in a single domain antibody will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.
Methods for obtaining single domain antibodies that bind to a particular antigen or epitope have been previously disclosed in the following references: van der linden et al, Journal of Immunological Methods,240(2000) 185-195; li et al, JBiol chem.,287(2012) 13713-13721; deffar et al, African Journal of Biotech biology Vol.8(12), pp.2645-2652,17June,2009 and WO 94/04678.
In addition, those 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.
In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the invention) molecule can bind. Specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity. The 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 the antigen binding site on the 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 the association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be determined in a known manner depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen binding molecule (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and an associated antigen. Affinity is related to both: affinity to its antigen binding site on the antigen binding molecule, and the number of relevant binding sites present on the antigen binding molecule.
The term "SARS-COV-2 Spike protein binding molecule (SARS-COV-2-Spike protein binding molecule)" as used herein means any molecule capable of specifically binding to SARS-COV-2 Spike protein. The SARS-COV-2 spike protein binding molecule may comprise a single domain antibody or conjugate thereof as defined herein directed against the SARS-COV-2 spike protein. SARS-COV-2 spike protein binding molecules also encompass so-called "SM IP" ("small modular immunopharmaceuticals"), or immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.
The "SARS-COV-2 spike protein binding molecule" of the invention may comprise at least one immunoglobulin single variable domain, such as a single domain antibody, that binds to the SARS-COV-2 spike protein. In some embodiments, a "SARS-COV-2 spike protein binding molecule" of the invention can comprise two immunoglobulin single variable domains, such as single domain antibodies, that bind to the SARS-COV-2 spike protein. SARS-COV-2 spike protein binding molecules containing more than one immunoglobulin single variable domain are also known as "formatted" SAR S-COV-2 spike protein binding molecules. The formatted SARS-COV-2 spike protein binding molecule may also comprise, in addition to the immunoglobulin single variable domain that binds to SARS-COV-2 spike protein, a linker and/or a moiety with effector function, for example a half-life extending moiety (such as an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as mpeg) and/or an Fc region. The "SARS-COV-2 spike protein binding molecule" of the invention also encompasses bispecific antibodies that contain immunoglobulin single variable domains that bind different antigens.
Generally, the SARS-COV-2 spike protein binding molecules of the invention will be preferably 10 as measured in a Biacore or Kin ExA assay-8To 10-12Mole/liter (M), more preferably 10-9To 10-11Mole/liter, even more preferably 10-10To 10-12Even more preferably 10-11To 10-12Or a lower dissociation constant (KD). Any greater than 10-4The KD value of M is generally considered to indicate non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, Surface Plasmon Resonance (SPR) assays, as described herein, and/or competitive binding assays (e.g., Enzyme Immunoassay (EIA) and sandwich competitive assays).
Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. Such conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably made where one amino acid within the following groups (1) - (5) is replaced with another amino acid residue within the same group: (1) smaller aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln; (3) polar positively charged residues: his, Ar g and Lys; (4) larger aliphatic non-polar residues: met, Leu, Ile, Val and Cys; and (5) aromatic residues: phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: ala substituted by Gly or Ser; arg is replaced by Lys; asn is replaced by Gln or His; asp substituted by Glu; cys is substituted with Ser; gln is substituted by Asn; glu is substituted with Asp; gly by Ala or Pro; his is substituted with Asn or Gln; ile is substituted by Leu or Val; leu is substituted by Ile or Val; lys is substituted with Arg, Gln, or Glu; met is substituted by Leu, Tyr or Ile; phe is substituted by Met, Leu or Tyr; ser substituted by T hr; thr is substituted by Ser; trp is substituted by Tyr; tyr is substituted with Trp or Phe; val is substituted by Ile or Leu.
"sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For the determination of sequence identity see, for example: sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,1987 and Se Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, Newyo rk, 1991.
A polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been separated from at least one other component with which it is normally associated in the source or medium (culture medium), such as another protein/polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component, as compared to the reaction medium or culture medium from which it is naturally derived and/or from which it is 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 than 1000-fold. The "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).
Example 1
Screening of Single Domain antibodies against SARS-COV-2-Spike protein
1.1 construction of the library
1.1.1 immunization
The alpaca is immunized by Spike-RBD protein of the novel coronavirus, and is immunized 4 times in 1, 2, 4 and 6 weeks respectively, and the dosage of each immunization is 300 ug.
1.1.2 extraction of Total RNA
Taking 50ml of peripheral blood of alpaca immunized in the 6 th week, separating lymphocytes, extracting total RNA of the lymphocytes by using Trizol, and detecting the extracted RNA by using an ultraviolet spectrophotometer to obtain a result: OD260/280 is 1.97, OD260/230 is 2.14, which shows that the extracted RNA is not obviously degraded and has better purity; the total RNA concentration was 937.5 ng/. mu.L. Agarose gel electrophoresis was performed using the extracted total RNA, and as a result, two bands, 28S and 18S, were observed as shown in FIG. 1.
1.1.3 reverse transcription of RNA
The RNA reverse transcription system is as follows:
Step 1：
Figure GDA0003448475940000101
Figure GDA0003448475940000111
mixing, keeping at 65 deg.C for 5min, and rapidly ice-cooling;
Step 2
Figure GDA0003448475940000112
after mixing evenly, carrying out reverse transcription to obtain cDNA, wherein the reverse transcription conditions are as follows: 30min at 42 ℃; 50 ℃ for 15 min; 70 ℃ for 15 min.
1.1.4 Single Domain antibody (VHH) Gene amplification
The VHH gene is amplified by adopting nested PCR, and the method comprises the following steps:
Step1
Figure GDA0003448475940000113
after mixing, carrying out PCR reaction under the following reaction conditions: 10s at 98 ℃, 30s at 50 ℃ and 1min at 72 ℃ for 20 cycles. The sequence of the amplification primers was: primer For-1: 5'-GTCCTGGCTGCTCTTCTAC AAGG-3' (SEQ ID NO: 108); primer Rev-1: 5'-GGTACGTGCTGTTGAACTG TTCC-3' (SEQ ID NO: 109).
Purifying and concentrating the PCR product by using a DNA purification kit, performing agarose gel electrophoresis, and obtaining an agarose gel electrophoresis image as shown in figure 2, wherein a 750bp strip is recovered by using a DNA product gel recovery kit, and quantifying by using an ultraviolet spectrophotometer to serve as a DNA template of Step 2;
Step 2
Figure GDA0003448475940000121
after mixing, carrying out PCR reaction under the following reaction conditions: 10s at 98 ℃, 30s at 55 ℃ and 30s at 72 ℃ for 20 cycles. The sequence of the amplification primers was: primer For-2: 5'-CTAGTGCGGCCGCTGGAGA CGGTGACCTGGGT-3' (SEQ ID NO: 110); primer Rev-2:5 '-GATGTGCAGC TGCAGGAGTCTGGRGGAGG-3' (SEQ ID NO: 111).
The obtained PCR product was subjected to agarose gel electrophoresis, and the agarose gel electrophoresis pattern is shown in FIG. 3, which was recovered using a DNA product gel recovery kit and quantified using an ultraviolet spectrophotometer. Finally, 200. mu.L of the target gene (VHH) of about 500bp was obtained at a concentration of 458 ng/. mu.L.
1.1.5 library transformation
The obtained target gene and the vector pHEN1 are subjected to double enzyme digestion by SfiI and Not1, the enzyme-digested target gene and the pHEN1 fragment are connected by T4 DNA ligase and then are transformed into an escherichia coli electroporation competent cell TG1, and a single-domain antibody gene library aiming at SARS-COV-2-Spike protein is constructed and named as S2-Lib. Co-transforming for 15 times, mixing and then uniformly spreading in 6 pieces of culture dish (LB solid culture medium containing ampicillin) with phi 150 mm.
The mixed solutions of 0.1. mu.L, 0.01. mu.L, 0.001. mu.L and 0.0001. mu.L were applied to a Phi 90mm petri dish (LB solid medium containing ampicillin) and used for calculation of library volume (counting on plates with a colony count of 30-300), as shown in Table 1, the library volume was calculated to be 1.425X 109cfu。
TABLE 1
Figure GDA0003448475940000131
Randomly selecting 8 colonies from the culture dish for calculating library capacity, performing colony PCR, performing agarose gel electrophoresis on PCR products, and calculating the target gene insertion rate of the library, wherein the agarose gel electrophoresis is shown in FIG. 4, which indicates that the library insertion rate is 100%, and the actual library capacity of the library is
1.425×109cfu。
The colony PCR system was as follows:
Figure GDA0003448475940000132
the colony PCR reaction conditions are as follows: 31 cycles of 98 ℃ for 10s, 50 ℃ for 30s and 72 ℃ for 1 min.
1.1.6 library rescue
Inoculating 10-100 times of live cells from the S2-Lib gene library, culturing to logarithmic phase, rescuing with M13K07 phage, centrifugally collecting phage, purifying with PEG-NaCl to obtain phage display library named S2-PDL with titer of 3.5 × 1013cfu/mL. Can be straightenedAnd then used for affinity screening of specific phages.
1.2 screening against the Single Domain antibody against SARS-COV-2-Spike protein
Coating the plate with 3. mu.g/well of Spike-RBD protein (Spike protein receptor binding domain protein), and standing at 4 ℃ overnight; blocking with 1 wt% skimmed milk powder at room temperature for 2h, adding 100. mu.l phage (8X 10)11tfu from the phage display library S2-PDL constructed at 1.1.6) at room temperature for 1 h. Followed by elution 5 times with PBST (0.05 vt% tween 20 in PBS) to wash away unbound phage; phages specifically bound to Spike-RBD protein were dissociated with triethylamine (100mM) and infected with E.coli TG1, which was grown in log phase, and phages were generated and purified for the next round of screening. The same screening process was repeated for 3 rounds. Thus, positive clones are enriched, and the aim of screening the Spike-RBD protein specific antibody in the antibody library by using the phage display library is fulfilled. Sequencing the obtained positive phage to obtain an antibody gene sequence.
The obtained antibody gene sequences are respectively constructed on pcDNA3.4 vectors, HEK-293 cells are used for expressing the antibodies, and the proteinA medium is used for purifying and collecting the antibodies in the culture medium supernatant. The purified antibody was incubated with a Spike-RBD coated plate for ELISA assay. Obtaining the antibody which can specifically bind to the Spike-RBD protein.
The obtained antibody sequences were analyzed according to the sequence alignment software Vector NTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered to be identical antibody strains, while clones with different CDR sequences were considered to be different antibody strains. A total of 21 different single domain antibody strains capable of specifically binding to the Spike-RBD protein were obtained, the single domain antibody sequences are shown in SEQ ID NO 64-84, and carry 21 sets of CDR1-3 sequences in SEQ ID NO 1-63, respectively, as shown in Table 2.
TABLE 2
Figure GDA0003448475940000141
Figure GDA0003448475940000151
The 21 different single domain antibody strains were incubated with the Spike-RBD coated plates for ELISA assay, and the values of OD450 in the duplicate wells after reaction of the single domain antibodies with Spike-RBD were determined as shown in Table 3.
TABLE 3
Figure GDA0003448475940000152
Figure GDA0003448475940000161
Wherein, blank is the OD450 value in the duplicate wells without the antibody.
From the data in Table 3, it is clear that the single domain antibody was bound to the Spike-RBD protein.
1.3 evaluation and identification of Single-Domain antibodies against SARS-COV-2-Spike protein
1.3.1 expression and purification of Single Domain antibodies in host bacteria Escherichia coli
The obtained gene coding sequences of 21 single-domain antibodies with different CDRs 1-3 were recombined into an expression vector PET32b (Novagen, product No.: 69016-3), and recombinant plasmids with correct sequencing identification were transformed into an expression host bacterium BL1(DE3) (Tiangen Biochemical technology, product No.: CB105-02), which was spread on LB plate containing 100. mu.g/mL ampicillin overnight at 37 ℃. Selecting single colony, inoculating, culturing overnight, transferring overnight strain for amplification next day, shake culturing at 37 deg.C until OD value reaches 0.5-1, adding 0.5mM IPTG for induction, shake culturing at 28 deg.C overnight. The next day, the cells were collected by centrifugation, and the collected cells were disrupted to obtain a crude antibody extract. Then, the 21-strain single-domain antibody protein is purified to achieve a purity of more than 90%.
1.3.2 Competition ELISA examination of the blocking Effect of 21 SARS-COV-2-Spike protein single-domain antibody on the binding of Spike-RBD protein and the receptor ACE2
The Spike-RBD protein and ACE2 protein were obtained by expression from HEK293 cells (pCDNA4, Invitrogen, CatV 86220). Then biotinylated ACE2 protein was obtained using the biotinylation kit from Thermo.
The plate was coated with 0.5. mu.g/well of Spike-RBD protein overnight at 4 ℃ after which 100ng of 1.3.1 purified single domain antibody and 5. mu.g of biotinylated ACE2 protein were added per well, and a control was set, in which no single domain antibody was added to the wells of control 1 and no biotinylated ACE2 protein was added to the wells of control 2 and reacted for 2h at room temperature. Then, SA-HRP (purchased from Sigma) was added, and after 1 hour of reaction at room temperature, a color developing solution was added, and the absorbance was read at a wavelength of 450 nm. When the OD value of the sample is less than 0.8 than that of the control, the single-domain antibody is considered to have a blocking effect.
As shown in Table 4, 21 different single domain antibody strains showed blocking effect on the interaction of Spike protein/ACE 2 protein.
TABLE 4
Figure GDA0003448475940000171
Figure GDA0003448475940000181
In a laboratory with a biological safety level of P3, the purified 21 strain single domain antibodies are respectively added into a culture system by infecting a VERO cell model with viruses, and the specific operation is as follows: will 104Adding/hole VERO cells into a 96-well plate, washing the cells 2 times by PBS after 24 hours, mixing 21 strains of single-domain antibodies with the viruses respectively, adding the mixture into the 96-well plate, wherein the initial concentration of the antibodies is 10 mu g/mL, diluting the mixture by 2 times for 10 gradients respectively, incubating the mixture for 5 multiple holes at 37 ℃ for 2 hours, and detecting whether the VERO cells are infected with the viruses on the 5 th day (if the cells are not diseased, the single-domain antibodies can neutralize the viruses and block the virus infection process on the VERO cells).
As shown in Table 5, the 21-strain single-domain antibody was effective in blocking the process of viral infection of cells at concentrations above 5. mu.g/ml, and some antibodies were still effective in blocking the process of viral infection of cells at concentrations below 0.02. mu.g/ml. The IC50(μ g/ml) data obtained in Table 5 show that the obtained 21-strain single-domain antibody can block the virus-infected cell process and is an effective neutralizing antibody.
TABLE 5
Figure GDA0003448475940000182
Figure GDA0003448475940000191
Note: "+" indicates that viral infection of cells can be blocked, and "-" indicates that viral infection of cells cannot be blocked.
Example 2
1.1 Fc fusion protein for preparing single domain antibody of SARS-COV-2-Spike protein
The amino acid sequence of the human IgG1-Fc region (SEQ ID NO:85) was obtained from the amino acid sequence of the constant region of human immunoglobulin (IgG1) on Unit protein database. A nucleic acid fragment encoding human IgG1-Fc (nucleic acid sequence shown in SEQ ID NO:107) was obtained from human PBMC total RNA by reverse transcription PCR, and a nucleic acid fragment encoding a fusion protein of a single domain antibody of SARS-COV-2-Spike protein and Fc was obtained by overlapping PCR and recombined into vector pCDNA4(Invitrogen, Cat V86220).
The constructed pCDNA4 plasmid containing the nucleic acid fragment of the fusion protein of the SARS-COV-2-Spike protein single domain antibody and Fc is transfected into HEK293 cells for expression. Specifically, the recombinant expression plasmid is diluted by Freestyle293 culture medium and added with PEI (polyethylenimine) solution required for transformation, and the plasmid/PEI mixture is respectively added into HEK293 cell suspension and placed at 37 ℃ with 10% CO2Culturing in a shaker at 100 rpm; adding 50 μ g/LIGF-1. After 4h, it was supplemented with EX293 medium, 2mM glutamine and 50. mu.g/LIGF-1, and shaken at 120 rpm. After 24h, 3.8mM VPA was added. After culturing for 5 days, collecting the expression culture supernatant, and purifying by a ProteinA affinity chromatography to obtain the fusion protein of the SARS-COV-2-Spike protein single domain antibody and Fc. The sequence of the fusion protein of 21 SARS-COV-2-Spike protein single-domain antibodies and Fc is shown in SEQ ID NO 86-106.
1.2 identification of the function of the fusion protein of the SARS-COV-2-Spike protein single domain antibody with Fc (SEQ ID NO:86-106)
The binding capacity of the SARS-COV-2-Spike protein single-domain antibody and the Fc fusion protein to the SARS-COV-2-Spike protein is identified by the SPR method. The specific operation is as follows: the binding kinetics of the obtained fusion protein of 21 strain SARS-COV-2-Spike protein single domain antibody and Fc against Spike-RBD was measured by the surface plasmon resonance (SRP) method using a BIAcoreX100 instrument, and the Spike-RBD protein was directly coated on a CM5 biosensor chip to obtain approximately 1000 Response Units (RU). For kinetic measurements, the fusion protein of the single domain antibody of SARS-COV-2-Spike protein and Fc was serially diluted three times (1.37nm to 1000nm) with HBS-EP +1 Xbuffer (GE, cat # BR-1006-69), injected at 25 ℃ for 120s with a dissociation time of 30min, and regenerated for 120s by addition of 10mM glycine-HCl (pH 2.0). The binding rate (kon), dissociation rate (koff) and equilibrium dissociation constant (kD) (calculated as the ratio koff/kon) of the fusion protein to SARS-COV-2-Spike protein were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2). The calculation results are shown in table 6.
TABLE 6
Figure GDA0003448475940000201
Figure GDA0003448475940000211
As shown in Table 6, the binding rate of the SARS-COV-2-Spike protein single domain antibody and the Fc fusion protein to the SARS-COV-2-Spike protein is high, the dissociation rate is low, and the equilibrium dissociation constant KD is less than 1.58E-10, which indicates that the fusion protein can more rapidly bind to the SARS-COV-2-Spike protein and is difficult to dissociate, and further indicates that the SARS-COV-2-Spike protein single domain antibody and the Fc fusion protein are used as a blocking antibody and have excellent blocking effect.
1.3 identification of the blocking Capacity of the SARS-COV-2-Spike protein Single Domain antibody and the Fc fusion protein for the interaction of Spike protein/ACE 2 by competitive ELISA
The ACE2 protein is obtained by expression of HEK293 cells. The biotinylated ACE2-Biotin protein was obtained using the Biotinylation kit from Thermo.
The plate was coated with 0.5. mu.g of Spike-RBD protein overnight at 4 ℃ and then, 200ng of the obtained 21 SARS-COV-2-Spike protein single domain antibody and Fc fusion protein and ACE2-Biotin 5ug were added to each well, and the reaction was carried out for 2 hours at room temperature without adding the fusion protein to control 1 and ACE2-Biotin to control 2. After washing, SA-HRP (purchased from Sigma) was added, the reaction was carried out at room temperature for 1 hour, and after washing, a color developing solution was added and absorbance was read at a wavelength of 450 nm. The results are shown in Table 7.
TABLE 7
Sample (I) OD
Control group
1 2.414
Control group 2 0.019
1 0.015
2 0.023
3 0.014
4 0.014
5 0.016
6 0.011
7 0.013
8 0.001
9 0.014
10 0.008
11 0.019
12 0.014
13 0.017
14 0.016
15 0.012
16 0.025
17 0.011
18 0.018
19 0.014
20 0.019
21 0.013
The result shows that the SARS-COV-2-Spike protein single-domain antibody and Fc fusion protein can effectively block the interaction between Spike protein and ACE 2.
1.4 analysis of the specificity of the binding of the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein to Spike protein
Plasmids (pCDNA4, Invitrogen, Cat V86220) carrying the full-length genes of the currently known 7 coronavirus (SARS-COV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV) Spike proteins were obtained by transient transfection using human HEK293 cells, and the Spike proteins were transiently expressed on the membrane. The plasmid enables the C end of the Spike protein to be fused with the EGFP protein, so that the expression level of the Spike protein on the membrane can be investigated through the green fluorescence intensity. The constructed cells were resuspended in 0.5% PBS-BSA Buffer, and SARS-COV-2-Spike protein single domain antibody and Fc fusion protein were added, while negative control was set, and incubated on ice for 20 min. After washing, the eBioscience secondary antibody anti-hIg-PE was added and the mixture was frozen for 20 min. After washing, the cells were resuspended in 500. mu.l of 0.5% PBS-BSABuffer and examined by flow cytometry. The results showed that the 21 strain SARS-COV-2-Spike protein single domain antibody-Fc fusion protein only specifically binds to SARS-COV-2-Spike protein, but not to Spike proteins of other coronaviruses.
1.5SARS-COV-2-Spike protein single domain antibody and Fc fusion protein block SARS-COV-2 infection of rhesus monkey
6 of 12 rhesus monkeys infected with SARS-COV-2 virus and showing symptoms (treatment group) were treated by administration of the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein (SARS-COV-2-Spike protein single domain antibody and Fc fusion protein 100. mu.g/rhesus monkey) provided by the present invention, and 6 (control group) were not treated by administration. The respiratory viral load was measured once daily within 6 days after treatment. The mean load of the new coronavirus in the respiratory tracts of 6 rhesus monkeys in the treatment group was significantly reduced compared to the control group, as shown in fig. 5. The treatment group was continued with 6 rhesus monkeys and their symptoms and respiratory viral load were measured every other week. After 2 weeks of continuous observation, the viral load in the respiratory tract was not detected and no corresponding symptoms of the disease were observed. The continuous observation for 3 months shows that the virus in 6 rhesus monkeys of the treatment group has no recurrence, which shows that the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein can play a long-acting role in vivo, and can avoid the relapse of positive after the living body infected with the novel coronavirus is cured. Wherein the detection process of the load of the new coronavirus is as follows: the throat swabs of the rhesus monkeys (treatment group) that were subjected to the administration treatment and the rhesus monkeys (control group) that were not subjected to the administration treatment were respectively collected, and nucleic acids of the viruses in the throat swabs were extracted and detected, and the detection process was: SARS-COV-2 RNA was extracted using an RNA extraction kit (Qiagen) according to the instructions, and the obtained RNA was dissolved in 50. mu.L of elution buffer and used as a template for RT-PCR amplification. The viral S region genes were amplified using primers RBD-qF1 (5'-CAATGGTTTAACAGGCACAGG-3', SEQ ID NO:112) and RBD-qR1 (5'-CTCAAGTGTCTGTGGATCACG-3', SEQ ID NO: 113). Adopting a HiScriptR II One Step qRT-PCR SYBRRGreen Kit (Vazyme Biotech Co., Ltd.) Kit, operating according to the Kit instruction, and setting the PCR amplification condition as follows; 50 ℃ for 3min, 95 ℃ for 10s, 60 ℃ for 30s, 40 cycles, and the PCR amplification instrument is an ABI quantitative PCR instrument.
The results of the in vivo experiments show that the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein of the invention show significant long-acting SARS-COV-2 infected cell inhibition and amplification effects in rhesus monkeys infected with SARS-COV-2, and the sun recovery rate of the treated rhesus monkeys is 0.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen Nennuosai Biotech Co., Ltd
<120> novel coronavirus (SARS-COV-2) spike protein binding molecule and use thereof
<130> 2020
<160> 113
<170> PatentIn version 3.5
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Gly Arg Ala Val Thr Ile Tyr His
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Ile Ser Pro Ser Arg Val Ile Thr
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Ala Ala Thr Arg Gly Ser Phe Asp Arg Asn Arg Val Asn Tyr Asp Tyr
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Gly Arg Thr Phe Ser Ser Tyr Ala
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Ile Ser Trp Thr Gly Asp Thr Thr
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Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Tyr Arg Thr Gln Asp Tyr
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Gly Phe Thr Leu Asp Tyr Tyr Thr
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Thr Ser Ser Ala Asp Gly Ser Thr
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Ala Ala Ala Pro Gly Ala Tyr Tyr Ser Gly Thr Tyr Tyr Arg Ala Cys
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His Ser Ala Gly Met Glu Tyr
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Gly Phe Thr Phe Asp Asp Tyr Ala
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Ile Gly Ser Arg Asp Gly Ser Thr
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Ala Ala Pro Asp Ser Val Ser Tyr Tyr Gly Trp Ser Leu Asn Pro Tyr
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Glu Tyr Asp Tyr
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Gly Gly Thr Phe Ser Arg Ser Gly
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Arg Ala Asp Val Gly Phe Pro
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Ala Ala Thr Ser Leu Gln Ser Gly Lys Tyr Asp Tyr
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Gly His Thr Phe Ser Arg Phe Gly
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Arg Asn Asp Gly Gly Phe Pro
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Ala Ala Thr Ser Pro Glu Ser Gly Arg Tyr Asp Tyr
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Arg Thr Phe Ser Val Gly Glu Val
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Ile Arg Trp Gly Ser Glu Ser Thr
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Ala Ala Thr Glu Lys Val Tyr Ser Ala Tyr Tyr Tyr Tyr Ser Arg Asp
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Arg Leu Tyr Asp Tyr
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Gly Phe Thr Phe Asp Asp Tyr Ala
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Ile Ser Ser Arg Asp Gly Arg Thr
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Ala Ala Asn His Leu Thr Tyr Tyr Ser Gly Ser Tyr Tyr Phe Pro Tyr
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Glu Tyr His Tyr
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Arg Cys Thr Phe Asn Trp Asp Gly
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Ile Ser Trp Ser Gly Gln Glu Pro
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Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
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Val Gly Tyr Asp Tyr
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Thr Arg Thr Phe Ser Asn Tyr Asn
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Ile Ser Arg Ala Gly Ala Glu Ile
1 5
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Ala Ala Ala Arg Val Met Gly Val Ser Pro Ala Ile Tyr Asn Tyr
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Gly His Thr Phe Ser Ser Tyr Ala
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Ile Asp Trp Ser Gly Asp Glu Ala
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Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Asp Arg Thr Gln Glu Tyr
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Gly Ile
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Gly Arg Thr Leu Arg Ser Phe Ala
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Ile Ser Trp Ser Gly Asp Asp Thr
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Ala Ala Gly Gly His Thr Ile Asn His Asp Ile Ile Ser Pro Asp Tyr
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Gly Leu Thr Gly Ser Thr Phe Val
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Ile Arg Trp Thr Tyr Gly Arg Thr
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Ala Ala Arg Glu Gly Tyr Arg Gly Ser Ala Tyr Gln Thr Ser Asp Phe
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Ala Ser Gly Arg Ser Thr His Arg
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<211> 8
<212> PRT
<213> CDR2
<400> 41
Ile Met Pro Gly Gly Gly Phe Pro
1 5
<210> 42
<211> 11
<212> PRT
<213> CDR3
<400> 42
Ala Ala Arg Ser Glu Thr Gly Glu Tyr Asp Asn
1 5 10
<210> 43
<211> 8
<212> PRT
<213> CDR1
<400> 43
Gly Phe Thr Leu Asp Tyr Tyr Arg
1 5
<210> 44
<211> 8
<212> PRT
<213> CDR2
<400> 44
Ile Ser Ser Ser Asp Gly Ser Thr
1 5
<210> 45
<211> 25
<212> PRT
<213> CDR3
<400> 45
Ala Thr Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Leu Gly Gly
1 5 10 15
Cys Phe Asp Thr Tyr Ala Met Asp Phe
20 25
<210> 46
<211> 8
<212> PRT
<213> CDR1
<400> 46
Gly Arg Thr Phe Ser Ser Tyr Thr
1 5
<210> 47
<211> 8
<212> PRT
<213> CDR2
<400> 47
Ile Ser Arg Ser Gly Gly Gly Thr
1 5
<210> 48
<211> 17
<212> PRT
<213> CDR3
<400> 48
Ala Val Ser Arg Arg Val Leu Leu Thr Asp Thr Pro Arg Asp Tyr Asp
1 5 10 15
Arg
<210> 49
<211> 8
<212> PRT
<213> CDR1
<400> 49
Gly Asn Thr Leu Asp Tyr Tyr Ala
1 5
<210> 50
<211> 8
<212> PRT
<213> CDR2
<400> 50
Ile Lys Ser Ser Asp Gly Ser Thr
1 5
<210> 51
<211> 20
<212> PRT
<213> CDR3
<400> 51
Ala Ile Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Phe Pro Tyr
1 5 10 15
Asp Tyr Asp Tyr
20
<210> 52
<211> 8
<212> PRT
<213> CDR1
<400> 52
Gly Arg Thr Ser Ser Arg Tyr Val
1 5
<210> 53
<211> 8
<212> PRT
<213> CDR2
<400> 53
Ile Thr Ser Thr Gly Phe Gly Thr
1 5
<210> 54
<211> 20
<212> PRT
<213> CDR3
<400> 54
Ala Ala Asp Thr Ser Leu Leu Thr Arg Arg Pro Ser Asp Ser Ala Ser
1 5 10 15
Pro Tyr Asp Tyr
20
<210> 55
<211> 8
<212> PRT
<213> CDR1
<400> 55
Gly Ser Ile Ala Ser Ile Thr Ala
1 5
<210> 56
<211> 7
<212> PRT
<213> CDR2
<400> 56
Ser Asn Ser Arg Gly Ser Thr
1 5
<210> 57
<211> 13
<212> PRT
<213> CDR3
<400> 57
Tyr Val Asn Thr Leu Arg Ala Glu Met Pro Glu Ala Tyr
1 5 10
<210> 58
<211> 8
<212> PRT
<213> CDR1
<400> 58
Gly Asp Thr Phe Asn Asn Tyr Ala
1 5
<210> 59
<211> 8
<212> PRT
<213> CDR2
<400> 59
Ile Ala Arg Ser Gly Arg Ser Thr
1 5
<210> 60
<211> 20
<212> PRT
<213> CDR3
<400> 60
Ala Ala Asp Pro Ala Pro Leu Trp Phe Ala Trp Thr Ser Asp Met Ala
1 5 10 15
Lys Tyr Lys Phe
20
<210> 61
<211> 8
<212> PRT
<213> CDR1
<400> 61
Gly Arg Ile Pro Ser Trp Asn Ile
1 5
<210> 62
<211> 8
<212> PRT
<213> CDR2
<400> 62
Ile Arg Trp Thr Ser Gly Thr Thr
1 5
<210> 63
<211> 19
<212> PRT
<213> CDR3
<400> 63
Gly Trp Arg Arg Gly Gly Val Gln Gln Ile Ser Thr Gly Ser His Asp
1 5 10 15
Tyr Asp Pro
<210> 64
<211> 123
<212> PRT
<213> VHH-1
<400> 64
Gln Leu 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 Thr Ser Gly Arg Ala Val Thr Ile Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Pro Ser Arg Val Ile Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Thr Arg Gly Ser Phe Asp Arg Asn Arg Val Asn Tyr Asp Tyr
100 105 110
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 65
<211> 126
<212> PRT
<213> VHH-2
<400> 65
Gln Leu 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 Thr Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Thr Gly Asp Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Tyr Arg Thr Gln Asp
100 105 110
Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 66
<211> 131
<212> PRT
<213> VHH-3
<400> 66
Gln Leu 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 Thr Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Thr Ser Ser Ala Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Pro Gly Ala Tyr Tyr Ser Gly Thr Tyr Tyr Arg Ala
100 105 110
Cys His Ser Ala Gly Met Glu Tyr Arg Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser
130
<210> 67
<211> 128
<212> PRT
<213> VHH-4
<400> 67
Gln Leu 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 Thr Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Ser Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Pro Asp Ser Val Ser Tyr Tyr Gly Trp Ser Leu Asn Pro
100 105 110
Tyr Glu Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 68
<211> 119
<212> PRT
<213> VHH-5
<400> 68
Gln Leu 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 Thr Ser Gly Gly Thr Phe Ser Arg Ser
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Arg Ala Asp Val Gly Phe Pro Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Thr Ser Leu Gln Ser Gly Lys Tyr Asp Tyr Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 69
<211> 119
<212> PRT
<213> VHH-6
<400> 69
Gln Leu 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 Thr Ser Gly His Thr Phe Ser Arg Phe
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Arg Asn Asp Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Thr Ser Pro Glu Ser Gly Arg Tyr Asp Tyr Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 70
<211> 129
<212> PRT
<213> VHH-7
<400> 70
Gln Leu 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 Thr Ser Arg Thr Phe Ser Val Gly Glu
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Arg Trp Gly Ser Glu Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Thr Glu Lys Val Tyr Ser Ala Tyr Tyr Tyr Tyr Ser Arg
100 105 110
Asp Arg Leu Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 71
<211> 128
<212> PRT
<213> VHH-8
<400> 71
Gln Leu 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 Thr Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Ser Arg Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn His Leu Thr Tyr Tyr Ser Gly Ser Tyr Tyr Phe Pro
100 105 110
Tyr Glu Tyr His Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 72
<211> 129
<212> PRT
<213> VHH-9
<400> 72
Gln Leu 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 Thr Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Ser Gly Gln Glu Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp
100 105 110
Gln Val Gly Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 73
<211> 123
<212> PRT
<213> VHH-10
<400> 73
Gln Leu 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 Thr Ser Thr Arg Thr Phe Ser Asn Tyr
20 25 30
Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ala Gly Ala Glu Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Arg Val Met Gly Val Ser Pro Ala Ile Tyr Asn Tyr
100 105 110
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 74
<211> 126
<212> PRT
<213> SP11
<400> 74
Gln Leu 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 Thr Ser Gly His Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Asp Trp Ser Gly Asp Glu Ala Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Asp Arg Thr Gln Glu
100 105 110
Tyr Gly Ile Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 75
<211> 124
<212> PRT
<213> VHH-12
<400> 75
Gln Leu 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 Thr Ser Gly Arg Thr Leu Arg Ser Phe
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Ser Gly Asp Asp Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gly Gly His Thr Ile Asn His Asp Ile Ile Ser Pro Asp
100 105 110
Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 76
<211> 126
<212> PRT
<213> VHH-13
<400> 76
Gln Leu 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 Thr Ser Gly Leu Thr Gly Ser Thr Phe
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Arg Trp Thr Tyr Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Arg Glu Gly Tyr Arg Gly Ser Ala Tyr Gln Thr Ser Asp
100 105 110
Phe Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 77
<211> 119
<212> PRT
<213> VHH-14
<400> 77
Gln Leu 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 Thr Ser Ala Ser Gly Arg Ser Thr His
20 25 30
Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Met Pro Gly Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Arg Ser Glu Thr Gly Glu Tyr Asp Asn Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 78
<211> 133
<212> PRT
<213> VHH-15
<400> 78
Gln Leu 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 Thr Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Thr Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Leu Gly
100 105 110
Gly Cys Phe Asp Thr Tyr Ala Met Asp Phe Arg Gly Gln Gly Thr Gln
115 120 125
Val Thr Val Ser Ser
130
<210> 79
<211> 125
<212> PRT
<213> VHH-16
<400> 79
Gln Leu 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 Thr Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Val Ser Arg Arg Val Leu Leu Thr Asp Thr Pro Arg Asp Tyr
100 105 110
Asp Arg Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 80
<211> 128
<212> PRT
<213> VHH-17
<400> 80
Gln Leu 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 Thr Ser Gly Asn Thr Leu Asp Tyr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Lys Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ile Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Phe Pro
100 105 110
Tyr Asp Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 81
<211> 128
<212> PRT
<213> VHH-18
<400> 81
Gln Leu 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 Thr Ser Gly Arg Thr Ser Ser Arg Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Thr Ser Thr Gly Phe Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Thr Ser Leu Leu Thr Arg Arg Pro Ser Asp Ser Ala
100 105 110
Ser Pro Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 82
<211> 120
<212> PRT
<213> VHH-19
<400> 82
Gln Leu 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 Thr Ser Gly Ser Ile Ala Ser Ile Thr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ser Asn Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Tyr Val Asn Thr Leu Arg Ala Glu Met Pro Glu Ala Tyr Arg Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 83
<211> 128
<212> PRT
<213> VHH-20
<400> 83
Gln Leu 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 Thr Ser Gly Asp Thr Phe Asn Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ala Arg Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Pro Ala Pro Leu Trp Phe Ala Trp Thr Ser Asp Met
100 105 110
Ala Lys Tyr Lys Phe Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 84
<211> 127
<212> PRT
<213> VHH-21
<400> 84
Gln Leu 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 Thr Ser Gly Arg Ile Pro Ser Trp Asn
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Arg Trp Thr Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Trp Arg Arg Gly Gly Val Gln Gln Ile Ser Thr Gly Ser His
100 105 110
Asp Tyr Asp Pro Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 85
<211> 232
<212> PRT
<213> Fc
<400> 85
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 86
<211> 355
<212> PRT
<213> VHH-1-Fc
<400> 86
Gln Leu 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 Thr Ser Gly Arg Ala Val Thr Ile Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Pro Ser Arg Val Ile Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Thr Arg Gly Ser Phe Asp Arg Asn Arg Val Asn Tyr Asp Tyr
100 105 110
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser
115 120 125
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
130 135 140
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
145 150 155 160
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
165 170 175
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
180 185 190
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
195 200 205
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
210 215 220
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
225 230 235 240
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
245 250 255
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
260 265 270
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
275 280 285
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
290 295 300
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
305 310 315 320
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
325 330 335
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
340 345 350
Pro Gly Lys
355
<210> 87
<211> 358
<212> PRT
<213> VHH-2-Fc
<400> 87
Gln Leu 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 Thr Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Thr Gly Asp Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Tyr Arg Thr Gln Asp
100 105 110
Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 88
<211> 363
<212> PRT
<213> VHH-3-Fc
<400> 88
Gln Leu 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 Thr Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Thr Ser Ser Ala Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Pro Gly Ala Tyr Tyr Ser Gly Thr Tyr Tyr Arg Ala
100 105 110
Cys His Ser Ala Gly Met Glu Tyr Arg Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
130 135 140
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
145 150 155 160
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
165 170 175
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
180 185 190
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
195 200 205
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
210 215 220
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
225 230 235 240
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
245 250 255
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
260 265 270
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
275 280 285
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
290 295 300
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
305 310 315 320
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
325 330 335
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
340 345 350
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 89
<211> 360
<212> PRT
<213> VHH-4-Fc
<400> 89
Gln Leu 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 Thr Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Ser Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Pro Asp Ser Val Ser Tyr Tyr Gly Trp Ser Leu Asn Pro
100 105 110
Tyr Glu Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 90
<211> 351
<212> PRT
<213> VHH-5-Fc
<400> 90
Gln Leu 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 Thr Ser Gly Gly Thr Phe Ser Arg Ser
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Arg Ala Asp Val Gly Phe Pro Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Thr Ser Leu Gln Ser Gly Lys Tyr Asp Tyr Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His
115 120 125
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
130 135 140
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
145 150 155 160
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
165 170 175
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
180 185 190
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
195 200 205
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
210 215 220
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
225 230 235 240
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
245 250 255
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
260 265 270
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
275 280 285
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
290 295 300
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
305 310 315 320
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
325 330 335
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 91
<211> 351
<212> PRT
<213> VHH-6-Fc
<400> 91
Gln Leu 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 Thr Ser Gly His Thr Phe Ser Arg Phe
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Arg Asn Asp Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Thr Ser Pro Glu Ser Gly Arg Tyr Asp Tyr Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His
115 120 125
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
130 135 140
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
145 150 155 160
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
165 170 175
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
180 185 190
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
195 200 205
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
210 215 220
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
225 230 235 240
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
245 250 255
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
260 265 270
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
275 280 285
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
290 295 300
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
305 310 315 320
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
325 330 335
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 92
<211> 361
<212> PRT
<213> VHH-7-Fc
<400> 92
Gln Leu 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 Thr Ser Arg Thr Phe Ser Val Gly Glu
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Arg Trp Gly Ser Glu Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Thr Glu Lys Val Tyr Ser Ala Tyr Tyr Tyr Tyr Ser Arg
100 105 110
Asp Arg Leu Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 93
<211> 360
<212> PRT
<213> VHH-8-Fc
<400> 93
Gln Leu 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 Thr Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Ser Arg Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn His Leu Thr Tyr Tyr Ser Gly Ser Tyr Tyr Phe Pro
100 105 110
Tyr Glu Tyr His Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 94
<211> 361
<212> PRT
<213> VHH-9-Fc
<400> 94
Gln Leu 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 Thr Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Ser Gly Gln Glu Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp
100 105 110
Gln Val Gly Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 95
<211> 355
<212> PRT
<213> VHH-10-Fc
<400> 95
Gln Leu 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 Thr Ser Thr Arg Thr Phe Ser Asn Tyr
20 25 30
Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ala Gly Ala Glu Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Arg Val Met Gly Val Ser Pro Ala Ile Tyr Asn Tyr
100 105 110
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser
115 120 125
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
130 135 140
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
145 150 155 160
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
165 170 175
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
180 185 190
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
195 200 205
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
210 215 220
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
225 230 235 240
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
245 250 255
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
260 265 270
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
275 280 285
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
290 295 300
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
305 310 315 320
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
325 330 335
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
340 345 350
Pro Gly Lys
355
<210> 96
<211> 358
<212> PRT
<213> VHH-11-Fc
<400> 96
Gln Leu 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 Thr Ser Gly His Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Asp Trp Ser Gly Asp Glu Ala Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Asp Arg Thr Gln Glu
100 105 110
Tyr Gly Ile Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 97
<211> 356
<212> PRT
<213> VHH-12-Fc
<400> 97
Gln Leu 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 Thr Ser Gly Arg Thr Leu Arg Ser Phe
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Ser Gly Asp Asp Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gly Gly His Thr Ile Asn His Asp Ile Ile Ser Pro Asp
100 105 110
Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser
115 120 125
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
260 265 270
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
290 295 300
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys
355
<210> 98
<211> 358
<212> PRT
<213> VHH-13-Fc
<400> 98
Gln Leu 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 Thr Ser Gly Leu Thr Gly Ser Thr Phe
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Arg Trp Thr Tyr Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Arg Glu Gly Tyr Arg Gly Ser Ala Tyr Gln Thr Ser Asp
100 105 110
Phe Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 99
<211> 351
<212> PRT
<213> VHH-14-Fc
<400> 99
Gln Leu 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 Thr Ser Ala Ser Gly Arg Ser Thr His
20 25 30
Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Met Pro Gly Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Arg Ser Glu Thr Gly Glu Tyr Asp Asn Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His
115 120 125
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
130 135 140
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
145 150 155 160
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
165 170 175
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
180 185 190
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
195 200 205
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
210 215 220
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
225 230 235 240
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
245 250 255
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
260 265 270
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
275 280 285
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
290 295 300
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
305 310 315 320
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
325 330 335
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 100
<211> 365
<212> PRT
<213> VHH-15-Fc
<400> 100
Gln Leu 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 Thr Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Thr Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Leu Gly
100 105 110
Gly Cys Phe Asp Thr Tyr Ala Met Asp Phe Arg Gly Gln Gly Thr Gln
115 120 125
Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
130 135 140
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
145 150 155 160
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
165 170 175
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
180 185 190
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
195 200 205
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
210 215 220
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
225 230 235 240
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
245 250 255
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
260 265 270
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
275 280 285
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
290 295 300
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
305 310 315 320
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
325 330 335
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
340 345 350
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360 365
<210> 101
<211> 357
<212> PRT
<213> VHH-16-Fc
<400> 101
Gln Leu 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 Thr Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Val Ser Arg Arg Val Leu Leu Thr Asp Thr Pro Arg Asp Tyr
100 105 110
Asp Arg Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 102
<211> 360
<212> PRT
<213> VHH-17-Fc
<400> 102
Gln Leu 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 Thr Ser Gly Asn Thr Leu Asp Tyr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Lys Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ile Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Phe Pro
100 105 110
Tyr Asp Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 103
<211> 360
<212> PRT
<213> VHH-18-Fc
<400> 103
Gln Leu 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 Thr Ser Gly Arg Thr Ser Ser Arg Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Thr Ser Thr Gly Phe Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Thr Ser Leu Leu Thr Arg Arg Pro Ser Asp Ser Ala
100 105 110
Ser Pro Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 104
<211> 352
<212> PRT
<213> VHH-19-Fc
<400> 104
Gln Leu 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 Thr Ser Gly Ser Ile Ala Ser Ile Thr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ser Asn Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Tyr Val Asn Thr Leu Arg Ala Glu Met Pro Glu Ala Tyr Arg Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr
115 120 125
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
130 135 140
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
165 170 175
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
180 185 190
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
195 200 205
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
210 215 220
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
225 230 235 240
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
245 250 255
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
260 265 270
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
275 280 285
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
290 295 300
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
305 310 315 320
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
325 330 335
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 105
<211> 360
<212> PRT
<213> VHH-20-Fc
<400> 105
Gln Leu 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 Thr Ser Gly Asp Thr Phe Asn Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ala Arg Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Pro Ala Pro Leu Trp Phe Ala Trp Thr Ser Asp Met
100 105 110
Ala Lys Tyr Lys Phe Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 106
<211> 359
<212> PRT
<213> VHH-21-Fc
<400> 106
Gln Leu 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 Thr Ser Gly Arg Ile Pro Ser Trp Asn
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Arg Trp Thr Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Trp Arg Arg Gly Gly Val Gln Gln Ile Ser Thr Gly Ser His
100 105 110
Asp Tyr Asp Pro Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 107
<211> 705
<212> DNA
<213> Fc DNA sequence
<400> 107
cgtacggagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660
aaccactaca cgcagaagag cctctccctg tctccgggta aatga 705
<210> 108
<211> 23
<212> DNA
<213> Primer For-1
<400> 108
gtcctggctg ctcttctaca agg 23
<210> 109
<211> 23
<212> DNA
<213> Primer Rev-1
<400> 109
ggtacgtgct gttgaactgt tcc 23
<210> 110
<211> 32
<212> DNA
<213> Primer For-2
<400> 110
ctagtgcggc cgctggagac ggtgacctgg gt 32
<210> 111
<211> 29
<212> DNA
<213> Primer Rev-2
<400> 111
gatgtgcagc tgcaggagtc tggrggagg 29
<210> 112
<211> 21
<212> DNA
<213> RBD-qF1
<400> 112
caatggttta acaggcacag g 21
<210> 113
<211> 21
<212> DNA
<213> RBD-qR1
<400> 113
ctcaagtgtc tgtggatcac g 21

Claims (17)

1. A SARS-COV-2 spike protein binding molecule, characterized by: is capable of specifically binding to the spike protein of SARS-COV-2 and comprises an immunoglobulin single variable domain in which CDR1, CDR2 and CDR3 are:
CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14 and CDR3 shown in SEQ ID NO. 15;
the immunoglobulin single variable domain is a single domain antibody.
2. The SARS-COV-2 spike protein binding molecule of claim 1, wherein: the single domain antibody comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 68.
3. The SARS-COV-2 spike protein binding molecule of claim 1, wherein: the single domain antibody comprises an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 68.
4. The SARS-COV-2 spike protein binding molecule of claim 1, wherein: the single domain antibody comprises an amino acid sequence having at least 99% sequence identity to the sequence of SEQ ID NO: 68.
5. The SARS-COV-2 spike protein binding molecule of claim 1, wherein: the single domain antibody comprises the amino acid sequence of SEQ ID NO 68.
6. A SARS-COV-2 spike protein binding molecule as claimed in any one of claims 1 to 5 wherein: further comprising an immunoglobulin Fc region.
7. The SARS-COV-2 spike protein binding molecule of claim 6, wherein: the immunoglobulin Fc region is a human immunoglobulin Fc region.
8. The SARS-COV-2 spike protein binding molecule of claim 7, wherein: the immunoglobulin Fc region is the Fc region of human IgG 1.
9. The SARS-COV-2 spike protein binding molecule of claim 8, wherein: the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO. 85.
10. The SARS-COV-2 spike protein binding molecule of claim 9, wherein: the binding molecule comprises the amino acid sequence of SEQ ID NO 90.
11. A nucleic acid molecule encoding the SARS-COV-2 spike protein binding molecule of any one of claims 1 to 10.
12. An expression vector comprising the nucleic acid molecule of claim 11 and expression control elements thereof.
13. A host cell comprising and expressing the nucleic acid molecule of claim 11.
14. A method of obtaining a SARS-COV-2 spike protein binding molecule according to any one of claims 1 to 10, comprising:
a. culturing the host cell of claim 13 under conditions that allow expression of the SARS-COV-2 spike protein binding molecule;
b. collecting the SARS-COV-2 spike protein binding molecule expressed by the host cell from the culture of step a.
15. A pharmaceutical composition comprising a SARS-COV-2 spike protein binding molecule according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.
16. Use of the pharmaceutical composition of claim 15 in the manufacture of a medicament for the treatment or prevention of pneumonia as a novel coronavirus disease.
17. A kit for detecting SARS-COV-2, comprising a SARS-COV-2 spike protein binding molecule of any one of claims 1 to 10.
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