阅读说明：本技术 靶向人IgE的单域抗体、人源化单域抗体及其应用 (Single-domain antibody targeting human IgE, humanized single-domain antibody and application thereof ) 是由 张海珍 杨正根 罗丽华 傅俊成 陈校园 于 2021-07-08 设计创作，主要内容包括：本发明涉及生物领域,公开了靶向人IgE的单域抗体、人源化单域抗体及其应用。特异性结合IgE的单域抗体,由框架区和抗原结合区CDR1～CDR3组成,其中CDR1～CDR3分如SEQ ID NO.：1～3、6～8所示。本发明一些实例的单域抗体,可以特异性结合人体免疫球蛋白IgE及其复合物,对其它免疫球蛋白或者血浆其它蛋白非特异性吸附量低,吸附性能稳定,吸附效率高且再生性能好,为I型变态反应病人提供了一种新的治疗途径。与传统抗体相比具有分子量小、稳定性强、高耐性、低免疫原性、在极端的温度和pH值下仍可保持稳定性、易于表达且表达量高适合于大规模生产,在临床治疗上安全性能好、经济效益高。(The invention relates to the field of biology, and discloses a single-domain antibody targeting human IgE, a humanized single-domain antibody and application thereof. A single domain antibody that specifically binds IgE, consisting of framework and antigen binding regions CDR 1-CDR 3, wherein CDR 1-CDR 3 are as set forth in SEQ ID No.: 1 to 3 and 6 to 8. The single domain antibody of some embodiments of the invention can be specifically combined with human immunoglobulin IgE and compounds thereof, has low non-specific adsorption quantity to other immunoglobulins or other plasma proteins, stable adsorption performance, high adsorption efficiency and good regeneration performance, and provides a new treatment way for patients with type I allergy. Compared with the traditional antibody, the antibody has the advantages of small molecular weight, strong stability, high tolerance, low immunogenicity, stability under extreme temperature and pH value, easy expression, high expression level, suitability for large-scale production, good safety performance in clinical treatment and high economic benefit.)

1. A single domain antibody that specifically binds IgE, consisting of framework regions and antigen binding regions CDR 1-CDR 3, characterized in that:

the amino acid sequence of CDR1 is as set forth in SEQ ID No.: 1, the sequence is GIRFSNYG; the amino acid sequence of CDR2 is as set forth in SEQ ID No.: 2, the sequence is ISSATSTT; the amino acid sequence of CDR3 is as set forth in SEQ ID No.: 3, the sequence is NARWRDYEY; or

The amino acid sequence of CDR1 is as set forth in SEQ ID No.: 6, the sequence is GTPLDYYT; the amino acid sequence of CDR2 is as set forth in SEQ ID No.: 7, the sequence is IGRLDTRV; the amino acid sequence of CDR3 is as set forth in SEQ ID No.: 8, sequence AADRGYSSATPLALGRYIW.

2. The single domain antibody of claim 1, characterized in that: the single domain antibody is an alpaca single domain antibody, and the amino acid sequence of the single domain antibody is shown in SEQ ID No.: 4 is shown in the specification; preferably, the nucleotide sequence corresponding to the amino acid sequence is as set forth in SEQ ID No.: 5, respectively.

3. The single domain antibody of claim 1, characterized in that: the single domain antibody is an alpaca single domain antibody, and the amino acid sequence of the single domain antibody is shown in SEQ ID No.: 9 is shown in the figure; preferably, the nucleotide sequence corresponding to the amino acid sequence is as set forth in SEQ ID No.: shown at 10.

4. The single domain antibody of claim 1, characterized in that: the single domain antibody is a humanized single domain antibody; preferably, the amino acid sequence of the humanized single domain antibody is as set forth in SEQ ID No.: 11 is shown in the figure; preferably, the nucleotide sequence of the humanized single domain antibody is as set forth in SEQ ID No.: shown at 12.

5. A plasmid for expressing a single domain antibody, characterized in that: the plasmid is inserted with a nucleotide sequence capable of expressing the single domain antibody of any one of claims 1 to 4.

6. A vector for expressing a single domain antibody, said vector being a bacterial, fungal or animal cell characterized in that: the single domain antibody is as defined in any one of claims 1 to 4.

7. The carrier of claim 6, wherein: the bacteria is selected from escherichia coli, and the fungi is selected from pichia pastoris; the animal cell is selected from CHO cell or 293fT cell.

8. Use of a single domain antibody for the preparation of an IgE and/or IgE complex adsorbent, characterized in that: the single domain antibody is as defined in any one of claims 1 to 4.

9. An adsorbent that specifically binds IgE and/or IgE complexes, comprising a solid support, wherein: the solid phase carrier carries the single domain antibody of any one of claims 1 to 4.

10. The sorbent of claim 9, wherein: the solid phase carrier is at least one selected from chitosan, agarose, cellulose, dextran, resin and cellulose.

Technical Field

The invention relates to a single domain antibody and application thereof, in particular to a single domain antibody specifically binding IgE protein and application thereof.

Background

Blood immunoadsorption therapy is a clinical medical technology emerging in recent 30 years, and difficult diseases with no obvious curative effect of some medicines, such as atopic dermatitis, autoimmune system diseases, nephrosis syndrome, severe hepatitis, malignant tumors and the like, can often obtain better effect when in vitro immunoadsorption blood purification is carried out. Blood immunoadsorption therapy techniques have been widely used in many clinical fields such as liver disease, acute toxicity, blood, urinary system, etc. The development and production of immunoadsorbents has also become an important high and new technology industry.

The clinical common allergic asthma, allergic urticaria, allergic dermatitis, allergic rhinitis and anaphylactic shock caused by penicillin lead the IgE concentration in a patient body to reach more than 10 times of the normal value, and particularly the concentration of allergen specific IgE can reach about 1000 times of the normal value. At present, immunosuppressant medicines are mainly used for clinical treatment, and the medicine therapy has good treatment effect on accidental anaphylactic reaction, but has great side effect on the treatment of chronic and intractable allergic diseases such as allergic asthma and the like which need to be taken for a long time, not only can the chronic and intractable allergic diseases not be cured, but also can generate medicine dependence. In recent years, the treatment of allergic reaction caused by high IgE by blood purification and adsorption is a new trend.

An IgE-specific adsorption product (WO2012140214A1) was developed by foreign companies, and a humanized single-chain antibody was purified by expression using Escherichia coli, and the purified protein was coupled with agarose using sodium periodate-oxidized agarose gel to synthesize an IgE adsorbent. Although a large amount of ligands can be coupled by the method for preparing the coupling adsorbent, the problem of easy falling of the ligands can be caused, and the adsorption quantity of the adsorbent is low due to the short coupling spacer arm. The Japanese scholars Sato H fixes sheep anti-human IgE IgG antibodies to glass beads, about 1g of solid phase carrier is coupled with 32.5mg of IgG protein to prepare an IgE adsorption column, and the effect of reducing the IgE amount in the blood of an adult patient from 10000IU to 3000IU is very obvious by using a blood perfusion method (Specific removal of IgE by therapeutic immunological adsorption system [ J ]. cervical of immunological methods.1989 (2); 61-168.) the adsorbent adopts IgE antibody protein as a ligand, can specifically recognize the IgE protein, has a remarkable effect of removing the IgE protein and causes less loss of other plasma proteins, and has the main defects that the glass beads are coupled with the antibody protein as the solid phase carrier, so that the ligand is easy to fall off, and the heterologous protein enters a patient to become a new allergen to cause the aggravation of anaphylactic reaction. Meanwhile, the authors also consider that an adsorption column using IgE as a ligand is connected in series to adsorb the fallen sheep antibodies, and the method undoubtedly increases the complexity of clinical treatment and increases the cost of the treatment. The giardia cloud of domestic scholars uses IgE receptor protein as an aglucon and agarose gel as a solid phase carrier to prepare an IgE adsorbent (CN102660569A), which well solves the problem of aggravated anaphylactic reaction caused by aglucon shedding, but the aglucon is escherichia coli prokaryotic expression protein and is insoluble inclusion body expression, protein renaturation and pyrogen removal are needed, so that the difficulty in obtaining the aglucon is improved, the production cost is increased, and the renatured protein is poor in stability and not easy to store for a long time.

Disclosure of Invention

The present invention aims to overcome at least one of the disadvantages of the prior art and to provide a single domain antibody specifically binding to the IgE protein and its use.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a single domain antibody that specifically binds IgE, consisting of framework and antigen binding regions CDR 1-CDR 3, wherein:

the amino acid sequence of CDR1 is as set forth in SEQ ID No.: 1, the sequence is GIRFSNYG; the amino acid sequence of CDR2 is as set forth in SEQ ID No.: 2, the sequence is ISSATSTT; the amino acid sequence of CDR3 is as set forth in SEQ ID No.: 3, the sequence is NARWRDYEY; or

The amino acid sequence of CDR1 is as set forth in SEQ ID No.: 6, the sequence is GTPLDYYT; the amino acid sequence of CDR2 is as set forth in SEQ ID No.: 7, the sequence is IGRLDTRV; the amino acid sequence of CDR3 is as set forth in SEQ ID No.: 8, sequence AADRGYSSATPLALGRYIW.

In some examples, the single domain antibody is an alpaca single domain antibody having an amino acid sequence as set forth in SEQ ID No.: 4 is shown in the specification; preferably, the nucleotide sequence corresponding to the amino acid sequence is as set forth in SEQ ID No.: 5, respectively.

In some examples, the single domain antibody is an alpaca single domain antibody having an amino acid sequence as set forth in SEQ ID No.: 9 is shown in the figure; preferably, the nucleotide sequence corresponding to the amino acid sequence is as set forth in SEQ ID No.: shown at 10.

In some examples, the single domain antibody is a humanized single domain antibody.

In some examples, the amino acid sequence of the humanized single domain antibody is as set forth in SEQ ID No.: shown at 11.

In some examples, the nucleotide sequence of the humanized single domain antibody is as set forth in SEQ ID No.: shown at 12.

In a second aspect of the present invention, there is provided:

a plasmid for expressing a single domain antibody, said plasmid having inserted therein a nucleotide sequence capable of expressing a single domain antibody according to the first aspect of the invention.

In some examples, the plasmid is a PET28a plasmid.

In some examples, the expression system of the plasmid is a bacterial, fungal or animal cell.

In some examples, the bacteria are selected from escherichia coli; the fungus is selected from pichia pastoris; the animal cell is selected from CHO cell or 293fT cell.

In a third aspect of the present invention, there is provided:

a vector expressing a single domain antibody, said vector being capable of expressing a single domain antibody according to the first aspect of the invention.

In some examples, the vector has inserted therein a nucleotide sequence that expresses a single domain antibody according to the first aspect of the invention.

In some examples, the vector is a bacterial, fungal, or animal cell.

In some examples, the bacteria are selected from escherichia coli; the fungus is selected from pichia pastoris; the animal cell is selected from CHO cell or 293fT cell.

In a fourth aspect of the present invention, there is provided:

use of a single domain antibody as described in the first aspect of the invention in the manufacture of an IgE and/or IgE complex adsorbent.

In a fifth aspect of the present invention, there is provided:

an adsorbent which specifically binds IgE and/or IgE complexes comprising a solid support having immobilised thereon a single domain antibody according to the first aspect of the invention.

In some examples, the solid support includes, but is not limited to, at least one of chitosan, agarose, cellulose, dextran, resin, cellulose.

The invention has the beneficial effects that:

the single domain antibody of some embodiments of the invention can be specifically combined with human immunoglobulin IgE and compounds thereof, has low non-specific adsorption capacity to other immunoglobulins or other plasma proteins, stable adsorption performance, high adsorption efficiency and good regeneration performance.

Some embodiments of the invention are humanized single domain antibodies, which have small molecular weight, strong stability, high tolerance, low immunogenicity, stability at extreme temperatures and pH values, easy expression, high expression level suitable for large-scale production, good safety in clinical treatment, and high economic benefit compared to conventional antibodies.

The adsorbent prepared from the single-domain antibody of some embodiments of the invention can effectively adsorb and remove excessive immunoglobulin IgE and complexes thereof in blood, and provides a new treatment way for patients with type I allergy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following description of the embodiments needs to be briefly described by using the drawings.

FIG. 1 is a protein electrophoresis diagram of purified anti-IgE single domain antibody expressed and purified by pronucleus, wherein the molecular weight of Marker is: 180. 130, 95, 72, 55, 43, 34, 26, 17 and 10 KD. The single domain antibody molecule has the size of 14KDa, and sequentially comprises a sequence 4, a sequence 9 and a sequence 11 from left to right.

FIG. 2 is a comparison of the binding activity of each anti-IgE single domain antibody expressed prokaryotically with IgE protein.

Detailed Description

In a first aspect of the present invention, there is provided:

the invention obtains the alpaca anti-human IgE phage library by immunizing alpaca with human IgE. Through screening, 2 sequences with higher adsorption performance are obtained from the phage library.

A single domain antibody that specifically binds IgE, consisting of framework and antigen binding regions CDR 1-CDR 3, wherein:

the amino acid sequence of CDR1 is as set forth in SEQ ID No.: 1, the sequence is GIRFSNYG; the amino acid sequence of CDR2 is as set forth in SEQ ID No.: 2, the sequence is ISSATSTT; the amino acid sequence of CDR3 is as set forth in SEQ ID No.: 3, the sequence is NARWRDYEY; or

The amino acid sequence of CDR1 is as set forth in SEQ ID No.: 6, the sequence is GTPLDYYT; the amino acid sequence of CDR2 is as set forth in SEQ ID No.: 7, the sequence is IGRLDTRV; the amino acid sequence of CDR3 is as set forth in SEQ ID No.: 8, sequence AADRGYSSATPLALGRYIW.

The two single-domain antibodies are obtained in the same phage library, and have certain similarity on amino acid sequences.

In some examples, the single domain antibody is an alpaca single domain antibody having an amino acid sequence as set forth in SEQ ID No.: 4, respectively.

SEQ ID NO.：4

MDVQLQESGGGLVQPGGSLRLSCVASGIRFSNYGLTWYRQAPGKSRELVAVISSATSTTSYGDSVRGRFTISRDNAKNTAFLQMNSLKPEDTAVYYCNARWRDYEYWGQGTQVTVSSHHHHHH (underlined respectively CDR1, CDR2, and CDR 3).

Preferably, the amino acid sequence of SEQ ID No.: 4 is as shown in SEQ ID No.: 5, respectively.

In some examples, the single domain antibody is an alpaca single domain antibody having an amino acid sequence as set forth in SEQ ID No.: shown at 9.

SEQ ID NO.：9

MDVQLQESGGGLVQSGGSLTLSCTASGTPLDYYTIGWFRQAPGKEREGVSSIGRLDTRVHYADSVKGRFTISRNNAENTVYLQMNSLKPEDTAIYYCAADRGYSSATPLALGRYIWWGQGTQVTVSSHHHHHH (underlined are CDR1, CDR2 and CDR3, respectively).

Preferably, the amino acid sequence of SEQ ID No.: 9 is as shown in SEQ ID No.: shown at 10.

In order to improve the safety of the single domain antibody and facilitate the adsorption of excess IgE and/or IgE complexes in blood, the single domain antibody can be subjected to humanized modification. In some examples, the single domain antibody is a humanized single domain antibody.

In some examples, the method of humanization is as follows: the human antibody sequences having higher similarity to the nucleotide sequences of the 2 selected single domain antibodies were searched by the IGBLAST database (www.ncbi.nlm.nih.gov/IGBLAST /), the human IGHV3 region sequences were selected, and the FR region sequences in the single domain antibodies were compared with the sequences of the human antibodies. And then carrying out random combination and replacement on individual amino acid residues according to different physicochemical properties of the amino acid residues, expressing corresponding protein, and screening out sequences with better indexes according to solubility, expression quantity, activity, stability and the like.

In some examples, the amino acid sequence of the humanized single domain antibody is as set forth in SEQ ID No.: shown at 11.

SEQ ID NO.：11

MEVQLQESGGGLVQSGGSLRLSCTASGTPLDYYTIGWVRQAPGKEREWVSSIGRLDTRVHYADSVKGRFTISRNNAENTLYLQMNSLRAEDTAVYYCAADRGYSSATPLALGRYIWWGQGTQVTVSSHHHHHH (underlined are CDR1, CDR2 and CDR3, respectively).

In some examples, the nucleotide sequence of the humanized single domain antibody is as set forth in SEQ ID No.: shown at 12.

In some examples, the N-terminus and/or C of the single domain antibody is supplemented with a signal peptide and/or an isolation tag. Facilitating better expression and purification of single domain antibodies. The signal peptide may be cleaved and/or the tag may be isolated as desired prior to use.

In a second aspect of the present invention, there is provided:

a plasmid for expressing a single domain antibody, said plasmid having inserted therein a nucleotide sequence capable of expressing a single domain antibody according to the first aspect of the invention. A nucleotide sequence of a signal peptide, a separation tag, etc. may be further added to the 5 'end and/or the 3' end of the nucleotide sequence of the single domain antibody to add a signal peptide, a separation tag, etc. to the N-terminus and/or C-terminus of the single domain antibody, facilitating better expression and purification of the single domain antibody.

The plasmid vector is not particularly limited and may be various commercially available plasmids, and in some examples, the plasmid is the PET28a plasmid.

In some examples, the expression system of the plasmid is a bacterial, fungal or animal cell.

In a third aspect of the present invention, there is provided:

a vector expressing a single domain antibody, said vector being capable of expressing a single domain antibody according to the first aspect of the invention.

The vector has inserted therein a nucleotide sequence for expressing a single domain antibody according to the first aspect of the invention to effect expression of the single domain antibody. For better expression and purification of the single domain antibody, a signal peptide, a separation tag, etc. may be further added to the N-terminus and/or C of the single domain antibody.

In some examples, the vector is a bacterial, fungal, or animal cell.

In some examples, the bacteria are selected from escherichia coli; the fungus is selected from pichia pastoris; the animal cell is selected from CHO cell or 293fT cell.

In a fourth aspect of the present invention, there is provided:

use of a single domain antibody as described in the first aspect of the invention in the manufacture of an IgE and/or IgE complex adsorbent.

In some examples, the IgE and/or IgE complex is an IgE and/or IgE complex in the blood of an animal.

In some examples, the IgE and/or IgE complex is an IgE and/or IgE complex in human blood.

In a fifth aspect of the present invention, there is provided:

an adsorbent which specifically binds IgE and/or IgE complexes comprising a solid support having immobilised thereon a single domain antibody according to the first aspect of the invention.

In some examples, the solid support includes, but is not limited to, at least one of chitosan, agarose, cellulose, dextran, resin, cellulose.

The method for coupling the single-domain antibody to the solid-phase carrier can be realized by sequentially introducing epoxy groups and carboxyl groups on the surface of the microsphere and then adding a catalyst to couple amino groups on the antibody and the carboxyl groups on the carrier.

The technical scheme of the invention is further explained by combining the examples.

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. All the technologies implemented based on the above-mentioned contents of the present invention should fall within the scope of the claims of the present application.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the kit biomaterials, if not specifically indicated, are commercially available.

EXAMPLE 1 prokaryotic expression and purification of Single Domain antibodies

The construction and screening method of the phage library is the same as CN111875706A, and sequencing is carried out on positive clones obtained by screening. The sequence of SEQ ID No.: : 5. SEQ ID No.: 10 and SEQ ID No.: 12 into pet-28a plasmid, designing DNA enzyme cutting sites as NcoI and XhoI. The plasmid was transformed into E.coli BL21(DE3), cultured in kanamycin-resistant LB medium, induced with 1mM IPTG for 4 hours, the disrupted bacteria were centrifuged to obtain a supernatant, and the anti-IgE single domain antibody adsorbed by cobalt ion filler was shown in FIG. 1. As can be seen from FIG. 1, the molecular weight of the single domain antibody is about 14kD, which is in line with the theoretical value.

Example 2 comparison of anti-IgE Single Domain antibody Activity

Diluting natural human IgE protein (abcam) to 1ug/ml, coating on an enzyme label plate, diluting each single-domain antibody with prokaryotic expression concentration of 1ug/ml to a certain concentration, adding the enzyme label plate, reacting at room temperature for 2 hours, washing the plate for 5 times, adding an anti-His antibody labeled with HRP, reacting at room temperature for 1 hour, adding TMB color developing solution and stop solution, and determining OD (optical density) by measuring₄₅₀Numerical values. The results are shown in FIG. 2 below. The results show that SEQ ID No.: 4 and SEQ ID No.: 9, the activity of the humanized single domain antibody was reduced by about 13%, which was within an acceptable range.

Example 3 Synthesis of adsorbent Filler

(1) Adding 2M NaOH solution into 5mL of agarose gel according to the volume ratio of 1:1, adding an activating agent (glycerol ether/epichlorohydrin) according to the volume ratio of 1:0.7, and reacting to obtain agarose gel with a large number of epoxy groups on the surface;

(2) washing the epoxy agarose by PBS, adding 0.5g of 6-aminocaproic acid, reacting for 2h in a shaking table at 37 ℃ and 180rpm, and reacting epoxy groups on the surface of the agarose with amino groups on the 6-aminocaproic acid to obtain agarose gel with carboxyl groups on the surface;

(3) after the carboxylated agarose was washed clean with PBS, 0.5g of EDC/NHS was added, and 100mg of each antibody solution (SEQ ID No.: 4, SEQ ID No.: 9, SEQ ID No.: 11) of pH 7.4 for prokaryotic expression was added simultaneously to perform a reaction, mixed and shaken at 30 ℃ and 120rpm for 14 hours, and after the synthesis, washed clean with PBS for use.

Example 4 measurement of adsorption Performance of adsorbent for respective immunoglobulins

The adsorbent synthesized by coupling in example 3 and a white ball agarose gel without coupling protein were measured 0.5ml of filler in 10ml of EP tube, 5ml of human plasma and 7500IU of recombinant human IgE protein were added to the plasma, and the reaction was carried out for 1 hour in a shaker at room temperature. And after the reaction is finished, pouring all the reaction liquid into a disposable affinity chromatography column, and collecting the reacted plasma. Detecting the content of IgE in blood plasma before and after adsorption by using an IgE detection kit; and detecting the content of each immunoglobulin in the blood plasma before and after adsorption by using a biochemical analyzer. The results are shown in table 1 below:

TABLE 1 adsorption of synthetic adsorbents for each immunoglobulin

The results show that the three coupling adsorbents synthesized have good clearing effect on the IgE protein, and the clearing rate is over 95 percent. The adsorption performance of the humanized antibody to IgE is not obviously different from that before the humanized antibody is humanized; but has low nonspecific adsorption to other immunoglobulins such as IgG, IgM and IgA, the adsorption rate is lower than 10 percent, and the cross reaction is small.

Example 5 stability testing of humanized Single Domain antibodies

The immunoadsorbent filler synthesized by the sequence 11 in the example 4 is stored in 20% ethanol, and is placed in a refrigerator at 4 ℃ for 7d, 14d and 30d respectively, and then the adsorption performance of the filler on the recombinant human IgE protein is repeatedly measured. As shown in Table 2, the inventors considered that the IgE adsorption performance of the filler did not decrease within the detection error range of the kit, and the IgE adsorption performance of the adsorbent did not decrease within one month of refrigerator storage at 4 ℃.

TABLE 2 stability determination of synthetic adsorbent packings

Time of standing	Pre-adsorption IgE protein (IU/ml)	Post-adsorption IgE protein (IU/ml)	IgE clearance (%)
				7d	1559	50	96.8
14d	1559	110	92.9
				30d	1559	80	94.9

Example 6 Long-term toxicity testing of humanized Single Domain antibodies

Carrying out long-term toxicity test on the humanized single-domain antibody with the sequence 11 by using a rat, observing the daily behavior state of the rat, and detecting the biochemical and blood conventional indexes of the blood of the rat before and after administration to evaluate whether the substance to be tested has toxic effect on the rat.

The doses administered were as follows:

negative control group: physiological saline; low dose group: humanized single domain antibody 0.25 u g/kg; the medium dose group: 1.375 mu g/kg of humanized single-domain antibody; high dose group: humanized single domain antibody 2.5 u g/kg.

TABLE 3 weight (unit: g) of rats at various administration times for each dose group

Time	Low dose group	Middle dose group	High dose group	Solvent set
					Before administration to rats	255.3±15.0	251.1±33.9	244.3±21.8	260.4±28.7
The drug is administered to rats for 30 days	346.6±40.6	325.9±45.1	349.1±45.8	335.1±43.9

TABLE 4 blood routine tests of rats in each dose group at different administration times

TABLE 5 Biochemical examination of blood in rats at different doses for different administration times

Through SPSS statistical analysis, before and after administration, each administration dose has no significant difference influence on the body weight, blood routine and blood biochemistry of rats, which indicates that the humanized single-domain antibody has good safety.

The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.

<110> Guangzhou Kangsheng Biotechnology GmbH

<120> Single domain antibody targeting human IgE, humanized Single domain antibody and uses thereof

<130>

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Met Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Thr Pro Leu Asp Tyr

20 25 30

Tyr Thr Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp

35 40 45

Val Ser Ser Ile Gly Arg Leu Asp Thr Arg Val His Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Glu Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Asp Arg Gly Tyr Ser Ser Ala Thr Pro Leu Ala Leu Gly

100 105 110

Arg Tyr Ile Trp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His

115 120 125

His His His His His

130

<210> 12

<211> 410

<212> DNA

<213> Artificial sequence

<400> 12

ccatggaagt gcagctgcag gagagcggcg gcggcctggt gcagtctggc ggcagcctgc 60

gcctgagctg cacagcgagc ggcaccccgc tggattatta tactataggc tgggtgcgcc 120

aggcgccggg caaagagcgt gaatgggtga gctcaattgg ccgcctggat acccgcgtgc 180

actatgcgga tagcgtgaaa ggccgcttta ccattagccg caacaacgcc gagaacaccc 240

tgtatctgca gatgaacagc ctgcgcgcgg aagataccgc ggtgtattat tgcgcggcgg 300

atcgcggcta tagcagcgcg accccgctgg cgctgggccg ctatatttgg tggggccagg 360

gcacccaggt gaccgtgagc agccatcatc atcatcatca ttaactcgag 410