CN114685664B

CN114685664B - Single-domain antibody for resisting human B lymphocyte surface antigen CD20 and application thereof

Info

Publication number: CN114685664B
Application number: CN202210394491.7A
Authority: CN
Inventors: 包福祥; 刘艳龙; 敖可心; 付山; 郝彦霞; 程艺; 张慧敏; 张磊
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2023-10-20
Anticipated expiration: 2042-04-12
Also published as: CN114685664A

Abstract

The invention relates to a single domain antibody of anti-human B lymphocyte surface antigen CD20 and application thereof. Amino acid sequences of the framework region FR and complementarity determining region CDRs of the VHH chain are disclosed, as are nucleotide sequences encoding the single domain antibodies and host cells capable of expressing the single domain antibodies. In addition, the invention also discloses a bispecific antibody containing an anti-CD20 VHH domain and an anti-CD3 VHH domain, wherein the bispecific antibody is formed by fusing two different single domain antibody sequences. The invention completely retains the biological activities of the anti-CD20 antibody and the anti-CD3 antibody, can identify tumor cells and effector cells, targets the effector cells to the tumor cells, and promotes the effector cells to play a remarkable role in killing. The invention can be used for researching and developing the medicine for treating B cell lymphoma.

Description

Single-domain antibody for resisting human B lymphocyte surface antigen CD20 and application thereof

Technical Field

The invention belongs to the technical field of molecular biology and biological medicine, and particularly relates to a single domain antibody for resisting a human B lymphocyte surface antigen CD20 and application thereof.

Background

B-cell lymphoma is a heterogeneous lymphoproliferative disease derived from lymphoid tissues with different clinical manifestations. It ranges from slow growing hodgkin's lymphoma (HD) to more aggressive non-hodgkin's lymphoma (NHL), such as diffuse large B-cell lymphoma (DLBCL). In recent years, the number of people suffering from non-hodgkin lymphomas is increasing, and the incidence of the diseases is also increasing. Of these non-hodgkin lymphoma (NHL) cases, more than 85% are B cell diseases. Meanwhile, more than 95% of B cell lymphomas are shown to be abnormally highly expressing CD20.

CD20 molecule, also known as B lymphocyte surface antigen B1, transmembrane 4 domain subfamily A member 1 and MS4A1, is a specific membrane protein on the surface of B lymphocytes, which exists in non-glycosylated form. The epitope is expressed in pre-B lymphocytes and mature B lymphocytes but not in hematopoietic stem cells, plasma cells and other tissues. Human CD20 consists of 297 amino acids in total and has a relative molecular weight of 33-35kDa. CD20 has four transmembrane domains, including two extracellular loops and an intracellular region. The research proves that the 47 amino acid outer loop of the third and fourth transmembrane regions is the epitope of CD20 and is recognized by most anti-CD20 antibodies. CD20 protein has no known natural ligand to interfere with its binding to antibodies, and CD20 does not significantly internalize and shed after binding to antibodies nor undergo antigen modulation by binding to antibodies, thus becoming an ideal target for the treatment of B cell lymphomas.

Currently, approved CD20 target antibody drugs, rituximab-rituximab (rituximab), have a large market share, and ofatuzumab, obituzumab, and oreuzumab are also marketed in 2009, 2013, 2017, respectively, for the treatment of leukemia and multiple myeloma. The anti-tumor activity of the targeted CD20 antibodies mainly includes induction of apoptosis, complement dependent cytotoxicity (Complement dependent cytotoxicity, CDC) and Antibody dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibodies will continue to be used in the treatment of a variety of cancers. However, because of their large molecular weight, carrying four polypeptide chains limits their access to tumor targets.

The antibody drug, in particular the genetic engineering antibody drug, has very good application prospect in diagnosis, prevention and passive immunotherapy of diseases as a passive immune preparation. In particular, the recent discovery of "single domain heavy chain antibodies" (VHHs) which naturally lack the antibody light and CH1 regions in camelids is one of the hot spots of current research. Because such a single domain heavy chain antibody VHH has a molecular weight of only 1/10 of that of a normal monoclonal antibody, and a size of only 2-5nm, it is also called a single domain antibody (Single domain antibody, sdAb), or Nanobody (Nb). The long CDR3 region of the heavy chain variable region may form a stable raised loop structure in which stable disulfide bonds may penetrate the interior of the antigen with higher affinity for VHH antibodies than for normal antibodies forming a concave topology with the antigen. The sdAb can be used for displaying hidden epitopes of traditional immunoglobulins, so that the sdAb can be widely applied in the fields of immune research, diagnostic detection, medical science, biological imaging, therapeutic antibody development and the like.

However, there is no report of single domain antibodies against CD20 molecules of B cell lymphomas.

Disclosure of Invention

In view of the problems of the prior art, the present invention provides a single domain antibody against the human B lymphocyte surface antigen CD20 and uses thereof.

The technical scheme for solving the technical problems is as follows:

the present invention provides a single domain antibody against human B lymphocyte surface antigen CD20, the single domain antibody VHH chain comprising framework region FR and complementarity determining region CDR;

the framework regions FR comprise FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 comprises the amino acid sequence shown in SEQ ID NO.1, the amino acid sequence of FR2 comprises the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of FR3 comprises the amino acid sequence shown in SEQ ID NO.3, and the amino acid sequence of FR4 comprises the amino acid sequence shown in SEQ ID NO. 4;

the complementarity determining regions CDR comprise CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 comprises the amino acid sequence shown in SEQ ID NO.5, the amino acid sequence of CDR2 comprises the amino acid sequence shown in SEQ ID NO.6, and the amino acid sequence of CDR3 comprises the amino acid sequence shown in SEQ ID NO. 7.

The beneficial effects of adopting the technical scheme include:

according to the invention, a human CD20 extracellular region polypeptide is adopted to immunize a bactrian camel, then a phage antibody library aiming at a CD20 single-domain heavy chain antibody is established by utilizing peripheral blood lymphocytes of the camel, an immune single-domain heavy chain antibody is screened by utilizing a phage display technology, a single-domain heavy chain antibody gene aiming at a CD20 protein is obtained unexpectedly, the gene is transferred into escherichia coli, a single-domain heavy chain antibody strain capable of being efficiently expressed in the escherichia coli is established, the gene sequence of each clone strain is analyzed according to sequence comparison software, the amino acid sequence of a single-domain heavy chain antibody VHH chain aiming at the CD20 protein is obtained, and the fact that the single-domain heavy chain antibody can be specifically combined with the CD20 protein is proved.

The single domain antibody of the human B lymphocyte surface antigen CD20 has the advantages of high affinity and high specificity.

Further, the amino acid sequence of the single domain antibody includes the amino acid sequence shown in SEQ ID NO. 8. Further, the amino acid sequence of the variable region of the single domain antibody includes the amino acid sequence shown in SEQ ID NO. 8.

Further, the nucleotide sequence of the single domain antibody includes the nucleotide sequence shown in SEQ ID NO. 9.

The single domain antibody provided by the invention has small molecular weight and better solubility and stability. Compared with the traditional antibody, the single-domain antibody provided by the invention has smaller immunogenicity, and is easy to carry out humanized reconstruction and clinical application. And the surface hydrophilicity is stronger, and the mismatch between the heavy chain and the light chain of the traditional antibody does not exist.

The invention provides a polypeptide, protein or bispecific antibody, wherein the amino acid sequence of the polypeptide, protein or bispecific antibody comprises the amino acid sequence of the VHH chain of the single domain antibody.

Further, the polypeptide, the protein, and the bispecific antibody all include the anti-CD20 specific single domain antibodies described above.

In an embodiment of the invention, a bispecific antibody is provided comprising a variable region as described above for a CD20 single domain antibody and a variable region for an anti-CD3 single domain antibody.

Further, bispecific antibodies include CD20 and CD3 single domain antibodies, with the CD20 and CD3 single domain antibodies linked by a Linker.

The beneficial effects of adopting the technical scheme include:

the invention connects the single-domain heavy chain antibody gene of the anti-CD20 protein and the single-domain antibody gene of the anti-CD3 through the connecting peptide, thereby avoiding the mismatch between the light chain and the heavy chain of the traditional bispecific antibody. The provided bispecific antibody can specifically bind with T cell surface antigen cluster 3, namely CD3 molecule, and tumor surface antigen cluster 20, namely CD20 molecule, and completely retains the biological activities of the anti-CD20 antibody and the anti-CD3 antibody, so that the bispecific single domain antibody can better recognize tumor antigen and effector cells, target the effector cells to the tumor cells, promote the effector cells to play a remarkable killing role, achieve a specific killing role, and have stronger killing capability, thereby laying a foundation for clinically treating B cell lymphoma diseases.

Further, the amino acid sequence of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID No. 10. The nucleotide sequence of the bispecific antibody may comprise the nucleotide sequence shown as SEQ ID NO. 11.

The gene engineering technology is utilized to construct two nano antibodies aiming at different targets of the tumor into a bispecific antibody for improving the activity of the anti-tumor antibody. Single domain antibodies do not take into account the problem of mismatch between the heavy and light chains of the antibody when constructing bispecific antibodies, and are advantageous when constructing bispecific antibodies. The sdAb is reasonably designed by a genetic engineering technology, and the constructed bispecific single domain antibody has stronger target binding force, longer serum half-life, lower drug resistance and lower serious adverse reaction, so that the bispecific single domain antibody has wide application prospect in diagnosis and treatment in the fields of infection, tumor and immunity.

The invention provides a nucleic acid which codes for one or a combination of a plurality of single domain antibodies, polypeptides, proteins and bispecific antibodies.

The nucleic acid may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

For example: the present invention provides a DNA molecule encoding the VHH chain of the above-described single domain antibody directed against CD20 extracellular region polypeptide. The nucleotide sequence of the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO. 9. The invention also provides a nucleic acid for encoding the bispecific antibody, wherein the nucleotide sequence of the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO. 11.

The invention also provides a nucleotide sequence or a combination thereof, which encodes a single domain antibody comprising the anti-human CD20 specificity.

The invention provides a vector comprising the nucleic acid.

The vector can be an expression vector or a cloning vector. The nucleotide sequence of the above DNA molecule may be included in the vector. The cloning vector carries the coded nucleic acid, so that the purpose of replication and amplification can be achieved. The expression can be achieved by means of expression elements on the expression vector.

For example: the invention provides an expression vector containing the nucleic acid, wherein the vector is pET-22b (+).

The invention provides a host cell comprising the vector. The single domain heavy chain antibodies described above for CD20 can be expressed. For example: the invention provides a host cell containing the expression vector, and the host cell is escherichia coli BL21 (DE 3) containing the expression vector.

The invention provides a medicine or a medicine composition, which comprises one or a combination of a plurality of single domain antibodies, polypeptides, proteins and bispecific antibodies.

The invention provides a reagent or a kit, which comprises one or a combination of a plurality of single domain antibodies, polypeptides, proteins and bispecific antibodies.

The invention provides the application of one or more of the single domain antibody, polypeptide, protein, bispecific antibody, nucleic acid, vector, host cell, medicine, pharmaceutical composition, reagent and kit in (1), (2) or (3);

(1) Preparing a reagent or a kit for detecting B cell lymphoma;

(2) Preparing a medicament or a pharmaceutical composition for treating malignant tumor diseases of the B lymphocyte system;

(3) A medicament or a pharmaceutical composition for treating diseases related to B lymphocytes.

For example: the invention provides application of the single domain antibody aiming at CD20 extracellular region polypeptide in preparing a reagent for detecting B cell lymphoma. Provides a bispecific single domain antibody of anti-human CD20 and anti-CD3, and further application thereof in preparing medicaments for treating malignant tumor diseases of B lymphocyte system.

For example: the bispecific antibody of the invention can be used for preparing therapeutic drugs for B lymphocyte-related diseases and tumor-related diseases which abnormally express CD20, wherein the tumor-related diseases are B cell malignant tumors positive to CD20 antigen.

Drawings

FIG. 1 is an electrophoresis chart of VHH gene fragments obtained by PCR amplification in example 1;

FIG. 2 shows the detection of CD 20-specific recombinant phages by Phage ELISA in example 3;

FIG. 3 is a SDS-PAGE electrophoresis of the single domain heavy chain antibody against CD20 of example 4 expressed and purified by nickel column affinity chromatography, M: the molecular weight marker of the protein is KDa; lane1-3, 4-6, 7-9: purifying the product;

FIG. 4 is a graph showing the results of Western-blot analysis of the expression and purification products of VHH-pET-25b-SBP in example 5, M: the molecular weight marker of the protein is KDa; lane 1: CD20 protein (i.e., anti-CD20 single domain heavy chain antibody);

FIG. 5 shows the binding results of the cell immunofluorescence assay single domain antibody of example 6 to cell surface CD20 protein, A: fluorescence image of sdAb group cells under uv excitation, B: fluorescence image of negative control cells under uv excitation;

FIG. 6 shows the results of the cleavage assay of recombinant plasmid pET-22b/BsNb in example 7; wherein, M.DNA Marker;1-2. Recombinant plasmid pET-22b/BsNb;

FIG. 7 shows the SDS-PAGE identification of Anti-CD20/CD3BsNb recombinant antibodies of example 7 by Ni affinity chromatography; wherein M: the molecular weight marker of the protein is KDa;1: crushing the crude extract by bacteria before induction; 2: crushing the crude extract by the induced bacteria; 3: crushing the supernatant; 4: crushing and precipitating; 5-7: a sample eluted by the eluent in the step 3;

FIG. 8 is a graph showing the results of ELISA assays for Anti-CD20/CD3BsNb binding activity to CD20 polypeptide in example 8;

FIG. 9 is a flow cytometer of example 9 to detect binding of Anti-CD20/CD3BsNb to target cells;

FIG. 10 shows the toxic effects of Anti-CD20/CD3BsNb mediated PBMCs on Raji cells in example 10;

FIG. 11 shows ELISA detection of Anti-CD20/CD3BsNb mediated secretion of cytokine hIFN-gamma by PBMCs in example 11.

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

Terms and abbreviations:

single-domain antibodies (sdabs);

CDR is an abbreviation of English Complementarity Determining Regions (CDRs), which refers to the antigen complementarity determining region of an antibody;

bispecific nanobodies (Bispecific nanobody, bsNb).

The pET-22b (+), pET-25b-SBP plasmid was stored in the national institutes of public health of the university of inner Mongolia agriculture. The pMECS vector is given a benefit from the university of brucella free, belgium Serge Muyldermans. The experiments described in the examples of the present invention can be repeated only for public non-commercial purposes to demonstrate the effects described in the examples.

E.coli BL21 (DE 3) competent cells were purchased from beijing all gold biotechnology limited; raji cells were purchased from zexin-free biotechnology limited; freund's adjuvant, neutralization buffer, FITC-labeled kit (FITC-Antibody conjugation Kit), ni-NTA purified filler were purchased from Shanghai Co., ltd; HRP-labeled Anti-His tag mab was purchased from Wohan Sanying antibody Co; PVDF membrane, chromogenic solution A and B, erythrocyte lysate from Beijing Soy Bao technology Co., ltd, and T4 DNA ligase from TaKaRa biological reagent company; 1640 cell culture medium was purchased from BI, trypan blue from yajin, wayo family biotechnology limited; fetal bovine serum FBS was purchased from Thermofisher scientific; LDH kit (LDH working solution), lysate from wuyiruite biotechnology, inc; IFN-gamma detection ELISA kit, biotinylated antibody working solution, ABC working solution and TMB stop solution are purchased from Wuhan New Dimmatory biotechnology Co., ltd; notI and NcoI restriction endonucleases were purchased from NEB; coli e.coli TG1 competent cells, helper phage were purchased from GE company. Ampicillin, kanamycin, IPTG, PBST, polylysine, BSA, PEG8000, PBS, triethylamine elution buffer, tris-HCl, paraformaldehyde were all purchased from soribao; TMB color development was purchased from Promega.

The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available.

The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.

As used herein, the terms "the present single domain antibody", "the present anti-CD20 single domain antibody", "the present anti-CD20 specific heavy chain single domain antibody" are used interchangeably and refer to a single domain antibody that specifically recognizes and binds to human CD20 protein.

The invention discloses a single domain antibody of an anti-CD20 antigen, discloses amino acid sequences of a framework region FR and a complementarity determining region CDR of a VHH chain of the single domain antibody, and also discloses a nucleotide sequence for encoding the single domain antibody and a host cell capable of expressing the single domain antibody. The invention also discloses a bispecific antibody containing an anti-CD20 VHH domain and an anti-CD3 VHH domain, which is formed by fusing two different single domain antibody sequences, and provides a nucleotide sequence for encoding the bispecific antibody, comprising a vector for expressing the nucleotide sequence and a host cell transformed by the nucleotide or the vector. The invention completely retains the biological activities of the anti-CD20 antibody and the anti-CD3 antibody, can identify tumor cells and effector cells, targets the effector cells to the tumor cells, and promotes the effector cells to play a remarkable role in killing. The invention is used for researching and developing a medicine for treating B cell lymphoma, and also provides a construction method of a bispecific antibody.

The invention is further illustrated below with reference to examples.

Example 1: construction of a single domain antibody phage library against CD20

(1) 5mg of solid human CD20 extracellular region polypeptide (CD 20 polypeptide for short) with the sequence of KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (amino acid sequence shown as SEQ ID NO. 12), which is synthesized by the division of biological engineering (Shanghai), 1mg of CD20 polypeptide (PBS dilution) is mixed with Freund's adjuvant in equal volume, and a healthy Bactrian camel is immunized once every two weeks for 4 times, so that B cells are stimulated to express antigen-specific single domain antibodies;

(2) After 4 times of immunization, extracting camel peripheral blood, separating lymphocytes and extracting total RNA;

(3) Reverse transcription into cDNA and amplification of VHH using nested PCR, the results are shown in FIG. 1, and it can be seen from FIG. 1 that VHH has been successfully amplified;

(4) Double-enzyme digestion is carried out by restriction enzymes NcoI and NotI, and the double-enzyme digestion is connected with a pMECS carrier digested by the same enzymes, so as to obtain a connection product;

(5) Transforming the connection product into E.coli TG1 competent cells to construct a phage antibody library against human CD20 extracellular region polypeptide;

(6) Diluting antibody library bacteria liquid with 10 times gradient by multiple ratio, calculating phagocytesThe library capacity of the body antibody library was 1.2X10 ⁸ And randomly picking 24 colonies to serve as templates for PCR detection, and identifying the recombination rate of the phage antibody library, wherein the result shows that the insertion rate of the constructed library reaches 91.7%.

Example 2: screening for CD20 Single Domain antibodies

(1) Human CD20 extracellular region polypeptide is coated with 10 mug/mL, 100 mug/well to 96-well ELISA plate.

Overnight at 4 ℃;

(2) Standing at room temperature for 30min, removing liquid in the holes, adding 300 μl of PBST into each hole, washing for 3min each time, and repeating the above steps (namely washing with PBST for 5 times, removing liquid in the holes after each washing, and removing liquid in the holes in the 5 th time);

(3) Adding a phage antibody library to be screened (namely the phage antibody library constructed in the embodiment 1) into the 96-well ELISA plate treated in the step (2), wherein the temperature is 37 ℃ and the time is 1h;

(4) 300 mu L of PBST is added into each hole, each time of washing is carried out for 3min, and the process is repeated for 5 times;

(5) On the basis of the previous step, 100. Mu.L of triethylamine elution buffer (100 mM) was added to each well and incubated at room temperature for 10min; then, 50. Mu.L of 1M Tris-HCl was added to each well, and neutralization was performed using a neutralization buffer having a pH of 9.7;

(6) The eluted recombinant phage is infected with E.coli TG1 again, helper phage M13K07 is added continuously, and the step is repeated for 3-4 rounds to obtain enrichment gradually.

Example 3: screening of CD 20-specific phages by Phage ELISA

(1) Diluting human CD20 extracellular region polypeptide to 2.5 mug/mL, 100 mug/well to 96-well ELISA plate, and 4 ℃ overnight;

(2) After standing at room temperature for 30min, removing the liquid in the hole, adding 300 μl of PBST into each hole, washing for 3min each time, and repeating the above steps (namely washing with PBST for 5 times, removing the liquid in the hole after each washing, and removing the liquid in the hole in the 5 th time). 300. Mu.L/well of 3% BSA was added at 37℃for 2h;

(3) 300 mu L of PBST is added into each hole, each time of washing is carried out for 3min, and the process is repeated for 5 times;

(4) E.coli TG1 infected with phage eluted by three screening was uniformly spread on 2 XYT-AG solid medium (ampicillin and glucose were added to the formulation of 2 XYT solid medium, ampicillin concentration was 100. Mu.g/mL, glucose concentration was 20%) with a spreader, and cultured overnight at 37 ℃. 92 clones were randomly picked and inoculated in 400. Mu.L of 2 XYT-AG liquid medium, 220rpm, and incubated overnight at 37 ℃;

(5) mu.L of the mixture was added to a liquid containing 400. Mu.L of 2 XYT-AG+M13K07 (phage M13K07 was added to a 2 XYT-AG liquid medium, the number of phage M13K07 was generally 20 times the number of E.coli), 37℃at 250rpm for 2 hours;

(6) And centrifuged at 14000rpm and 25℃for 5min. The pellet was resuspended in 400. Mu.L of 2 XYT-AK liquid (ampicillin and kanamycin were added to the formulation of 2 XYT liquid medium at a concentration of 100. Mu.g/mL, kanamycin at a concentration of 100. Mu.g/mL) and incubated overnight at 37℃at 250 rpm;

(7) Centrifuging at 14000rpm and 4deg.C for 5min, collecting supernatant, mixing with 20% PEG8000, and standing at 4deg.C for 30min. Centrifuging at 4deg.C and 10000rpm for 20min, centrifuging, discarding supernatant, and taking 100 μl PBS to resuspend precipitate;

(8) Adding 50 mu L/hole heavy suspension into a 96-hole ELISA plate, setting negative control and blank control which are auxiliary phage M13K07 and PBS respectively, and incubating for 2h at room temperature;

(9) Removing liquid in the wells, adding 100 μl of TMB into each well, reacting at room temperature in the dark for 15-30min, adding 50 μl of 2M H into each well ₂ SO ₄ Terminating the reaction, and measuring OD450nm by using an enzyme-labeled instrument;

(10) When the OD450nm value of the sample hole is more than 2.1 times of the OD value of the negative control hole, the sample hole is judged to be positive; otherwise, judging as negative; the experimental results of the Phage ELISA for detecting CD20 specific recombinant Phage are shown in FIG. 2;

(11) Positive clones were inoculated into 5mL of LB liquid containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced;

(12) The gene sequences of all clone strains are analyzed according to sequence alignment software Vector NTI, the strains with the same CDR1, CDR2 and CDR3 sequences are regarded as the same clone strain, the strains with different sequences are regarded as different clone strains, 54 clones in randomly selected clones are far higher than a positive judgment value of 0.220, and 1 positive clone is selected from the clones and named sdAb11. Antibodies produced by this clone may be referred to as CD20 single domain antibodies or anti-CD20 single domain heavy chain antibodies, having the nucleotide sequence shown in SEQ ID NO.9 and the amino acid sequence shown in SEQ ID NO.8, including VHH, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Wherein the amino acid sequence of FR1 is the amino acid sequence shown as SEQ ID NO.1, the amino acid sequence of CDR1 is the amino acid sequence shown as SEQ ID NO.5, the amino acid sequence of FR2 is the amino acid sequence shown as SEQ ID NO.2, the amino acid sequence of CDR2 is the amino acid sequence shown as SEQ ID NO.6, the amino acid sequence of FR3 is the amino acid sequence shown as SEQ ID NO.3, the amino acid sequence of CDR3 is the amino acid sequence shown as SEQ ID NO.7, and the amino acid sequence of FR4 is the amino acid sequence shown as SEQ ID NO. 4.

Example 4: anti-CD20 single domain heavy chain antibody expressed and purified in host E.coli

(1) Adding 50 mu L of a strain solution of a phage ELISA positive clone (namely clone sdAb11 obtained in example 3) into 5mL of a 2 XYT liquid culture medium, culturing overnight at 37 ℃ and at 250rpm, extracting plasmids from the cultured strain solution, and sequencing genes;

(2) After sequence analysis, the correct cloning plasmid is digested by Not I and Nco I restriction enzymes, and is connected with pET-25b-SBP plasmid which is digested by the same restriction enzymes to obtain VHH-pET-25b-SBP plasmid, and the VHH-pET-25b-SBP plasmid is transformed into E.coli BL21 (DE 3) competent cells, and cultured overnight at 37 ℃;

(3) Inoculating 1mL of overnight strain into 100mL of LB culture medium, culturing at 37 ℃ until the OD600nm value reaches 0.4-0.6, adding 1mM IPTG with final concentration, and culturing at 30 ℃ overnight;

(4) The next day, centrifugally collecting thalli, crushing bacterial liquid by ultrasonic waves, and collecting supernatant and sediment;

(5) Analysis was performed by SDS-PAGE gel electrophoresis, the crushed supernatant was purified using Ni-NTA purification packing, and the purified recombinant single domain antibody (i.e., anti-CD20 single domain heavy chain antibody) was collected, as shown in the results of FIG. 3.

Example 5: western-blot identification recombinant single domain antibody

(1) Separating the human CD20 extracellular region polypeptide by SDS-PAGE gel electrophoresis;

(2) After electrophoresis, transferring the protein to a PVDF membrane, putting the PVDF membrane into 3% BSA, and blocking overnight at 4 ℃;

(3) The next day the membranes were removed and washed 4 times with 5mL of PBST for 10min each;

(4) Recombinant single domain antibody (prepared by the method of example 3) diluted with PBS to a final concentration of 1. Mu.g/mL was added as primary antibody and incubated at 37℃for 1h. The membrane was removed and washed 4 times with 5mL of PBST for 10min each;

(5) HRP-labeled Anti-His tag monoclonal antibody diluted with PBS 1:10000 was added as secondary antibody and incubated at 37℃for 1h. The membrane was removed and washed 4 times with 5mL of PBST for 10min each;

(6) Mixing the developing solution A and the developing solution B uniformly by 500 mu L (light-shielding operation), dripping the developing solution A and the developing solution B on a PVDF membrane, putting the membrane into a Western-blot imager, checking the result, and recording by photographing, wherein the result is shown in FIG. 4, and the anti-CD20 single domain heavy chain antibody prepared by the invention can be specifically combined with the CD20 extracellular region polypeptide as shown in FIG. 4.

Example 6: cell immunofluorescence detection of single domain antibody binding to cell surface CD20 protein

(1) Absolute ethyl alcohol is prepared in advance, and the cover glass is soaked for 5min and then is clamped out, and after the cover glass is naturally dried, the cover glass is placed into a 6-hole cell culture plate.

(2) Polylysine (0.01 mg/mL) was added to the 6-well plate, and after 10min, the liquid in the 6-well plate was aspirated and discarded, and the solution was dried in an ultra clean bench.

(3) The Raji cells after passage are added into a 6-hole cell culture plate, and then the cell culture plate is placed into a 37 ℃ and 5% carbon dioxide incubator for culture. Cells were grown to cover slips, washed 3 min/time with PBS, and repeated 3 times.

(4) 4% paraformaldehyde was added to the cell culture plate, incubated for 20min, rinsed 3 min/time with PBS, and repeated 3 times.

(5) 5% BSA was added to the cell culture plate and blocked for 1h. An experimental group, to which sdAb was added as primary antibody (100 μl/well at a concentration of 10 μg/mL), and a negative control group, to which PBS (100 μl/well) was added, were set, and incubated overnight at 4 ℃.

(6) The cells were rinsed 5 times with PBST for 3min each. Adding 100 mu L of secondary antibody into each well488-Conjugated 6 his, his-Tag Mouse Monoclonal antibody, operating in the dark), room temperature for 1h.

(7) The cells were rinsed 5 times with PBST for 3min each. The results of the sample were checked under a fluorescence inversion microscope and photographed for storage, and the results are shown in fig. 5, in which fig. 5, a non-fluorescent image of the negative control cells under ultraviolet light excitation is shown, and a fluorescent image of the cells of the experimental group sdAb group under ultraviolet light excitation is shown, which indicates that the anti-CD20 single domain heavy chain antibody prepared by the present invention can bind to the cell surface CD20 protein.

Example 7: preparation of anti-CD20 and CD3 bispecific Single domain antibodies

The Anti-CD20 sdabs required for this set of experiments were prepared by the phage antibody library technique of examples 1-3, and the corresponding gene sequences and amino acid sequences were obtained by sequencing of example 3; the nucleic acid sequence of the Anti-CD3 sdAb may be the nucleotide sequence shown in SEQ ID No. 13.

(1) Connecting anti-CD20 and CD3 single domain antibody gene fragments by connecting peptide (Linker) by utilizing SnapGene Viewer software to construct a CD20 sdAb-Linker-CD3 sdAb sequence, which is named as BsNb, the nucleotide sequence is shown as SEQ ID NO.11, and the antibody amino acid sequence of the gene is shown as SEQ ID NO. 10;

(2) The constructed BsNb gene sequence (CD 20 sdAb-linker-CD3 sdAb sequence) was sent to the division of biological engineering (Shanghai) for gene synthesis. Two restriction enzyme sites of Not I and Nco I are reserved at two ends of the synthesized gene, and the synthesized gene and pET-22b (+) are subjected to double enzyme digestion by the restriction enzymes Not I and Nco I;

(3) The T4 DNA ligase is connected overnight to obtain recombinant plasmid pET-22b/BsNb, and the result is shown in figure 6 after double enzyme digestion identification, and the result of enzyme digestion can be shown initially that the recombinant plasmid pET-22b/BsNb is successfully prepared;

(4) Converting recombinant plasmid pET-22b/BsNb into E.coli BL21 (DE 3) competent cells, inducing and expressing for 6 hours by using IPTG with a final concentration of 1mM, and performing SDS-PAGE analysis on the expression condition after ultrasonic disruption, wherein the result is shown in a graph in FIG. 7 (M: protein molecular weight marker, unit is kDa;1: uninduced pre-bacterial disruption crude extract; 2: induced bacterial disruption crude extract; 3: post-disruption supernatant; 4: post-disruption precipitation; 5-7: sample eluted by 3 steps of eluent);

(5) The target protein in inclusion bodies is purified by Ni-NTA agarose purification resin under the denaturation condition, and the target protein is dialyzed and renatured to obtain the double antibody protein with biological functions, which is named as Anti-CD20/CD3 BsNb.

Example 8: detecting the binding Activity of purified Anti-CD20/CD3BsNb to CD20 antigen

(1) Diluting the CD20 extracellular region polypeptide to a final concentration of 2.5 mug/mL, coating 100 mug/hole to a 96-hole ELISA plate, and performing overnight at 4 ℃;

(2) Standing at room temperature for 30min, discarding the liquid in the hole, adding 300 μl of PBST,3 min/time, and repeating the process for 5 times;

(3) Then adding 300 mu L of 3% BSA blocking solution, incubating for 2 hours at 37 ℃, and washing by PBST for 5 times;

(4) The purified Anti-CD20/CD3BsNb (prepared by the method of example 7) was gradient diluted from 30. Mu.g/mL to 5. Mu.g/mL as primary antibody, 100. Mu.L per well. A Negative control (Negative control) was E.coli disruption solution (1:1000) after pET-22b (+) empty vector transformation, and a blank control (PBS) was set. Incubation is carried out for 1h at 37 ℃, and PBST is washed for 5 times;

(5) HRP-labeled Anti-His tag (1:10000) was added, 100. Mu.L/well, 37℃for 1h, and PBST was washed 5 times;

(6) Then, 100. Mu.L/well of TMB was added thereto, the reaction was developed in a dark place for 10 minutes, and 50. Mu.L/well of a stop solution was added thereto to terminate the reaction. The result is judged by the reading of the enzyme labeling instrument OD450nm (positive is judged when the OD450nm value of the experimental group is 2.1 times of the average value of the negative control, otherwise, the result is judged as negative). As shown in fig. 8, it can be seen from fig. 8 that both Negative control (Negative control) and blank control (PBS) test results were Negative, the Anti-CD20/CD3BsNb of the experimental group could bind to the CD20 antigen, and the higher the binding capacity of the Anti-CD20/CD3BsNb to the CD20 antigen as the Anti-CD20/CD3BsNb concentration increased.

Example 9: binding of bispecific antibody molecules to CD 20-and CD 3-expressing cells

The invention adopts a lymphoma cell line Raji as a CD20 positive cell; freshly isolated healthy human lymphocytes were used as CD3 positive cells.

FITC was dissolved in DMSO according to FITC-Antibody conjugation Kit instructions and added to Anti-CD20/CD3BsNb (1.2 mg/ml) (prepared using the method of example 7), placed on a horizontal shaker, incubated in an ice bath in the dark for 1h, antibody FITC conjugate was aspirated and placed on the surface of the packing in the purification column, centrifuged at 3000rpm for 2min, and the labeled antibody was collected for use at 4 ℃.

In an ultra clean bench, peripheral blood of healthy volunteers is diluted by normal saline and then slowly added above an equal amount of human lymphocyte separation liquid; centrifugation conditions: 800g, room temperature, acceleration 3, deceleration 0, centrifuging for 30min; after centrifugation, sucking middle layer (white) mononuclear cells into a new 15ml centrifuge tube, adding PBS, and uniformly mixing; centrifuging at 1500rpm for 10min; removing supernatant, adding red blood cell lysate (for example, 2ml red blood cell lysate obtained from Soy Bioreagent company if the initial separation of fresh blood is 1 ml) with twice the initial blood volume for 3-5min, adding PBS, mixing, centrifuging at 1200rpm for 10min; removing supernatant, adding 10ml PBS, mixing, centrifuging at 1000rpm for 10min; cells were resuspended in 1640 cell culture medium containing 10% fetal bovine serum FBS and inoculated at 25cm ² In the flask, trypan blue staining was counted and the total human Peripheral Blood Mononuclear Cells (PBMCs) were recorded for use.

The binding activity of bispecific antibodies to Raji cells and PBMCs cells was examined using a flow cytometer.

(1) Raji cells and PBMCs cells were washed 2 times with 1 XPBS containing 1% BSA and split into Ep tubes of each group for use, ensuring that the total number of cells per tube was 1X 10 ⁶ A plurality of;

(2) Adding FITC-Anti-CD20/CD 3BsNb to the Raji cells, the PBMCs cells and the mixed group of the Raji cells and the PBMCs cells to a final concentration of 100 mug/mL, setting a negative control group, adding 100 mug PBS only to the negative control group, and incubating for 60min at 4 ℃ by rotating an ice bath;

(3) Cells were washed 3 times with PBS containing 1% bsa; cells were then resuspended with 200 μl PBS;

(4) Cells were loaded onto a BD flow cytometer and examined, as shown in FIG. 9, and it was seen from FIG. 9 that purified Anti-CD20/CD3BsNb specifically bound to Raji cell surface CD20 molecules and PBMCs cell surface CD3 molecules.

Example 10: anti-CD20/CD3BsNb mediated killing of tumor cells by PBMCs

Determining the cytotoxic effect of the Anti-CD20/CD3BsNb by using an LDH kit, wherein the target cells are Raji, the effector cells are PBMCs, and the concentration of the Anti-CD20/CD3BsNb is 0 mu g/mL to 40 mu g/mL;

(1) Raji cells were diluted to 1×10 per well with 1640 medium ⁴ Spreading the cells into PerkinElmer Cell Carrier, and culturing for 12h in each group of three compound holes;

(2) The following effects are achieved: target = 5:1 and effect: target = 10:1 adding PBMCs, adding Anti-CD20/CD3BsNb respectively to final concentration (40. Mu.g/mL, 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0. Mu.g/mL), setting up blank wells (i.e. 100. Mu.L 1640 culture medium), sample control wells (i.e. 50. Mu.L Raji cells+50. Mu.L PBMCs cells), maximum enzyme activity control wells (i.e. 50. Mu.L Raji cells+50. Mu.L PBMCs cells+10. Mu.L lysate) respectively, culturing at 37℃for 24h;

(3) Adding 10 mu L of lysate into the maximum release hole of the target cells 1h before incubation, fully mixing, centrifuging for 5min at 1200r/min, taking supernatant, and adding into a labeled new 96-well plate;

(4) Adding 50 mu L of LDH working solution into each hole, uniformly mixing, and incubating for 30min at room temperature in a dark place;

(5) The OD490 nm value was measured with a microplate reader, and the death rate (Killing rate) of Raji cells was calculated [ death rate (%) = (sample measurement well OD value-blank well OD value-sample control well OD value-blank well OD value)/(maximum enzyme activity control well OD value-blank well OD value) ×100% ]. As a result, as shown in FIG. 10, it can be seen from FIG. 10 that Anti-CD20/CD3BsNb can mediate the killing of tumor cells by PBMCs, and that Anti-CD20/CD3BsNb can mediate the killing of tumor cells by PBMCs with increasing Anti-CD20/CD3BsNb concentration.

Example 11: ELISA detection of the Effect of Anti-CD20/CD3BsNb on the secretion of cytokines by PBMCs cells

(1) Raji cells are spread on PerkinElmer Cell Carrier 96-well plates, 5000 cells are plated on each well, and incubated for 12 hours at 37 ℃;

(2) PBMCs cells were added at 5:1 and 10:1 target-to-target ratios, and wells without antibody were used as Control groups with final concentrations of 0 μg/mL, 5 μg/mL,10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, anti-CD20/CD3BsNb, respectively. Three compound holes are arranged in each group, and the culture is carried out for 24 hours;

(3) Absorbing the cell supernatant after co-incubation, and detecting secretion of the cytokine IFN-gamma by using an ELISA kit;

(4) Adding prepared samples and standard substances, wherein the samples are cell supernatant in each hole sucked in the step (3), and the standard substances are human IFN-gamma of ELISA kit boxes, so as to establish a standard curve and calculate the content of IFN-gamma in the samples after the test. Reacting for 90min at 37 ℃;

(5) Washing the plate for 2 times, adding biotinylated antibody working solution, and reacting at 37 ℃ for 60min;

(6) Washing the plate for 3 times, adding ABC working solution, and reacting for 30min at 37 ℃;

(7) Washing the plate for 5 times, adding TMB color developing solution, and reacting at 37 ℃;

(8) Adding TMB stopping solution;

(9) Measuring an OD450nm value by using an enzyme label instrument within 10min;

(10) And drawing a standard curve, and calculating the IFN-gamma content of the factor to be detected of the specimen. The experimental results are shown in FIG. 11, and the IFN-. Gamma.content of the Anti-CD20/CD3 BsNb-mediated T cell secretion of the present invention is significantly different from that of the control group at different target ratios.

The embodiment proves that the bispecific antibody of the invention can enhance the effect of specific killing of immune effector cells on tumor cells by targeting the immune effector cells to the tumor cells while maintaining the original biological activities of the anti-CD20 antibody and the anti-CD3 antibody. The bispecific antibody of the invention can be used for preparing therapeutic drugs for B lymphocyte-related diseases and tumor-related diseases which abnormally express CD20, wherein the tumor-related diseases are B cell malignant tumors positive to CD20 antigen.

The foregoing embodiments are merely illustrative of the technical solution of the present invention, and the present invention is not limited to the above-mentioned preferred embodiments, but the above-mentioned embodiments and the description are merely illustrative of the principles of the present invention, and any person can obtain other various forms of products without departing from the spirit and scope of the technical solution of the embodiments of the present invention, regardless of any modification or substitution of the shape or structure thereof.

Sequence listing

<110> university of inner Mongolia agriculture

<120> single domain antibody against human B lymphocyte surface antigen CD20 and use thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR1

<400> 1

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Pro

20 25

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR2

<400> 2

Met Asp Trp Trp Arg Gln Ala Ala Gly Lys Gln Arg Glu Trp Val Ser

1 5 10 15

Ser

<210> 3

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR3

<400> 3

Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Lys

1 5 10 15

Ala Lys Asp Thr Val Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Asn Cys

35

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR4

<400> 4

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 5

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR1

<400> 5

Gly Phe Thr Ser Asn Asn Cys Gly

1 5

<210> 6

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR2

<400> 6

Ile Ala Thr Asp Gly Ser Thr

1 5

<210> 7

<211> 20

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR3

<400> 7

Ala Met Arg Arg Trp Gly Trp Gly Cys Leu Gly Arg Leu Trp Gln Ser

1 5 10 15

Asp Phe Gly Tyr

20

<210> 8

<211> 135

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CD20 Single Domain antibody

<400> 8

Met Ala Gln Val Gln Leu Gln Met Ala Asp Val Gln Leu Val Glu Ser

1 5 10 15

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr

20 25 30

Ala Pro Gly Phe Thr Ser Asn Asn Cys Gly Met Asp Trp Trp Arg Gln

35 40 45

Ala Ala Gly Lys Gln Arg Glu Trp Val Ser Ser Ile Ala Thr Asp Gly

50 55 60

Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys

65 70 75 80

Asp Lys Ala Lys Asp Thr Val Phe Leu Gln Met Asn Ser Leu Lys Pro

85 90 95

Glu Asp Thr Ala Met Tyr Asn Cys Ala Met Arg Arg Trp Gly Trp Gly

100 105 110

Cys Leu Gly Arg Leu Trp Gln Ser Asp Phe Gly Tyr Trp Gly Gln Gly

115 120 125

Thr Gln Val Thr Val Ser Ser

130 135

<210> 9

<211> 405

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<230> CD20 Single Domain antibody

<400> 9

atggcccagg tgcagctgca gatggctgat gtccagctgg tggagtctgg gggaggcttg 60

gtgcagcctg gggggtctct gagactctcc tgcacagccc ctggattcac ctccaataac 120

tgcggcatgg actggtggcg ccaggctgca gggaagcagc gcgagtgggt ctcatctatt 180

gcaactgatg gtagcacaag ctatgcagac tccgtgaagg gccgattcac catctccaaa 240

gacaaagcca aggacacggt gtttctgcaa atgaacagcc tgaaacctga ggacactgcc 300

atgtacaact gtgcaatgcg gcggtgggga tggggatgtt tgggtcggct ttggcagagt 360

gactttggtt actggggcca ggggacccag gtcaccgtct cctca 405

<210> 10

<211> 277

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> bispecific single domain antibodies against CD20 and CD3

<400> 10

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

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Trp Leu Glu Trp Val Ser Asp Ile Ser Trp Asn Gly Gly Ser Thr

50 55 60

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Glu Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Ala Lys Met Gly Glu Gly Gly Trp Gly Ala

100 105 110

Asn Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala

130 135 140

Gln Val Gln Leu Gln Met Ala Asp Val Gln Leu Val Glu Ser Gly Gly

145 150 155 160

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Pro

165 170 175

Gly Phe Thr Ser Asn Asn Cys Gly Met Asp Trp Trp Arg Gln Ala Ala

180 185 190

Gly Lys Gln Arg Glu Trp Val Ser Ser Ile Ala Thr Asp Gly Ser Thr

195 200 205

Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Lys

210 215 220

Ala Lys Asp Thr Val Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

225 230 235 240

Thr Ala Met Tyr Asn Cys Ala Met Arg Arg Trp Gly Trp Gly Cys Leu

245 250 255

Gly Arg Leu Trp Gln Ser Asp Phe Gly Tyr Trp Gly Gln Gly Thr Gln

260 265 270

Val Thr Val Ser Ser

275

<210> 11

<211> 831

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<230> bispecific single domain antibodies against CD20 and CD3

<400> 11

gaggtgcagc tgctcgagga ggtgcagctg gtggagtctg ggggaggctt ggtgcagcct 60

ggggggtctc tgagactctc ctgtgcagcc tctggattca cttttgatga ttatggcatg 120

agctgggtcc gacaggctcc agggaagtgg ctggagtggg tctcagatat tagctggaat 180

ggtggtagca catactatgc agactccgtg aagggccggt tcaccatctc cagagacaac 240

gccgagaaca cgctgtatct gcaaatgaac agcctgaaac ctgacgacac ggccgtgtat 300

tactgtgcaa aaatgggtga agggggatgg ggtgcaaatg actactgggg ccaggggacc 360

caggtcaccg tctcctcctc aggtggaggc ggttctggcg gaggtggctc aggcggtgga 420

ggctcgatgg cccaggtgca gctgcagatg gctgatgtcc agctggtgga gtctggggga 480

ggcttggtgc agcctggggg gtctctgaga ctctcctgca cagcccctgg attcacctcc 540

aataactgcg gcatggactg gtggcgccag gctgcaggga agcagcgcga gtgggtctca 600

tctattgcaa ctgatggtag cacaagctat gcagactccg tgaagggccg attcaccatc 660

tccaaagaca aagccaagga cacggtgttt ctgcaaatga acagcctgaa acctgaggac 720

actgccatgt acaactgtgc aatgcggcgg tggggatggg gatgtttggg tcggctttgg 780

cagagtgact ttggttactg gggccagggg acccaggtca ccgtctcctc a 831

<210> 12

<211> 47

<212> PRT

<213> person (Homo sapiens)

<230> human CD20 extracellular region polypeptide

<400> 12

Lys Ile Ser His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala

1 5 10 15

His Thr Pro Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser

20 25 30

Glu Lys Asn Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser

35 40 45

<210> 13

<211> 378

<212> DNA

<213> filial generation of alpaca and llama (Vicugna pacos, lama glama)

<230> Anti-CD3 sdAb

<400> 13

gaggtgcagc tgctcgagga ggtgcagctg gtggagtctg ggggaggctt ggtgcagcct 60

ggggggtctc tgagactctc ctgtgcagcc tctggattca cttttgatga ttatggcatg 120

agctgggtcc gacaggctcc agggaagtgg ctggagtggg tctcagatat tagctggaat 180

ggtggtagca catactatgc agactccgtg aagggccggt tcaccatctc cagagacaac 240

gccgagaaca cgctgtatct gcaaatgaac agcctgaaac ctgacgacac ggccgtgtat 300

tactgtgcaa aaatgggtga agggggatgg ggtgcaaatg actactgggg ccaggggacc 360

caggtcaccg tctcctcc 378

Claims

1. A single domain antibody against human B lymphocyte surface antigen CD20, characterized in that the single domain antibody VHH chain comprises framework region FR and complementarity determining region CDR;

the framework region FR comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 is shown as SEQ ID NO.1, the amino acid sequence of FR2 is shown as SEQ ID NO.2, the amino acid sequence of FR3 is shown as SEQ ID NO.3, and the amino acid sequence of FR4 is shown as SEQ ID NO. 4;

the complementarity determining regions CDR comprise CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown as SEQ ID NO.5, the amino acid sequence of the CDR2 is shown as SEQ ID NO.6, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 7.

2. The single domain antibody of claim 1, wherein the amino acid sequence of the single domain antibody comprises the amino acid sequence set forth in SEQ ID No. 8.

3. A polypeptide, protein or bispecific antibody, characterized in that the amino acid sequence of the polypeptide, protein or bispecific antibody comprises the VHH chain amino acid sequence of the single domain antibody of claim 1 or 2.

4. A polypeptide, protein or bispecific antibody according to claim 3, characterized in that the amino acid sequence of the bispecific antibody comprises the amino acid sequence shown in SEQ ID No. 10.

5. A nucleic acid encoding one or more of the single domain antibody of claim 1 or 2, the polypeptide of claim 3 or 4, the protein of claim 3 or 4, and the bispecific antibody of claim 3 or 4.

6. A vector comprising the nucleic acid of claim 5.

7. A host cell comprising the vector of claim 6.

8. A pharmaceutical or pharmaceutical composition comprising one or a combination of several of the single domain antibody of claim 1 or 2, the polypeptide of claim 3 or 4, the protein of claim 3 or 4, the bispecific antibody of claim 3 or 4.

9. A reagent or kit comprising one or more of the single domain antibody of claim 1 or 2, the polypeptide of claim 3 or 4, the protein of claim 3 or 4, and the bispecific antibody of claim 3 or 4.

10. Use of one or more of the single domain antibody of claim 1 or 2, the polypeptide of claim 3 or 4, the protein of claim 3 or 4, the bispecific antibody of claim 3 or 4, the nucleic acid of claim 5, the vector of claim 6, the host cell of claim 7, the drug of claim 8, the pharmaceutical composition of claim 8, the agent of claim 9, the kit of claim 9 in (1) or (2);

(1) Preparing a reagent or a kit for detecting B cell lymphoma;

(2) Preparing a medicine or a medicine composition for treating malignant tumor diseases of the B lymphocyte system.