Single-domain antibody of anti-CD99 protein as well as coding gene and application of single-domain antibody

Abstract

The invention discloses an anti-CD99 protein single-domain antibody as well as a coding gene and application thereof. The single-domain antibody Nab99-30 of the anti-CD99 protein comprises three complementary determining regions CDR1, CDR2 and CDR3, and is characterized in that the amino acid sequences of the single-domain antibody Nab99-30 are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence; the amino acid sequence of the single-domain antibody Nab99-30 is as shown in SEQ ID NO.4, and the nucleotide sequence of the coding gene of the single-domain antibody Nab99-30 is as shown in SEQ ID NO.5. Compared with a common monoclonal antibody, the single-domain antibody for resisting the CD99 protein obtained through screening has the advantages of being small in size, good in stability, high in activity and the like, has high binding capacity with the CD99 protein, high in specificity and high in affinity, and can be applied to preparation of a reagent for detecting the CD99 protein or treatment of tumors with abnormal extracellular expression CD99.

Description

Technical Field

[0001] The present invention relates to single domain antibodies, in particular to single domain antibodies against CD99 protein and their encoding genes. The present invention further relates to their use in preparing drugs or reagents for treating or detecting Ewing's sarcoma, belonging to the field of single domain antibodies against CD99 protein and their application. Background Art

[0002] Ewing's sarcoma is a poorly differentiated malignant tumor of small round cells and the second most common bone tumor in children and adolescents. It accounts for 6% to 8% of all primary bone tumors. Its early symptoms are mainly pain and swelling, followed by neurological impairment. In the past 30 years, the diagnosis and treatment of localized disease have made significant progress, but there is still much room for improvement. Advances in its treatment have improved the survival rate of patients with localized tumors from about 10% before the introduction of chemotherapy, which has now increased to about 75%. However, it is still not effective for patients with tumor metastasis, and the treatment has short-term and long-term toxicity. For these patients, multidisciplinary care is essential, and the development of immunotherapy has brought new hope for the treatment of Ewing's sarcoma.

[0003] An Ewing sarcoma-specific chromosomal translocation fuses the EWS gene on chromosome 22 to a subset of ETS transcription factor family members, generating an aberrant transcription factor, EWS-FLI1, which induces CD99 expression either by direct binding to the CD99 gene promoter or by regulation of microRNAs (miRNAs). As with most developmental cancers, additional recurrent mutations are rare in Ewing sarcoma, suggesting that substantial changes in the Ewing sarcoma transcriptome occur not as DNA alterations but through fundamental epigenomic mechanisms. The EWS-ETS family of gene fusions and their downstream effects in Ewing sarcoma offer opportunities for novel therapeutic approaches. These include inhibition of the fusion gene or its protein product, as well as pathways involving IGF1 and mTOR. Inhibition of tyrosine kinases, interference with angiogenesis, and exploitation of non-apoptotic cell death are promising new approaches.

[0004] Growth differentiation factor 6 (GDF6) is a member of the bone morphogenetic protein (BMP) cytokine family, but Ewing sarcoma is dependent on the prodomain of GDF6, not the BMP domain. The prodomain of GDF6 is a ligand for CD99, and binding of the prodomain of GDF6 to the extracellular domain of CD99 leads to the recruitment of CSK (C-terminal Src kinase) to the YQKKK motif of the CD99 intracellular domain, inhibiting Src activity and maintaining the growth of Ewing sarcoma in an autocrine manner.

[0005] CD99 protein is a glycosylated protein located on the cell membrane, encoded by the gene mic2, with a molecular weight of 32kd. It is highly expressed in normal tissue cells such as thymic epithelial cells, pancreatic islet cells, ovarian granulosa cells and testicular supporting cells. It participates in many basic cell functions, including cell adhesion and migration, cell death and differentiation, intracellular protein transport, endocytosis and exocytosis. CD99 is abnormally expressed in tumors including osteosarcoma, hematological malignancies, malignant gliomas, breast cancer, epithelial cancer and pancreatic cancer. CD99 can be used as a potential therapeutic target for hematological malignancies, such as T-lineage acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Ewing sarcoma cells express high levels of CD99, which is necessary to maintain the malignancy of Ewing sarcoma independent of the oncogenic driver EWS-FLI 1. Ewing sarcoma cells that lack CD99 but still express EWS-FLI 1 show significantly inhibited growth, migration and metastasis, and tend to differentiate into the neural lineage. Targeting CD99 using specific antibodies severely reduced cell proliferation and migration in vitro and tumor growth and metastasis formation in vivo, making anti-CD99 antibodies a more viable therapeutic approach than anti-EWS-FLI 1 strategies.

[0006] Nanobodies, also known as single-domain antibodies, were first discovered by a research team led by Professor Raymond Hamers of Belgium to exist naturally in camel blood. They have strong antigen targeting and binding capabilities, and have many unique properties and advantages over ordinary antibodies. Their molecular weight is only one-tenth of that of ordinary antibodies. Nanobodies are considered to be the next generation of antibody-derived tools in antigen-related recognition and regulation. Currently, nanoantibody technology is becoming increasingly mature, and many nanoantibodies have been developed and evaluated at different stages of clinical trials for cancer treatment.

[0007] According to the current status of Ewing's sarcoma treatment, the development of new methods has become an urgent problem to be solved. Therefore, the application of nano-antibody technology platform to screen CD99 protein-specific single-domain antibodies will be of considerable value in the treatment of Ewing's sarcoma. Summary of the invention

[0008] One of the purposes of the present invention is to provide a single domain antibody against CD99 protein and a gene encoding the same;

[0009] The second object of the present invention is to couple the single domain antibody with one or more of an enzyme phase, a radioisotope, a fluorescent compound or a chemiluminescent compound to obtain a conjugate;

[0010] The third object of the present invention is to use the single domain antibody and conjugate to prepare drugs or reagents for treating or detecting Ewing's sarcoma;

[0011] The above object of the present invention is achieved through the following technical solutions:

[0012] The present invention first provides a single-domain antibody Nab99-30 against CD99 protein, comprising a framework region and three complementary determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3, respectively.

[0013] Furthermore, the amino acid sequence of the single-domain antibody Nab99-30 is selected from any one of (1)-(3):

[0014] (1) the amino acid sequence shown in SEQ ID No. 1;

[0015] (2) a protein mutant obtained by deleting, replacing, inserting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 1, wherein the protein mutant has the same function as the protein before mutation;

[0016] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID No. 1, preferably, an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID No. 1, and more preferably, an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID No. 1.

[0017] The present invention further provides the single domain antibody encoding gene.

[0018] As a preferred embodiment of the present invention, the nucleotide sequence of the gene encoding the single-domain antibody Nab99-30 is selected from any one of (1) to (3):

[0019] (1) the polynucleotide sequence shown in SEQ ID NO.5; or (2) a polynucleotide sequence that can hybridize with the complementary sequence of the polynucleotide sequence shown in SEQ ID NO.5 under stringent hybridization conditions; or (3) a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence shown in SEQ ID NO.5; preferably, a polynucleotide sequence that is at least 80% identical to the polynucleotide sequence shown in SEQ ID NO.5; further preferably, a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence shown in SEQ ID NO.5; more preferably, a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence shown in SEQ ID NO.5; most preferably, a polynucleotide sequence that is at least 99% identical to the polynucleotide sequence shown in SEQ ID NO.5.

[0020] The present invention further fuses the single-domain antibody Nab99-30 against CD99 protein with Fc to obtain an Fc fusion protein; wherein the Fc gene sequence can be an Fc gene sequence derived from IgG, IgA, IgM or any one of human IgG1, IgG2, IgG3 or IgG4 gene sequences.

[0021] The present invention further couples the single-domain antibody with one or more of an enzyme (such as horseradish peroxidase, alkaline phosphatase, etc.), a radioactive isotope, a fluorescent compound or a chemiluminescent compound to obtain a conjugate, and these conjugates can be used to prepare reagents for detecting Ewing's sarcoma or.

[0022] The present invention also provides an expression vector containing a gene encoding the single-domain antibody Nab99-30 and an expression vector containing a gene encoding an Fc fusion protein; the expression vector may be a prokaryotic expression vector, a eukaryotic expression vector or other expression vectors.

[0023] The present invention also discloses a recombinant host cell containing the expression vector, wherein the host cell is a prokaryotic expression cell, a eukaryotic expression cell, a fungal cell or a yeast cell, and the eukaryotic expression cell is preferably a CHO cell.

[0024] Compared with common monoclonal antibodies, the single-domain antibodies against CD99 protein screened by the present invention have the advantages of small size, good stability, high activity, etc., have strong binding ability with CD99 protein, strong specificity, and high affinity, and can be used to prepare reagents for detecting CD99 protein or treat tumors that abnormally express CD99 extracellularly.

[0025] Definitions of terms used in this invention

[0026] The term "single domain antibody (sdAb)" used in this article refers to a fragment containing a single variable domain of an antibody, also known as a nanobody. Like a complete antibody, it can selectively bind to a specific antigen. Compared with the mass of 150-160kDa of a complete antibody, a single domain antibody is much smaller, only about 12-15kDa. The first single domain antibody was artificially engineered from a camel's heavy chain antibody, called a "VHH segment."

[0027] The term "framework region" refers to the skeleton region. There are about 110 amino acid sequences near the N-terminus of the H and L chains of immunoglobulins, which vary greatly, while the amino acid sequences of other parts are relatively constant. Based on this, the light chain and heavy chain can be divided into variable regions (V) and constant regions (C). The variable region contains the hypervariable region HVR (hypervariable region) or complementary determining region CDR (Complementarity-determining region) and the FR skeleton region.

[0028] The term "amino acid sequence" refers to the order in which amino acids are linked to form a peptide chain (or polypeptide). An amino acid sequence can only be read in one direction. There are more than 100 different types of amino acids, 20 of which are commonly used. The present invention does not exclude the possibility that the amino acid chain is modified by other substances, such as sugars, lipids, etc., and the present invention is not limited to the 20 commonly used amino acids.

[0029] The term "nucleotide sequence" or "polynucleotide sequence" refers to the order of bases in DNA or RNA, i.e., the order of A, T, G, C in DNA, or the order of A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA, and mRNA. It should be understood that the antibody gene for which protection is sought in the present invention also covers RNA (rRNA, tRNA, mRNA) and their complementary sequences in addition to DNA sequences.

[0030] The substitution described in the present invention may be a conservative substitution, i.e., a specific amino acid residue is replaced by a residue having similar physical and chemical characteristics. Non-limiting examples of conservative substitutions include substitutions between amino acid residues containing aliphatic groups (e.g., mutual substitutions between Ile, Val, Leu or Ala), substitutions between polar residues (e.g., mutual substitutions between Lys and Arg, Glu and Asp, Gln and Asn), etc. Mutants resulting from the deletion, substitution, insertion and / or addition of amino acids can be prepared by subjecting DNA encoding wild-type proteins to site-directed mutagenesis, which is a well-known technique (see, e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, which is incorporated herein by reference in its entirety).

[0031] The term "identity" of a sequence can be used interchangeably with "sameness" and refers to the degree of similarity between sequences as determined by sequence alignment software such as BLAST. Methods and software for sequence alignment are well known to those skilled in the art. A modified nucleotide sequence can be obtained by substituting, deleting and / or adding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more) amino acids or bases to a known sequence. For example, by conventional means (e.g., conservative substitution, etc.), the amino acid or nucleotide sequence shown in one or more of the sequences SEQ ID NO: 1-198 of the present invention is modified to obtain a sequence identity greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% with these sequences, and having substantially the same properties, which are all within the scope of protection of the present invention. Preferably, the present invention obtains sequence identity by conservative substitution, but is not limited to conservative substitution.

[0032] The term "complementary" herein refers to two nucleotide sequences including antiparallel nucleotide sequences that can pair with each other after forming hydrogen bonds between the complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary chains are reverse complementary to each other when both sequences are viewed from the 5' to 3' direction. It is also known in the art that two sequences that can hybridize to each other under a given set of conditions do not necessarily have to be 100% completely complementary.

[0033] The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, under stringent conditions, the detectable degree of hybridization of a probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times greater than background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. By controlling the stringency of hybridization or washing conditions, a target sequence that is 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference may be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes," Overview of principles of hybridization and the strategy of nucleic acid assays, 1993). More specifically, the stringent conditions are usually selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a specified ionic strength and pH. Tm is the temperature (under specified ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in a state of equilibrium (because the target sequence is present in excess, 50% of the probes are occupied in a state of equilibrium at Tm). The stringent conditions may be the following conditions: wherein the pH is 7.0 to 8.3, the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salts), and the temperature is at least about 30°C for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The wash can be performed for 5, 15, 30, 60, 120 minutes or longer.

[0034] In this specification, "one or more amino acids" refers to the amino acids of the degree that can be deleted, substituted, inserted and / or added by the site-directed mutagenesis method, which is not limited, but preferably less than 20, less than 15, less than 10 or less than 7, more preferably less than 5. With regard to the site-directed mutagenesis method, for example, except for the desired variation, i.e., specific inconsistency, it is also possible to use a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated in the following manner. That is, the above-mentioned synthetic oligonucleotide is used as a primer to synthesize a chain complementary to the phage, and the obtained double-stranded DNA is used to transform the host cell. The culture of the transformed bacteria is spread on agar, and plaques are formed by a single cell containing the phage. Then, the plaques hybridized with the probe are collected, cultured, and DNA is recovered. Furthermore, methods for subjecting the amino acid sequence of a biologically active peptide such as an enzyme to deletion, substitution, insertion and / or addition of one or more amino acids while maintaining its activity include, in addition to the above-mentioned site-directed mutagenesis, methods for treating genes with a mutagenic source, and methods for selectively disconnecting genes, then deleting, substituting, inserting or adding selected nucleotides, and then connecting them.

[0035] The term "expression vector" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) to the basic skeleton of a cloning vector to enable the expression of a target gene. An expression vector has four parts: target gene, promoter, terminator, and marker gene. The present invention includes but is not limited to prokaryotic cell expression vectors, eukaryotic cell expression vectors, or other cell expression vectors.

[0036] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally known to those skilled in the art.

[0037] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the SDS-PAGE electrophoresis diagram of the anti-CD99 protein single domain antibody.

[0039] Figure 2 These are the activity test results of the purified anti-CD99 protein single domain antibody specifically binding to the CD99 protein antigen.

[0040] Figure 3 The results of affinity determination of single-domain antibody Nab99-30. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0042] Example 1 Design and construction of phage display immune alpaca nanoantibody gene library

[0043] (1) Isolation of alpaca peripheral blood lymphocytes and extraction and purification of RNA: Whole blood was collected from more than 5 alpacas, and alpaca peripheral blood lymphocytes were separated. Total RNA was extracted from the alpaca peripheral blood lymphocytes using an RNA extraction kit (QIAGEN).

[0044] (2) Nested PCR method to obtain the variable region of alpaca heavy chain antibody - VHH: In order to improve the specificity of amplification, the reverse transcription primer uses the specific primer of the heavy chain antibody, and the extracted total RNA is used to synthesize the first chain of cDNA. Using this template, the nested PCR method is used to PCR amplify the heavy chain antibody VHH gene fragment with two sets of primers. In the first PCR amplification, the fragments larger than 800 bp are ordinary heavy chain gene fragments, and the fragments between 800 and 500 bp are heavy chain antibody gene fragments lacking light chains. The light chain and heavy chain antibody gene fragments lacking light chains are cut and recovered from the gel. Using this as a template, VHH specific primers are used to amplify the VHH target gene (500 bp) by PCR.

[0045] (3) The VHH fragment and the phage display vector were then digested with restriction endonuclease Sfi I, respectively, and ligated with T4 ligase (NEB) in an appropriate ratio. The ligated plasmid was then electroporated into the TG1 competent cell.

[0046] (4) Identification and preservation of the capacity and diversity of the VHH antibody gene library: The library capacity was calculated by multiplying the total amount of transformation by the titer measured after transformation. 90 electroporation transformations were performed, and the library capacity was 9×10 10 ; 50 clones growing on the plate for titer determination after electroporation were randomly selected for PCR and sequencing identification. All 50 colony PCRs were positive, and the size of the amplified fragment was the same as the size of the inserted VHH; the sequencing results of the 50 clones showed that there were no repeated VHH sequences, and the library capacity and diversity of the antibody gene library met the design requirements.

[0047] Example 2 Screening and Sequencing of Single Domain Antibodies Against CD99

[0048] (1) Screening of CD99-specific single-domain antibodies

[0049] Immunotubes were coated with CD99 protein (homemade), and 25ug / ml, 15ug / ml and 10ug / ml antigens were used for three rounds of screening, respectively. The tubes were blocked with PBST (0.1% Tween-20) containing 4% skim milk powder, and the above-mentioned phage library was added for binding for a certain period of time, and non-specific binding phages were removed by washing, and the specifically bound phages were eluted with TEA, and then amplified for three rounds of screening.

[0050] Table 1 Affinity screening for enrichment of phage antibodies

[0051] Number of screening rounds Input phage titer Output phage titer 1 <![CDATA[2.0×10 12 ]]> <![CDATA[2.6×10 4 ]]> 2 <![CDATA[1.2×10 12 ]]> <![CDATA[5.1×10 6 <!-- 5 -->]]> 3 <![CDATA[1.0×10 12 ]]> <![CDATA[1.89×10 8 ]]>

[0052] (2) Enzyme-linked immunosorbent assay (ELISA) was used to measure the OD value of the culture supernatant of a single clone and screen for positive clones

[0053] A single colony was randomly picked from an agar plate with good growth and separation of a single colony, inoculated in a 96-well culture plate containing 2YT liquid medium containing Amp, cultured overnight, centrifuged, and the supernatant was separated. The CD99 protein was used as an antigen to coat a 96-well ELISA plate, and a phage ELISA assay was performed to select positive well clones for CD99, and DNA sequencing was performed to identify the gene sequence of the CD99-specific single domain antibody clone, wherein the amino acid sequences of the three complementary determining regions of the Nab99-30 single domain antibody are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, and its amino acid sequence is shown in SEQ ID No. 4, and the nucleotide sequence of the Nab99-30 single domain antibody gene is shown in SEQ ID No. 5. This example also screened another specific single domain antibody Nab99-38 against CD99 protein.

[0054] Example 3 Construction of the expression plasmid of the specific single domain antibody Nab99-30

[0055] The specific Nab99-30 single-domain antibody gene and the single-domain antibody Nab99-38 gene obtained in Example 2 were amplified by PCR to obtain PCR products with restriction endonucleases BbsI and BamHI sites. The PCR products and vector (pSJF2 vector) were treated with restriction endonucleases BbsI and BamHI, respectively, and recombined by T4 ligase to obtain plasmid Nab-pSJF2 that can be efficiently expressed in Escherichia coli, and the gene sequence was determined to determine the correctness of its sequence.

[0056] Example 4 Expression, purification and activity determination of specific CD99 single domain antibody Nab99-30

[0057] (I) Expression and purification of the specific CD99 single domain antibody Nab99-30

[0058] (1) Inoculate the bacterial strain containing plasmid Nab-pSJF2 described in Example 3 on a 2YT culture plate containing ampicillin at 37°C overnight. (2) Select a single colony and inoculate it in 2 ml of LB culture medium containing ampicillin and culture it in a shaking incubator at 37°C overnight. (3) Transfer the strain to 100 ml of 2YT culture medium containing ampicillin and culture it in a shaking incubator at 37°C at 220 rpm. When the OD value reaches 0.6-1.0, add 0.1-0.5 M IPTG and continue to culture overnight. Centrifuge at 5000 rpm for 20 minutes to harvest the bacteria. (5) Add 0.05 M Tris buffer to wash the bacteria twice, use hypertonic sucrose to extract the soluble single domain antibody expressed in the bacterial periplasm, and centrifuge to collect the soluble single domain antibody protein in the supernatant. (6) Single domain antibody protein with a purity of more than 90% was obtained by separation using Ni+ ion affinity chromatography magnetic beads (BeaverBeadsTM His-tag Protein Purification, Suzhou Beaver Biomedical Engineering Co., Ltd.). Figure 1 SDS-PAGE electrophoresis of Nab99-30-pSJF2 and Nab99-38-pSJF2 carry S and His6 tags, and the expressed protein molecule is about 13.7 kd.

[0059] (II) Activity determination of expressed single domain antibodies

[0060] 1. Experimental materials: ELISA plate (Thermofisher), CD99 antigen protein, coating solution pH 9.6, blocking solution, HRP Anti-S tag antibody (abcam, Cat: ab19324), TMB colorimetric solution (Beijing Mecowand, Cat: 1001), TMB stop solution (Beijing Mecowand, Cat: 1001SA).

[0061] 2. Experimental process:

[0062] 2.1 Coat the cells with CD99 protein at a concentration of 2ug / ml, 100ul / well, and incubate at 4°C overnight.

[0063] 2.2 Add 2% skim milk PBS to block, 300ul / well. Incubate at 37℃ for 1h.

[0064] 2.3 Dilute CD99 single domain antibodies with different numbers to final concentrations of 50.0ug / ml, 10.0ug / ml, 2ug / ml, 0.4ug / ml, 0.08ug / ml, 0.016ug / ml, 0.0032ug / ml, 0.00064ug / ml, 100ul / well.

[0065] 2.4 Dilute Anti-S tag antibody (HRP) (1:2000), 100ul / well, incubate at 37℃ for 1h.

[0066] 2.5 Add TMB colorimetric solution, 100ul / well, and react for 8 minutes in the dark.

[0067] 2.6 Add 50ul / well TMB stop solution to terminate the reaction.

[0068] 2.7 Measure the OD value at a wavelength of 450nm.

[0069] Figure 2 The figures are the experimental results of the activity of purified Nab99-30 single domain antibody and Nab99-38 single domain antibody in specific binding to CD99 antigen.

[0070] according to Figure 2 The experimental results show that Nab99-30 single domain antibody and Nab99-38 single domain antibody have high specific binding activity with CD99 antigen.

[0071] Experimental Example 1 Affinity determination experiment of specific CD99 single domain antibody Nab99-30

[0072] In this experiment, Octet-BLI was used to determine the affinity of the anti-CD99 single-domain antibody Nab99-30.

[0073] 1. Experimental Materials and Methods

[0074] 1. Experimental Materials

[0075] Biotin-labeled CD99 protein, anti-CD99 single domain antibody Nab99-30 (sample), PBST (PBS+0.02% Tween, pH 7.4), SA biosensor, black 96-well plate greiner, Regeneration buffer, 10Mm Gly-HCl, and an Octet instrument.

[0076] 2. Experimental methods

[0077] (1) Prepare 1000 nM, 500 nM, and 100 nM of anti-CD99 single domain antibody Nab99-30; prepare 10 mg / ml of biotin-labeled CD99 protein.

[0078] (2) Prepare and initialize the Octet device. Place the pre-wet plate in the blue base plate, then place the sensor on the green plate at the corresponding position of the pre-wet hole, then insert the green plate into the blue plate, and add 200uL PBST to the corresponding pre-wet plate. Place the sensor plate and sample plate in the instrument and close the instrument door. Set up the program for detection.

[0079] (3) The detection parameters of the molecular interaction instrument based on BLI technology were set as follows: Run the following programs: ① Baseline 60s, baseline step; ② Loading 180, sensor captures biotin-labeled CD99 protein; ③ Baseline 2180s, baseline step; ④ Association 300s, biotin-labeled CD99 protein on the sensor binds to CD99 single domain antibody Nab99-30, and the final binding signal of this step is detected; ⑤ Dissociation 600s, dissociation step. ⑥ Regeneration 30s, regeneration step.

[0080] (4) The contact conditions of CD99 single domain antibody Nab99-30 and biotin-labeled CD99 protein captured on the biosensor surface were: 30° C. for 10 min. Rotation speed: 1000 rpm; operating temperature: 30° C.; acquisition frequency: Standard kinetics (5.0 Hz, averaging by 20).

[0081] (5) The concentration of the biotin-labeled CD99 protein solution was 10 μg / mL, and 200 μL was added to each well of the Load array. The CD99 single domain antibody Nab99-30 was added to the analysis sample array wells at 1000 nM, 500 nM, and 100 nM, respectively.

[0082] (6) Start testing.

[0083] 2. Experimental Results

[0084] The results of the single domain antibody Nab99-30 affinity test are shown in Figure 3 According to the affinity experiment results, the affinity constant of the single domain antibody Nab99-30 is: K D =3.031×10 -8 M (30.31 nM).

Claims

1. A single domain antibody against CD99 protein, comprising a framework region and three complementary determining regions CDR1, CDR2, and CDR3, characterized in that: The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively.

2. The single domain antibody according to claim 1, characterized in that The amino acid sequence of the single-domain antibody Nab99-30 is selected from any one of the amino acid sequences (1) to (3): (1) the amino acid sequence shown in SEQ ID NO.4; or (2) a protein mutant obtained by deleting, replacing, inserting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.4, wherein the protein mutant has the same function as the protein before mutation; or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID No.4, preferably, an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID No.4, and more preferably, an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID No.

4.

3. A gene encoding the single domain antibody according to claim 1 or 2.

4. The coding gene according to claim 3, characterized in that The nucleotide sequence encoding the gene is selected from any one of (1) to (3): (1) the polynucleotide sequence shown in SEQ ID NO.5; or (2) a polynucleotide sequence that can hybridize with the complementary sequence of the polynucleotide sequence shown in SEQ ID NO.5 under stringent hybridization conditions; or (3) A polynucleotide sequence that is at least 75% identical to the polynucleotide sequence shown in SEQ ID NO.5; preferably, a polynucleotide sequence that is at least 80% identical to the polynucleotide sequence shown in SEQ ID NO.5; further preferably, a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence shown in SEQ ID NO.5; more preferably, a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence shown in SEQ ID NO.5; most preferably, a polynucleotide sequence that is at least 99% identical to the polynucleotide sequence shown in SEQ ID NO.

5.

5. A fusion protein obtained by fusing the single-domain antibody of claim 1 or 2 with an Fc protein, wherein the coding gene sequence of the Fc protein is derived from an Fc gene sequence of IgG, IgA, IgM or any one gene sequence selected from IgG1, IgG2, IgG3 or IgG4. The gene encoding the fusion protein of claim 5 .

7. A conjugate, characterized in that The single-domain antibody according to claim 1 or 2 is coupled to one or more of an enzyme, a radioisotope or a chemiluminescent compound to obtain a conjugate.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the coding gene according to claim 3 or the coding gene according to claim 6.

9. Use of the single domain antibody according to claim 1 or 2 in the preparation of a drug or reagent for treating or detecting a tumor that abnormally expresses CD99 extracellularly; preferably, the tumor that abnormally expresses CD99 extracellularly is Ewing's sarcoma.

10. Use of the encoding gene according to claim 3 or 6, the fusion protein according to claim 5 or the conjugate according to claim 7 in the preparation of a drug or reagent for treating or detecting a tumor that abnormally expresses CD99 extracellularly; preferably, the tumor that abnormally expresses CD99 extracellularly is Ewing's sarcoma.

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