Single-domain antibody against cd99 protein, encoding gene and application thereof

Abstract

The application discloses a single-domain antibody against CD99 protein and a coding gene and application thereof. The single-domain antibody Nab99-30 against CD99 protein comprises three complementarity determining regions CDR1, CDR2 and CDR3, characterized in that the amino acid sequences are sequentially shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; the amino acid sequence of the single-domain antibody Nab99-30 is shown in SEQ ID NO. 4, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 5. The single-domain antibody against CD99 protein screened by the application has the advantages of small volume, good stability, high activity and the like compared to common monoclonal antibodies, has strong binding capacity with CD99 protein, has high specificity and high affinity, and can be applied to preparation of a reagent for detecting CD99 protein or treatment of tumors with extracellular abnormal expression of CD99.

Description

TECHNICAL FIELD

[0001] The present application relates to single domain antibody, especially relates to the single domain antibody of anti-CD99 protein and its coding gene, and the application further relates to the application of the single domain antibody in the preparation of drugs or reagents for treating or detecting Ewing's sarcoma, and belongs to the single domain antibody of anti-CD99 protein and the application field thereof. BACKGROUND

[0002] Ewing's sarcoma is a small round cell poorly differentiated malignant tumor, and is the second most common bone tumor in children and adolescents, accounting for 6-8% of all primary bone tumors. The early symptoms mainly manifest as pain and swelling, followed by nerve function damage. In the past 30 years, remarkable progress has been made in the diagnosis and treatment of localized disease, but there is still much room for improvement. The progress in treatment has increased the survival rate of patients with localized tumors from about 10% before the introduction of chemotherapy to about 75%. However, the treatment is still ineffective for patients with tumor metastasis, and the therapy has short-term and long-term toxicity. Multidisciplinary care is essential for these patients, and the development of immunotherapy brings new hope for the treatment of Ewing's sarcoma.

[0003] Ewing's sarcoma-specific chromosomal translocation fuses the EWS gene on chromosome 22 to a subset of the ETS transcription factor family, producing an abnormal transcription factor, EWS-FLI1, which induces CD99 expression by directly binding to the CD99 gene promoter or by modulating microRNA (miRNA). Like most developmental cancers, additional recurrent mutations are rare in Ewing's sarcoma, which indicates that the substantial changes in the Ewing's sarcoma transcriptome do not occur through DNA alterations but through basic epigenomic mechanisms. The EWS-ETS gene fusion family and its downstream effects in Ewing's sarcoma provide opportunities for new therapeutic approaches. These include inhibition of the fusion gene or its protein product, as well as pathways related to 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's sarcoma depends on the prodomain of GDF6 rather than the BMP domain. The prodomain of GDF6 is a ligand for CD99, and the binding of the GDF6 prodomain to the extracellular domain of CD99 leads to the recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity and maintaining the growth of Ewing's sarcoma in an autocrine manner.

[0005] CD99 protein is a glycosylated protein encoded by the gene mic2, which is located on the cell membrane, with a molecular weight of 32 kd, and is mainly highly expressed in normal tissue cells such as thymic epidermal cells, pancreatic islet cells, ovarian granulosa cells and testicular supporting cells. It is involved 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 glioma 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 essential for maintaining Ewing sarcoma malignancy 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 towards the neural lineage. Targeting CD99 with specific antibodies can severely reduce cell proliferation and migration in vitro and tumor growth and metastasis formation in vivo, so anti-CD99 antibodies are a more feasible treatment than anti-EWS-FLI 1 strategies.

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

[0007] According to the current status of Ewing sarcoma treatment, developing new methods is a pressing problem. Therefore, using the nanobody technology platform to screen CD99 protein-specific single-domain antibodies will have considerable value for the treatment of Ewing sarcoma. SUMMARY

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

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

[0010] The third purpose of the present application is to apply the single-domain antibody and the conjugate to the preparation of a drug or reagent for treating or detecting Ewing sarcoma;

[0011] The above purposes of the present application are achieved by the following technical solutions:

[0012] The present application provides a single-domain antibody Nab99-30 against CD99 protein, comprising a framework region and three complementarity determining regions CDR1, CDR2 and CDR3, wherein 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.

[0013] Further, 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, substituting, inserting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 1, which has the same function as the protein before mutation;

[0016] (3) an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID No. 1, preferably an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID No. 1, more preferably an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 1.

[0017] The present application further provides a gene encoding the single-domain antibody.

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

[0019] (1) the polynucleotide sequence shown in SEQ ID No. 5; or (2) a polynucleotide sequence capable of hybridizing to the complement of the polynucleotide sequence shown in SEQ ID No. 5 under stringent hybridization conditions; or (3) a polynucleotide sequence having at least 75% identity with the polynucleotide sequence shown in SEQ ID No. 5; preferably a polynucleotide sequence having at least 80% identity with the polynucleotide sequence shown in SEQ ID No. 5; further preferably a polynucleotide sequence having at least 85% identity with the polynucleotide sequence shown in SEQ ID No. 5; more preferably a polynucleotide sequence having at least 95% identity with the polynucleotide sequence shown in SEQ ID No. 5; most preferably a polynucleotide sequence having at least 99% identity with the polynucleotide sequence shown in SEQ ID No. 5.

[0020] The application further fuses the anti-CD99 protein single-domain antibody Nab99-30 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 a human IgG1, IgG2, IgG3 or IgG4 gene sequence.

[0021] The application further couples the single-domain antibody with one or more of an enzyme (such as horseradish peroxidase, alkaline phosphatase, etc.), a radioisotope, a fluorescent compound or a chemiluminescent compound to obtain a conjugate, which can be used to prepare a reagent for detecting Ewing's sarcoma or.

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

[0023] The application 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] The anti-CD99 protein single-domain antibody screened by the application has the advantages of small size, good stability, high activity, strong binding capacity with CD99 protein, high specificity and high affinity, and can be applied to prepare a reagent for detecting CD99 protein or a treatment for tumors with abnormal extracellular expression of CD99.

[0025] Definitions of terms in which the invention is concerned

[0026] The term "single-domain antibody (sdAb)" as used herein 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 150-160 kDa of a complete antibody, a single-domain antibody is much smaller, about 12-15 kDa. The first single-domain antibody was artificially engineered from a heavy chain antibody of a camel, called "VHH segment".

[0027] The term "framework region" (FW), i.e., the skeleton region, is a region of about 110 amino acid sequences at the N-terminal of H and L chains of immunoglobulin, and the other part of the amino acid sequence is relatively constant, whereby the light chain and the heavy chain can be distinguished into variable region (V) and constant region (C). The variable region contains hypervariable region (HVR) or complementarity-determining region (CDR) and FR skeleton region.

[0028] The term "amino acid sequence" means the order of amino acids connected to each other to form a peptide chain (or polypeptide), and the amino acid sequence can be read in only one direction. There are more than 100 different types of amino acids, among which 20 are commonly used. The present application does not exclude other substances such as sugars and lipids from modifying the amino acid chain, and the present application is not limited to the 20 commonly used amino acids.

[0029] The term "nucleotide sequence" or "polynucleotide sequence" means the order of bases in DNA or RNA, i.e., the order of A, T, G, and C in DNA, or the order of A, U, G, and C in mRNA, and also includes the order of bases in rRNA, tRNA, and mRNA. It should be understood that the antibody-based claims of the present application encompass not only DNA sequences, but also RNA (rRNA, tRNA, mRNA) and their complementary sequences.

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

[0031] The term "identity" of a sequence can be used interchangeably with "homology" and refers to the extent to which sequences are similar as determined by sequence alignment software such as BLAST. Methods and software for sequence alignment are well known in the art. Engineered nucleotide sequences can be obtained by substitution, deletion, and / or addition of one or several (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, it is within the scope of the application to obtain a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity to one or more of the amino acid or nucleotide sequences set forth in SEQ ID NOS: 1-198, and having substantially the same properties, by making modifications to the sequences by routine means (e.g., conservative substitutions, etc.). Preferably, the sequence identity is obtained by conservative substitutions, but is not limited to conservative substitutions.

[0032] The term "complementary" refers herein to two nucleotide sequences comprising antiparallel nucleotide sequences that are capable of pairing with each other upon formation of 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 strands are reverse complements of each other when both sequences are viewed in the 5' to 3' direction. It is also known in the art that two sequences that are capable of hybridizing to each other under a given set of conditions do not necessarily have to be 100% perfectly complementary.

[0033] The term "stringent hybridization conditions" means conditions known in the art that are low in ionic strength and high in temperature. Typically, under stringent conditions, a detectable degree of hybridization of a probe to its target sequence is higher (e.g., at least 2-fold over background) than to other sequences. Stringent hybridization conditions are sequence dependent, and will be different in varying environmental conditions, with longer sequences hybridizing specifically at higher temperatures. A target sequence 100% complementary to a probe can be identified by controlling stringency or wash conditions of hybridization. For a detailed discussion of nucleic acid hybridization, see 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 typically chosen to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequences at the specified ionic strength pH. Tm is the temperature (under a defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes hybridize to their targets at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 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 with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least twice the background hybridization, and more preferably 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 x SSC and 1% SDS, at 42°C, or 5 x SSC, 1% SDS at 65°C, with wash in 0.2 x SSC and 0.1% SDS at 65°C. The washes can be for 5, 15, 30, 60, 120 minutes or more.

[0034] In the present specification, "one or more amino acids" means the extent of deletion, substitution, insertion and / or addition of amino acids by site-directed mutagenesis method, and is not limited, but preferably 20 or less, 15 or less, 10 or less, or 7 or less, more preferably 5 or less. In the case of site-directed mutagenesis method, for example, in addition to the desired variation, i.e., a specific mismatch, a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated can be used as follows. That is, a strand complementary to the phage is synthesized using the above synthetic oligonucleotide as a primer, and the resulting double-stranded DNA is used to transform host cells. The culture of the transformed bacteria is spread on agar, and plaques are formed from single cells containing phage. Then, the plaques hybridized with the probe are collected, cultured, and the DNA is recovered. Furthermore, in addition to the above-described site-directed mutagenesis, there are methods of treating the gene with a mutagen source while maintaining its activity, and methods of selectively cleaving the gene, and then deleting, substituting, inserting, or adding selected nucleotides, and then ligating.

[0035] The term "expression vectors" refers to vectors that have expression elements (e.g., promoters, RBS, terminators, etc.) added to the basic backbone of a cloning vector, enabling the expression of a gene of interest. An expression vector has four parts: a gene of interest, a promoter, a terminator, and a marker gene. The present application 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 usual meaning herein, referring to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and have the same meaning as is generally known by those skilled in the art.

[0037] The term "host cell" or "recombinant host cell" means a cell which contains a polynucleotide of the present application, regardless of the methodology used to introduce the polynucleotide into the host cell, such as direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide can remain, for example, as a non-integrated vector, such as a plasmid, or can integrate into the host genome. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 SDS-PAGE electrophoretogram of anti-CD99 protein single-domain antibody.

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

[0040] Figure 3 Results of affinity determination of single-domain antibody Nab99-30. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0042] Example 1: Design and construction of a phage-display-immunized alpaca nanobody gene library

[0043] (1) Isolation of alpaca peripheral blood lymphocytes and extraction and purification of RNA: collect whole blood from more than 5 alpacas, isolate alpaca peripheral blood lymphocytes, and extract total RNA from alpaca peripheral blood lymphocytes using an RNA extraction kit (QIAGEN).

[0044] (2) Obtaining the variable region-VHH of alpaca heavy chain antibody using nested PCR: To improve amplification specificity, specific primers for heavy chain antibodies were used for reverse transcription. The first strand of cDNA was synthesized using extracted total RNA. Using this template, nested PCR was performed with two sets of primers to amplify the VHH gene fragment of the heavy chain antibody. In the first PCR amplification, the fragment larger than 800 bp was the ordinary heavy chain gene fragment, and the fragment between 800 and 500 bp was the heavy chain antibody gene fragment lacking the light chain. The heavy chain antibody gene fragment lacking the light chain was recovered by gel excision and used as a template to amplify the VHH target gene (500 bp) using VHH-specific primers.

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

[0046] (4) Identification and preservation of the VHH antibody gene library's capacity and diversity: The library capacity was calculated by multiplying the total amount of transformations by the titer measured after transformation. Ninety electrotransformations were performed, resulting in a library capacity of 9 × 10⁻⁶. 10 Fifty clones grown on plates after electroporation and titer determination were randomly selected and identified by PCR and sequencing. All 50 colonies were positive by PCR, and the size of the amplified fragment was the same as the size of the inserted VHH. Sequencing results of the 50 clones showed that there were no duplicate VHH sequences. 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 single-domain antibody specific to CD99

[0049] The immunotubes were coated with CD99 protein (self-made), and 25 ug / ml, 15 ug / ml and 10 ug / ml antigens were used for coating in 3 rounds of screening. After blocking with PBST (0.1% Tween-20) containing 4% skim milk, the phage library was added and combined for a certain time. After washing to remove the non-specifically combined phages, the specifically combined phages were eluted with TEA, and then amplified and subjected to 3 rounds of screening.

[0050] Table 1 Enrichment effect of phage antibody by affinity screening

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

[0052] (2) Determination of OD value of supernatant of single clone culture by enzyme-linked immunosorbent assay (ELISA) for positive clone screening

[0053] The single colonies were randomly picked from the agar plate with good growth and separation, inoculated in 2YT liquid medium containing Amp in a 96-well plate and cultured overnight, centrifuged, and the supernatant was separated. The 96-well ELISA plate was coated with CD99 protein as antigen, and phage ELISA assay was performed. The positive well clones against CD99 were selected, and the gene sequence of the specific single-domain antibody clone against CD99 was identified by DNA sequencing. The amino acid sequences of the three complementarity 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 the amino acid sequence is shown in SEQ ID No. 4. The nucleotide sequence of the Nab99-30 single-domain antibody gene is shown in SEQ ID No. 5. Another specific single-domain antibody Nab99-38 against CD99 protein was also screened in this embodiment.

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

[0055] The specific Nab99-30 single-domain antibody gene obtained in Example 2 and the single-domain antibody Nab99-38 gene were amplified by PCR to obtain a PCR product with restriction endonuclease BbsI and BamHI sites. The PCR product and the vector (pSJF2 vector) were treated with restriction endonuclease BbsI and BamHI, respectively, and then linked by T4 ligase to obtain a plasmid Nab-pSJF2 that can be highly expressed in E. coli. The sequence of the plasmid was determined to confirm its correctness.

[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 onto a 2YT culture plate containing ampicillin and incubate overnight at 37°C. (2) Select a single colony and inoculate it into 2 ml of LB culture medium containing ampicillin and incubate overnight at 37°C on a shaker. (3) Transfer the colony to 100 ml of 2YT culture medium containing ampicillin and incubate at 37°C on a shaker at 220 rpm. When the OD value reaches 0.6-1.0, add 0.1-0.5 M IPTG and continue incubating overnight. Centrifuge at 5000 rpm for 20 minutes to collect the bacteria. (5) Wash the bacterial cells twice with 0.05 M Tris buffer, extract the soluble single-domain antibody expressed in the bacterial pericyte with hypertonic sucrose, and centrifuge to collect the soluble single-domain antibody protein from the supernatant. (6) Single-domain antibody proteins with a purity of over 90% were obtained by Ni+ ion affinity chromatography using magnetic beads (BeaverBeads™ His-tag Protein Purification, Suzhou Beaver Biomedical Engineering Co., Ltd.). Results are shown below. Figure 1 SDS-PAGE electrophoresis image. Nab99-30-pSJF2 and Nab99-38-pSJF2 are tagged with S and His6, and their expressed protein molecules are approximately 13.7 kDa.

[0059] (ii) Assay of the activity of expressed single-domain antibodies

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

[0061] 2. Experimental procedure:

[0062] 2.1 Coat CD99 protein separately at a concentration of 2ug / ml, 100ul / well, and incubate overnight at 4℃.

[0063] 2.2 Add 2% skim milk PBS for blocking, 300 μL / well. Incubate at 37°C for 1 hour.

[0064] 2.3 Dilute the CD99 single-domain antibodies with different numbers to a final concentration of 50.0 ug / ml, 10.0 ug / ml, 2 ug / ml, 0.4 ug / ml, 0.08 ug / ml, 0.016 ug / ml, 0.0032 ug / ml, and 0.00064 ug / ml, 100 μl / well.

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

[0066] 2.5 Add TMB color developing solution, 100ul / well, avoid light, 8min.

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

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

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

[0070] According to the experimental results Figure 2 It can be seen that the Nab99-30 single domain antibody and the Nab99-38 single domain antibody have high specific binding activity to the CD99 antigen.

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

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

[0073] I. Experimental materials and experimental 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, Octet instrument.

[0076] 2. Experimental method

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

[0078] (2) Octet instrument is prepared and initialized, put the pre-wet plate into the blue base plate, then put the sensor into the green plate corresponding to the pre-wet hole, then insert the green plate into the blue plate, and the corresponding pre-wet plate is added with 200uL PBST. Put the sensor plate and sample plate into the instrument, close the instrument door. Set the program for detection.

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

[0080] (4) The contact conditions of CD99 single domain antibody Nab99-30 contacting with biotin-labeled CD99 protein captured on the surface of the biosensor are as follows: 30℃ for 10min. Rotation speed: 1000rpm; running temperature: 30℃; acquisition frequency: Standard kinetics (5.0Hz, averaging by 20).

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

[0082] (6) Start detection.

[0083] II. Experimental results

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

Claims

1. A single-domain antibody against CD99 protein, comprising a framework region and three complementarity-determining regions CDR1, CDR2, and CDR3, characterized in that, 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.

2. The single-domain antibody according to claim 1, characterized in that, The amino acid sequence of the single-domain antibody is selected from either (1) or (2): (1) The amino acid sequence shown in SEQ ID NO.4; or (2) a protein mutant obtained by deleting, substituting, inserting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.4, the protein mutant having the same function as the original protein.

3. The single-domain antibody according to claim 1, characterized in that, The amino acid sequence of the single-domain antibody is an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID No.

4.

4. The encoding gene of the single-domain antibody as described in claim 1 or 2.

5. The encoding gene according to claim 4, characterized in that, The nucleotide sequence of the encoded gene is the polynucleotide sequence shown in SEQ ID NO.

5.

6. The encoding gene according to claim 4, characterized in that, The nucleotide sequence encoding the gene is a polynucleotide sequence that has at least 75% identity with the polynucleotide sequence shown in SEQ ID NO.

5.

7. A fusion protein obtained by fusing the single-domain antibody of claim 1 or 2 with the Fc protein, wherein, The gene sequence encoding the Fc protein is derived from the Fc gene sequence of IgG, IgA, or IgM.

8. The fusion protein according to claim 7, characterized in that, The IgG gene sequence is selected from any one of the gene sequences IgG1, IgG2, IgG3 or IgG4.

9. The gene encoding the fusion protein of claim 7.

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

11. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the coding gene of claim 4 or the coding gene of claim 9.

12. The use of the single-domain antibody according to claim 1 or 2 in the preparation of a reagent for detecting tumors with abnormal extracellular expression of CD99; wherein, The tumor that abnormally expresses CD99 in the extracellular space is Ewing's sarcoma.

13. Use of the encoding gene of claim 4 or 9, the fusion protein of claim 7, or the conjugate of claim 10 in the preparation of a reagent for detecting tumors with abnormal extracellular expression of CD99; wherein, The tumor that abnormally expresses CD99 in the extracellular space is Ewing's sarcoma.

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