CN115286715B - anti-CD3 nano antibody or antigen binding portion thereof and preparation method thereof - Google Patents

Abstract

The invention discloses an anti-CD3 nano antibody or an antigen binding portion thereof and a preparation method thereof, wherein the antibody comprises complementarity determining regions CDR1-CDR3 in a heavy chain variable region shown in SEQ ID NO. 1. Firstly, constructing a CD3 antigen to immunize alpaca for four times to obtain alpaca PBMC cells; secondly, screening and separating PBMC cells by a microfluidic technology; capturing RNA of a single cell, obtaining an antibody gene fragment by a nested PCR method, and constructing a eukaryotic expression vector; then inducing high-flux expression of the antibody by a mammalian cell high-flux expression system; finally, the antibody with high sensitivity and specificity is obtained through ELISA and FACS detection and sequencing result analysis. The antibodies are useful in the treatment of tumors and hyperproliferative disorders.

Description

anti-CD3 nano antibody or antigen binding portion thereof and preparation method thereof

Technical Field

The invention belongs to the fields of cell immunology and molecular biology, and relates to an anti-CD3 antibody and application thereof.

Background

CD3 molecules are important differentiation antigens on T cell membranes and are characteristic markers for mature T cells. It forms a complex with the T cell surface receptor TCR and plays an important role in intracellular signal transduction. CD3 antibodies specifically recognize T cell surface CD3 molecules, causing cross-linking of the T cell TCR-CD3 complex, directly producing an activation signal, resulting in T cell activation and proliferation. Current research indicates that CD3 antibodies play an important role in tumor therapy. However, antibody drug applications have a number of problems, such as long development cycles and excessive production costs; difficult to mass produce; the stability is poor, the degradation is easy, and the storage cost is high; the pollution is easy, and the maintenance cost is high; and has immunogenicity and the like, thereby limiting the application range in clinic. Nanobody technology is an antibody engineering revolution by biomedical scientists based on traditional antibodies by combining molecular biology technology with the concept of nanoscience, so that the latest and smallest antibody molecules are developed.

Hamers et al, 1993, reported that the alpaca immune system produced two types of antibodies when detecting foreign invaders such as bacteria and viruses: the other is equivalent to one tenth of the size of a normal antibody, and these smaller antibodies are called single domain antibodies or nanobodies (i.e., light chain-deleted "heavy chain antibodies"), which have only a small fragment of the light chain deleted heavy chain antibody to bind antigen as normal IgG and the like, and have high specific strong affinity. Nanobodies have a fully functional, minimal antigen-binding fragment with an oval crystal structure, 2.5nm in diameter and 4nm in length. Nanobodies are much simpler in chemical composition and shape than antibodies, are not chemically hydrophobic, are more resistant to heat and acid and alkali, are more easily combined with each other or other compounds, can be encoded by a single gene, and are easily synthesized by microorganisms. The nano antibody has good tolerance to environment, high conformational stability, smaller molecular mass and better clinical treatment effect, and simultaneously, the small protein molecules are easier to synthesize and lower in price. The unique property of the nano antibody makes the nano antibody show wider application prospect in the aspects of accurate diagnosis of diseases, immune targeting treatment and the like.

Disclosure of Invention

The invention aims to provide an anti-CD3 nano antibody which has short research and development period, can be produced in a large scale, has strong stability and is not easy to degrade, and a development method thereof.

The invention adopts the following technical scheme:

in one aspect, the invention provides an anti-CD3 antibody or antigen binding portion thereof, which comprises complementarity determining regions CDR1-CDR3 in a heavy chain variable region shown in SEQ ID NO.1, wherein the amino acid sequence of CDR1 is amino acids 31-35 of SEQ ID NO.1 in a sequence table; the amino acid sequence of the CDR2 is amino acids 49-55 of SEQ ID No.1 in a sequence table; the amino acid sequence of the CDR3 is the 99 th to 105 th amino acid of SEQ ID No.1 in a sequence table.

Further, the antibody or antigen binding portion thereof further comprises framework regions FR1-FR4 in a heavy chain variable region shown in SEQ ID NO.1, wherein the amino acid sequence of the FR1 is amino acids 1-30 of SEQ ID NO.1 in the sequence Listing; the amino acid sequence of the FR2 is 36 th-49 th amino acid of SEQ ID No.1 in a sequence table; the amino acid sequence of the FR3 is amino acids 67-98 of SEQ ID No.1 in a sequence table; the amino acid sequence of FR4 is amino acids 106-116 of SEQ ID No.1 in the sequence table.

Further, the antibody is a nano antibody, and the amino acid sequence of the nano antibody is shown as SEQ ID No.1 in a sequence table.

In another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or antigen binding portion thereof as hereinbefore described, the nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 2.

Once the DNA fragments encoding the VH segments are obtained, these DNA fragments are further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, or antigen-binding portion DNA fragments. In these manipulations, a DNA fragment encoding a VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or flexible linker. The term "operably linked" as used herein is intended to mean that two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame (in-frame).

The isolated DNA encoding the VH region may be converted to a full length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions (CH 1, CH2, and CH 3). The sequence of a human heavy chain constant region gene is known in the art.

In a further aspect the invention provides a vector of the nucleic acid molecule as hereinbefore described.

Vectors useful in the present invention include plasmids, expression vectors, cloning vectors, viral vectors, and the like. Various vectors known in the art may be used. For example, expression vectors can be formed by selecting commercially available vectors and then operably linking the nucleotide sequences encoding the antibodies of the invention to expression control sequences.

In a further aspect the invention provides a host cell of a nucleic acid molecule as hereinbefore described or of a vector as hereinbefore described.

Host cells useful in the present invention include prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, bacillus subtilis, and the like. Host cells for expressing the antibodies include E.coli, yeast cells, insect cells, COS cells, CHO cells, and the like. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell, most preferably a HEK-293T cell or a CHO cell.

In another aspect, the invention provides a method of preparing an anti-CD3 antibody, or antigen binding portion thereof, comprising any one of the following:

1) A method of preparing an anti-CD3 antibody, or antigen-binding portion thereof, by transfecting the vector described previously into a host cell, culturing the host cell, and isolating or recovering the anti-CD3 antibody.

Further, the host cell is a mammalian cell.

2) A method for developing an anti-CD3 antibody or antigen-binding portion thereof based on a SCMES (monoclonal mammalian cell expression screening system) platform, the method comprising the steps of:

a) After the CD3 antigen is used for immunizing alpaca, obtaining alpaca PBMC cells;

b) Sorting the PBMC cells by a droplet microfluidic technology to obtain functional antibody secreting cells capable of binding CD3 antigen;

c) Capturing RNA of a functional antibody secretion cell which is obtained by sorting and can be combined with a CD3 antigen, obtaining an antibody gene fragment by a PCR method, and constructing a eukaryotic expression vector;

d) Inducing high-throughput expression of anti-CD3 antibodies by a mammalian cell high-throughput expression system;

e) The anti-CD3 antibody is obtained by an antibody detection technology and sequencing result analysis.

Further, the PCR method is a nested PCR method.

Further, the antibody detection technique is ELISA or FACS detection.

In another aspect, the invention provides a product for detecting CD3, the product comprising an antibody or antigen-binding portion thereof as hereinbefore described.

Such functional linkages include, for example, linkages by chemical coupling, gene fusion, non-covalent binding, or other means.

Further, the product may be a conjugate or conjugate comprising:

1) An antibody or antigen-binding portion thereof as described above;

2) A diagnostic agent conjugated or conjugated to an antibody or antigen binding portion thereof as described above;

further, the diagnostic agent includes radionuclides, contrast agents, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, photosensitive diagnostic agents.

Paramagnetic ions useful in the present invention include: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

Fluorescent agents useful in the present invention include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.

Chemiluminescent agents useful in the present invention include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalic esters.

Bioluminescent agents useful in the present invention include luciferin, luciferase and aequorin.

Enzymes useful in the present invention include horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-galactosidase, urease, catalase, or glucoamylase.

Specific examples of such products include kits. The kit also comprises a solid phase carrier, and the antibody is fixed on the solid phase carrier (such as a porous plate, a cover glass and microbeads) or exists in a free mode.

In another aspect, the present invention provides a pharmaceutical composition, characterized in that it comprises any one of the following:

1) An antibody or antigen-binding portion thereof as described above;

2) A conjugate or conjugate, the conjugate or conjugate comprising:

a) An antibody or antigen-binding portion thereof as described above;

b) A therapeutic agent conjugated or conjugated to an antibody or antigen binding portion thereof as hereinbefore described.

The therapeutic agent comprises cytotoxin, medicine and radionuclide.

Cytotoxins or cytotoxic agents include any agent that is detrimental to cells (e.g., kills cells). Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine (decacabazine), alkylating agents (e.g., mechlorethamine, thioepa), chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide (cyclosphamide), busulfan, dibromomannitol, streptavidin, mitomycin C, and cis-dichlorodiamplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin D (formerly actinomycin), bleomycin, mithramycin, and anthranilic), and antimitotics (e.g., neomycin and the bases) duromycin, calicheamicin, and amastatin, and derivatives thereof.

Cytotoxins may be conjugated to antibodies of the invention using linker technology known in the art. Examples of types of linkers that have been used to couple cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers.

The antibodies of the invention may also be conjugated to a radioisotope to produce a cytotoxic radiopharmaceutical. Examples of radioisotopes that may be conjugated to antibodies for treatment include, but are not limited to, iodine¹³¹ Indium (indium)¹¹¹ Yttrium⁹⁰ And lutetium¹⁷⁷ 。

Radionuclides useful in the present invention include¹¹⁰ In、¹¹¹ In、¹⁷⁷ Lu、¹⁸ F、⁵² Fe、⁶² Cu、⁶⁴ Cu、⁶⁷ Cu、⁶⁷ Ga、⁶⁸ Ga、⁸⁶ Y、⁹⁰ Y、⁸⁹ Zr、^94m Tc、⁹⁴ Tc、^99m Tc、¹²⁰ I、¹²³ I、¹²⁴ I、¹²⁵ I、¹³¹ I、^154-158 Gd、³² F、¹¹ C、¹³ N、¹⁵ O、¹⁸⁶ Re、¹⁸⁸ Re、⁵¹ Mn、^52m Mn、⁵⁵ Co、⁷² As、⁷⁵ Br、⁷⁶ Br、^82m Rb、⁸³ Sr or other gamma emitter, beta emitter or positron emitter.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, physiologically compatible carriers. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (antibody or antigen binding portion thereof, conjugate/conjugate or bispecific molecule) may be coated in a substance to protect the active ingredient from acids and other natural conditions that may inactivate the active ingredient.

In another aspect the present invention provides a method for detecting the presence of CD3 in a sample for non-therapeutic purposes, said method comprising the steps of: the antibody or antigen-binding portion thereof described above is used as a detection antibody for CD3, and the presence of CD3 in a sample to be tested is detected by an antigen-antibody reaction.

The CD3 sequence is full length or two-segment active ingredient from public databases: asp 23-Asp 126 (CD 3E): access P07766-1 (CD 3E); phe 22-Ala 105 (CD 3D): access P04234-1 (CD 3D).

The method comprises the steps of detecting the reaction of antigen and antibody by using an enzyme-linked immunosorbent method, an immunofluorescence detection method, a radioimmunoassay, a luminescent immunoassay method, a colloidal gold immunochromatography method, an agglutination method and an immunoturbidimetry method.

In another aspect the invention provides an application comprising an application as described in any one of the following:

1) Use of the antibody or antigen binding portion thereof as described above in combination with a diagnostic agent as described above or a conjugate thereof for the preparation of a CD3 assay product;

2) Use of an antibody or antigen binding portion thereof as described hereinbefore, a conjugate or conjugate of a therapeutic agent as described hereinbefore, in the manufacture of a medicament for the treatment of a CD3 mediated disease;

CD3 mediated diseases as described herein include hyperproliferative diseases, particularly cancers of any tissue or organ, particularly for the treatment of cancers of the head, neck, breast, liver, skin, stomach, bladder, kidney, esophagus, gynaecology, bronchi, nasopharynx, thyroid, prostate, colorectal, ovary, pancreas, lung and fibrosarcoma.

The nucleotide sequence of the nanobody of the invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation methods. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the nanobody of the invention are derived from the nucleotide sequence of the invention and are equivalent to the sequence of the invention as long as the nanobody is encoded.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence encoding the protein shown in SEQ ID No.1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

The 75% or more identity may be 75%, 80%, 85%, 90% or more than 95% identity.

In the present application, the term "nanobody" refers to a single domain antibody obtained by cloning its variable region from a heavy chain antibody (e.g., derived from camelid body) lacking a light chain, which is the smallest functional antigen binding fragment, and has a relative molecular mass (Mr) of only about 15000. The nano antibody has the characteristics of small molecular mass, strong stability, good solubility, easy expression, low immunogenicity and the like.

In the present application, the term "antigen binding portion" generally refers to an immunoglobulin or polypeptide fragment of an antibody that competes with an antigen or with an intact antibody that binds to an antigen (i.e., specifically binds to) that they are derived from. The antigen binding fragments may include, but are not limited to: fab, fab ', F (ab') 2, and Fv fragments, linear antibodies, single chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

Drawings

FIG. 1 shows a graph of the results of the antiserum titers after 4 days;

FIG. 2 shows the results of nested PCR of cDNA fragments of anti-CD3 antibody, wherein A is the target band pattern obtained by the first PCR and B is the target band pattern obtained by the second PCR;

FIG. 3 shows a graph of monoclonal ELISA results;

FIG. 4 shows a graph of FACS positive antibody detection results;

figure 5 shows a graph of anti-CD3 binding experiments performed on FACS positive antibodies after expression purification.

Detailed Description

The present invention will be described with reference to the following specific examples, but the present invention is not limited thereto.

Reagents, biological materials, etc. used in the examples described below are commercially available unless otherwise specified.

Firstly, constructing a CD3 antigen to immunize alpaca for four times to obtain alpaca PBMC cells; secondly, screening and separating PBMC cells by a microfluidic technology; capturing RNA of a single cell, obtaining an antibody gene fragment by a nested PCR method, and constructing a eukaryotic expression vector; then inducing high-flux expression of the antibody by a mammalian cell high-flux expression system; finally, the antibody with high sensitivity and specificity is obtained through ELISA and FACS detection and sequencing result analysis.

Example 1 preparation of CD3 immunogen

1. Preparation of immunogens

We based on the protein sequence and gene sequence information of CD3, the CD3 sequence was from the public database, asp 23-Asp 126 (CD 3E): access P07766-1 (CD 3E); phe 22-Ala 105 (CD 3D): access P04234-1 (CD 3D) is analyzed and designed to effectively induce alpaca to produce specific antibody aiming at human CD3, and His-tag is connected at C end for subsequent purification and detection.

EXAMPLE 2 alpaca immunization and antiserum obtained

1. Alpaca immunization and antiserum acquisition

Priming alpaca with an emulsified mixture of 200 μg of human CD3/His protein and 200 μl Freund's complete adjuvant, boosting 3 times with human CD3/His protein and 200 μl Freund's incomplete adjuvant ondays 21, 42, 63, 1 week after each immunization, and blood sampling to detect Anti-CD3/His serum titer; after 1 week ofimmunization 4, 50ml of blood was collected for single cell sorting by the microfluidic platform.

The Anti-CD3/His serum titers were detected by ELISA, the ELISA plate was coated with CD3/His protein at a concentration of 2. Mu.g/ml, 2-fold gradient diluted serum or 100. Mu.l of purified antibody (control was immune alpaca serum) was added to each well, and incubated at 37℃for 1.5hWash 2 times, add 1 per well: 10000-diluted horseradish peroxidase-labeled anti-Alpaca IgG (H+L) secondary antibody, incubating at 37 ℃ for 1H, washing 5 times, adding 100 μl TMB substrate, incubating at 37 ℃ for 10min, and incubating at 50 μl 0.1M H₂ SO₄ The reaction was stopped to determine OD450nm. ELISA detection serum titers were defined as 2-fold or more over OD450nm compared to the blank, and the results are shown in FIG. 1, where FIG. 1 shows an antiserum titer of 102400 after 4-priming. Thus, the antigen can induce alpaca to produce high titer antisera specific to CD3 protein.

EXAMPLE 3 construction and screening of VHH monoclonal expression library construction and screening

1. Constructing and screening a VHH monoclonal expression library:

1) Sorting cells:

after immunization for 1 week for 4 times, 50ml of blood is taken for carrying out functional antibody secretion cell sorting by a microfluidic platform, the cell and the antigen can be wrapped in picoliter-level monodisperse oil drops by a liquid drop microfluidic technology, the generation speed of thousands of monodisperse liquid drops per second can be reached, each micro drop of a single cell is obtained independently, the environmental independence between the cells is realized, and the mutual pollution is avoided; VHH is identified in oil drops through a fluorescence-labeled alpaca secondary antibody, and if the VHH antibody secreted by cells can identify a fluorescence-labeled antigen, FRET signals can be formed to be separated, and B cells which can secrete VHH and have binding activity are obtained through screening.

2) Building a warehouse:

RNA is extracted by using magnetic beads, and is inverted into cDNA by using oligo (dT), and the cloning result of the target fragment is shown in figure 2 (partial result) by the technologies of primer amplification, molecular cloning and the like; cloning the VHH gene of alpaca into eukaryotic expression vector, and transforming competent cells to obtain VHH monoclonal expression library. In order to further identify whether the CD3-VHH monoclonal expression library is successfully constructed, 10 clones are selected from a coated monoclonal bacterial plate for sequencing, and the sequencing result shows that the positive cloning rate and the sequence diversity are 100%; the alignment shows that the difference sequences are mostly in the CDR binding regions. The construction was examined to obtain a CD3-VHH monoclonal expression library.

3) Extracting plasmid:

picking a monoclonal bacterial colony into a 96-hole deep hole plate by using a CD3-VHH monoclonal expression library-coated bacterial plate, and culturing for 8 hours at 37 ℃ by shaking a shaking table for 800 r/min; placing a 96-well deep-hole plate for culturing bacterial liquid in a horizontal centrifuge, centrifuging at 4000r/min for 10min, discarding the supernatant, adding 300 mu L of solution I, and vibrating to uniformly suspend the bacterial body; adding 300 mu L of solution II, gently and fully reversing the solution for 4 to 6 times, and uniformly mixing to ensure that bacterial liquid is fully cracked until a transparent solution is formed; finally, 400 mu L of solution III is added, the solution III is gently and fully turned upside down for 6 to 8 times, and the solution III is centrifuged for 10min at 4000 r/min; after centrifugation, absorbing 800 mu L of supernatant from each hole into a 96-hole filter plate (the lower part of the filter plate is connected with a 96-hole deep hole plate), and centrifuging for 2min at 4000 r/min; collecting filtrate, adding 300 μl of isopropanol into each well, centrifuging at 4000r/min for 15min, and discarding supernatant; adding 500 mu L of 70% absolute ethanol into each hole, centrifuging for 10min at 4000r/min, and discarding the supernatant; the 96-well deep-hole plate is placed for airing ethanol at room temperature, and 70 mu L of ddH is added into each well₂ And O, shaking and mixing uniformly, and measuring the plasmid concentration.

4) Cell transfection and selection:

the antibiotic-free medium DMEM was added to each of the 96-well cell culture plates, 75 μl per well. Mu.l of plasmid was added to each well (no wells were strung corresponding to the plasmid positions); at least 75mL of transfection reagent (1 mL of PEI is added into 75mL of DMEM culture medium) is prepared by co-transfecting a 10-plate 96-cell plate, and 75 mu L of PEI transfection reagent diluted by DMEM is added into each hole of the 96-hole cell culture plate with plasmids; incubating the plasmid and the transfection reagent for 15min at room temperature; HEK-293T cells in good cell status were digested with 0.05% pancreatin during preparation of transfection reagent, and resuspended in DMEM (containing 1% diabody) with 2.4% FBS for cell blow-off count; the cell suspension was added drop-wise to the corresponding 96-well cell culture plate, 5×10 per well⁴ mu.L of the cell suspension was added to each cell, and 5% CO at 37℃was used₂ Culturing in a cell culture box, and continuously culturing for 72 hours. The transfected cell supernatants were tested for binding to CD3 protein. Monoclonal ELISA results showed that a total of 960 monoclonal cells from ten 96-well cell plates gave 396 positive clones (as shown in fig. 3, part of the results were shown) and these sequences were sequenced and aligned to eliminate the repeated sequences. To further verify binding of CD3-VHH eggs in the libraryWhite positive antibodies, cell supernatants were examined by flow cytometry, and the results showed 15 FACS positive antibodies among 396 ELISA positive clones (as shown in fig. 4, a part of the results were shown). After expression purification of the FACS positive antibody detection results, cell binding assay detection is carried out, and the results show that the Emax of the finally obtained 1 VHH antibody through Cell binding assay screening is obviously higher than that of a control antibody (shown in FIG. 5), the VHH antibody is ANb-M249-4M-905, and the sequence of the VHH antibody is shown as SEQ ID No. 1. The nano antibody is verified by ELISA and flow cytometry, and the antibody with high sensitivity and specificity is obtained.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Sequence listing

<110> Shanghai Baiying Biotech Co., ltd

<120> an anti-CD3 nanobody or antigen-binding portion thereof and method for preparing the same

<141> 2022-05-18

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

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Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Gly Ile Ser Asn Tyr

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His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile Val

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

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Tyr

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

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

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gaggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

acctgtgcag cctctggacg cggaatcagt aactatcaca tgggctggtt ccgccaggct 120

ccaggaaagg agcgtgaaat tgtagcagcg attacgagca ctggtgctcg tacatactat 180

gtagactccg tgaagggccg attcaccatc tccagagaca accccaagaa cacgctgtat 240

ctggaaatga acagcctgaa acctgaagac acggccatgt actactgtgt cgcaaattct 300

gtcgacgact cctactgggg ccaggggacc caagtcaccg tctcctca 348

Claims (23)

1. An anti-CD3 nanobody comprising complementarity determining regions CDR1-CDR3 in the heavy chain variable region as set forth in SEQ ID No. 1.

2. The nanobody of claim 1, wherein the amino acid sequence of CDR1 is amino acids 31-35 of SEQ ID No. 1.

3. The nanobody of claim 2, wherein the amino acid sequence of CDR2 is amino acids 49-55 of SEQ ID No. 1.

4. The nanobody of claim 3, wherein the amino acid sequence of CDR3 is amino acids 99-105 of SEQ ID No. 1.

5. The nanobody of claim 4, further comprising framework regions FR1-FR4 in the heavy chain variable region shown in SEQ ID No. 1.

6. The nanobody of claim 5, wherein the amino acid sequence of FR1 is amino acids 1-30 of SEQ ID No. 1.

7. The nanobody of claim 6, wherein the amino acid sequence of FR2 is amino acids 36-49 of SEQ ID No. 1.

8. The nanobody of claim 7, wherein the amino acid sequence of FR3 is amino acids 67-98 of SEQ ID No. 1.

9. The nanobody of claim 8, wherein the amino acid sequence of FR4 is amino acids 106-116 of SEQ ID No. 1.

10. An isolated nucleic acid molecule encoding the nanobody of any of claims 1-9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.

13. A method for preparing anti-CD3 nanobodies, comprising transfecting the vector of claim 11 into a host cell, culturing the host cell, and isolating or recovering the anti-CD3 nanobodies.

14. The method of claim 13, wherein the host cell is a mammalian cell.

15. A product for detecting CD3, characterized in that it comprises a nanobody according to any one of claims 1 to 9.

16. The product of claim 15, wherein the product is a conjugate or conjugate.

17. The product of claim 16, wherein the conjugate or conjugate comprises:

1) The nanobody of any of claims 1-9;

2) A diagnostic agent coupled or conjugated to the nanobody.

18. The product of claim 17, wherein the diagnostic agent comprises a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, a photosensitive diagnostic agent.

19. A pharmaceutical composition, characterized in that it comprises any one of the following:

1) The nanobody of any of claims 1-9;

2) A conjugate or conjugate, the conjugate or conjugate comprising:

a) The nanobody of any of claims 1-9;

b) A therapeutic agent coupled or conjugated to the nanobody.

20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

21. A method for non-therapeutic destination detection of the presence of CD3 in a sample, the method comprising the steps of: contacting the nanobody of any of claims 1-9 with a test sample, and detecting the presence of CD3 in the test sample by antigen-antibody reaction.

22. Use of the nanobody of any of claims 1-9, the conjugate or the conjugate of claim 16 for the preparation of a CD3 detection product.

23. Use of a nanobody according to any of claims 1-9, a conjugate or conjugate according to claim 19 in the manufacture of a medicament for the treatment of a CD3 mediated disease.

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