A nanobody targeting TfR1 and its application

Abstract

The present invention discloses a nano antibody targeting TfR1 and its application, belonging to the field of biomedicine. The nano antibody targeting TfR1 disclosed in the present invention has three unique complementary determining regions CDR1 (SEQ ID NO.1), CDR2 (SEQ ID NO.2), and CDR3 (SEQ ID NO.3). The present invention also provides an expression vector containing the variable region coding sequence of the nano antibody, and a host cell containing the expression vector, as well as the application of the nano antibody in the preparation of a drug targeting TfR1. The nano antibody provided by the present invention has specific recognition and binding ability to TfR1, and the affinity of the nano antibody can reach 2.28E-9. The nano antibody can bind to cell surface TfR1 and neutralize its activity, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to nano antibodies targeting TfR1, their coding sequences and applications in detection. Background Art

[0002] Transferrin receptor protein 1 (TfR1), a 90 kDa type II transmembrane protein, is a key regulator of intracellular iron transport. TfR1 is composed of two homodimeric subunits cross-linked by disulfide bonds. The TfR1 monomer consists of a large extracellular C-terminal domain (671 amino acids) containing the transporter binding site, a transmembrane domain (28 amino acids), and an intracellular N-terminal domain. The C-terminal extracellular domain contains three N-linked glycosylation sites at asparagine residues 251, 317, and 727, and one O-linked glycosylation site at threonine 104, all of which are required for the receptor to function properly.

[0003] Under normal physiological conditions, TfR1 interacts with ferroportin, mediating the entry of iron ions into cellular channels and playing a key role in regulating cellular iron metabolism. Iron is essential for numerous cellular processes, including DNA synthesis and cell proliferation. Iron deficiency inhibits cell growth and leads to cell death. Cancer cells are more sensitive to iron deficiency than normal cells. After becoming malignant, cells require large amounts of iron to maintain a high cell proliferation rate. Excessive iron promotes tumor development, and abnormal iron metabolism has been considered a specific marker of tumors. Under normal circumstances, TfR1 itself is tightly regulated, with only basal expression in many tissues. Increased expression has been found only in brain capillary endothelial cells, hepatocytes, and rapidly proliferating cells. Furthermore, TfR1 expression is significantly increased on the surface of many malignant tumor cells, often at levels approximately 20-fold higher than in normal cells. Therefore, TfR1 is considered a potential tumor marker, and targeted therapies targeting TfR1 can effectively inhibit tumor growth and metastasis.

[0004] Currently, several clinical drugs targeting TfR1 are under development, primarily for the treatment of cancer, anemia, iron metabolism disorders, and neurodegenerative diseases. Among them, PPMX-T003, CX-2029, DYNE-251, and Trontinemab are already in Phase 1 / 2 clinical trials. In recent years, TfR1-targeted therapeutic strategies have continued to advance, and TfR1 is expected to become an effective target molecule in the clinical treatment of a variety of diseases.

[0005] Whether used in atopic diseases or tumors, the immunogenicity of monoclonal antibodies has always been one of the most important issues for researchers. While ensuring efficacy, minimizing the immunogenicity of drugs can greatly reduce side effects. Therefore, many researchers have modified and recombined monoclonal antibodies to reduce their molecular size, and then humanized them through methods such as amino acid mutations at key sites or affinity maturation. However, most humanization modifications will reduce the affinity activity or stability of the antibody itself.

[0006] There is an antibody that naturally lacks light chains in the peripheral blood of alpacas. This antibody only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other as artificially modified single-chain antibody fragments (scFv), or even aggregate into clumps. More importantly, the VHH structure cloned and expressed separately has a structural stability and antigen binding activity comparable to the original heavy chain antibody, and is the smallest unit known to bind to the target antigen. The VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only 15kDa. It is also called a nanobody (Nb). Compared with the scFv of conventional four-chain antibodies, nanobodies are comparable to their corresponding scFv in terms of affinity, but surpass scFv in solubility, stability, resistance to aggregation, refoldability, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination. And because nanobodies are small molecules and have high stability, they can be administered by atomization, which not only improves the convenience of administration, but also broadens the application scenarios of drugs. Therefore, providing nanoantibodies targeting TfR1 antigen and the application of their coding sequences in detection have important practical significance. Summary of the Invention

[0007] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a nanobody targeting TfR1 and its application. The nanobody targeting TfR1 provided by the present invention has excellent specific antigen binding ability and can reduce its own immunogenicity compared with traditional monoclonal antibodies.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] In a first aspect of the present invention, a nanobody targeting TfR1 is provided, wherein the nanobody comprises three complementary determining regions CDR1, CDR2, and CDR3, wherein:

[0010] I. The complementarity determining region CDR1 of the Nanobody has the amino acid sequence shown in SEQ ID NO.1, the complementarity determining region CDR2 has the amino acid sequence shown in SEQ ID NO.2, and the complementarity determining region CDR3 has the amino acid sequence shown in SEQ ID NO.3;

[0011] or II. an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in I, and having the same function as the amino acid sequence described in I;

[0012] or III, an amino acid sequence that has more than 80% identity with the amino acid sequence described in I or II and exerts similar physiological activities.

[0013] In some embodiments of the present invention, the nanobody targeting TfR1 further comprises four framework regions FR1, FR2, FR3, and FR4, wherein:

[0014] The amino acid sequence of the framework region FR1 of the Nanobody is shown in SEQ ID NO.4;

[0015] The amino acid sequence of the framework region FR2 of the Nanobody is shown in SEQ ID NO.5;

[0016] The amino acid sequence of the framework region FR3 of the Nanobody is shown in SEQ ID NO.6;

[0017] The amino acid sequence of the framework region FR4 of the nanobody is shown in SEQ ID NO.7.

[0018] In some embodiments of the invention, the amino acid sequence of the variable region of the Nanobody is selected from any one of the following:

[0019] I. the amino acid sequence shown in SEQ ID NO.8;

[0020] or II. an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in I, and having the same function as the amino acid sequence described in I;

[0021] or III, an amino acid sequence that has more than 80% identity with the amino acid sequence described in I or II and exerts similar physiological activities.

[0022] In some embodiments of the present invention, a preferred embodiment of the Nanobody having the variable region sequence obtained by screening is Nanobody 1B6, the variable region amino acid sequence of 1B6 is shown in SEQ ID NO.8, wherein the amino acid sequence at positions 1-25 is FR1 (as shown in SEQ ID NO.4), the amino acid sequence at positions 26-33 is CDR1 (as shown in SEQ ID NO.1), the amino acid sequence at positions 34-50 is FR2 (as shown in SEQ ID NO.5), the amino acid sequence at positions 51-58 is CDR2 (as shown in SEQ ID NO.2), the amino acid sequence at positions 59-96 is FR3 (as shown in SEQ ID NO.6), the amino acid sequence at positions 97-114 is CDR3 (as shown in SEQ ID NO.3), and the amino acid sequence at positions 115-126 is FR4 (as shown in SEQ ID NO.7).

[0023] In some embodiments of the present invention, the Nanobody further comprises a derivative polypeptide modified from the amino acid sequence, wherein the modification includes, but is not limited to, functional group modification or the addition of a molecular label. Further preferably, the functional group modification includes, but is not limited to, modification of the FR region with a hydrophilic group or replacement of the hydrophobic residues in the FR region. Further preferably, the molecular label includes, but is not limited to, polyethylene glycol, streptavidin, biotin, a radioisotope, or a fluorescent agent.

[0024] The second aspect of the present invention provides a nucleic acid molecule encoding the nanobody targeting TfR1.

[0025] In some embodiments of the present invention, the nucleic acid molecule comprises a coding nucleic acid that can be translated into the aforementioned Nanobody due to codon degeneracy, and the coding nucleic acid is not limited to DNA or RNA. Preferably, the coding nucleic acid is DNA, including cDNA, genomic DNA, or artificially synthesized DNA; the DNA may be single-stranded or double-stranded, and may be a coding strand or a non-coding strand.

[0026] In some embodiments of the present invention, the nucleic acid sequence of the nucleic acid molecule is shown as SEQ ID NO.9.

[0027] In the third aspect of the present invention, the present invention also provides an expression vector comprising the nucleic acid molecule.

[0028] In some embodiments of the present invention, the expression vector includes but is not limited to a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or other vectors. Preferably, the expression vector is a bacterial plasmid or a yeast plasmid.

[0029] In a fourth aspect, the present invention also provides a host cell transformed or transfected with the expression vector.

[0030] In some embodiments of the present invention, the host cell is a plant cell or a microbial cell. Preferably, the host cell is a microbial cell. More preferably, Escherichia coli is used as the host cell.

[0031] In a fifth aspect, the present invention further provides a conjugate or a coupling comprising a chemically labeled or biologically labeled Nanobody as claimed in claim 1 and an acceptable excipient or carrier.

[0032] In the present invention, the chemical label is an isotope, an immunotoxin and / or a chemical drug; the biological label is a biotin, avidin or an enzyme label.

[0033] In the sixth aspect of the present invention, based on the above research, the present invention also provides the use of the nanoantibody targeting TfR1 or the conjugate or conjugate in the preparation of drugs targeting TfR1.

[0034] In the seventh aspect of the present invention, the present invention also provides the use of the nanobody targeting TfR1 or the conjugate or conjugate in the preparation of TfR1 detection antibody reagents and / or kits.

[0035] In the eighth aspect of the present invention, based on the above research, the present invention also provides a TfR1 detection antibody reagent, including the nanoantibody targeting TfR1 and / or the conjugate or coupling and an acceptable adjuvant and / or carrier.

[0036] The beneficial effects of the present invention are:

[0037] The nanobody targeting TfR1 provided by the present invention has unique CDR1, CDR2, and CDR3 regions, which enable the nanobody to specifically recognize and bind to the TfR1 antigen. The nanobody has an affinity of up to 2.28E-9, showing highly specific binding activity. In addition, the nanobody provided by the present invention can bind to cell surface TfR1 and inhibit its activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0039] Figure 1 This is the SDS-PAGE image of the recombinant TfR1 antigen purification;

[0040] Figure 2 This is the electrophoresis identification diagram of the extracted total RNA;

[0041] Figure 3 This is the electrophoresis identification diagram of the first and second rounds of PCR amplification of the antibody variable region gene;

[0042] Figure 4 This is the electrophoresis identification diagram of the double enzyme digestion reaction products of the pMES4 vector;

[0043] Figure 5 Estimate the storage capacity results for single colony counts;

[0044] Figure 6 This is the electrophoresis identification diagram of transformants identified by colony PCR;

[0045] Figure 7 This is the SDS-PAGE image of nanobody purification;

[0046] Figure 8 This is a diagram showing the Tf-TfR1 binding inhibitory activity of the nanobody. DETAILED DESCRIPTION

[0047] The present invention discloses a nano antibody targeting TfR1 and the application of its coding sequence in detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] The present invention first immunizes alpacas with the recombinant antigen TfR1. After four immunizations, peripheral blood lymphocytes are isolated from the immunized alpacas, and a specific nanobody gene library targeting TfR1 is constructed. The recombinant TfR1 antigen is coated on an ELISA plate, and phage display technology is used to screen the nanoantibody library for nanoantibodies targeting the recombinant TfR1 antigen. The constructed nanoantibody expression vector is introduced into Escherichia coli, expressed, and purified. The purified nanoantibody is able to specifically bind to the cell surface TfR1 protein and neutralize its activity.

[0049] The present invention provides a nano antibody coding sequence that can specifically bind to TfR1 protein, and a preparation method and application thereof.

[0050] Specifically:

[0051] The variable region of the nanobody has three complementary determining regions CDR1, CDR2, and CDR3, wherein:

[0052] The CDR1 sequence consists of the amino acid sequence described in SEQ ID NO.1;

[0053] The CDR2 sequence consists of the amino acid sequence described in SEQ ID NO.2;

[0054] The CDR3 sequence consists of the amino acid sequence described in SEQ ID NO.3.

[0055] The amino acid sequence of the variable region of the nanobody is shown in SEQ ID NO.8.

[0056] The present invention also provides a nucleic acid molecule encoding the above-mentioned nanobody, the nucleic acid sequence of which is shown in SEQ ID NO.9.

[0057] The present invention also provides the use of the above-mentioned nanobody in the preparation of a drug targeting TfR1.

[0058] The present invention also provides the use of the above-mentioned nanobody in the preparation of a TfR1 detection antibody reagent and / or a kit.

[0059] The sequences involved in the present invention are as follows:

[0060] Amino acid sequence of the CDR1 region of Nanobody 1B6:

[0061] 5'-GRTFSSYA-3' (SEQ ID NO. 1).

[0062] Amino acid sequence of the CDR2 region of Nanobody 1B6:

[0063] 5'-ISWSADNT-3' (SEQ ID NO. 2).

[0064] Amino acid sequence of the CDR3 region of Nanobody 1B6:

[0065] 5'-AADPTPLHTIVVVTPEYD-3' (SEQ ID NO. 3).

[0066] Amino acid sequence of the FR1 region of Nanobody 1B6:

[0067] 5'-QVQLQESGGGLVQAGGSLRLSCAAS-3' (SEQ ID NO. 4).

[0068] Amino acid sequence of the FR2 region of Nanobody 1B6:

[0069] 5'-MGWFRQAPGKEREFLAA-3' (SEQ ID NO. 5).

[0070] Amino acid sequence of the FR3 region of Nanobody 1B6:

[0071] 5'-YYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC-3' (SEQ ID NO. 6).

[0072] Amino acid sequence of the FR4 region of Nanobody 1B6:

[0073] 5'-YWGQGTQVTVSS-3' (SEQ ID NO. 7).

[0074] The amino acid sequence of the variable region of Nanobody 1B6:

[0075] 5'–QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFLAAISWSADNTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCAADPTPLHTIVVVTPEYDYWGQGTQVTVSS–3' (SEQ ID NO. 8).

[0076] The nucleic acid sequence of Nanobody 1B6:

[0077] 5'–caggtgcagctgcaggagtctgggggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcaccttcagtagct atgccatgggctggttccgccaggctccagggaaggagcgtgagtttctagcagctattagctggagtgctgataacacatactatgcagactccgt gaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacaacctgaaacctgaggacacggccgtttattactgtgcagcagatccgaccccactgcatactatagtggtagttactcctgagtatgactactggggccaggggacccaggtcaccgtctcctca–3'(SEQ ID NO.9).

[0078] CALL001 primer sequence:

[0079] 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO. 10).

[0080] CALL002 primer sequence:

[0081] 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO. 11).

[0082] VHH-Back primer sequence:

[0083] 5'-GATGGTGCAGCTGCAGGAGTCTGGRGGAGG-3' (SEQ ID NO. 12).

[0084] VHH-For primer sequence:

[0085] 5'-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3' (SEQ ID NO. 13).

[0086] pMES-F primer sequence:

[0087] 5'-GCCGCTGGATTGTTATTACTC-3' (SEQ ID NO. 14).

[0088] pMES-R primer sequences:

[0089] 5'-CTTTCAACAGTGGAACCGTAG-3' (SEQ ID NO. 15).

[0090] The nanobody targeting TfR1 provided by the present invention has unique CDR1, CDR2, and CDR3 regions, which enable the nanobody to specifically recognize and bind to the TfR1 antigen. The nanobody has an affinity of up to 2.28E-9, showing highly specific binding activity. In addition, the nanobody provided by the present invention can bind to cell surface TfR1 and inhibit its activity.

[0091] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0092] An "antibody" is a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic structural unit. This form is a tetramer, composed of two identical pairs of antibody chains, each consisting of a heavy chain and a light chain. Within each pair, the variable regions of the light and heavy chains work together to bind to the antigen, while the constant regions are responsible for the antibody's effector functions.

[0093] The "variable region" is the N-terminal mature region of the chain. Currently known antibody types include kappa and lambda light chains, and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu heavy chains or their equivalents. A full-length immunoglobulin "light chain" (approximately 25 kDa or approximately 214 amino acids) comprises a variable region formed by approximately 110 amino acids at the NH2-terminus, and a kappa or lambda constant region at the COOH-terminus. A full-length immunoglobulin "heavy chain" (approximately 50 kDa or approximately 446 amino acids) also comprises a variable region (approximately 116 amino acids) and one of the heavy chain constant regions, such as gamma (approximately 330 amino acids).

[0094] "Antibodies" include antibodies or immunoglobulins of any isotype, or antibody fragments that retain specific antigen binding, including but not limited to Fab, Fy, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be labeled and detected, for example, using radioisotopes, enzymes that produce detectable substances, fluorescent proteins, biotin, and the like. Antibodies can also be bound to solid supports, including but not limited to polystyrene plates or beads, and the like.

[0095] "Humanized antibody" refers to an antibody that comprises a CDR region derived from a non-human antibody and the rest of the antibody molecule is derived from one (or several) human antibodies. In addition, some residues of the framework (called FR) segment may be modified to retain binding affinity.

[0096] The "nanobody" refers to an antibody that is naturally deficient in light chains and exists in the peripheral blood of alpacas. The antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other as the artificially modified single-chain antibody fragment (scFv), or even aggregate into clumps. More importantly, the VHH structure cloned and expressed separately has a structural stability and antigen binding activity comparable to the original heavy chain antibody, and is the smallest unit known to bind to the target antigen. The VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only 15kDa. It is also called a nanobody (Nanobody, Nb). Compared to the scFv of a conventional four-chain antibody, the nanobody is comparable to its corresponding scFv in terms of affinity, but surpasses scFv in terms of solubility, stability, resistance to aggregation, refoldability, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination. Moreover, due to their small molecules and high stability, nanoantibodies can be administered through atomization, which not only improves the convenience of administration but also broadens the application scenarios of drugs.

[0097] The medicament contains at least one functional ingredient and also includes a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or a polyalkylene glycol such as polypropylene glycol, triglyceride, etc. The type of pharmaceutically acceptable carrier used depends in particular on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present invention may contain a wetting agent, an emulsifier or a buffer substance as an additive.

[0098] As used herein, "CDR region" or "CDR" refers to the Complementary Determining Regions of a Nanobody. There are three CDRs. Depending on the circumstances, the term CDR or CDRs as used herein is intended to indicate one of these regions, or several or even all of these regions, which contain the majority of the amino acid residues responsible for binding through the affinity of the antibody to the antigen or its recognized epitope.

[0099] "FR region" or "FR" as used herein refers to the framework region of a Nanobody. There are four FRs. Depending on the circumstances, the term FR as used herein is intended to indicate one of these regions, or several of these regions, or even all of them.

[0100] The nanobodies provided by the present invention and the raw materials and reagents used in their applications can all be purchased from the market.

[0101] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0102] Example 1 Expression of TfR1 recombinant protein

[0103] According to the amino acid sequence of human TfR1 on NCBI (Sequence ID: AAA61153.1), Cys88-Phe760 was synthesized, and a histidine tag was added to the N-terminus of the sequence and connected to the vector pCDNA3.1(+). After endotoxin-free plasmid extraction, 293 cells in logarithmic growth were used for transfection. After culturing the transfected cells for 36 hours, the cell culture medium was poured into a 50mL centrifuge tube, centrifuged at 12000g for 5 minutes, the supernatant was collected, filtered with a 0.22μm filter membrane, and the culture supernatant was purified by nickel column affinity chromatography. Protein expression was detected by SDS-PAGE. The results are shown in Figure 1 (M is a protein marker, and lane 1 is the purified TfR1 antigen protein).

[0104] Example 2 Construction and screening of anti-TfR1 nanobody phage display library

[0105] 2.1 Alpaca Immunity

[0106] Adult alpacas of moderate size, good health, no signs of injury or discomfort, and in good spirits were selected. Recombinant TfR1 protein was mixed with Gerbu adjuvant at a 1:1 ratio and injected bilaterally near the cervical lymph nodes, with 0.5 mL injected at four sites on each side. Four immunizations were performed, with two weeks between each. Peripheral blood was then collected for the construction of a phage display library.

[0107] 2.2 Isolation of alpaca lymphocytes

[0108] The alpaca peripheral blood was collected and lymphocytes were separated using the camel peripheral blood lymphocyte separation reagent kit (Tianjin Haoyang Company, product number LTS1076) according to the instructions. 7 Add 1 mL of RNA isolation reagent to each living cell, take 1 mL for RNA extraction, and store the rest at -80℃.

[0109] 2.3 RNA extraction

[0110] Repeatedly pipette 1mL Tipure Isolation Reagent containing lymphocytes and let it stand for 5 minutes; add 200μL chloroform, shake vigorously vertically up and down for 30 seconds and then let it stand for another 5 minutes; centrifuge at 4℃, 12000g for 15 minutes, aspirate the aqueous phase and transfer it to a new EP tube; add an equal amount of isopropanol and let it stand for 10 minutes; centrifuge at 4℃, 12000g for 10 minutes, discard the supernatant; wash with 1mL pre-cooled 70% ethanol, centrifuge at 4℃, 7500g for 5 minutes, discard the supernatant and dry for 5 minutes; add 30μL RNase-free water to dissolve the precipitate and adjust the concentration to 1μg / μL for gel electrophoresis detection. The results are shown in the figure. Figure 2 , lane 1.

[0111] 2.4 Reverse transcription and cDNA synthesis

[0112] According to the instructions of the reverse transcription kit (abm Bio All-In-One 5x RT Mastermix), reverse transcription of cDNA was performed using the RNA obtained in step 2.3 as a template.

[0113] 2.5 Amplification of Antibody Variable Region Genes

[0114] The cDNA obtained by reverse transcription was used as a template for the first round of PCR reaction. The primer sequences of the PCR reaction were as follows:

[0115] CALL001:GTCCTGGCTGCTCTTCTACAAGG(SEQ ID NO.10)

[0116] CALL002:GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO.11)

[0117] PCR reaction conditions and procedures are as follows:

[0118] 95℃5min;

[0119] 95℃30s, 57℃30s, 72℃30s, 25cycles;

[0120] 72℃7min.

[0121] Use agarose gel recovery kit to recover the band of about 700 bp, and finally adjust the nucleic acid concentration to 5 ng / μL with water (see the first round PCR product identification). Figure 2 , where M is a molecular weight marker and 1 is the first-round PCR product).

[0122] The second round of PCR was performed using the first round PCR product as a template. The primer sequences are as follows:

[0123] VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO.12)

[0124] VHH-For:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO.13)

[0125] PCR reaction conditions and procedures are as follows:

[0126] 95℃5min;

[0127] 95℃30s, 55℃30s, 72℃30s, 25cycles;

[0128] 72℃5min.

[0129] Purify the PCR product using a PCR product recovery kit (see the second round of PCR product identification for details). Figure 2 , where M is a molecular weight marker and 1 is the product of the second round of PCR).

[0130] 2.6 Vector Construction

[0131] The pMES4 vector (purchased from Biovector) and the second PCR product were double-digested with PstI and BstEII, respectively. 2 μg of the digested vector and 2 μg of the second-round PCR product were added to 40 μL of T4 DNA ligase, supplemented with buffer and water to a total volume of 150 μL. The ligation reaction was incubated overnight at 16°C and the ligated product was recovered using a PCR product recovery kit and eluted with 20 μL of water.

[0132] Figure 3 This is the electrophoresis identification of the pMES4 vector double-enzyme digestion reaction products. M: molecular weight marker; 1: pMES4 vector double-enzyme digestion product; 2: undigested pMES4 vector.

[0133] 2.7 Electroporation and Storage Capacity Determination

[0134] Take 20 μL of the purified ligation product and add it to the bottom of a pre-cooled 2 mm electroporation cup containing 200 μL of E. coli TG1 competent cells. Mix and place in an ice bath for 30 minutes. Wipe the electroporation cup clean and place it in an electroporator (Gene Pulser Xcell). TM , BIO-RAD Company) for electroporation, with the electroporation parameters set at 2.5 kV, 25 μF, 200 Ω. Immediately after electroporation, 800 μL of SOC medium was added, mixed, and transferred to a sterile EP tube. The cells were revived at 37°C and 200 rpm for 2 h. After the resuscitation, 100 μL of bacterial solution was aspirated and the cells were resuspended in 10% PBS. -1 , 10 -2 , to 10-6 10 -4 , 10 -5 , 10 -6 100 μL of each gradient bacterial solution was aspirated and applied to 2YT-A plates and cultured overnight in a 37°C incubator. Count the single colonies on each dilution plate, select the plate with the appropriate number of single colonies, and calculate the library capacity according to the dilution (see the results). Figure 4 ). Use a sterile pipette tip to randomly pick 20 single clones and perform colony PCR identification (see electrophoresis identification results for details). Figure 5 , where M is a molecular weight marker; 1-20 are PCR identification products that were not randomly selected; and N is a negative control).

[0135] Primer sequences:

[0136] pMES-F: GCCGCTGGATTGTTATTACTC (SEQ ID NO.14)

[0137] pMES-R:CTTTCAACAGTGGAACCGTAG(SEQ ID NO.15)

[0138] The conditions and procedures for the PCR reaction are as follows:

[0139] 95℃5min;

[0140] 95℃30s, 55℃30s, 72℃30s, 30cycles;

[0141] 72℃5min.

[0142] The PCR positive rate was calculated based on the electrophoresis results and the reservoir capacity was estimated.

[0143] Library capacity = number of clones × dilution factor × positive rate × 10 × library volume.

[0144] Storage capacity = 150 × 10 6 ×95%×10×2=2.85*10 9 CFU. After calculation, the storage capacity is 2.85*10 9 CFU.

[0145] Example 3 Screening and expression of nanobodies

[0146] 3.1 Phage display of nanobodies

[0147] Take the recovered bacterial solution and inoculate it into four 10 mL 2YT-AG mediums, and culture at 37°C, 200 rpm until the culture OD600 = 0.5. Add 4×10 10Inoculate with pfu VCSM13 at 37°C for 30 minutes. Centrifuge at 4000 rpm for 10 minutes at room temperature, and discard the supernatant. Resuspend the cells in 100 mL of 2×YT-AK medium (containing ampicillin and kanamycin) and culture overnight at 37°C, 200 rpm. Centrifuge the overnight culture at 10,800 g for 15 minutes at 4°C, collect the supernatant, add 10 mL of PEG / NaCl (20% / 2.5 M) solution to each 40 mL of supernatant, mix thoroughly, incubate on ice for 2 hours, centrifuge at 10,800 g for 30 minutes at 4°C, discard the supernatant, resuspend the pellet in 8 mL of ice-cold PBS, add 2 mL of pre-chilled PEG / NaCl, mix thoroughly, incubate on ice for 1 hour, centrifuge at 3300 g for 30 minutes at 4°C, and resuspend the pellet in 1 mL of PBS.

[0148] Determination of phage titer: culture TG1 to OD600 = 0.4, gradient dilution of phage with sterile PBS, mix the serially diluted phage TG1 culture (1:20), culture at 30°C for 30 min, take 100 μL and apply it to 2YT-AG solid plate, observe the plaque formation in the culture plate the next day, count the plaques on the dilution gradient plates with 30-300 and calculate the displayed phage titer (cfu) according to the following formula.

[0149] Phage titer (cfu / mL) = dilution factor × 2 × number of plaques × 100

[0150] 3.2 Solid-phase panning of phage display libraries

[0151] Dilute TfR1 recombinant antigen to 10 μg / mL with CBS, coat the ELISA plate with 100 μL per well, incubate at 4°C overnight, and wash the plate 5 times with PBST; add 250 μL of 1% BSA to each well, block at 37°C for 2 h, and wash the plate 5 times with PBST; add 100 μL of the diluted solution to 10 11 Incubate the displayed phage at 100 cfu / mL at 37°C for 2 hours, then wash the plate 15-25 times with PBST. After the final wash, add 100 μL of glycine solution (0.2 M, pH 2.2) to each well and incubate on a horizontal shaker for 15 minutes. Add the eluate from each well to an EP tube pre-filled with 15 μL of Tris solution, combine, and titer. Increase the stringency of the panning appropriately based on the results of each round, repeating 3-4 times in total.

[0152] 3.3 Phage Elisa screening of positive clones

[0153] Dilute TfR1 recombinant antigen to 5 μg / mL in CBS, coat ELISA plates with 100 μL per well, incubate at 4°C overnight, and wash five times with 0.05% PBST. Add 250 μL of 5% BSA to each well, block at 37°C for 1 hour, and wash five times with 0.05% PBST. Add 100 μL of overnight monoclonal phage supernatant to each well, incubate at 37°C for 1 hour, and wash five times with 0.05% PBST. Add 100 μL of HRP-labeled mouse anti-M13 secondary antibody to each well, incubate at 37°C for 1 hour, and wash five times with 0.05% PBST. Add 100 μL of TMB colorimetric solution to each well, incubate at room temperature in the dark for 15-30 minutes, and add 100 μL of 2M sulfuric acid stop solution to each well. Read at 450 nm using a microplate reader. Select clones with positive phage ELISA results and send for sequencing.

[0154] 3.4 Amplification of the original nanobody strain TG1 and transformation of the nanobody recombinant plasmid into Escherichia coli BL21 (DE3)

[0155] Select the clones with positive results, and inoculate the original strain TG1 glycerol stock containing the nanobody nucleic acid into 5 mL of fresh LB-A medium at a ratio of 1:1000, and culture at 37°C 200 rpm overnight. The next day, use the Plasmid mini kit (OMEGA) according to the instructions to extract the plasmid. After verification, 1 μL of the above plasmid is transformed into 100 μL of competent cells, gently mixed, placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45 seconds, and cooled in an ice bath for 2 minutes. Add 600 μL of LB medium to the centrifuge tube and culture at 37°C with shaking for 60 minutes. Take 100 μL of the supernatant, spread it on an LB-A plate, and culture it upside down at 37°C overnight.

[0156] 3.5 Inducible expression of nanobodies

[0157] Pick the above monoclonal colony and culture in LB-A medium at 37°C with shaking overnight. The next day, add 100 mL of fresh LB-A medium at a 1:100 ratio and culture at 37°C with shaking for 3 hours until the OD600 of the culture is approximately 0.8. Add IPTG to a final concentration of 1 mM and induce overnight at 30°C, 200 rpm. The next day, collect the cells by centrifugation at 8000 rpm at 4°C for 10 minutes and resuspend the pellet in 1.5 mL of pre-chilled TES buffer. Incubate on ice for 2 minutes, gently shake for 30 seconds, and repeat this cycle six times. Add 3.0 mL of TES / 4 (dilute TES 4-fold with water), gently shake for 30 seconds, and then incubate on ice for 2 minutes. Repeat the shaking and ice-incubation steps six times. Centrifuge at 9000 rpm at 4°C for 10 minutes and collect approximately 4.5 mL of supernatant (periplasmic extract).

[0158] 3.6 Purification and identification of nanobodies

[0159] After resuspending IMAC Sepharose (GE), take 2mL and add it to the gravity column. Let it stand for 30 minutes to allow the sepharose to naturally settle at the bottom of the gravity column and flow out the preservation buffer. Add 2 times the column volume of nickel sulfate solution (0.1M) and flow out the nickel sulfate solution at a flow rate of about 8 seconds / drop; add 10 times the column volume of equilibrium buffer to balance and wash the sepharose, and keep the flow rate unchanged; dilute the sample 2 times with equilibrium buffer, add it to the gravity column, adjust the flow rate to 6 seconds / drop, and collect the penetration liquid; add 10 times the column volume of washing buffer to wash the sepharose, maintain the flow rate unchanged, and collect the washing liquid; add 3 times the column volume of elution buffer, maintain the flow rate at 6 seconds / drop, and collect the eluate containing the target protein; finally, add 10 times the column volume of equilibrium buffer, 10 times the column volume of pure water and 10 times the column volume of 20% ethanol to wash the sepharose, and finally retain 4mL of 20% ethanol to preserve the column. The above collected samples were respectively subjected to SDS-PAGE detection ( Figure 7 : M is Thermo Fisher protein marker, product number 26616; lanes 1-3 are the purified nanoantibodies 1A5, 1B6, and 1H9, respectively). The results are shown in Figure 7 It was shown that all three nanobodies were expressed.

[0160] The amino acid sequence of 1B6 was analyzed for the antibody heavy chain using Vector NTI software to determine the framework regions (FR) and complementary determining regions (CDR) of the variable region.

[0161] A preferred embodiment of the Nanobody screened by the present invention is named "1B6". By DNA sequencing, the heavy chain nucleic acid sequence of the Nanobody 1B6 is shown in SEQ ID NO.9, and the variable region amino acid sequence is shown in SEQ ID NO.8, wherein the amino acid sequence at positions 1-25 is FR1 (as shown in SEQ ID NO.4), the amino acid sequence at positions 26-33 is CDR1 (as shown in SEQ ID NO.1), the amino acid sequence at positions 34-50 is FR2 (as shown in SEQ ID NO.5), the amino acid sequence at positions 51-58 is CDR2 (as shown in SEQ ID NO.2), the amino acid sequence at positions 59-96 is FR3 (as shown in SEQ ID NO.6), the amino acid sequence at positions 97-114 is CDR3 (as shown in SEQ ID NO.3), and the amino acid sequence at positions 115-126 is FR4 (as shown in SEQ ID NO.7).

[0162] Example 4 Determination of the affinity of nanobodies to antigens

[0163] 4.1 Chip Antigen Coupling

[0164] TfR1 was prepared into a 50 μg / mL working solution using sodium acetate buffer at different pH values ​​(pH 5.5, pH 5.0, pH 4.5, and pH 4.0). A 50 mM NaOH regeneration solution was also prepared. Electrostatic binding between the antigen and the surface of a chip (GE Biosystems) at different pH values ​​was analyzed using the template method in the Biacore T100 Protein Interaction Analysis System. The most neutral pH system was selected as the coupling condition, with the antigen concentration adjusted as needed, based on a signal increase of 5-fold relative to the RL. The chip was coupled using the instrument's built-in template method: blank coupling mode was selected for channel 1, and target coupling mode was selected for channel 2, with the target set to the designed theoretical coupling amount. The coupling process took approximately 60 minutes.

[0165] 4.2 Exploration of analyte concentration setting conditions and optimization of regeneration conditions

[0166] Use manual injection mode, select 2-1 mode injection for channels 1 and 2, and set the flow rate to 30μL / min. The injection conditions are all 120s, 30μL / min. The regeneration conditions are all 30s, 30μL / min. First, continue to empty the running buffer until all baselines are stable. Prepare nanoantibody solutions with a large concentration span, and configure them with running buffer. It is recommended to set them to 200μg / mL, 150μg / mL, 100μg / mL, 50μg / mL, 20μg / mL, 10μg / mL, and 2μg / mL. Prepare the regeneration solution and select the regeneration solution with four pH gradients of the glutamic acid hydrochloric acid system: 1.5, 2.0, 2.5, and 3.0. Manually inject 200μg / mL of analyte sample, observe channel 2, and regenerate from the regeneration buffer with the most neutral pH until the response line of channel 2 after regeneration returns to the same height as the baseline. Manually inject another 200 μg / mL analyte sample, observe the signal change in channel 2-1, and record the binding amount. After regeneration with the regeneration solution used to return the response line to baseline in the previous step, manually inject another 200 μg / mL analyte sample, observe the signal change in channel 2-1, and record the binding amount. Compare the binding amount with the previous value. If the deviation is less than 5%, the regeneration solution at this pH is considered optimal. If the binding amount of the second injection is low, continue the experiment with a regeneration buffer at a lower pH. Use the selected optimal regeneration solution as the chip surface regeneration reagent after each injection. Inject samples at the analyte concentrations set above and analyze the binding amount at each concentration to ultimately determine the concentration gradient required for affinity testing.

[0167] 4.3 Affinity test

[0168] Following the optimized sample concentration gradient, the solution was regenerated and the affinity between the nanobody and the antigen was tested using the instrument's built-in template method (with injection conditions set to 60 seconds at 30 μL / min, dissociation time: 600 seconds, and regeneration conditions: 30 seconds at 30 μL / min). The signal in channel 2-1 was monitored continuously. The affinity test process took approximately 200 minutes.

[0169] 4.4 Results Analysis

[0170] Several appropriate concentration gradient binding and dissociation curves were selected and fitted using a 1:1 binding model. Finally, the affinity values ​​and important parameters such as binding constants and dissociation constants were obtained. The results are shown in Table 1. The results showed that all three nanobodies could specifically bind to the TfR1 protein coupled to the chip, with 1B6 having an affinity of 2.28E-9.

[0171] Table 1 Affinity values, binding constants and dissociation constants of nanobodies

[0172] Antibody <![CDATA[k on (M -1 ·s -1 )]]> <![CDATA[k off (s -1 )]]> <![CDATA[K D (M)]]> 1A5 <![CDATA[8.084×10 4 ]]> <![CDATA[1.32×10 -3 ]]> <![CDATA[1.6×10 -8 ]]> 1B6 <![CDATA[5.726×10 4 ]]> <![CDATA[1.310×10 -4 ]]> <![CDATA[2.28×10 -9 ]]> 1H9 <![CDATA[2.742×10 4 ]]> <![CDATA[1.866×10 -3 ]]> <![CDATA[6.806×10 -8 ]]>

[0173] Example 5 Activity Analysis of Nanobodies

[0174] 5.1 Determination of antibody binding affinity to target cells

[0175] Human breast cancer cells (MCF7) express TfR1 at high levels, and the expression level is positively correlated with their proliferation state. This example uses these cells as an experimental model to verify the ability of the three nanobodies in Example 4 to specifically target and bind to cell surface TfR1 in vitro.

[0176] Experimental method: Three nanoantibodies 1A5, 1B6 and 1H9 were cloned into the pFUSE-hIgG1-Fc vector and fused with IgG1-Fc for expression. At the same time, an unrelated nanoantibody Nb control As a negative control. MCF7 cells in the logarithmic growth phase were digested and resuspended in FACS buffer (PBS containing 1% FBS). A negative control or gradient-diluted anti-TfR1 antibody (initial concentration was 10 μg / mL, 2-fold dilution, 8 concentration gradients) was added for incubation; after incubation on ice for 30 minutes, the supernatant was discarded, and after washing once with PBS, anti-human IgG flow cytometry antibody anti-human Fc-PE (Invitrogen, 12-4998-82) was added. After incubation on ice for 30 minutes, the supernatant was discarded after centrifugation. After washing twice with PBS, flow cytometry was performed, and MFI was calculated using Flowjox software, and data were processed using Graphpad software.

[0177] The results are shown in Table 2. Antibodies 1B6 and 1H9 bound to MCF7 cells, while 1A5 and the negative control Nb NGF Does not bind to MCF7 cells.

[0178] Table 2 1B6, 1H9, 1A5 and Nb control Effective concentration for binding to MCF7 cells

[0179] Antibody 1A5 1B6 1H9 <![CDATA[Nb control ]]> <![CDATA[EC 50 (nM)]]> 57.8 64.9 NA NA

[0180] 5.2 Elisa detection of Tf-TfR1 binding inhibitory activity of nanoantibodies

[0181] The TfR1 described in Example 1 was adjusted to 5.0 μg / mL in CBS and TM Dispense the dilution into a 96-well plate at 100 μL / well and incubate at 4°C overnight. The next day, discard the supernatant and wash the plate five times with 0.05% PBST. Add 250 μL of 1% BSA to each well, block at 37°C for 1 hour, and wash the plate five times with PBST. Add 50 μL of HRP-labeled Tf (2 μg / mL) to each well, followed by 50 μL of 1A5, 1B6, and 1H9 (10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, and 0.001 μg / mL) per well. As a positive control, add 50 μL of HRP-labeled Tf (2 μg / mL) to each well, followed by the corresponding concentrations of A24 antibody, as described in patent document US2008 / 0193453. Incubate at 37°C for 1 hour, and wash the plate five times with PBST. Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 15-30 min. Then add 100 μL of 2 M sulfuric acid stop solution to each well and measure the absorbance at 450 nm using a microplate reader.

[0182] The results are as follows Figure 8 The results showed that 1B6 completely inhibited Tf-TfR1 binding at a relatively low concentration (0.01 μg / mL); 1A5 had a certain degree of inhibitory effect, but the effect was weaker than that of the A24 antibody; and 1H9 had no inhibitory effect. These results indicate that 1B6 can specifically inhibit Tf-TfR1 binding and is significantly superior to the A24 antibody.

[0183] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A nanobody targeting TfR1, characterized in that The nanobody comprises three complementary determining regions CDR1, CDR2, and CDR3; The amino acid sequence of the complementary determining region CDR1 of the nanobody is shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO.

3.

2. The nanobody targeting TfR1 according to claim 1, characterized in that The nanobody targeting TfR1 also includes four framework regions FR1, FR2, FR3, and FR4, wherein: The amino acid sequence of the framework region FR1 of the Nanobody is shown in SEQ ID NO.4; The amino acid sequence of the framework region FR2 of the Nanobody is shown in SEQ ID NO.5; The amino acid sequence of the framework region FR3 of the Nanobody is shown in SEQ ID NO.6; The amino acid sequence of the framework region FR4 of the nanobody is shown in SEQ ID NO.

7.

3. The nanobody targeting TfR1 according to claim 1, characterized in that The variable region amino acid sequence of the Nanobody is selected from any one of the following: I. the amino acid sequence shown in SEQ ID NO.8; or II. an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in I, and having the same function as the amino acid sequence described in I; or III, an amino acid sequence that has more than 80% identity with the amino acid sequence described in I or II and exerts similar physiological activity.

4. The nanobody targeting TfR1 according to claim 3, characterized in that The nanobody also includes a derivative polypeptide obtained by modifying the amino acid sequence of the variable region, wherein the modification method includes functional group modification; The functional group modification includes modifying the FR region with a hydrophilic group or replacing the hydrophobic residues in the FR region.

5. The nanobody targeting TfR1 according to claim 3, characterized in that The nanobody also includes a derivative polypeptide obtained by modifying the amino acid sequence of the variable region, wherein the modification includes adding a molecular tag; The molecular labels include polyethylene glycol, streptavidin, biotin, radioisotopes or fluorescent agents.

6. A nucleic acid molecule encoding a nanobody targeting TfR1 as described in any one of claims 1 to 3.

7. The nucleic acid molecule according to claim 6, wherein The nucleic acid sequence of the nucleic acid molecule is shown as SEQ ID NO.

9.

8. An expression vector, characterized in that Comprising the nucleic acid molecule according to claim 6 or 7.

9. The expression vector according to claim 8, wherein The expression vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or mammalian cell virus.

10. A host cell transformed or transfected with the expression vector according to claim 8 or 9; The host cell is a microbial cell.

11. A conjugate or a coupling substance, characterized in that Comprising a chemically labeled or biologically labeled nanobody targeting TfR1 as described in any one of claims 1 to 5 and an acceptable excipient or carrier.

12. Use of the nanobody targeting TfR1 according to any one of claims 1 to 5 or the conjugate or conjugate according to claim 11 in the preparation of a TfR1 detection antibody reagent and / or kit.

13. A TfR1 detection antibody reagent or detection kit, characterized in that: It comprises the nanobody targeting TfR1 as described in any one of claims 1 to 5 and / or the conjugate or coupling as described in claim 11 and an acceptable adjuvant and / or carrier.

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