A nanoantibody targeting CD36 and its application

Abstract

The present invention discloses a nano antibody targeting CD36 and its application, which belongs to the field of biomedicine. The nano antibody targeting CD36 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 CD36. The nano antibody provided by the present invention has specific recognition and binding ability to CD36, and the affinity of the nano antibody can reach 5.408E-09. The nano antibody can bind to cell surface CD36 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 a nano antibody targeting CD36, its coding sequence and its application in detection. Background Art

[0002] CD36 (cluster of differentiation 36) belongs to the class B scavenger receptor family and is a multifunctional membrane protein widely present on the surfaces of various cells. The human CD36 gene is located on chromosome 7q11. It is approximately 46 kb long, includes 15 exons, and is transcribed and translated into 472 amino acids with a theoretical molecular mass of 53 kDa. Structurally, the CD36 protein consists of two transmembrane domains, a large extracellular domain containing the ligand binding site, and short cytoplasmic tails at the N- and C-termini. The extracellular domain contains two hydrophobic pockets. Pocket 1 primarily functions to transport oxidized low-density lipoprotein (oxLDL) and advanced glycation end products (AGEs), while pocket 2 serves as a transport channel for fatty acids (FAs).

[0003] Studies have found that CD36 is closely related to abnormal lipid metabolism in cancer. CD36 is highly expressed on the surface of many cancer cells, including ovarian cancer, breast cancer, gastric cancer, melanoma, oral squamous cell carcinoma, etc. In cancer cells, CD36 acts as a fatty acid transport receptor protein, mediating the uptake of fatty acids outside cancer cells, providing huge energy for the growth and metastasis of cancer cells. In addition, cancer cells use CD36 to mediate excessive uptake of fatty acids, increase the oxidation of fatty acids, thereby changing the metabolic mechanism of cancer cells and leading to drug resistance. In addition, many studies have shown that CD36 is also involved in immune regulation in the tumor microenvironment, mediating CD8 + The accumulation of lipid peroxides in T cells reduces cytotoxic factors. Therefore, given the widespread expression of CD36 in cancer cells and its role in promoting the development of various cancers, CD36 is expected to become a very promising broad-spectrum cancer therapeutic target, producing anti-cancer effects by inhibiting the function of CD36.

[0004] 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.

[0005] 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 against CD36 antigen and the application of their coding sequences in detection has important practical significance. Summary of the Invention

[0006] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a nanobody targeting CD36 and its application. The nanobody targeting CD36 provided by the present invention has excellent specific antigen binding ability and can reduce its own immunogenicity compared with traditional monoclonal antibodies.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] In a first aspect of the present invention, a nanobody targeting CD36 is provided, wherein the variable region of the nanobody has a CDR1 region as shown in SEQ ID NO.1, a CDR2 region as shown in SEQ ID NO.2, and a CDR3 region as shown in SEQ ID NO.3.

[0009] In some embodiments of the present invention, the Nanobody further comprises four framework regions FR1, FR2, FR3, and FR4, wherein:

[0010] The amino acid sequence of the framework region FR1 of the Nanobody is shown in SEQ ID NO.4;

[0011] The amino acid sequence of the framework region FR2 of the Nanobody is shown in SEQ ID NO.5;

[0012] The amino acid sequence of the framework region FR3 of the Nanobody is shown in SEQ ID NO.6;

[0013] The amino acid sequence of the framework region FR4 of the nanobody is shown in SEQ ID NO.7.

[0014] 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:

[0015] (1) the amino acid sequence shown in SEQ ID NO. 8;

[0016] (2) An amino acid sequence having a sequence similarity of 80% and still exhibiting similar physiological activity after adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO. 8.

[0017] In some embodiments of the present invention, a preferred embodiment of the Nanobody having the variable region sequence obtained by screening is Nanobody 1E5, the variable region amino acid sequence of said 1E5 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 34-49 is FR2 (as shown in SEQ ID NO.5), the amino acid sequence at positions 50-56 is CDR2 (as shown in SEQ ID NO.2), the amino acid sequence at positions 57-94 is FR3 (as shown in SEQ ID NO.6), the amino acid sequence at positions 95-105 is CDR3 (as shown in SEQ ID NO.3), and the amino acid sequence at positions 106-116 is FR4 (as shown in SEQ ID NO.7).

[0018] In some embodiments of the present invention, the Nanobody further comprises a derivative polypeptide obtained by modifying the amino acid sequence described in (1) or (2), 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 modifying the FR region with a hydrophilic group or replacing 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.

[0019] The second aspect of the present invention provides a nucleic acid molecule encoding the Nanobody.

[0020] 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.

[0021] In some embodiments of the present invention, the nucleic acid sequence of the nucleic acid molecule is shown as SEQ ID NO.9.

[0022] In the third aspect of the present invention, the present invention also provides an expression vector comprising the nucleic acid molecule.

[0023] 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.

[0024] In a fourth aspect, the present invention also provides a host cell transformed or transfected with the expression vector.

[0025] 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.

[0026] In a fifth aspect, the present invention further provides a conjugate or a coupling comprising the chemically labeled or biologically labeled Nanobody and an acceptable excipient or carrier.

[0027] 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.

[0028] In a sixth aspect of the present invention, based on the above research, the present invention also provides the use of the nanobody or the conjugate or the conjugate in the preparation of a drug targeting CD36.

[0029] In a seventh aspect, the present invention further provides the use of the nanobody or the conjugate or the conjugate in the preparation of CD36 detection antibody reagents and / or kits.

[0030] In an eighth aspect of the present invention, based on the above research, the present invention also provides a CD36 detection antibody reagent, comprising the nanobody and / or the conjugate or coupling and an acceptable adjuvant and / or carrier.

[0031] The beneficial effects of the present invention are:

[0032] The CD36-targeting Nanobody provided by the present invention has unique CDR1, CDR2, and CDR3 regions, which enable the Nanobody to specifically recognize and bind to the CD36 antigen. The Nanobody has an affinity of up to 5.408E-09, demonstrating highly specific binding activity. Furthermore, the Nanobody provided by the present invention can bind to cell surface CD36 and inhibit its activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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.

[0034] Figure 1 This is the SDS-PAGE image of the recombinant CD36 antigen purification;

[0035] Figure 2 This is the electrophoresis identification diagram of the extracted total RNA;

[0036] Figure 3 This is the electrophoresis identification diagram of the first round of PCR amplification of the antibody variable region gene;

[0037] Figure 4 This is the electrophoresis identification diagram of the second round of PCR amplification of the antibody variable region gene;

[0038] Figure 5 This is the electrophoresis identification diagram of transformants identified by colony PCR;

[0039] Figure 6 This is the SDS-PAGE image of nanobody purification;

[0040] Figure 7 This is a diagram showing the activity of nanoantibodies in neutralizing cell surface CD36. DETAILED DESCRIPTION

[0041] The present invention discloses a nanobody targeting CD36 and its coding sequence for use in detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions 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. It is obvious that relevant personnel can 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.

[0042] The present invention first immunizes alpacas with the recombinant CD36 antigen. After four immunizations, peripheral blood lymphocytes are isolated from the immunized alpacas, and a specific CD36-targeting nanobody gene library is constructed. The recombinant CD36 antigen is coated on an ELISA plate, and phage display technology is used to screen the immunized nanobody library for nanobodies targeting the recombinant CD36 antigen. A nanobody expression vector is constructed, introduced into Escherichia coli, and purified after expression. The purified nanobody is able to specifically bind to the cell surface CD36 protein and neutralize its activity.

[0043] The present invention provides a nano antibody capable of specifically binding to CD36 protein, a coding sequence, a preparation method and an application thereof.

[0044] Specifically:

[0045] The variable region of the nanobody has three complementary determining regions CDR1, CDR2, and CDR3, wherein:

[0046] The CDR1 sequence consists of the amino acid sequence described in SEQ ID NO.1;

[0047] The CDR2 sequence consists of the amino acid sequence described in SEQ ID NO.2;

[0048] The CDR3 sequence consists of the amino acid sequence described in SEQ ID NO.3.

[0049] The amino acid sequence of the variable region of the nanobody is shown in SEQ ID NO.8.

[0050] 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.

[0051] The present invention also provides the use of the above nanobody in the preparation of drugs targeting CD36.

[0052] The present invention also provides the use of the above-mentioned nanobody in preparing CD36 detection antibody reagents and / or kits.

[0053] The sequences involved in the present invention are as follows:

[0054] Amino acid sequence of the CDR1 region of Nanobody 1E5:

[0055] 5'-GSILNFYA-3' (SEQ ID NO. 1).

[0056] Amino acid sequence of the CDR2 region of Nanobody 1E5:

[0057] 5'-VIAGGST-3' (SEQ ID NO. 2).

[0058] Amino acid sequence of the CDR3 region of Nanobody 1E5:

[0059] 5'-NAQVVFDRTNY-3' (SEQ ID NO. 3).

[0060] Amino acid sequence of the FR1 region of Nanobody 1E5:

[0061] 5'-QVQLQESGGGLVQPGGSLRLSCAAS-3'(SEQ ID NO.4)

[0062] Amino acid sequence of the FR2 region of Nanobody 1E5:

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

[0064] The amino acid sequence of the FR3 region of Nanobody 1E5:

[0065] 5'-KYEDSVKGRFTISTDVSKNTVYMEMNNLKPEDTAVYYC-3'(SEQ ID NO.6)

[0066] The amino acid sequence of the FR4 region of Nanobody 1E5:

[0067] 5'-WGQGTQVTVSS-3'(SEQ ID NO.7)

[0068] The amino acid sequence of the variable region of Nanobody 1E5:

[0069] 5'-

[0070] QVQLQESGGGLVQPGGSLRLSCAASGSILNFYAMGWYRQAPGKQRELVAVIAGGST

[0071] KYEDSVKGRFTISTDVSKNTVYMEMNNLKPEDTAVYYCNAQVVFDRTNYWGQGTQ VTVSS-3' (SEQ ID NO. 8).

[0072] The nucleic acid sequence of Nanobody 1E5:

[0073] 5'-

[0074] caggtgcagctgcaggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggaagcatcctc

[0075] aatttctatgccatgggctggtaccgccaggctccagggaagcagcgcgagttggtcgcagttattgccggtggtagtacaaagtatg

[0076] aagactccgtgaagggccgattcaccatctccacagacgtgtccaagaacacagtgtatatggagatgaacaacctgaaacctgaggacacggccgtctattactgtaatgcacaagtggttttcgaccggacgaactactggggccaggggacccaggtcaccgtctcctca-3' (SEQ ID NO. 9).

[0077] CALL001 primer sequence:

[0078] 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO. 10).

[0079] CALL002 primer sequence:

[0080] 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO. 11).

[0081] VHH-Back primer sequence:

[0082] 5'-GATGGTGCAGCTGCAGGAGTCTGGRGGAGG-3' (SEQ ID NO. 12).

[0083] VHH-For primer sequence:

[0084] 5'-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3' (SEQ ID NO. 13).

[0085] pMES-F primer sequences:

[0086] 5'-GCCGCTGGATTGTTATTACTC-3' (SEQ ID NO. 14).

[0087] pMES-R primer sequences:

[0088] 5'-CTTTCAACAGTGGAACCGTAG-3' (SEQ ID NO. 15).

[0089] The CD36-targeting Nanobody provided by the present invention has unique CDR1, CDR2, and CDR3 regions, which enable the Nanobody to specifically recognize and bind to the CD36 antigen. The Nanobody has an affinity of up to 5.408E-09, demonstrating highly specific binding activity. Furthermore, the Nanobody provided by the present invention can bind to cell surface CD36 and inhibit its activity.

[0090] 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.

[0091] 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.

[0092] 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).

[0093] "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.

[0094] "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 in the framework (called FR) segment can be modified to retain binding affinity.

[0095] 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, so 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.

[0096] The medicament contains at least one functional ingredient and 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 the pharmaceutically acceptable carrier depends, among other things, on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration.

[0097] 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.

[0098] "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.

[0099] The nanobodies provided by the present invention and the raw materials and reagents used in their applications can all be purchased from the market.

[0100] 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.

[0101] Example 1. Screening of anti-CD36 nanobodies

[0102] 1.1 Expression of CD36 recombinant protein

[0103] According to the amino acid sequence of human CD36 on NCBI (NP_000063.2), Gly30-Asn439 was synthesized, and a histidine tag was added to the C-terminus of the sequence and connected to the vector pCDNA3.1(+). After the plasmid was extracted without endotoxin, it was transfected into 293 cells in logarithmic growth. 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 protein marker, lane 1 is the purified CD36 antigen protein).

[0104] 1.2 Alpaca Immunity

[0105] A healthy adult alpaca was selected and immunized with a recombinant CD36 antigen mixed with Freund's adjuvant at a 1:1 ratio. 6-7 μg / kg was administered subcutaneously to the alpaca via multiple injections at the back for four immunizations, with a two-week interval between immunizations. Subsequently, 10 mL of peripheral blood was collected from the alpaca for the construction of a phage display library.

[0106] 1.3 Isolation of alpaca lymphocytes

[0107] 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℃.

[0108] 1.4 RNA extraction

[0109] Repeatedly pipette 1 mL of Tipure Isolation Reagent containing lymphocytes and let it sit for 5 minutes; add 200 μL of chloroform, vortex for 30 seconds, and let it sit for another 5 minutes; centrifuge at 12,000 g at 4°C for 15 minutes, aspirate the aqueous phase and transfer it to a new EP tube; add an equal amount of isopropanol and let it sit for 10 minutes; centrifuge at 12,000 g at 4°C for 10 minutes, discard the supernatant; wash with 1 mL of pre-cooled 70% ethanol, centrifuge at 7,500 g at 4°C for 5 minutes, discard the supernatant and dry for 5 minutes; add 30 μL of RNase-free water to dissolve the precipitate and adjust the concentration to 1 μg / μL for gel electrophoresis detection. The results are shown in the figure. Figure 2 , lane 1.

[0110] 1.5 Reverse transcription and cDNA synthesis

[0111] According to the instructions of the reverse transcription kit (Transcripor First Stand cDNA Synthesis KIT from Roche), reverse transcription of cDNA was performed using the RNA obtained in step 1.4 as a template.

[0112] 1.6 Amplification of Antibody Variable Region Genes

[0113] The cDNA obtained by reverse transcription was used as a template for PCR reaction. Two rounds of amplification were performed. The primer sequences for the first round of PCR were as follows:

[0114] CALL001:GTCCTGGCTGCTCTTCTACAAGG(SEQ ID NO.10)

[0115] CALL002:GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO.11)

[0116] PCR reaction conditions and procedures were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds,

[0117] 7 cycles; 72℃ for 7 minutes. Use agarose gel recovery kit to recover the 700bp band, and finally adjust the nucleic acid concentration to 5ng / μL with water ( Figure 3 : M is a marker; 1 is the first-round PCR product).

[0118] The primer sequences for the second round of PCR are as follows:

[0119] VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO.12)

[0120] VHH-For:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO.13)

[0121] PCR reaction conditions and procedures were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 15

[0122] 7 cycles; 72°C for 7 minutes. Purify the PCR product using a PCR product recovery kit ( Figure 4 : M is a marker; 1 is the second-round PCR product).

[0123] 1.7 Vector Construction

[0124] The pMES4 vector (purchased from Biovector) and the second PCR product were double-digested with PstI and BstEII, respectively. 1.5 μg of the digested vector and 450 ng of the second PCR product were added to 15 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. The product was recovered using a PCR product recovery kit and eluted with 20 μL of water.

[0125] 1.8 Electroporation and storage capacity determination

[0126] Take 10 μL of the purified ligation product and add it to a pre-cooled electroporation cup containing 50 μL of E. coli TG1 competent cells and place it in an electroporator (ECM630 electroporator from BTX, USA) for electroporation. Remove the electroporation cup, recover and culture the transformants. Randomly select clones and perform colony PCR identification ( Figure 5 M is a marker; N is a negative control; 1-20 are randomly selected monoclonal PCR identification products. The reservoir capacity was calculated based on the PCR positive rate (reservoir capacity = number of clones × dilution factor × PCR positive rate × 10). The primer sequences are as follows:

[0127] pMES-F: GCCGCTGGATTGTTATTACTC (SEQ ID NO.14)

[0128] pMES-R: CTTTCAACAGTGGAACCGTAG (SEQ ID NO. 15).

[0129] Amplification of 1.9M13 phage

[0130] Take the recovered bacterial solution and inoculate it into YT-AG medium, and culture it at 37℃200rpm until the culture OD 600 =0.5. Take out 10mL of bacterial solution and add 4×10 10VCSM13 cells were statically infected at 37°C for 30 minutes. Centrifuge at 4000 rpm for 10 minutes at room temperature and discard the supernatant. Resuspend the cells in 2×YT-AK medium (containing ampicillin and kanamycin) and incubate overnight at 37°C at 200 rpm. After centrifugation, transfer 40 mL of the supernatant to a tube and add 10 mL of PEG / NaCl (20% / 2.5 M) solution, mix thoroughly, and discard the supernatant after centrifugation. Wash the pellet with 1 mL of ice-cold PBS and centrifuge. Remove 250 μL of the supernatant in pre-chilled PEG / NaCl, mix thoroughly, and resuspend.

[0131] Determine the phage titer: culture TG1 to OD 600 =0.4, gradient dilution of phage was performed with LB medium, and the doubly diluted phage TG1 culture was mixed and cultured. The next day, the plaque formation in the culture plate was observed, and the number of plaques on the dilution gradient plates ranging from 30 to 300 was counted and the phage titer (pfu) was calculated according to the following formula.

[0132] Phage titer (pfu / mL) = dilution factor × number of plaques × 100

[0133] 1.10 Phage Display of Nanobodies

[0134] Take 1 mL of the bacterial solution of the nanobody immune library and inoculate it into two 10 mL 2×YT-AG medium, and culture at 37°C and 200 rpm until OD 600 =0.5, add 4×10 10 Infect with pfu helper phage at 37°C for 30 minutes. Centrifuge at 4000 rpm for 10 minutes at room temperature, discard the supernatant, and resuspend in 3 mL of 2×YT-AK medium. Finally, add the suspension to 100 mL of 2YT-AK medium and incubate overnight at 37°C, 200 rpm. Concentrate and precipitate the displayed phage the next day, and determine the titer.

[0135] Displayed phage titer (cfu / mL) = dilution factor × number of colonies × 100 × 2

[0136] 1.11 Solid-phase panning of phage display libraries

[0137] All EP tubes used in this example were pre-filled with PBS buffer containing 1% BSA and allowed to stand at room temperature for 10 minutes to seal the tube walls to reduce the adsorption of trace proteins by the tube walls and the impact on screening results.

[0138] Dilute CD36 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 100 μL of 1% BSA to each well, block at 37°C for 1 hour, and wash the plate 5 times with PBST; add 100 μL of 1% BSA to each well, incubate at 37°C for 1 hour, and wash the plate 5 times with PBST;11 Incubate the cfu-displaying phage 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 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. Perform panning 3-4 times in total.

[0139] 1.12 Phage ELISA screening of positive clones

[0140] Screen positive clones by ELISA. Coat an ELISA plate with CD36 recombinant antigen, block with 5% BSA, and wash with PBST. Add 100 μL of phage supernatant to each well and incubate at 37°C for 1 hour. Discard the supernatant and add HRP-conjugated mouse anti-M13 secondary antibody. Incubate at 37°C for 1 hour. Discard the supernatant and add TMB solution. Incubate at room temperature for 5 hours. Add 2 M sulfuric acid stop buffer to each well and read at 450 nm using a microplate reader. Select clones with positive phage ELISA results and send for sequencing.

[0141] Example 2. Expression and purification of anti-CD36 nanobody

[0142] 2.1 Amplification of the original nanobody strain TG1 and transformation of the nanobody recombinant plasmid into Escherichia coli BL21 (DE3)

[0143] Select the clones with positive results, inoculate the original strain TG1 glycerol bacteria containing nanobody nucleic acid into 5mL fresh LB-A medium at a ratio of 1:1000, and culture at 37℃ 200rpm overnight. The next day, use Plasmid mini kit (OMEGA) to extract the plasmid according to the instructions. After verification, 1μL of the above plasmid was transformed into 100μl competent cells, gently mixed, placed on ice for 30 minutes, heat-shocked in a 42℃ water bath for 90 seconds, and cooled in an ice bath for 3 minutes. Add 600μL LB medium to the centrifuge tube and shake-culture at 37℃ for 60 minutes. Take 100μL of the supernatant, spread it on the LB-A plate with a triangular spreader, and culture it upside down at 37℃ overnight.

[0144] 2.2 Inducible Expression of Nanobodies

[0145] Pick the positive monoclonal colony obtained in step 2.1 and culture it in LB-A medium at 37℃ overnight. The next day, take the bacterial solution and add 100mL fresh LB-A medium at a ratio of 1:100, and culture it at 37℃ for 3h until the bacterial solution OD reaches 600= around 0.8, add IPTG to a final concentration of 1 mM, and induce overnight at 30°C. On the third day, collect the cells by centrifugation at 8000 rpm for 10 minutes, and resuspend the pellet in 1.5 mL of pre-chilled TES buffer. After a 2-minute ice bath, gently shake for 30 seconds, and repeat this cycle six times. Add 3.0 mL of TES / 4 (TES diluted 4-fold with water), gently shake for 30 seconds, and let stand on ice for 2 minutes. Repeat the shaking and standing steps six times. Centrifuge at 9000 rpm at 4°C for 10 minutes, and collect approximately 4.5 mL of supernatant (periplasmic extract).

[0146] 2.3 Purification and identification of nanobodies

[0147] After resuspending IMAC Sepharose (GE), take 2mL and add it to the gravity column. Let it stand for 30min 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 8s / 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 6s / 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 6s / 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 6 :M is Thermo Fisher protein marker, product number 26616; lanes 1-3 are purified nanoantibodies 1E5, 1C4, and 2H5). The results are shown in Figure 6 As shown, all three nanobodies were expressed.

[0148] The amino acid sequence of the nanoparticle 1E5 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.

[0149] A preferred embodiment of the Nanobody screened by the present invention is named "1E5". By DNA sequencing, the heavy chain nucleic acid sequence of the Nanobody 1E5 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 34-49 is FR2 (as shown in SEQ ID NO.5), the amino acid sequence at positions 50-56 is CDR2 (as shown in SEQ ID NO.2), the amino acid sequence at positions 57-94 is FR3 (as shown in SEQ ID NO.6), the amino acid sequence at positions 95-105 is CDR3 (as shown in SEQ ID NO.3), and the amino acid sequence at positions 106-116 is FR4 (as shown in SEQ ID NO.7).

[0150] Example 3 Determination of the affinity of nanobodies to antigens

[0151] 3.1 Chip Antigen Coupling

[0152] CD36 was prepared at a working concentration of 50 μg / mL in 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) was analyzed using the template method in the Biacore T100 Protein Interaction Analysis System at different pH values. The most neutral pH was selected as the coupling condition, with the antigen concentration adjusted as needed, based on a signal increase of 5-fold 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.

[0153] 3.2 Exploration of analyte concentration setting conditions and optimization of regeneration conditions

[0154] 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.

[0155] 3.3 Affinity test

[0156] 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.

[0157] 3.4 Results Analysis

[0158] Several appropriate concentration gradient binding and dissociation curves were selected and fitted using a 1:1 binding model. Finally, affinity values ​​and important parameters such as binding and dissociation constants were obtained. The results are shown in Table 1. The results showed that the 1E5 nanobody could specifically bind to the CD36 protein coupled to the chip with an affinity of 5.408E-09.

[0159] Table 1 Affinity values, binding constants and dissociation constants of nanobodies

[0160] Antibody <![CDATA[k on (M -1 ·s -1 )]]> <![CDATA[k off (s -1 )]]> <![CDATA[K D (M)]]> 1E5 <![CDATA[4.550×10 4 ]]> <![CDATA[2.461×10 -4 ]]> <![CDATA[5.408×10 -9 ]]> 1C4 <![CDATA[2.727×10 4 ]]> <![CDATA[4.310×10 -4 ]]> <![CDATA[1.580×10 -8 ]]> 2H5 <![CDATA[3.130×10 4 ]]> <![CDATA[6.347×10 -4 ]]> <![CDATA[2.028×10 -8 ]]>

[0161] Example 4. Activity analysis of nanobodies

[0162] 4.1 Determination of antibody binding affinity to target cells

[0163] Human colon cancer cells (SW48) highly express CD36 protein on their cell membranes. In this example, these cells were used as an experimental model to verify the binding ability of three specific CD36-targeting nanoantibodies to CD36 protein molecules on the cell membrane surface.

[0164] Experimental method: Three nanoantibodies 1E5, 1C4 and 2H5 were cloned into the pFUSE-hIgG1-Fc vector and fused with IgG1-Fc for expression. At the same time, the nanoantibody Nb that specifically targets β-NGF was selected. NGF (Preliminary laboratory screening) served as a negative control. SW48 cells in the logarithmic growth phase were digested and resuspended in FACS buffer (PBS containing 1% FBS). A negative control or serially diluted anti-CD36 antibody (initial concentration 10 μg / mL, 2-fold dilution, 8 concentration gradients) was added and incubated. After incubation on ice for 30 minutes, the supernatant was discarded. After washing once with PBS, anti-human IgG flow cytometry antibody anti-human Fc-PE (Invitrogen, 12-4998-82) was added and incubated on ice for 30 minutes. The cells were then centrifuged and the supernatant discarded. After washing twice with PBS, flow cytometry analysis was performed. MFI was calculated using Flowjox software, and data were processed using Graphpad software.

[0165] The results are shown in Table 2. Nanobodies 1E5 and 1C4 bind to SW48 cells, while 2H5 and negative control Nb NGF Does not bind to SW48 cells.

[0166] Table 2 1E5, 1C4, 2H5 and Nb NGF Effective concentration for binding to SW48 cells

[0167] Antibody 1E5 1C4 2H5 <![CDATA[Nb NGF ]]> <![CDATA[EC 50 (nM)]]> 53.8 66.3 NA NA

[0168] 4.2 Determination of antibody neutralization activity of cell surface CD36

[0169] CD36 on the cell surface has the function of taking up oxidized low-density lipoprotein (oxLDL). In this example, human colon cancer cells (SW48) were used as a model to verify the neutralizing effects of two antibodies, 1E5 and 1C4, after binding to SW48 cells.

[0170] Experimental method: SW48 cells were cultured in culture medium containing oxLDL for 24 hours in advance, and then the cells were incubated with 1E5 and 1C4 antibodies (20 μg / mL). After 2 hours, the cell supernatant was collected and the oxLDL content in the cell supernatant was detected using an oxLDL ELISA kit.

[0171] The results are as follows Figure 7 As shown, antibody 1E5 has a significant neutralizing effect on cellular uptake of oxLDL, while the neutralizing effect of antibody 1C4 is not obvious.

[0172] 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 CD36, characterized in that The variable region of the nanobody has a CDR1 region as shown in SEQ ID NO.1, a CDR2 region as shown in SEQ ID NO.2, and a CDR3 region as shown in SEQ ID NO.

3.

2. The CD36-targeting Nanobody according to claim 1, wherein The nanobody 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 CD36-targeting Nanobody according to claim 1, wherein The amino acid sequence of the Nanobody is selected from any one of the following: (1) the amino acid sequence shown in SEQ ID NO. 8; (2) An amino acid sequence having a sequence similarity of 80% and still exhibiting similar physiological activity after adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO.

8.

4. The CD36-targeting Nanobody according to claim 3, wherein The nanobody also includes a derivative polypeptide obtained by modifying the amino acid sequence described in (1) or (2), wherein the modification method includes functional group modification.

5. The CD36-targeting Nanobody according to claim 4, wherein The functional group modification includes modifying the FR region with a hydrophilic group or replacing the hydrophobic residues in the FR region.

6. The CD36-targeting Nanobody according to claim 3, wherein The nanobody also includes a derivative polypeptide obtained by modifying the amino acid sequence described in (1) or (2), wherein the modification method includes adding a molecular tag.

7. The CD36-targeting Nanobody according to claim 6, wherein The molecular labels include polyethylene glycol, streptavidin, biotin, radioisotopes or fluorescent agents.

8. A nucleic acid molecule encoding a Nanobody according to any one of claims 1 to 3.

9. The nucleic acid molecule according to claim 8, wherein The nucleic acid sequence of the nucleic acid molecule is shown as SEQ ID NO.

9.

10. An expression vector, characterized in that Comprising the nucleic acid molecule according to claim 8 or 9.

11. The expression vector according to claim 10, wherein The expression vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or mammalian cell virus.

12. A host cell transformed or transfected with the expression vector according to claim 10 or 11; The host cell is a microbial cell.

13. A conjugate or a coupling substance, characterized in that Comprising a chemically labeled or biologically labeled Nanobody as claimed in any one of claims 1 to 7 and an acceptable excipient or carrier.

14. Use of the Nanobody according to any one of claims 1 to 7 or the conjugate or conjugate according to claim 13 in the preparation of a CD36 detection antibody reagent and / or kit.

15. A CD36 detection antibody reagent, characterized in that: Comprising the Nanobody according to any one of claims 1 to 7 and / or the conjugate or coupling according to claim 13 and an acceptable adjuvant and / or carrier.

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