CN118638235B

CN118638235B - Nanometer antibody targeting TfR1 and application thereof

Info

Publication number: CN118638235B
Application number: CN202410908439.8A
Authority: CN
Inventors: 刘金燕; 刘文帅; 年锐; 刘晓萌
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2024-07-08
Filing date: 2024-07-08
Publication date: 2025-09-19
Anticipated expiration: 2044-07-08
Also published as: CN118638235A

Abstract

The invention discloses a TfR 1-targeted nano antibody and application thereof, and belongs to the field of biological medicine. The invention discloses a TfR1 targeting nanobody which has unique 3 complementarity determining regions CDR1 (SEQ ID NO. 1), CDR2 (SEQ ID NO. 2) and CDR3 (SEQ ID NO. 3), and also provides an expression vector containing a variable region coding sequence of the nanobody, a host cell containing the expression vector and application of the nanobody in preparing TfR1 targeting drugs. The nano antibody provided by the invention has specific recognition and binding capacity to TfR1, the affinity of the nano antibody can reach 2.28E-9, and the nano antibody can be combined with TfR1 on the cell surface and neutralize the activity of the TfR1, so that the nano antibody has good application prospect.

Description

Nanometer antibody targeting TfR1 and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a nanometer antibody for targeting TfR1, a coding sequence thereof and application thereof in detection.

Background

Transferrin receptor 1 (TRANSFERRIN RECEPTOR PROTEIN, tfr 1) is a type II transmembrane protein with a molecular weight of 90KDa, an important transmembrane protein that regulates the intracellular iron transport process. TfR1 is formed by disulfide cross-linking of two homodimeric subunits, and TfR1 monomers consist of a large extracellular C-terminal domain (671 amino acids) comprising a site of binding to a transporter, a transmembrane domain (28 amino acids) and an intracellular N-terminal domain. The C-terminal extracellular domain contains three n-chain glycosylation sites at asparagine residues 251, 317 and 727 and one o-chain glycosylation site at threonine 104, all of which are necessary for the receptor to function adequately.

Under normal physiological conditions, tfR1 interacts with iron transporters, mediating the entry of iron ions into cellular channels, playing a key role in regulating cellular iron metabolism. Iron is necessary for a variety of cellular processes and is also essential for DNA synthesis and cell proliferation. Iron deficiency inhibits cell growth, which leads to cell death. Cancer cells are more sensitive to iron deficiency than normal cells. After the cells become cancerous, a large amount of iron is needed to maintain a high cell proliferation rate, and excessive iron promotes the development of tumor cells, so that abnormal iron metabolism is considered as one of specific markers of tumors. Normally, tfR1 itself is tightly regulated with only basal expression in many tissues of the body. Only increased expression has been found on brain capillary endothelial cells, hepatocytes and rapidly proliferating cells. In addition, tfR1 expression on the surface of many malignant tumor cells is obviously increased, and the expression level is about 20 times that of normal cells, so that TfR1 is considered as a potential tumor marker, and polarity treatment aiming at TfR1 can effectively inhibit tumor growth and metastasis.

At present, a plurality of clinical medicines aiming at TfR1 are being developed, and the medicines are mainly used for treating cancers, anemia, iron metabolic disorder diseases, neurodegenerative diseases and the like. Wherein, PPMX-T003, CX-2029, DYNE-251, trontinemab and other medicaments are already in clinical stage 1/2. In recent years, targeting therapeutic strategies based on TfR1 are continuously developed, and TfR1 is expected to become an effective target molecule to participate in clinical treatment of various diseases.

In the application of both atopic diseases and tumor diseases, the immunogenicity of monoclonal antibodies has been one of the most important problems of developers, and the side effects of drugs can be reduced to a great extent by reducing the immunogenicity of the drugs to the greatest extent while ensuring the efficacy, so that many researchers reform monoclonal antibodies by reforming, reducing the molecular size of the antibodies, and then humanizing by amino acid mutation or affinity maturation at key sites. Most humanizations, however, reduce the affinity or stability of the antibody itself.

In alpaca peripheral blood there is a naturally deleted light chain antibody comprising only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but not as easily sticking to each other or even agglomerating as artificial engineered single chain antibody fragments (scFv). More importantly, the VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity to the antigen, the smallest unit known to bind the antigen of interest. The VHH crystals were 2.5nm long by 4nm and had a molecular weight of only 15kDa, which is also known as Nanobody (Nb). Nanobodies are comparable in affinity to their corresponding scFv relative to conventional four-chain antibodies, but exceed scFv in terms of solubility, stability, resistance to aggregation, refolding, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination. The nanometer antibody has small molecules and high stability, can be administrated in an atomization mode, improves the administration convenience and widens the application scene of the medicine. Therefore, the application of the nanometer antibody aiming at the TfR1 antigen and the coding sequence thereof in detection has important practical significance.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide the TfR 1-targeting nano antibody and the application thereof, and the TfR 1-targeting nano antibody provided by the invention has excellent specific antigen binding capacity and can reduce the immunogenicity of the antibody compared with the traditional monoclonal antibody.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

In a first aspect of the invention, there is provided a TfR 1-targeting nanobody comprising three complementarity determining regions CDR1, CDR2, CDR3, wherein:

I. The complementarity determining region CDR1 of the nano antibody has an amino acid sequence shown as SEQ ID NO.1, the complementarity determining region CDR2 has an amino acid sequence shown as SEQ ID NO.2, and the complementarity determining region CDR3 has an amino acid sequence shown as SEQ ID NO. 3;

or II, amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in I, and the amino acid sequence has the same function as the amino acid sequence shown in I;

Or III, an amino acid sequence which has more than 80% identity with the amino acid sequence described in I or II and which exhibits similar physiological activity.

In some embodiments of the invention, the TfR 1-targeting nanobody further comprises four framework regions FR1, FR2, FR3, FR4, wherein,

The amino acid sequence of the framework region FR1 of the nano antibody is shown as SEQ ID NO. 4;

the amino acid sequence of the framework region FR2 of the nano antibody is shown as SEQ ID NO. 5;

The amino acid sequence of the framework region FR3 of the nano antibody is shown as SEQ ID NO. 6;

the amino acid sequence of the framework region FR4 of the nano antibody is shown as SEQ ID NO. 7.

In some embodiments of the invention, the variable region amino acid sequence of the nanobody is selected from any one of the following:

I. An amino acid sequence as shown in SEQ ID NO. 8;

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

In some embodiments of the invention, the nanobody further comprises a derivative polypeptide modified with the amino acid sequence, including but not limited to functional group modification or addition of a molecular marker. Further preferred, the functional group modification includes, but is not limited to, modification of the FR region with a hydrophilic group or substitution of hydrophobic residues of the FR region. Further preferred, the molecular markers include, but are not limited to, polyethylene glycol, streptavidin, biotin, a radioisotope, or a fluorescent agent.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding said TfR 1-targeting nanobody.

In some embodiments of the invention, the nucleic acid molecules include nucleic acids encoding the nanobodies described above, not limited to DNA or RNA, that can be translated due to codon degeneracy. Preferably, the coding nucleic acid is DNA, including cDNA, genomic DNA or synthetic DNA, which may be single-stranded or double-stranded, and may be coding or non-coding.

In some embodiments of the invention, the nucleic acid molecule has a nucleic acid sequence as shown in SEQ ID NO. 9.

In a third aspect of the invention, the invention also provides an expression vector comprising said nucleic acid molecule.

In some embodiments of the invention, the expression vector includes, but is not limited to, a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Preferably, the expression vector is a bacterial plasmid or a yeast plasmid.

In a fourth aspect of the invention, the invention also provides a host cell transformed or transfected with said expression vector.

In some embodiments of the invention, the host cell is a plant cell or a microbial cell. Preferably, the host cell is a microbial cell. It is further preferred to use E.coli as host cell.

In a fifth aspect of the invention, the invention also provides a conjugate or conjugate comprising a chemically or biologically labelled nanobody as claimed in the claims and an acceptable adjuvant or carrier.

In the present invention, the chemical label is an isotope, an immunotoxin and/or a chemical drug, and the biomarker is biotin, avidin or an enzyme label.

Based on the research, the invention also provides the application of the TfR1 targeting nano antibody or the conjugate in preparing TfR1 targeting medicaments.

In a seventh aspect, the invention also provides an application of the TfR 1-targeting nanobody or the conjugate in preparing a TfR1 detection antibody reagent and/or a kit.

In an eighth aspect of the present invention, based on the above study, the present invention further provides a TfR1 detection antibody reagent, comprising said TfR 1-targeting nanobody and/or said conjugate or conjugate, and acceptable adjuvants and/or carriers.

The beneficial effects of the invention are as follows:

The TfR 1-targeted nanobody provided by the invention has a unique CDR1 region, a CDR2 region and a CDR3 region, so that the nanobody has specific recognition and binding capacity to TfR1 antigen, and the affinity of the nanobody can reach 2.28E-9, and has high specific binding activity. In addition, the nano antibody provided by the invention can be combined with the TfR1 on the cell surface and inhibit the activity of the TfR 1.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a SDS-PAGE diagram of recombinant TfR1 antigen purification;

FIG. 2 is an electrophoretically identified map of total RNA extracted;

FIG. 3 shows a first and second round of PCR amplified antibody variable region gene electrophoresis;

FIG. 4 is a diagram showing the identification of pMES4 vector double cleavage reaction products by electrophoresis;

FIG. 5 is a single colony count estimation library capacity result;

FIG. 6 is an electrophoretically identified map of colony PCR identified transformants;

FIG. 7 is a SDS-PAGE of nanobody purification;

FIG. 8 is a graph of the binding inhibition activity of Tf-TfR1 of nanobodies.

Detailed Description

The invention discloses application of a TfR1 targeting nano antibody and a coding sequence thereof in detection, and a person skilled in the art can refer to the content of the nano antibody and the coding sequence and properly improve the implementation of technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

According to the invention, recombinant antigen TfR1 is immunized with alpaca, 4 times of immunization are carried out, peripheral blood lymphocytes of the immunized alpaca are separated, and a specific TfR 1-targeted nanobody gene library is constructed. Coating the TfR1 recombinant antigen on an ELISA plate, and screening the immunized nanobody library by utilizing phage display technology to obtain the nanobody targeting the TfR1 recombinant antigen. The constructed nanobody expression vector is introduced into escherichia coli, and purified after expression, the purified nanobody can be specifically combined with cell surface TfR1 protein and neutralize the activity of the TfR1 protein.

The invention provides a nanometer antibody coding sequence capable of specifically binding TfR1 protein, a preparation method and application thereof.

Specifically:

The nanobody variable region has 3 complementarity determining regions CDR1, CDR2, CDR3, wherein,

The CDR1 sequence consists of the amino acid sequence shown in SEQ ID NO. 1;

The CDR2 sequence consists of the amino acid sequence shown in SEQ ID NO. 2;

the CDR3 sequence consists of the amino acid sequence set forth in SEQ ID NO. 3.

The amino acid sequence of the variable region of the nano antibody is shown as SEQ ID NO. 8.

The invention also provides a nucleic acid molecule for encoding the nano antibody, and the nucleic acid sequence of the nucleic acid molecule is shown as SEQ ID NO. 9.

The invention also provides application of the nano antibody in preparing a targeting TfR1 drug.

The invention also provides application of the nano antibody in preparing TfR1 detection antibody reagents and/or kits.

The sequence related to the invention is as follows:

CDR1 region amino acid sequence of nanobody 1B 6:

5’–GRTFSSYA–3’(SEQ ID NO.1)。

CDR2 region amino acid sequence of nanobody 1B 6:

5’–ISWSADNT–3’(SEQ ID NO.2)。

CDR3 region amino acid sequence of nanobody 1B 6:

5’–AADPTPLHTIVVVTPEYD–3’(SEQ ID NO.3)。

amino acid sequence of FR1 region of nanobody 1B 6:

5’–QVQLQESGGGLVQAGGSLRLSCAAS–3’(SEQ ID NO.4)。

amino acid sequence of FR2 region of nanobody 1B 6:

5’–MGWFRQAPGKEREFLAA–3’(SEQ ID NO.5)。

Amino acid sequence of FR3 region of nanobody 1B 6:

5’–YYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC–3’(SEQ ID NO.6)。

Amino acid sequence of FR4 region of nanobody 1B 6:

5’–YWGQGTQVTVSS–3’(SEQ ID NO.7)。

variable region amino acid sequence of nanobody 1B 6:

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

nucleic acid sequence of nanobody 1B 6:

5'–caggtgcagctgcaggagtctgggggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcaccttcagtagctatgccatgggctggttccgccaggctccagggaaggagcgtgagtttctagcagctattagctggagtgctgataacacatactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacaacctgaaacctgaggacacggccgtttattactgtgcagcagatccgaccccactgcatactatagtggtagttactcctgagtatgactactggggccaggggacccaggtcaccgtctcctca–3'(SEQ ID NO.9).

CALL001 primer sequence:

5’–GTCCTGGCTGCTCTTCTACAAGG–3’(SEQ ID NO.10)。

CALL002 primer sequence:

5’–GGTACGTGCTGTTGAACTGTTCC–3’(SEQ ID NO.11)。

VHH-Back primer sequence:

5’–GATGTGCAGCTGCAGGAGTCTGGRGGAGG–3’(SEQ ID NO.12)。

VHH-For primer sequences:

5’–CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT–3’(SEQ ID NO.13)。

pMES-F primer sequence:

5’–GCCGCTGGATTGTTATTACTC–3’(SEQ ID NO.14)。

pMES-R primer sequence:

5’–CTTTCAACAGTGGAACCGTAG–3’(SEQ ID NO.15)。

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, reference is made specifically to Current Protocols in MolecularBiology (Ausubel) by the expert. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

An "antibody" refers to a protein composed of one or more polypeptides capable of specifically binding an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer that is composed of two identical pairs of antibody chains, each pair having one heavy chain and one light chain. In each pair of antibody chains, the variable regions of the light and heavy chains are taken together to be responsible for binding to the antigen, while the constant regions are responsible for the effector functions of the antibody.

The "variable region" is the N-terminal mature region of the chain. Currently known antibody types include kappa and lambda light chains, as well as alpha, gamma (IgGl, igG2, igG3,1gG 4), delta, epsilon and mu heavy chains or other types of equivalents thereof. The full length immunoglobulin "light chain" (about 25kDa or about 214 amino acids) comprises a variable region formed of about 110 amino acids at the NH 2-terminus, and a kappa or lambda constant region at the COOH-terminus. The full length immunoglobulin "heavy chain" (about 50kDa or about 446 amino acids) also comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, e.g., gamma (about 330 amino acids).

"Antibody" includes antibodies or immunoglobulins of any isotype, or antibody fragments that remain specifically bound to an antigen, including but not limited to Fab, fy, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be labeled and detected, for example, by radioisotope, enzyme capable of producing a detectable substance, fluorescent protein, biotin, and the like. Antibodies may also be bound to solid supports including, but not limited to, polystyrene plates or beads, and the like.

"Humanized antibody" refers to an antibody that comprises CDR regions derived from a non-human antibody, and the remainder of the antibody molecule is derived from a human antibody (or antibodies). Furthermore, to preserve binding affinity, some residues of the backbone (referred to as FR) segment may be modified.

By "nanobody" is meant an antibody that has a naturally deleted light chain in the peripheral blood of alpaca, which comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but is not as easily sticky to each other or even aggregates as an artificially engineered single chain antibody fragment (scFv). More importantly, the VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity to the antigen, the smallest unit known to bind the antigen of interest. The VHH crystals were 2.5nm long by 4nm and had a molecular weight of only 15kDa, which is also known as Nanobody (Nb). Nanobodies are comparable in affinity to their corresponding scFv relative to conventional four-chain antibodies, but exceed scFv in terms of solubility, stability, resistance to aggregation, refolding, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination. The nanometer antibody has small molecules and high stability, can be administrated in an atomization mode, improves the administration convenience and widens the application scene of the medicine.

The medicine contains at least one functional component and also comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline solution such as PBS (phosphate buffered saline), dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the invention may comprise as additives wetting agents, emulsifiers or buffer substances.

"CDR region" or "CDR" as used herein refers to the complementarity determining regions of nanobodies, complementary Determining Regions. There are three CDRs. The term CDR or CDRs as used herein is intended to indicate one of these regions, or several or even all of these regions, as the case may be, comprising the majority of amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope.

As used herein, "FR region" or "FR" refers to the framework region of a nanobody, framework Regions. There are four FRs. The term FR is used herein to indicate one of these regions, or several or even all of these regions, as the case may be.

The nano antibody and the raw materials and reagents used in the application of the nano antibody can be purchased from the market.

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

EXAMPLE 1 expression of TfR1 recombinant proteins

Cys88-Phe760 was synthesized based on the amino acid Sequence of human TfR1 at NCBI (Sequence ID: AAA 61153.1), and a histidine tag was added to the N-terminus of the Sequence and ligated to vector pCDNA3.1 (+). The 293 cells in logarithmic growth were transfected with endotoxin-free large plasmids. After the transfected cells were obtained and cultured for 36 hours, the cell culture solution was poured into a 50mL centrifuge tube, 12000g was centrifuged for 5 minutes, the supernatant was collected, filtered with a 0.22 μm filter membrane, and the culture supernatant was purified by nickel column affinity chromatography. SDS-PAGE detects protein expression. The results are shown in FIG. 1 (M is protein Marker, lane 1 is purified TfR1 antigen protein).

EXAMPLE 2 construction and screening of anti-TfR 1 nanobody phage display library

2.1 Alpaca immunization

An adult alpaca with moderate body type, healthy and strong body, no injury uncomfortable symptoms and good mental state is selected. The recombinant protein TfR1 and Gerbu adjuvant are uniformly mixed according to the proportion of 1:1, and are injected at the left side and the right side near the alpaca neck lymph node each time, 4 points are injected at each side, and 0.5mL is injected at each point. The total immunization was four times, each two weeks apart. Then, alpaca peripheral blood was collected for constructing phage display library.

2.2 Isolation of alpaca lymphocytes

The collected alpaca peripheral blood is subjected to lymphocyte separation by using a camel peripheral blood lymphocyte separation liquid kit (Tianjin line ocean company, product number LTS 1076) instruction manual operation, 1mL of RNA separation reagent is added into every 2.5X10 ⁷ living cells, 1mL of RNA is taken for RNA extraction, and the rest is preserved at-80 ℃.

2.3 Extraction of RNA

Repeatedly blowing 1mL Tipure Isolation Reagent containing lymphocytes, standing for 5min, adding 200 μl chloroform, shaking vertically and vigorously for 30 s, standing for 5min, centrifuging at 4deg.C for 15min, absorbing water phase, transferring to new EP tube, adding equal amount of isopropanol, standing for 10 min, centrifuging at 4deg.C for 10 min 12000g, discarding supernatant, washing with 1mL precooled 70% ethanol, centrifuging at 4deg.C for 5min 7500g, discarding supernatant and drying for 5min, adding 30 μl RNase-free water to dissolve precipitate, adjusting concentration to 1 μg/μl, and performing gel electrophoresis detection to obtain lane 1 as shown in FIG. 2.

2.4 Reverse transcription to cDNA

The cDNA was reverse transcribed using the RNA obtained In the 2.3 step as a template according to the reverse transcription kit instructions (abm company biological All-In-One 5X RT Mastermix).

2.5 Amplification of antibody variable region genes

The cDNA obtained by reverse transcription is used as a template to carry out a first round of PCR reaction, and the primer sequences of the PCR reaction are as follows:

CALL001:GTCCTGGCTGCTCTTCTACAAGG(SEQ ID NO.10)

CALL002:GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO.11)

the PCR reaction conditions and procedures were:

95°C5min;

95°C30s,57°C30s,72°C30s,25cycles;

72°C7min。

The band of about 700bp was recovered using agarose gel recovery kit gel, and finally the nucleic acid concentration was adjusted to 5 ng/. Mu.L with water (first round PCR product identification see FIG. 2, where M: molecular weight marker; 1: first round PCR product).

The first round PCR product is used as a template to carry out the second round PCR, and the sequences of the primers are as follows:

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

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

the PCR reaction conditions and procedures were:

95°C5min;

95°C30s,55°C30s,72°C30s,25cycles;

72°C5min。

The PCR products were purified using a PCR product recovery kit (second round PCR product identification see FIG. 2, where M: molecular weight marker; 1: second round PCR product).

2.6 Vector construction

PMES4 vector (from Biovector) and the second PCR product were subjected to PstI and BstEII double digestion, respectively, 2. Mu.g of the digested vector and 2. Mu.g of the digested second PCR product were taken, 40. Mu. L T4 DNA ligase was added, buffer and water were supplemented to a total volume of 150. Mu.L, and ligation was performed overnight at 16℃and the ligation product was recovered. The PCR product was recovered using a PCR product recovery kit, and eluted with 20. Mu.L of water.

FIG. 3 is a diagram showing the identification of pMES4 vector double cleavage reaction products by electrophoresis. Wherein, M is a molecular weight mark, 1 is a double enzyme digestion product of a pMES4 vector, and 2 is an unencleaved pMES vector.

2.7 Electric conversion and storage Capacity determination

20 Mu L of the purified ligation product was added to the bottom of a pre-chilled 2mm electric rotating cup containing 200 mu L of E.coli TG1 competent cells, mixed with an ice bath for 30min, wiped clean by the electric rotating cup and placed into an electric rotating instrument (Gene Pulser Xcell ^TM, BIO-RAD company) for electric conversion, the electric conversion parameters were set to 2.5kV,25 mu F,200 omega, 800 mu L of SOC medium was added immediately after the electric conversion was completed, mixed and transferred to a sterile EP tube, and resuscitated at 37℃for 2h at 200 r/min. After resuscitating, 100. Mu.L of the bacterial liquid was aspirated and diluted in a gradient of 10 ^-1,10^-2 to 10 ^-6. mu.L of each 10 ^-4,10^-5,10^-6 -gradient bacteria solution was aspirated and plated onto 2YT-A plates and incubated overnight in a 37℃incubator. The single colonies on each dilution plate were counted, the plate with the appropriate number of single colonies was selected, and the library capacity was calculated from the dilutions (see FIG. 4 for results). 20 individual clones were randomly picked using a sterile gun head for colony PCR identification (see FIG. 5 for electrophoretic identification results, where M: molecular weight markers; 1-20 non-randomly selected PCR identification products; N: negative control).

Primer sequences:

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

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

the conditions and procedures for the PCR reaction were:

95°C5min;

95°C30s,55°C30s,72°C30s,30cycles;

72°C5min。

and calculating the PCR positive rate according to the electrophoresis result of the time, and calculating the storage capacity.

Library capacity = clone number x dilution x positive rate x 10 x library volume.

Reservoir capacity = 150 x 10 ⁶×95％×10×2＝2.85*10⁹ CFU. The calculated storage capacity was 2.85 x 10 ⁹ CFU.

EXAMPLE 3 selection and expression of nanobodies

Phage display of 3.1 nanobodies

Resuscitated bacterial solution was inoculated into 410 ml of 2yt-AG medium, cultured at 37 ℃,200rpm to culture od600=0.5. 4X 10 ¹⁰ pfu VCSM13 was added per 10mL of bacterial liquid and the infection was allowed to stand at 37℃for 30 minutes. Centrifugation at 4000rpm for 10min at normal temperature and removal of supernatant. The cells were resuspended in 100mL of 2 XYT-AK (ampicillin and kanamycin) medium and incubated overnight at 37℃at 200 rpm. The supernatant was collected by centrifugation at 4℃for 15min at 10800g overnight, 10mL of PEG/NaCl (20%/2.5M) solution was added to each 40mL of supernatant and mixed well, the supernatant was discarded by centrifugation at 4℃for 30min with 2h ice bath, 10800g, the pellet was resuspended in 8mL of ice PBS, 2mL of pre-chilled PEG/NaCl was added, mixed well thoroughly, and after 1h ice bath 3300g, centrifugation at 4℃for 30min,1mL of LPBS was resuspended.

Phage titer was determined by culturing TG1 to OD600 = 0.4, gradient diluting phage with sterile PBS, mixing phage TG1 cultures (1:20) diluted in a doubling ratio, standing at 30℃for 30min, plating 100. Mu.L on 2YT-AG solid plates, observing plaque formation in the plates the next day, counting the number of plaques on diluted gradient plates of 30-300 and calculating phage titer (cfu) according to the following formula.

Phage titer (cfu/mL) =dilution x 2 x number of plaques x 100

3.2 Solid phase panning of phage display libraries

The TfR1 recombinant antigen was diluted with CBS to 10. Mu.g/mL, 100. Mu.L of coated ELISA plate per well, left standing overnight at 4℃for 5 times, PBST wash plate was added with 250. Mu.L of 1% BSA per well, blocked at 37℃for 2h, PBST wash plate was added 5 times, 100. Mu.L of display phage diluted to 10 ¹¹ cfu/mL per well, incubated at 37℃for 2h, PBST wash plate was 15-25 times, 100. Mu.L of glycine solution (0.2M, pH 2.2) per well was added at the end of the last wash, incubated for 15min on a horizontal shaker, the eluate per well was added to an EP tube with 15. Mu.L Tris solution in advance, and the titer was detected after combining. The panning severity was increased appropriately depending on the results of each round, amounting to 3-4 panning times.

3.3 Screening of phage Elisa for Positive clones

TfR1 recombinant antigen was diluted with CBS to 5 μg/mL, 100 μl of coated elisa plate per well, left standing overnight at 4 ℃ and the plates were washed 5 times with PBST (0.05%), 250 μl of 5% BSA per well, blocked 1h at 37 ℃ and 5 times with PBST (0.05%). mu.L of overnight cultured monoclonal phage supernatant was added to each well, incubated at 37℃for 1h, and the plates were washed 5 times with PBST (0.05%). mu.L of HRP-labeled mouse anti-M13 secondary antibody was added to each well, incubated at 37℃for 1h, and plates were washed 5 times with PBST (0.05%). 100. Mu.L of TMB color development solution is added to each well, incubated at room temperature for 15-30min in the dark, and 100. Mu.L of 2M sulfuric acid stop solution is added to each well. Read with a microplate reader at 450 nm. Clones positive for phage ELISA were selected and sequenced.

3.4 Nanobody original Strain TG1 amplification and nanobody recombinant plasmid transformation E.coli BL21 (DE 3)

Clones with positive results were selected, and original strain TG1 glycerol bacteriA containing nanobody nucleic acids were inoculated in 5mL of fresh LB-A medium at A ratio of 1:1000, and cultured overnight at 37℃at 200 rpm. The following day, plasmids were extracted using PLASMID MINI KIT (OMEGA) according to the instructions. After verification, 1. Mu.L of the above plasmid was transformed into 100. Mu.L of competent cells, gently mixed, placed on ice for 30 minutes, heat-shocked in a 42℃water bath for 45 seconds, and cooled in an ice bath for 2 minutes. 600 μLLB medium was added to the centrifuge tube and incubated at 37℃for 60 minutes with shaking. 100. Mu.L of the supernatant was spread on LB-A plates and cultured overnight at 37℃in an inverted state.

3.5 Induction of expression of nanobodies

The above monoclonal colonies were picked up in LB-A medium and cultured overnight at 37℃with shaking. The next day, 100mL of fresh LB-A medium was added to the bacterial liquid at A ratio of 1:100, and the bacterial liquid was cultured with shaking at 37℃for 3 hours until the bacterial liquid OD600 = 0.8, and then 1mM IPTG was added at A final concentration of 30℃and induced at 200rpm overnight. The following day, cells were collected by centrifugation at 5000 rpm for 10 minutes at 4℃and the pellet was resuspended in 1.5mL of pre-chilled TES buffer. After 2 minutes of ice bath, the cycle was repeated 6 times with gentle shaking for 30 seconds. 3.0mL of TES/4 (4-fold dilution of TES with water) was added, and after gentle shaking for 30 seconds, the ice bath was allowed to stand for 2 minutes, and the shaking and ice bath steps were repeated 6 times as much. Centrifugation was performed at 9000rpm at 4℃for 10 minutes, and about 4.5mL of the supernatant (periplasmic extract) was collected.

3.6 Purification and identification of nanobodies

After the IMAC Sepharose (GE company) was resuspended, 2mL was added to the gravity column and allowed to stand for 30 minutes to allow the Sepharose to naturally settle to the bottom of the gravity column and the preservation buffer was drained. Adding 2 times of column volume of nickel sulfate solution (0.1M), flowing out of the nickel sulfate solution at a flow rate of about 8 seconds/drop, adding 10 times of column volume of balance buffer solution for balancing and washing sepharose, keeping the flow rate unchanged, diluting a sample with the balance buffer solution 2 times, adding the sample into a gravity column, adjusting the flow rate to 6 seconds/drop, collecting penetrating liquid, adding 10 times of column volume of washing buffer solution for washing sepharose, keeping the flow rate unchanged, collecting washing liquid, adding 3 times of column volume of elution buffer solution, keeping the flow rate to 6 seconds/drop, collecting the elution liquid containing target protein, and finally sequentially adding 10 times of column volume of balance buffer solution, 10 times of column volume of pure water and 10 times of column volume of 20% ethanol for washing sepharose, and finally keeping 4mL of 20% ethanol for preserving the column. SDS-PAGE was performed on the collected samples (FIG. 7:M is a Thermo fisher protein Marker, lane 26616; lanes 1-3 are purified nanobodies 1A5, 1B6, 1H9, respectively). The results are shown in FIG. 7, where all three nanobodies were expressed.

Antibody heavy chain analysis of the amino acid sequence of 1B6 was performed using Vector NTI software to determine the framework regions (Framework Regions, FR) and complementarity determining regions (Complementary Determining Regions, CDR) of the variable regions.

The nanobody of one preferred embodiment selected by the present invention is designated as "1B6". Through DNA sequencing, the heavy chain nucleic acid sequence of the nano antibody 1B6 is shown as SEQ ID NO.9, the amino acid sequence of the variable region is shown as SEQ ID NO.8, wherein the amino acid sequences at positions 1-25 are FR1 (shown as SEQ ID NO. 4), the amino acid sequences at positions 26-33 are CDR1 (shown as SEQ ID NO. 1), the amino acid sequences at positions 34-50 are FR2 (shown as SEQ ID NO. 5), the amino acid sequences at positions 51-58 are CDR2 (shown as SEQ ID NO. 2), the amino acid sequences at positions 59-96 are FR3 (shown as SEQ ID NO. 6), the amino acid sequences at positions 97-114 are CDR3 (shown as SEQ ID NO. 3), and the amino acid sequences at positions 115-126 are FR4 (shown as SEQ ID NO. 7).

Example 4 determination of affinity of nanobodies to antigen

4.1 Chip antigen coupling

TfR1 was formulated with sodium acetate buffers of different pH (pH 5.5, pH5.0, pH4.5, pH 4.0) to 50. Mu.g/mL working solution, while 50mM NaOH regenerating solution was prepared, and electrostatic binding between antigens of different pH conditions and the surface of the chip (GE company) was analyzed by a template method in a Biacore T100 protein interaction analysis system instrument, with the amount of signal increase reaching 5 times RL as a standard, a suitable most neutral pH system was selected and the antigen concentration was adjusted as required as conditions at the time of coupling. And (3) coupling the chip according to a template method in the instrument, wherein a 1 channel selects a blank coupling mode, a 2 channel selects a Target coupling mode, and the Target is set to be the designed theoretical coupling amount. The coupling process takes approximately 60 minutes.

4.2 Analyte concentration setting Condition exploration and regeneration Condition optimization

And adopting a manual sample injection mode, selecting a 1, 2-channel 2-1 mode sample injection mode, and setting the flow rate to be 30 mu L/min. Sample injection conditions were 120s and 30. Mu.L/min. The regeneration conditions were 30s, 30. Mu.L/min. First, running buffer was kept empty until all baselines were stable. Nanobody solutions with large concentration spans were prepared for running buffer configurations, suggesting that 200 μg/mL,150 μg/mL,100 μg/mL,50 μg/mL,20 μg/mL,10 μg/mL,2 μg/mL were set. A regenerating solution is prepared, and four pH gradient regenerating solutions of a glutamic acid hydrochloric acid system are selected to be 1.5,2.0,2.5,3.0. Samples of 200 μg/mL analyte were manually injected and 2 channels were observed for regeneration from the most neutral pH regeneration buffer until the response line after 2 channel regeneration returned to the same height as baseline. And manually feeding the analyte sample once again by 200 mug/mL, observing the signal change of the 2-1 channel, recording the binding amount, recovering the analyte sample again by 200 mug/mL after regenerating the regeneration solution of which the response line is returned to the baseline in the last step, observing the signal change of the 2-1 channel, recording the comparison between the binding amount and the value of the binding amount just before, and if the deviation is less than 5%, confirming that the regeneration solution with the pH value is the optimal regeneration solution, and if the binding amount of the reinjection is lower, continuing to perform experiments by using the regeneration buffer solution with the lower pH value. And taking the selected optimal regeneration solution as a chip surface regeneration reagent after each sample injection. The analyte concentration samples set forth above were separately sampled and the binding capacity for each concentration was analyzed to finally determine the concentration gradient required for the affinity test.

4.3 Affinity test

According to the optimized sample concentration gradient, regenerating the solution, and testing the affinity between the nano antibody and the antigen by using a template method (wherein the sampling condition is set to 60s,30 mu L/min, the dissociation time is 600s, and the regeneration condition is 30s,30 mu L/min) carried out by the instrument. The signal condition of the 2-1 channel is observed at any time. The affinity test procedure takes approximately 200min.

4.4 Analysis of results

The binding dissociation curves of the appropriate concentration gradients were selected and all curves were fitted in a 1:1binding mode to finally obtain the affinity values and important parameters such as binding constant and dissociation constant, and the results are shown in Table 1. The results show that all three nanobodies can specifically bind to TfR1 protein coupled on a chip, wherein the 1B6 affinity reaches 2.28E-9.

TABLE 1 affinity values, binding and dissociation constants for nanobodies

Antibodies to	k_on(M^-1·s^-1)	k_off(s^-1)	K_D(M)
				1A5	8.084×10⁴	1.32×10^-3	1.6×10^-8
1B6	5.726×10⁴	1.310×10^-4	2.28×10^-9
				1H9	2.742×10⁴	1.866×10^-3	6.806×10^-8

EXAMPLE 5 Activity analysis of nanobodies

5.1 Determination of binding force of antibody to target cells

Human breast cancer cells (MCF 7) express TfR1 at high levels, with expression levels positively correlated with their proliferation status. In the embodiment, the cell is taken as an experimental model, and the capability of the three nano antibodies in the embodiment 4 to specifically target and bind to the TfR1 on the cell surface in vitro is verified.

The test method comprises cloning three nanobodies 1A5, 1B6 and 1H9 to pFUSE-hIgG1-Fc carrier to be fused and expressed with IgG1-Fc, and selecting an irrelevant nanobody Nb _control as a negative control. MCF7 cells in the logarithmic growth phase were resuspended using FACS buffer (PBS containing 1% FBS), incubated with negative control or gradient diluted anti-TfR 1 antibody (initial concentration 10. Mu.g/mL, 2-fold dilution, 8 concentration gradients) at a final concentration of 10. Mu.g/mL, centrifuged off supernatant after incubation on ice for 30min, washed once with PBS, anti-human IgG flow antibody anti-human Fc-PE (Invitrogen, 12-4998-82) was added, and the supernatant centrifuged off ice for 30 min. PBS was washed twice for post-flow detection, MFI was counted using Flowjox software, and data was processed using Graph pad software.

The results are shown in table 2, with antibodies 1B6 and 1H9 binding to MCF7 cells, whereas 1A5 and negative control Nb _NGF did not bind to MCF7 cells.

TABLE 2 effective concentrations of 1B6, 1H9, 1A5 and Nb _control binding to MCF7 cells

Antibodies to	1A5	1B6	1H9	Nb_control
					EC₅₀(nM)	57.8	64.9	NA	NA

5.2Elisa detection of Tf-TfR1 binding inhibition Activity of nanobody

TfR1 described in example 1 was adjusted to 5.0. Mu.g/mL in CBS, and dilutions were dispensed in Nunc ^TM well plates at 100. Mu.L/well and allowed to stand at 4℃overnight. The next day, the supernatant was discarded and the plates were washed 5 times with PBST (0.05%). mu.L of 1% BSA was added to each well, blocked at 37℃for 1h, and the plates were washed 5 times with PBST. 50. Mu. LHRP labeled Tf (2. Mu.g/mL) was added to each well, followed by 50. Mu.L of 1A5, 1B6, 1H9 (10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001. Mu.g/mL) respectively, and the corresponding concentrations of A24 antibody were added sequentially after 50. Mu. LHRP labeled Tf (2. Mu.g/mL) was added to each well using the A24 antibody described in U.S. Pat. No. 2008/0193453 as a positive control. Incubate at 37 ℃ for 1h, wash the plate 5 times with pbst. 100 mu LTMB color development solution is added to each hole, incubated for 15-30min at room temperature and in a dark place, then 100 mu L of 2M sulfuric acid stop solution is added to each hole, and the absorbance at 450nm is measured by an enzyme-labeling instrument.

As shown in FIG. 8, 1B6 can completely inhibit Tf-TfR1 binding at a lower concentration (0.01. Mu.g/mL), 1A5 has a certain inhibition effect, but the effect is weaker than that of the A24 antibody, and 1H9 has no inhibition effect. This result indicates that 1B6 specifically blocks Tf-TfR1 binding and is significantly better than the a24 antibody.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanobody targeting TfR1, wherein the nanobody comprises three complementarity determining regions CDR1, CDR2, CDR3;

the amino acid sequence of a CDR1 of the nano antibody is shown as SEQ ID NO.1, the amino acid sequence of a CDR2 of the nano antibody is shown as SEQ ID NO.2, and the amino acid sequence of a CDR3 of the nano antibody is shown as SEQ ID NO. 3.

2. The TfR 1-targeting nanobody of claim 1, wherein said TfR 1-targeting nanobody further comprises four framework regions FR1, FR2, FR3, FR4, wherein,

3. A TfR 1-targeting nanobody as claimed in claim 1, wherein the variable region amino acid sequence of said nanobody is selected from any one of:

I. An amino acid sequence as shown in SEQ ID NO. 8;

4. A TfR 1-targeting nanobody according to claim 3, wherein said nanobody further comprises a derivative polypeptide modified in the amino acid sequence of said variable region, said modification comprising functional group modification;

The functional group modification includes modification of the FR region with a hydrophilic group or substitution of hydrophobic residues of the FR region.

5. A TfR 1-targeting nanobody as claimed in claim 3, further comprising a derivative polypeptide modified in the amino acid sequence of said variable region, said modification comprising the addition of a molecular tag;

The molecular marker comprises polyethylene glycol, streptavidin, biotin, radioisotope or fluorescent agent.

6. A nucleic acid molecule encoding the TfR 1-targeting nanobody of any of claims 1-3.

7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule has a nucleic acid sequence as set forth in SEQ ID No. 9.

8. Expression vector comprising a nucleic acid molecule according to claim 6 or 7.

9. The expression vector of claim 8, wherein the expression vector comprises a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, or a mammalian cell virus.

10. Transforming or transfecting a host cell with the expression vector of claim 8 or 9;

The host cell is a microbial cell.

11. A conjugate or conjugate comprising a chemically or biologically labeled TfR 1-targeting nanobody as claimed in any one of claims 1 to 5 and an acceptable adjuvant or carrier.

12. Use of a TfR 1-targeting nanobody as defined in any one of claims 1-5 or a conjugate or conjugate as defined in claim 11 for the preparation of a TfR1 detection antibody reagent and/or kit.

A TfR1 detection antibody reagent or detection kit comprising a TfR 1-targeting nanobody as claimed in any one of claims 1 to 5 and/or a conjugate or conjugate as claimed in claim 11 and an acceptable adjuvant and/or carrier.