CN113121692B

CN113121692B - Alpaca-derived antibodies that bind to the extracellular domain of human RAGE

Info

Publication number: CN113121692B
Application number: CN202110475381.9A
Authority: CN
Inventors: 金腾川; 艾哈迈德·穆罕默德
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2022-12-06
Anticipated expiration: 2041-04-29
Also published as: CN113121692A

Abstract

The invention discloses an alpaca antibody capable of binding with high affinity to the extracellular domain of human RAGE, which is also called nanobody, and can be used for preventing, treating and/or diagnosing RAGE-related disorders.

Description

Alpaca-derived antibodies that bind to the extracellular domain of human RAGE

Technical Field

The present invention is in the field of biopharmaceuticals and in particular relates to nanobodies against the extracellular domain of human RAGE (hRAGE) for use in therapy and diagnosis.

Background

Advanced glycation end product Receptors (RAGE) are members of the immunoglobulin superfamily. RAGE is not only involved in the development and progression of chronic complications of diabetes, but also in inflammatory responses, tumor invasion and migration, nerve regeneration, nephropathy, arteriosclerosis, alzheimer's disease. RAGE is a multiligand receptor with important functions, and ligands for RAGE include advanced glycation end products (AGE), high mobility box 1 proteins, family of S100 proteins, carboxymethyllysine, amyloid beta peptide, and the like.

The structure of RAGE includes the extracellular domain (amino acid residues 23-342), the transmembrane domain (19 amino acid residues) and the intracellular domain (41 amino acid residues). <xnotran> V (23-116 , AQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVR) (SEQ ID NO: 1) C1 (124-221 , PEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALR) (SEQ ID NO: 2) C2 (227-317 , PRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVS) (SEQ ID NO: 3). </xnotran>

Antagonizing binding of physiological ligands to RAGE may down-regulate pathophysiological changes caused by excessive concentrations of AGE and other RAGE ligands. Symptoms associated with RAGE-mediated disorders may be reduced by reducing the binding of endogenous ligands to RAGE. Soluble RAGE is effective in antagonizing the binding of RAGE ligands to RAGE. However, the half-life of soluble RAGE upon administration in vivo may be too short to be suitable for the treatment of one or more disorders. Therefore, there is a need to develop drugs that antagonize binding of AGEs and other physiological ligands to RAGE.

The V variable domain of RAGE is considered to be the primary ligand binding domain, and therefore small molecule inhibitors targeting the V variable domain of RAGE may be of clinical value, for example single domain antibodies. Single domain antibodies, also known as nanobodies (Nbs), are small antibodies produced by camelids (camel, llama, alpaca). The nanobody has a minimal variable antigen-binding fragment, called the VHH domain, with a molecular weight of about 15kDa. Compared with conventional antibodies, nanobodies have unique advantages: 1) The nano antibody has simple structure and small molecular weight, and is more beneficial to expression and use; 2) More convenient to express in large quantities and high efficiency in escherichia coli and various eukaryotic systems; 3) The antibody has only one binding site, belongs to a single-domain antibody, and has better permeability, specificity and detection linearity when used as a diagnostic reagent; 4) Convenient for coupling with various fusion proteins or easier for labeling with various labels; 5) The bifunctional antibody is easier to prepare, and is more beneficial to targeted drug development and directional transportation of cell targets; 6) As a medicine development, the medicine has small immunogenicity for human and is not easy to generate immunological rejection.

Disclosure of Invention

The present invention provides alpaca-derived antibodies, also known as nanobodies, that bind with high affinity to the extracellular domain of human RAGE and can be used for the prevention, treatment and/or diagnosis of RAGE-related disorders.

The inventors immunized alpaca 4 times with recombinant RAGE extracellular domain, then isolated peripheral blood lymphocytes and extracted total RNA of the cells followed by reverse transcription into cDNA, which was used as template to amplify the nanobody sequence. We isolated 3 nanobodies named 3CNB, 4BNB and 5ENB. The amino acid sequences of these nanobodies are as follows:

3CNB amino acid sequence:

QVQLVESGGGLVQAGGSLRLSCGASGSIFSIHTMGWYRQAPGQERELVAAISSFGSTYYADSVKGRFTISRDNAKNTLYLQVNSLKPEDTAMYYCVGREHHNGPYDYWGQGTQVTVSS(SEQ ID NO:6)

amino acid sequence of 4 BNB:

QVQLVESGGGLVQAGGSLRLSCAASGRTFSDYVMGWFQQAPGKEREFVAAISWSGGKTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGSGRGTYDYWGQGTQVTVSS(SEQ ID NO:7)

5 amino acid sequence of ENB:

QLQLVESGGGLVQAGGSLRLSCAASTRAFSNYAIGWFRQAPGKEREFVAAISWSGASTYYADSVKGRCTISRDDSKNTVYLQMNSLKPEDTGVYYCAAQEGDTRRYDYWGQGTQVIVSS(SEQ ID NO:8)

the three antigen complementarity determining regions (CDR 1, CDR2 and CDR 3) of these 3 nanobodies are shown in underlined section, specifically as follows:

3CNB antigen complementarity determining region:

CDR1:GSIFSIHTM(SEQ ID NO:9)

CDR2:LVAAISSFGS(SEQ ID NO:10)

CDR3:VGREHHNGP(SEQ ID NO:11)

4BNB antigen complementarity determining region:

CDR1:GRTFSDYVM(SEQ ID NO:12)

CDR2:FVAAISWSGGK(SEQ ID NO:13)

CDR3:AAGSGRGT(SEQ ID NO:14)

5 antigen complementarity determining region of ENB:

CDR1:TRAFSNYAI(SEQ ID NO:15)

CDR2:FVAAISWSGAS(SEQ ID NO:16)

CDR3:AAQEGDTRR(SEQ ID NO:17)

specifically, the invention provides the following technical schemes:

in one aspect, the invention provides an antibody or antigen-binding fragment thereof, preferably that specifically binds to the extracellular domain of RAGE (e.g. human), preferably the antibody or antigen-binding fragment thereof is derived from an alpaca, preferably the antibody is a nanobody comprising only heavy chain antibodies, characterized in that the antibody comprises:

CDR1 comprising or consisting of a sequence depicted in SEQ ID NO 9 or 12 or 15, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

CDR2 comprising or consisting of a sequence shown as SEQ ID NO 10 or 13 or 16, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

CDR3 comprising or consisting of a sequence depicted in SEQ ID NO 11 or 14 or 17, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

Further, the antibody comprises a sequence selected from the group consisting of:

(a) CDR1 as shown in SEQ ID NO. 9, CDR2 as shown in SEQ ID NO. 10 and CDR3 as shown in SEQ ID NO. 11;

(b) CDR1 as shown in SEQ ID NO. 12, CDR2 as shown in SEQ ID NO. 13 and CDR3 as shown in SEQ ID NO. 14;

(c) CDR1 as shown in SEQ ID NO. 15, CDR2 as shown in SEQ ID NO. 16 and CDR3 as shown in SEQ ID NO. 17;

further wherein the antibody comprises a sequence selected from one or more of the group consisting of:

(a) As shown in SEQ ID NO:6 with a sequence of amino acids as shown in SEQ ID NO,

(b) And the nucleotide sequence shown as SEQ ID NO:6, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in fig. 6;

(c) As shown in SEQ ID NO:7 (c) or (c) to the amino acid sequence shown in (7),

(d) And the nucleotide sequence as shown in SEQ ID NO:7, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in seq id no;

(e) As shown in SEQ ID NO:8 with a sequence of amino acids as set forth in SEQ ID NO,

(f) And the nucleotide sequence as shown in SEQ ID NO:8, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in figure 8.

Further, the antibody or antigen binding fragment thereof, further has an Fc domain, preferably an IgG1Fc domain, more preferably a human IgG1Fc domain, the sequence of the human IgG1Fc domain being, for example, as set forth in SEQ ID NO:4, respectively.

In another aspect, the invention provides a polynucleotide encoding an antibody or antigen-binding fragment thereof as described above.

In another aspect, the present invention provides an expression vector comprising a polynucleotide as described above.

In another aspect, the invention provides a host cell comprising an expression vector as described above, said host cell being a host cell for the expression of a foreign protein, e.g. a bacterium, a yeast, an insect cell, a mammalian cell.

In another aspect, the invention provides a method of making an antibody or antigen-binding fragment thereof as described above, comprising the steps of culturing a host cell as described above, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable carrier.

Further wherein the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

In another aspect, the invention provides the use of an antibody or antigen-binding fragment thereof as described above in the manufacture of a kit or medicament for the prevention, treatment and/or diagnosis of a disorder associated with RAGE.

Further, the RAGE-associated disorder is selected from the group consisting of: chronic complications of diabetes, inflammatory reactions, invasion and migration of tumors, nerve regeneration, nephropathy, arteriosclerosis, alzheimer's disease.

In another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof as described above, preferably the antibody further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof, optionally the second antibody comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

In another aspect, the invention provides a multispecific antibody, preferably a bispecific antibody, comprising an antibody or antigen-binding fragment thereof as described above.

In another aspect, the invention provides a fusion protein comprising an antibody or antigen-binding fragment thereof as described above.

Advantages and positive effects of the invention

(1) The antibody provided by the invention is derived from a natural alpaca heavy chain antibody, so that the antibody has the characteristics of high expression level, high affinity and high stability.

(2) The antibody provided by the invention, which binds to the extracellular domain of human RAGE, is expressed and secreted by mammalian cells (293F), the antibody is fused with human IgG1Fc, cloned into a mammalian expression vector pTT5, the vector is transfected into the mammalian cells 293F, and the supernatant is collected after 5 days of culture. The Protein A column is adopted to purify the fusion Protein in the supernatant, and the yield of 3 kinds of nano antibodies is more than 60mg/L. Circular Dichroism (CD) experiments show that the three kinds of nano antibodies have higher thermal stability. All 3 nanobodies showed high affinity binding to the extracellular domain of human RAGE.

Drawings

FIG. 1 ELISA results of monoclonal phages specifically binding to the V-C1 domain of human RAGE.

FIG. 2 shows the results of SDS-PAGE analysis. Lane M is Marker, and lanes 1 to 3 are Fc fusion proteins of nanobodies 3CNB, 4BNB and 5ENB in this order. Lanes 4 to 6 are 3CNB, 4BNB and 5ENB nanobodies without FC.

FIG. 3.3 results of circular dichroism spectroscopy (CD) experiments on denaturation temperature of nanobodies. FIGS. 3A-3C show the denaturation Temperatures (TM) for 3CNB, 5ENB and 4BNB, respectively.

FIG. 4 analysis of Nanobody binding to the V-C1 domain of human RAGE by ELISA. 3CNB-FC: EC50=106.9 ± 0.05nM;4BNB-FC: EC50=67.8 ± 0.07nM;5ENB-FC: EC50=54.7 ± 0.04nM.

FIG. 5 analysis of the recognition specificity of Nanobodies to the V domain-GB 1 tag, C1 domain and V-C1 domain of human RAGE and to unrelated proteins, namely TEV protease, NTPase, caspase, MBP, human NLRP1-CARD, GB 1. TEV protease: a protease encoded in tobacco mosaic virus; NTPase: a nucleoside hydrolase; caspase: a cysteine-containing aspartate hydrolase; MBP: a mannose binding protein; human NLRP1-CARD: caspase activation and recruitment domains of human NLRP1 proteins; the GB1 sequence is shown as SEQ ID NO: 18) shown in fig. 18).

FIG. 6 detection of the binding affinity of Nanobodies to the V-C1 and V domains of human RAGE by SPR. KD: a dissociation equilibrium constant; ka, kinetic association rate; kd, kinetic off-rate. FIGS. 6A-6C show the binding affinity results for the V-C1 domain of human RAGE for 3CNB-FC,4BNB-FC, and 5 ENB-FC. FIGS. 6D-6F show SPR binding kinetics for the V domain of human RAGE for 3CNB-FC,4BNB-FC, and 5 ENB-FC.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

A. Purification of the V-C1 domain of human RAGE for immunization of alpaca

1) The V-C1 domain (residues 23-237) of the human RAGE protein was expressed by the E-Coli, BL21-Codon Plus (DE 3) -RIPL strain (Stratagene) (see SEQ ID NO: 5) and purifying the V-C1 domain by affinity chromatography and size exclusion chromatography. The primary immunization was performed with Freund's complete adjuvant, followed twice by Freund's incomplete adjuvant. The alpaca was injected subcutaneously 3 times 3 weeks apart, each time with a 200 μ g/dose of antigen mixed at an antigen to adjuvant volume ratio of 1.

2) At week 16, blood was collected to isolate lymphocytes and the immune response to the antigen of our interest (the V-C1 domain of human RAGE) was analyzed.

3) Total RNA was extracted using the Omega Biotek RNA extraction kit and genomic DNA was removed. PrimeScript using Takara ^TM II first Strand cDNA Synthesis kit, RNA is reverse transcribed into cDNA.

4) Construction of a nano antibody phage display library: and using PCR to amplify the cDNA as a template to obtain the coding sequence of the nano antibody. The amplified nanobody sequences were cloned into the NcoI and NotI sites of phagemid pR2 using Gibson assembly (Gibson assembly). The Gibbson assembly product is the initial nanometer antibody phage library.

5) Coli TG1 competent cells were transformed using BTX ECM 399 electroporation: phage pR2 Nanobody genes (Gibbon assembly products) were transformed into E.coli TG1 competent cells in a 0.1cm electroporation cuvette. The electroporated product (500. Mu.L) was resuspended in 5mL LB medium and incubated at 37 ℃ for 45 min at 220 rpm. Bacteria were plated on 5 150mm TY (formulation 1l. Next, the transformed colonies were scraped off the plate, mixed well with a final concentration 25% glycerol vortex, snap frozen in 1mL aliquots of liquid nitrogen and stored at-80 ℃ and the size of the phage library was calculated (pfu/mL = OD) ₂₆₀ ×100×22.14×10 ¹⁰ )。

6) Amplification of the nanobody phage display library: to amplify the phage library of nanobodies, 0.2ml of the frozen library was thawed on ice, diluted into 200ml of TY medium supplemented with 100. Mu.g/ml ampicillin and 2% glucose, andthe culture was carried out at 37 ℃. Next, 1 × 10 ¹² pfu of KM13 helper phage (purchased from MRC molecular biology laboratories) was added to the culture and incubated at 37 deg.C (water bath) for 45 min. The cell pellet was separated by high speed centrifugation and resuspended in 200mL of 2 × TY medium supplemented with 0.1% glucose, 50 μ g/mL kanamycin and 100 μ g/mL ampicillin. Cells were incubated at 25 ℃ for 20 hours at 220rpm to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate phage particles. The precipitated phage particle pellet was dissolved in PBS and stored in 1mL aliquots at-80 ℃ in the presence of 25% glycerol.

7) Translation: the V-C1 domain of purified human RAGE was diluted with PBS to a final concentration of 0.1mg/ml and coated into one well of a 96-well immunoplate ELISA (Nunc maxsorp plate) and one well ELISA plate was left for use as a negative control. After 3 washes with PBS, 300 μ l MPBS (PBS with 5% skim milk) was added to each well of the ELISA plate and incubated for 3 hours at room temperature to block unbound sites. Next, the plate was washed 3 times with PBS and would contain 1X 10 for V-C1 ¹¹ pfu (dilution in 100. Mu.l MPBS) phage pools were added to each well and after one hour incubation at room temperature, washed 3 times with PBST (0.1% Tween 20 in PBS). Phage displaying specific V-C1 domain nanobodies were eluted by incubation with trypsin at a final concentration of 0.5mg/ml for one hour at room temperature. mu.L of the eluted phage was added to 1ml of E.coli TG1 competent cells and incubated for 45 min at 37 deg.C (water bath) for infection, and then the bacterial culture was plated on 2 XTY supplemented with 100. Mu.g/ml ampicillin and 2% glucose and incubated overnight at 37 deg.C.

8) Preparation of monoclonal phage: after one round of panning, 48 individual colonies were picked into a 96-well round bottom plate containing 100. Mu.l of 2 XTY medium supplemented with 100. Mu.g/ml ampicillin and 2% glucose (w/v). Incubation was continued at 37 deg.C/180 rpm for 12 hours. Next, 5. Mu.l of this culture broth was inoculated into a new 96-well round-bottom plate containing 200. Mu.l of 2 XTY medium supplemented with100. Mu.g/ml ampicillin and 2% glucose (w/v). Freshly inoculated plates were incubated at 37 ℃/250rpm for 1.5 hours until the OD260 was about 0.5. 50 μ l of the solution containing 4X 10 ⁸ pfu KM13 helper phage 2 XTY medium was added to each well of the plate and incubated at 37 ℃ for 45 minutes without shaking for infection. After infection, 150. Mu.l of supernatant was discarded and the remaining volume was centrifuged at 3500g for 15 min, the supernatant discarded, and the bacterial pellet resuspended in 200ml of 2 XTY medium supplemented with 100. Mu.g/ml ampicillin, 50. Mu.g/ml kanamycin and 0.1% glucose (w/v) and incubated overnight at 25 ℃/250rpm for a maximum of 20 hours. The next day, the culture was centrifuged at 3500g for 30 minutes, and then 150. Mu.l of the supernatant was transferred to a new 96-well plate and stored at 4 ℃ in preparation for screening for nanobodies.

8. Phage ELISA test: the dilution was performed by using PBS, the 96-well round-bottom immuno-ELISA plate was coated with 100. Mu.l streptavidin at a final concentration of 0.5. Mu.g/ml, and incubated at 4 ℃ for 16 hours. Next, the ELSA plates were washed 3 times with wash buffer containing TPBS (PBS with 0.1% tween 20). The plate was blocked with MPBS (PBS containing 5% skim milk) for 3 hours at room temperature. Then, the wells were washed 4 times with PBS-0.1

% Tween

20, 100. Mu.l of 1. Mu.g/ml biotinylated V-C1 domain was added to each well and incubated for one hour at room temperature with slow agitation (80 rpm) to bind biotinylated V-C1-streptavidin, and then the ELSA plates were washed 5 times with PBS-0.1% Tween 20. The supernatant containing soluble fragments of monoclonal phage displaying nanobodies (25. Mu.l of monoclonal phage per 75. Mu.l of 5% MPBS) was diluted, 100. Mu.l were transferred to each well and the C1 domain of RAGE was coated with 0.5. Mu.g/ml streptavidin-Biotin-V-, incubated for one hour at room temperature with slow agitation (180 rpm), after which the phage were discarded after one hour, and the ELISA plate was washed 5 times with PBS-0.1% Tween 20. KM13 was measured in 5% MPBS at a ratio of 1:8000 dilutions were added and gently stirred (80 rpm) at room temperature for one hour and then 100. Mu.l of 3,3', 5' -Tetramethylbenzidine (TMB) per 100 ml of substrate was added and washed 5 times with PBS and incubated in the dark for 10 min to develop color. 50 μ l of 1M sulfuric acid was added to reactShould be stopped and OD450 measured by ELISA spectrophotometer at 24 ℃ _nm The value is obtained.

9. Sequencing all positive clones with OD450nm values greater than 1, analyzing sequence results, and finally determining 3 positive phage nano antibodies specific to the V-C1 structural domain of the hRAGE protein, wherein the positive phage nano antibodies are respectively named; 3CNB (amino acid sequence shown as SEQ ID NO: 6), 4BNB (amino acid sequence shown as SEQ ID NO: 7) and 5ENB (amino acid sequence shown as SEQ ID NO: 8), the 3 nanobodies having specific CDR region binding to V-C1 domain of hRAGE.

B. Expression and purification of Nanobody-FC fusion proteins

1) In addition to fusing human IgG1Fc to the C end of the nano antibody gene, we can recombine the nano antibody gene and human IgG1Fc, and then clone into a mammalian expression vector pTT 5. Transfection of the construction vector into HEK293F (density about 2.5X 10) with Polyethylenimine (PEI) ⁶ Cells/ml), in Freestyle ^TM 293 expression Medium (purchased from Unionvern), mammalian cells after transfection were cultured in suspension. Mixing CO at 37 DEG C ₂ The incubator (incubator should not be overdried) is rotated at 150rpm to incubate the mammalian cells for 5 days and then centrifuged at 2000rpm for 10 minutes to collect the supernatant of the mammalian cell culture. And (3) purifying the nano antibody-FC fusion protein by using a protein A column, and performing SDS-PAGE electrophoretic analysis, wherein the result shows that the high-purity nano antibody-FC fusion protein is obtained from the supernatant.

2) The purified nanobody-FC fusion Protein was subjected to TEV enzyme digestion to digest the recombination sites of human IgG1FC, and then the mixture products were placed on a Protein a column and a Ni column, respectively, and nanobodies without FC products flowed through the Protein a column. And removing undigested complete nano antibody-Fc protein, fc and TEV enzyme by using a nickel column respectively, collecting flow-through liquid, concentrating, and performing SDS-PAGE electrophoresis to obtain the high-purity nano antibody in the flow-through liquid.

C. Stability of Nanobodies at high temperatures

Circular Dichroism (CD) was used to analyze the secondary structure (far ultraviolet CD spectroscopy) and thermal stability (temperature 20-95 ℃) of nanobodies. The far-ultraviolet CD spectra of each sample were obtained in a quartz cuvette at 190-250nm, with optical paths of 0.1cm and 1cm, respectively. CD spectra were collected by continuous scanning at 0.1nm intervals, scanning speed of 100nm/min, response time of 1s, bandwidth of 1 nm. The protein concentration of the nanobody is 0.3mg/ml. Baseline was corrected in all experiments using protein-free PBS solution as control. Data were analyzed using GraphPad Prism software, selecting spectral values at 217nm as a function of temperature, and further fitting Tm values. The TM values for each nanobody are shown in each nanobody curve, as shown in fig. 3A-3C, the TM values for 3CNB, 4BNB, and 5ENB are: 79.09 +/-0.29 ℃, 77.29 +/-0.02 ℃ and 86.89 +/-0.39 ℃.

D. Binding affinity analysis of Nanobodies to V-C1 Domain

Immuno MaxiSorb plates (Nunc) were coated with 2. Mu.g/ml of V-C1 domain, unbound sites were blocked with MPBS for 3 hours at room temperature, and then flowed by washing 4 times with PBS-0.1% Tween 20. Next, serial dilutions (1000-0.5 nM) of nanobody-FC protein were added to each well and incubated for 1 hour at room temperature with gentle stirring (80 rpm). After incubation for 1 hour at room temperature with slow stirring (80 rpm), nanobody-FC was detected using anti-FC-HRP secondary antibody (Beijing Yinqiao), and color development was observed after addition of TMB by using 50ml 1M H ₂ SO ₄ Development was stopped and absorbance at 450nM was measured. Next, we investigated the binding affinity of anti-V-C1 nanobody-FC against the variable V domain or C1 domain of hRAGE. Thus, in addition to the unrelated proteins (i.e., TEV, NTPase, caspase, MBP and hNLRP1-CARD, GB 1), variable V, C1 and V-C1 domains were coated at a final concentration of 5. Mu.g/ml into Immuno MaxiSorb plates (Nunc) for blocking and washing as described above. Next, 100ng/ml of 3CNB-FC,4BNB-FC and 5ENB-FC were added and incubated at room temperature for 1 hour, after washing 5 times with PBST, the nanobodies were detected using an anti-FC-HRP secondary antibody, color development was observed when TMB was added, by using 50ml of 1M H ₂ SO ₄ The reaction was stopped and the absorbance at 450nM was measured. FIG. 5 showsThe three kinds of nano antibodies have stronger binding affinity with a V structural domain and a V-C1 structural domain of human RAGE.

The results in FIGS. 6A-6C show that ka (1/Ms) =1.53E10 for 3CNB-FC ⁺⁶ ，Kd(1/s)＝8.37E ^-4 ，KD(M)＝5.478E ^-10 (ii) a Ka (1/Ms) =1.21E10 of 4BNB-FC ⁺⁶ ，Kd(1/s)＝7.52E10 ^-4 ，KD(M)＝6.21E10 ^-10 (ii) a Ka (1/Ms) =2.02E10 of 5ENB-FC ⁺⁶ ，Kd(1/s)＝8.33E10 ^-4 ，KD(M)＝4.14E10 ^-10 。

E. Characterization of affinity between Nanobodies and hRAGE proteins by SRP

Reagents were used; sensor chip CM5, phosphate buffer plus 0.05% Tween 20 (0.05% TPBS), acetate, pH4.5, V-C1 and variable V domains of human RAGE, anti-hRAGE Nanobody proteins (3 CNB-FC,4BNB-FC, and 5 ENB-FC), ethanolamine-HCl blocker, 20mM and 10mM NaOH regeneration solutions, and activation solutions. SPR experimental analysis was done using the Biacore T200 System (GE Healthcare) and 0.05% TPBS was used as sample buffer and running buffer. All assay temperatures were set at 25 ℃. Immobilization of the human RAGE V-C1 domain was performed using an amine coupling kit, pH 4.5. The V-C1 domain is immobilized in flow cell 3-4, and the other domains serve as reference cells for the nanobodies captured in flow cell 3-4, respectively. In kinetic studies, the capture level of nanobodies is typically in the range of 400-800 Reaction Units (RU).

The V-C1 domain was injected for 60s to increase the concentration of the reference flow cell and the active flow cell using a single cycle kinetic approach at seven concentrations (1000-15.625 nM). The surface was regenerated by injecting 120s of regeneration solution (20 mM NaOH) from the capture kit to remove the bound nanobodies. Nanobody +0.05% tpbs buffer injection + regeneration (blank cycle) was completed in each nanobody. The response is first subtracted from the reference flow-through cell and then the data for the blanking period is subtracted as a double reference. By using Biacore ^TM The T200 evaluation software 2.0 selected a one-to-one binding model to fit the data. SPR analysis of variable V-domain parameters was identical to V-C1 domains of the desired regeneration solution (10 mM NaOH injection time 40 s).

The results in FIGS. 6D-6F show that ka (1/Ms) =1.57E10 for 3CNB-FC ⁺⁶ ，Kd(1/s)＝0.0017，KD(M)＝1.09E10 ^-9 (ii) a Ka (1/Ms) =2.21E10 of 4BNB-FC ⁺⁶ ，Kd(1/s)＝0.0041，KD(M)＝1.61E10 ^-9 (ii) a Ka (1/Ms) =2.51E10 of 5ENB-FC ⁺⁶ ，Kd(1/s)＝0.0032，KD(M)＝1.28E10 ^-9 。

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds the extracellular domain of human RAGE comprising CDR1 as set forth in SEQ ID NO. 9, CDR2 as set forth in SEQ ID NO. 10, and CDR3 as set forth in SEQ ID NO. 11.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: and 6, respectively.

3. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A host cell comprising the expression vector of claim 4, said host cell being a host cell for expression of a foreign protein.

6. The host cell of claim 5, wherein the host cell is a bacterium, yeast, insect cell, or mammalian cell.

7. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-2, comprising the steps of culturing the host cell of claim 5 or 6, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2 and a pharmaceutically acceptable carrier.

9. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-2 in the manufacture of a kit or medicament for the detection of RAGE.