Anti-procalcitonin nano antibody and application thereof

Abstract

Based on the current situation that no nano antibody aiming at a procalcitonin (PCT) target spot is disclosed in the field, the invention develops an anti-PCT nano antibody which has relatively good binding activity and relatively good binding sensitivity and is simple and convenient to produce, and the anti-PCT nano antibody comprises a detection nano antibody and a coating nano antibody; the nano antibody for specifically targeting PCT provided by the invention meets the requirements on diagnosis sensitivity and precision, and has relatively high application potential in the field of detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals, and particularly relates to an anti-procalcitonin nanobody and its application. Background Art

[0002] Procalcitonin (PCT) is a hormone precursor produced by thyroid C cells. Under normal circumstances, its concentration in healthy human bodies is very low. Viral infections and non-specific inflammations do not cause an increase in procalcitonin levels, but bacterial infections, severe shock, and multiple organ failure syndrome can all lead to a significant increase in procalcitonin levels, and the increase amplitude is closely related to the severity of the disease and the prognosis of patients. Procalcitonin detection has high clinical application value in the differential diagnosis of infectious diseases, monitoring of critically ill patients, evaluation of their treatment effects, prognosis judgment, and medication guidance. PCT has become an important biomarker for diagnosing and monitoring infectious diseases, inflammatory states, and systemic inflammatory response syndrome.

[0003] Nanobodies are the structures of the variable regions of heavy-chain antibodies (VHH) in camels. The single VHH has the activity of binding to antigens. Due to its special structural characteristics, compared with traditional monoclonal antibodies, nanobodies have advantages such as high solubility, not easy to aggregate, high temperature resistance, strong acid and strong alkali resistance, etc. The relatively small volume of nanobodies endows them with stronger tissue penetration ability. At the same time, nanobodies are easy to be expressed and amplified in prokaryotic / eukaryotic cells in vitro in microbial systems such as bacteria, yeast, and fungi, so that they can be mass-produced at a relatively low cost; they have wide applications in biomedical research and clinical medicine such as molecular diagnosis, drug delivery, and tumor immunotherapy.

[0004] Up to now, no nanobody targeting the PCT target has been disclosed. Therefore, there is an urgent need in this field to develop an anti-PCT nanobody with good binding activity, good diagnostic sensitivity, and simple production. Based on the current requirements for diagnostic sensitivity and accuracy, nanobodies specifically targeting PCT have been developed, which have high application potential in the detection field. Summary of the Invention

[0005] The first aspect of the present invention provides an anti-PCT nanobody, and the complementarity-determining regions (CDR regions) of the VHH chain in the nanobody are as follows:

[0006] (1) CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7; or

[0007] (2) CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:14.

[0008] In a preferred embodiment, for the anti-PCT nanobody, the CDR1, CDR2, and CDR3 are separated by framework regions FR1, FR2, and FR3 of the VHH chain.

[0009] In a preferred embodiment, the VHH chain of the anti-PCT nanobody further includes a framework region FR, and the framework region FR is as follows:

[0010] (1) FR1 shown in SEQ ID NO:2, FR2 shown in SEQ ID NO:3, and FR3 shown in SEQ ID NO:4; or

[0011] (2) FR1 shown in SEQ ID NO:9, FR2 shown in SEQ ID NO:10, and FR3 shown in SEQ ID NO:11.

[0012] In a preferred embodiment, the amino acid sequence of the VHH chain of the anti-PCT nanobody is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:8, or a combination thereof.

[0013] The second aspect of the present invention provides an anti-PCT antibody, which is an antibody against a PCT epitope and has the anti-PCT nanobodies described in each preferred embodiment of the first aspect.

[0014] The third aspect of the present invention provides an anti-PCT antibody, which comprises one or more anti-PCT nanobodies described in each preferred embodiment of the first aspect.

[0015] In a preferred embodiment, the PCT antibody of the third aspect of the present invention comprises monomers, bivalent antibodies, and / or multivalent antibodies.

[0016] The fourth aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of: the anti-PCT nanobodies described in each preferred embodiment of the first aspect of the present invention, and the anti-PCT antibodies described in the third and fourth aspects of the present invention.

[0017] The fifth aspect of the present invention provides an expression vector containing the polynucleotide provided in the fourth aspect of the present invention.

[0018] The sixth aspect of the present invention provides a host cell, which contains the expression vector provided in the fifth aspect of the present invention, or a polynucleotide provided in the fourth aspect of the present invention is integrated into its genome.

[0019] The seventh aspect of the present invention provides a method for generating an anti-PCT nanobody, and the method includes the following steps:

[0020] (a) Culturing the host cell provided in the sixth aspect of the present invention under conditions suitable for generating nanobodies, so as to obtain a culture containing the anti-PCT nanobody;

[0021] (b) Separating or recovering the anti-PCT nanobody from the culture; and

[0022] (c) Optionally, purifying and / or modifying the anti-PCT nanobody obtained in step (b).

[0023] The seventh aspect of the present invention provides an immunoconjugate, which contains:

[0024] (a) The anti-PCT nanobodies described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibodies described in the third and fourth aspects of the present invention;

[0025] (b) A conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a magnetic nanoparticle, a virus coat protein or VLP, or a combination thereof.

[0026] The eighth aspect of the present invention provides a multispecific antibody, and the multispecific antibody contains: the anti-PCT nanobodies described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibodies described in the third and fourth aspects of the present invention.

[0027] The ninth aspect of the present invention provides a recombinant protein, and the recombinant protein has:

[0028] (i) The anti-PCT nanobodies described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibodies described in the third and fourth aspects of the present invention; and

[0029] (ii) Optionally, a tag sequence for assisting expression and / or purification.

[0030] The tenth aspect of the present invention provides a pharmaceutical composition, and the pharmaceutical composition contains:

[0031] (i) The anti-PCT nanobodies described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibodies described in the third and fourth aspects of the present invention; and

[0032] (ii) A pharmaceutically acceptable carrier.

[0033] The eleventh aspect of the present invention provides the use of the anti-PCT nanobody described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibody described in the third and fourth aspects of the present invention, the immunoconjugate provided by the seventh aspect of the present invention, the multispecific antibody provided by the eighth aspect of the present invention, the recombinant protein provided by the ninth aspect of the present invention, or the pharmaceutical composition provided by the tenth aspect of the present invention in the preparation of a drug for preventing and / or treating PCT-related diseases.

[0034] The twelfth aspect of the present invention provides the use of the anti-PCT nanobody described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibody described in the third and fourth aspects of the present invention, the immunoconjugate provided by the seventh aspect of the present invention, the multispecific antibody provided by the eighth aspect of the present invention, the recombinant protein provided by the ninth aspect of the present invention, or the pharmaceutical composition provided by the tenth aspect of the present invention in the preparation of a reagent, test strip or kit for detecting PCT.

[0035] The eleventh aspect of the present invention provides a method for detecting PCT protein in a sample, the method being for non-diagnostic purposes and comprising the steps of:

[0036] (1) contacting the sample with the anti-PCT nanobody described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibody described in the third and fourth aspects of the present invention, the immunoconjugate provided by the seventh aspect of the present invention, the multispecific antibody provided by the eighth aspect of the present invention, the recombinant protein provided by the ninth aspect of the present invention;

[0037] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PCT protein in the sample.

[0038] The twelfth aspect of the present invention provides a PCT protein detection reagent, the detection reagent comprising:

[0039] (i) the anti-PCT nanobody described in each preferred embodiment of the first aspect of the present invention, the anti-PCT antibody described in the third and fourth aspects of the present invention, the immunoconjugate provided by the seventh aspect of the present invention, the multispecific antibody provided by the eighth aspect of the present invention, the recombinant protein provided by the ninth aspect of the present invention; and

[0040] (ii) a detection-acceptable carrier.

[0041] The thirteenth aspect of the present invention provides a kit for detecting PCT protein, the kit containing the immunoconjugate provided by the seventh aspect of the present invention or the detection reagent provided by the twelfth aspect of the present invention, and an instruction manual.

[0042] The fourteenth aspect of the present invention provides a use of an immunoconjugate in the preparation of a contrast agent for detecting PCT protein in vivo, and the immunoconjugate is the immunoconjugate provided in the seventh aspect.

[0043] The antibody or its antigen-binding fragment provided by the present invention has one or more of the following advantages:

[0044] 1. The present application provides a PCT-specific nanobody for the first time. This nanobody can specifically bind to the PCT antigen and has strong affinity.

[0045] 2. By using the combination of the detection nanobody and the coating nanobody shown in Table 2 of the present invention, the PCT immunogen can be effectively detected, with high detection sensitivity, significantly higher than that of the commercial anti-PCT antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Shows the binding sensitivity experiment of the antibody of the present application and the commercial control antibody to the PCT immunogen shown in Example 2. Terms:

[0047] As used herein, the terms "single domain", "VHH", "nanobody", and "heavy chain antibody" (singledomain antibody, sdAb, or nanobody) have the same meaning and can be used interchangeably, referring to cloning the variable region of the heavy chain of an antibody and constructing a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region (CH1) of the heavy chain, the variable region of the heavy chain of the antibody is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention will be further described below in conjunction with specific embodiments. The described embodiments are some, but not all, of the embodiments of the present invention. It should be understood that the following embodiments are provided to give a complete disclosure and description to those of ordinary skill in the art of the present invention on how to use the methods and compositions of the present invention, and are not used to limit the scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0049] Example 1. Generation of Nanobody

[0050] 1.1 Preparation of Immunogen

[0051] Synthesize the PCT gene sequence (see Table 1), construct the eukaryotic expression vector PCDNA3.1-PCT, transfect HEK293 cells, after culturing for 6 days, centrifuge to obtain the supernatant, and purify it with HIS filler.

[0052] Table 1. PCT gene sequence

[0053]

[0054] 1.2 Immunization protocol

[0055] 1) Select adult alpacas over 8 months old, with an immunization dose of 2 mg / time.

[0056] 2) Primary immunization: Mix 2 mg of antigen with Freund's complete adjuvant and emulsify it, then perform subcutaneous multi-point immunization injection on the back of the alpaca.

[0057] 3) Boost immunization: 14 days after the primary immunization, perform the boost immunization. Change Freund's complete adjuvant to incomplete adjuvant, and the other steps are the same as the primary immunization; collect blood 7 days later and detect the serum titer.

[0058] 4) Second boost immunization: 14 days after the boost immunization, perform the second boost immunization in the same way as the boost immunization, collect blood 7 days later and detect the serum titer.

[0059] 5) Third boost immunization: 14 days after the second boost immunization, perform the third boost immunization in the same way as the boost immunization, collect blood 7 days later and detect the serum titer.

[0060] 1.3 Antibody library construction:

[0061] Extract RNA from alpaca peripheral blood; amplify the nanobody gene; construct the nanobody gene and pcomb3x vector; electrotransform the pcomb3x vector ligated with the nanobody into XL1-Blue competent cells to prepare the antibody bacterial liquid library; add VCSM13 helper phage to infect the cultured bacterial liquid library, and centrifuge after shaking culture with 4% PEG800 precipitation to obtain the expressed phage library. 1.4 Screening of phage library:

[0062] Coat the ELISA plate with 10 μg / ml of PCT antigen overnight, wash the plate with PBST, block it with skim milk powder, wash the plate with PBST, then add 5*10^13 phages and incubate for 2 h. After washing the plate, elute with Glycine-Hcl. Mix the eluate with Escherichia coli XL1-Blue and culture for 30 min, then add helper phage and culture overnight to obtain the first-round library. Repeat the above steps 2 times to obtain the third-round library. Detect the third-round amplified library by ELISA and perform monoclonal screening.

[0063] 1.5 Monoclonal screening:

[0064] Select 192 monoclonal clones from the third-round library, coat the ELISA plate with the antigen, after blocking and washing, add the phage expression supernatant, detect by ELISA, and sequence the positive clones.

[0065] 1.6 Nanobody Expression and Identification

[0066] Construct an expression vector for the positive clone, express it in HEK293 cells, and perform paired detection of PCT on the obtained nanobodies. Finally, obtain the coated nanobodies and the detection nanobodies.

[0067] After identification, the amino acid sequences of the coated nanobodies and the detection nanobodies are obtained and shown in Table 2.

[0068] Table 2. Amino Acid Sequences of Coated Nanobodies and Detection Nanobodies

[0069]

[0070] Example 2 Verification of the Detection Effect of Nanobodies

[0071] 2.1 ELISA Detection Effect

[0072] 1) Preparation of experimental liquids:

[0073] Coating solution (CBS): 0.8 g of Na2CO3, 1.46 g of NaHCO3, dissolved in water and made up to 500 ml.

[0074] Washing solution PBST: 4 g of KH2PO4, 58 g of Na2HPO4·12H2O, 160 g of NaCl, 4 g of KCl, 10 mL of Tween-20, made up to 2 L with water.

[0075] 2) Experimental procedure:

[0076] Set up experimental groups and control groups; the experimental group is: using the coated nanobody-detection nanobody shown in Table 2 of this application form, and the control group is: commercially available PCT murine monoclonal antibody.

[0077] Coating: Dilute the coated nanobody with a PH9.6 carbonate buffer (CBS) coating solution at 1 μg / ml, 100 μL per well, coat overnight at 4°C, wash twice with PBST, and pat dry.

[0078] Blocking: Prepare 3% skim milk powder, 340 μL per well, incubate at room temperature for 1 h, wash twice with PBST, and pat dry.

[0079] Sample addition: Dilute the standard sample PCT to 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.8, 12.6, 25.2 ng / ml, 100 μL per well, incubate at room temperature for 1 h, wash twice with PBST, and pat dry.

[0080] The HRP-labeled detection nanobody was diluted to the working concentration of 2 μg / ml, 100 μL per well, incubated at room temperature for 1 h, washed 3 times with PBST, and patted dry.

[0081] Color development: TMB color development solution, 100 μL per well, reacted at room temperature for 20 min.

[0082] Termination: 2 M sulfuric acid, 50 μL per well.

[0083] Reading with an enzyme-linked immunosorbent assay reader: OD450nm

[0084] The detection process of the commercial PCT murine monoclonal antibody is the same as the above operation.

[0085] The experimental result graph is as Figure 1 shown. It can be seen from the figure that when detecting with the VHH antibody in Table 2, the OD45 value is more significant, reflecting a more sensitive detection effect, far superior to the commercial PCT murine monoclonal antibody in the control group.

Claims

1. An anti-procalcitonin (PCT) nanobody that binds to PCT, characterized in that the complementarity-determining regions (CDR regions) of the VHH chain in the nanobody are as follows: (1) CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7; or (2) CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:

14.

2. The anti-PCT nanobody according to claim 1, characterized in that, The CDR1, CDR2, and CDR3 are separated by the framework regions FR1, FR2, and FR3 of the VHH chain; Preferably, the VHH chain of the anti-PCT nanobody further includes a framework region FR, and the framework region FR is as follows: (1) FR1 shown in SEQ ID NO:2, FR2 shown in SEQ ID NO:3, and FR3 shown in SEQ ID NO:4; or (2) FR1 shown in SEQ ID NO:9, FR2 shown in SEQ ID NO:10, and FR3 shown in SEQ ID NO:

11.

3. The anti-PCT nanobody according to claim 1 or 2, characterized in that, The amino acid sequence of the VHH chain of the anti-PCT nanobody is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:8, or a combination thereof.

4. A polynucleotide, characterized in that, The polynucleotide encodes the anti-PCT antibody according to any one of claims 1-3.

5. An expression vector, characterized in that, The expression vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector according to claim 5, or the polynucleotide according to claim 4 is integrated into the genome of the host cell.

7. A method for producing an anti-PCT nanobody, the method comprising the following steps: (a) Culturing the host cell according to claim 6 under conditions suitable for producing nanobodies, thereby obtaining a culture containing the anti-PCT nanobody; (b) Separating or recovering the anti-PCT nanobody from the culture; and (c) Optionally, purifying and / or modifying the anti-PCT nanobody obtained in step (b).

8. An immunoconjugate, the immunoconjugate containing: (a) The anti-PCT nanobody according to any one of claims 1-3, or the anti-PCT nanobody prepared by the method according to claim 7; (b) A conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a magnetic nanoparticle, a virus capsid protein or VLP, or a combination thereof.

9. A method for in vitro detecting PCT protein in a sample, the method is for non-diagnostic purposes, and the method comprises the steps of: (1) Contacting the sample with the anti-PCT nanobody according to any one of claims 1-3, the anti-PCT nanobody prepared by the method according to claim 7, or the immunoconjugate according to claim 8; (2) Detecting whether an antigen-antibody complex is formed, and the formation of the complex indicates the presence of PCT protein in the sample.

10. A PCT protein detection kit, wherein the detection reagent comprises: (i) The anti-PCT nanobody according to any one of claims 1-3, the anti-PCT nanobody prepared by the method according to claim 7, or the immunoconjugate according to claim 8; and the specification.

11. Use of the anti-PCT nanobody according to any one of claims 1-3, the anti-PCT nanobody prepared by the method according to claim 7, the immunoconjugate according to claim 8, or the PCT protein detection kit according to claim 10 in the preparation of a PCT protein preparation for detection.

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