Anti-procalcitonin antibody and detection kit containing the antibody

Abstract

The present invention provides an anti-procalcitonin antibody. The present invention also provides a detection kit comprising the anti-procalcitonin antibody. The anti-procalcitonin antibody provided by the present invention has high affinity and specificity for the binding of procalcitonin and can be used for detecting the level of anti-procalcitonin in serum.

Description

Technical Field

[0001] The present invention relates to an anti-procalcitonin antibody and also relates to a detection kit comprising the anti-procalcitonin antibody. Background Art

[0002] Procalcitonin (PCT) is a 116 amino acid glycoprotein without hormone activity, with a molecular weight of about 13KD, encoded by the CALC-I gene located on chromosome 11. Procalcitonin (PCT) is the precursor of calcitonin (CT), including the 57 amino acid N-terminus (N-PCT), the 32-peptide active CT and the C-terminal 21-peptide anti-calcitonin (Katacalcin, PDN 21). Under normal conditions, PCT is produced by thyroid C cells and cleaved into calcitonin. The content of PCT in human serum is extremely low, at the picogram level per milliliter. However, in patients with systemic inflammatory response caused by bacterial infection, sepsis or septic shock, chronic renal failure, etc., serum PCT and its components are significantly increased, which can reach hundreds to tens of thousands of times the normal level, but the content of CT remains unchanged or slightly increased. Therefore, PCT can be used as a specific indicator of bacterial infection. Compared with other markers, PCT has higher accuracy and specificity. For example, PCT can be detected 2 hours after infection, reaches a peak value in 6-12 hours, and decreases in concentration after 24 hours, with a half-life of about 20-24 hours, which can reflect the treatment effect in a timely manner, while C-reactive protein (CRP) reaches a peak value 24-48 hours after infection, and the concentration remains at a high level after inflammation is controlled, which is not conducive to early diagnosis and treatment evaluation. In addition, PCT can distinguish between bacterial and viral infections, and can also assess the severity of infection based on PCT levels.

[0003] Commonly used PCT detection methods include immunochromatography, electrochemiluminescence immunoassay, enzyme-linked immunosorbent assay and latex immunoturbidimetry, most of which require the use of anti-PCT antibodies for detection. Since the PCT content in healthy human serum is extremely low and may be interfered by CT during detection, it is necessary to develop anti-PCT antibodies with high affinity and specificity. Summary of the invention

[0004] The object of the present invention is to provide an anti-procalcitonin antibody and a detection kit comprising the antibody.

[0005] The present invention adopts the following technical scheme.

[0006] The present invention provides a single domain antibody targeting human procalcitonin, wherein the complementarity determining region sequence of the single domain antibody is as follows:

[0007] 1) the sequence of HCDR1 is shown in SEQ ID NO: 5, the sequence of HCDR2 is shown in SEQ ID NO: 6, and the sequence of HCDR3 is shown in SEQ ID NO: 7; or

[0008] 2) The sequence of HCDR1 is shown in SEQ ID NO: 9, the sequence of HCDR2 is shown in SEQ ID NO: 10, and the sequence of HCDR3 is shown in SEQ ID NO: 11.

[0009] In some embodiments, the heavy chain variable region of the single domain antibody comprises the sequence shown in SEQ ID NO: 4 or 8.

[0010] In some embodiments, the heavy chain variable region is linked to a human Fc fragment at its C-terminus.

[0011] In some embodiments, the human Fc fragment comprises the sequence shown in SEQ ID NO:3.

[0012] In some embodiments, the single domain antibody binds to human procalcitonin. 50 The value was no higher than 30 ng / mL.

[0013] In some embodiments, the single domain antibody binds to calcitonin, anticalcitonin and / or N-PCT. 50 The value was higher than 2 μg / mL.

[0014] In another aspect, the present invention provides a procalcitonin detection kit, which comprises the above-mentioned single domain antibody.

[0015] The single domain antibody provided by the present invention can bind to procalcitonin with high affinity, but does not bind to calcitonin, and has high binding specificity. DETAILED DESCRIPTION

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0017] The term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In the text, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the situation consisting of the elements, integers or steps described. For example, when it is mentioned that an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0018] The term "about" generally refers to a variation within a range of 10% above or below a specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0019] The term "or" refers to a single element of the listed optional elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed optional elements.

[0020] "Antibody" refers to an immunoglobulin or other type of molecule including one or more antigen-binding domains that specifically bind to an antigen, which is a protein or polypeptide that exhibits binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are usually tetramers composed of 2 identical heavy chains and 2 identical light chains interconnected by disulfide bonds. According to the conservative differences in the amino acid sequences, the heavy chain and the light chain are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable region is used to recognize and bind to the antigen, and the constant region (such as the Fc fragment) is used to initiate downstream effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC). In the variable regions of the heavy and light chains, there are three local regions whose amino acid composition and arrangement order have a higher degree of variation, which are key positions for antibody-antigen binding, and are therefore also referred to as complementary determining regions (CDRs). The amino acid sequence of the CDR can be determined using a numbering scheme recognized in the art, such as Kabat, Chothia, IMGT, or Contact. The three complementarity determining regions of the heavy chain are called HCDR1, HCDR2 and HCDR3, and the three complementarity determining regions of the light chain are called LCDR1, LCDR2 and LCDR3. Each heavy chain variable region (VH) and light chain variable region (VL) can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0021] Unlike classical antibody molecules, "single domain antibodies (sdAb)", also known as "VHH antibodies", nanoantibodies, etc., refer to antibody molecules with antigen binding ability, including heavy chain variable regions but no light chains. Structurally, single domain antibodies can also be considered as fragments of classical four-chain antibody molecules. Single domain antibodies were first discovered in camelids, and then researchers discovered more single domain antibodies with antigen binding ability through screening of antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (e.g., classical antibody molecules), such as but not limited to: smaller molecular weight, easy to reach tissues or parts that ordinary antibody molecules cannot reach when used in the human body, or able to contact antigen epitopes in proteins or polypeptides that ordinary antibody molecules cannot reach; more stable, able to withstand changes in temperature and pH, as well as the effects of denaturants and proteases.

[0022] "Fc fragment" refers to the handle region of the classic "Y"-shaped antibody molecule, i.e., the crystallizable fragment, including the second and third constant domains (CH2 and CH3 domains) of the heavy chain. The antibody Fc region can be obtained by hydrolyzing the antibody molecule with a proteolytic enzyme (such as papain). The effector functions of the "Fc fragment" may include binding to Fc receptors, Clq binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediated phagocytosis, etc.

[0023] For antibodies or their antigen-binding fragments, "targeting" or "specific binding" means that one molecule (such as an antibody or its antigen-binding fragment) has a higher binding affinity for another molecule (such as an antigen) than other molecules present in the environment. The binding affinity of an antibody to an antigen can be measured by some parameters, such as the EC of the antibody binding to the antigen. 50 value or KD value.

[0024] “EC 50 EC50 refers to the concentration that causes 50% of the maximum effect. When used to indicate the binding ability of an antibody molecule to the corresponding antigen in an enzyme-linked immunosorbent assay (ELISA), it refers to the concentration of the antibody molecule that produces half of the maximum detection signal (such as colorimetric or fluorescence intensity). The lower the EC50 value, the greater the binding affinity to the antigen.

[0025] Provided herein are single domain antibodies that bind to PCT with high affinity, which can be used in various detection methods and detection reagents for detecting the content of PCT in samples based on antigen-antibody reactions. For example, the single domain antibodies provided herein can be present in a PCT detection kit with a secondary antibody (targeting the Fc segment of the single domain antibody) coupled with a protease (such as horseradish peroxidase) that is convenient for detection. Alternatively, the single domain antibodies provided herein can also be coupled to the above-mentioned protease or other detectable labels (such as radioisotopes, fluorophores, etc.) to detect PCT by direct ELISA.

[0026] The present invention is further illustrated by the following specific examples.

[0027] Example 1 Preparation of phage antibody library and affinity panning of specific clones

[0028] Fusion proteins PCT-huFc and CT-huFc linked to human IgG1 Fc fragment were expressed in 293T cells using eukaryotic expression vectors. The PCT sequence is shown in SEQ ID NO: 1, the CT sequence is shown in SEQ ID NO: 2, and the huFc sequence is shown in SEQ ID NO: 3. Protein A chromatography column was used for separation and purification.

[0029] PCT sequence:

[0030] APFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRSKRCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPGKKRDMSSDLERDHRPHVSMPQNAN (SEQ ID NO: 1)

[0031] CT sequence:

[0032] CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO: 2)

[0033] IgG1 Fc sequence

[0034] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3)

[0035] Alpacas were immunized with purified PCT-huFc by multiple injections at the back and neck for a total of 4 times, with an interval of 3 weeks between each immunization. One week after the last immunization, 100 mL of blood was collected to separate peripheral blood mononuclear cells (PBMCs), and total RNA was extracted using an RNA extraction kit and reversely transcribed into cDNA. Using degenerate primers, the nucleic acid fragment encoding the heavy chain single domain antibody (VHH) was amplified by PCR, and then connected to the phage display plasmid pComb3XSS and transformed into competent Escherichia coli TG1 cells, and a phage antibody display library was constructed in the presence of helper phage M13K07.

[0036] Biotinylated PCT-huFc was coated with magnetic beads coupled with streptavidin for positive panning of phages. Biotinylated PC-huFc was coated with magnetic beads coupled with streptavidin for negative panning of phages. A total of four rounds of liquid phase panning were performed, with negative panning first and positive panning next in each round. After the last round of panning, monoclonal clones were randomly selected from the enriched phage pool, packaged into phages, and the binding of monoclonal phages to PCT-huFc and PC-huFc was detected by ELISA to find clones that bind to PCT-huFc but not PC-huFc. The encoding nucleic acid sequence was obtained by sequencing, and then the antibody amino acid sequence was obtained. A total of 23 monoclonal phages were screened. In subsequent studies, it was found that the antibodies corresponding to two clones had relatively high affinity and specificity with PCT. The VHH sequences of the two antibodies (numbered 13# and 20#) are as follows.

[0037] Antibody 13# VHH:

[0038] EVQLAESGGGLVQAGGALRLSCAASGRTFSLNASKWFRQAPGKERDFVAVSDASASYTYSAVAGQFAISRDNSKNTVYLQMSSLKPEDTAVYYCAADPSLMGGKYSLLREVDYWGRGTQVTVSS (SEQ ID NO: 4)

[0039] Antibody 13# HCDR1: LNASK (SEQ ID NO: 5)

[0040] Antibody 13# HCDR2: VSDASASYTYSAVAG (SEQ ID NO: 6)

[0041] Antibody 13# HCDR3: DPSLMGGKYSLLREVDY (SEQ ID NO: 7)

[0042] Antibody 20# VHH:

[0043] DVQLVESGGGLVQAGGALRLSCAASGRTFSSYAMALWFRQAPGKERDFVATYSLSWSGSRIFAISRDNSKNTVYLQMNSLKPEDTAVYYCAAAADVVETEWYARLDEWGRGTQVTVSS (SEQ ID NO: 8)

[0044] Antibody 20# HCDR1: SYAMAL (SEQ ID NO: 9)

[0045] Antibody 20# HCDR2: TYSLSWSGSRI (SEQ ID NO: 10)

[0046] Antibody 20# HCDR3: AADVVETEWYARLDE (SEQ ID NO: 11).

[0047] Example 2 Expression and purification of single domain antibodies in eukaryotic cells

[0048] The coding sequences of the heavy chain variable regions of antibodies 13# and 20# obtained in Example 1 were codon optimized and cloned into the vector pCDNA3.4 together with the human Fc gene sequence, and the correctness of the clones was identified by enzyme digestion. 293T cells were transfected, cultured for 72 hours, and the supernatant was collected by centrifugation, purified using a Protein A chromatography column, and the molecular weight and purity of the purified antibodies were identified by SDS-PAGE. The molecular weight of the two antibodies was about 38kD and the purity was about 95%.

[0049] Example 3 Determination of EC binding of antibodies to PCT and its fragments by ELISA 50 value

[0050] The binding ability of antibodies 13# and 20# prepared in Example 2 to PCT, CT, Katacalcin (amino acid sequence as shown in SEQ ID NO: 12) and N-PCT (amino acid sequence as shown in SEQ ID NO: 13) was evaluated by indirect ELISA (PCT, CT, Katacalcin were purchased commercially, and N-PCT was obtained by commissioned synthesis). ELISA plates were coated with 0.5 μg / mL PCT, CT, Katacalcin or N-PCT at 4°C overnight. The plates were washed with PBS-T (0.05% Tween) and blocked with PBS-T containing 1% BSA at 37°C for 2 hours. The blocking solution was discarded, and 100 μl of 2 μg / mL antibody 13# or 20# was added to the first well, and the antibody was added to the subsequent wells in a 3-fold gradient dilution, for a total of 11 wells. After incubation at room temperature for 1 hour, the plate was washed three times with PBS-T, and goat anti-human Fc antibody coupled to horseradish peroxidase was added and incubated at 37°C for 1 hour. The plate was washed 4 times with PBS-T, then TMB colorimetric solution was added and incubated at room temperature in the dark for 20 minutes. The reaction was terminated by adding 50 μL of 1 M HCl stop solution. The plate was read at 450 nm using a microplate reader, and a curve was plotted based on the OD450 value to obtain the EC 50 The results are shown in Table 1.

[0051] Katacalcin sequence:

[0052] DMSSDLERDHRPHVSMPQNAN (SEQ ID NO: 12)

[0053] N-PCT sequence:

[0054] APFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRS (SEQ ID NO: 13)

[0055] Table 1 EC50 values ​​of antibody binding to various antigens (ng / mL)

[0056] antibody PCT CT Catacalcin N-PCT 13# 28.3 > 2000 > 2000 > 2000 20# 12.7 > 2000 > 2000 518.4

[0057] Antibody 13# has high affinity to PCT, but basically does not bind to the fragments after PCT degradation. It is speculated that its binding epitope should involve amino acids in at least two of the fragments. Antibody 20# has high affinity to PCT, basically does not bind to CT and Katacalcin, and has a lower binding affinity to N-PCT. It is speculated that its binding epitope is related to the N-PCT part, but also involves amino acids in other parts of PCT.

Claims

1. A single domain antibody targeting human procalcitonin, wherein the complementarity determining region sequence of the single domain antibody is as follows: 1) the sequence of HCDR1 is shown in SEQ ID NO: 5, the sequence of HCDR2 is shown in SEQ ID NO: 6, and the sequence of HCDR3 is shown in SEQ ID NO: 7; or 2) The sequence of HCDR1 is shown in SEQ ID NO: 9, the sequence of HCDR2 is shown in SEQ ID NO: 10, and the sequence of HCDR3 is shown in SEQ ID NO:

11.

2. The single-domain antibody according to claim 1, wherein the heavy chain variable region of the single-domain antibody comprises the sequence shown in SEQ ID NO: 4 or 8.

3. The single domain antibody according to claim 2, wherein the heavy chain variable region is connected to a human Fc fragment at its C-terminus.

4. The single-domain antibody according to claim 3, wherein the human Fc fragment comprises the sequence shown in SEQ ID NO:

3.

5. The single domain antibody according to claim 1, wherein the single domain antibody binds to human procalcitonin. 50 The value was no higher than 30 ng / mL.

6. The single domain antibody according to any one of claims 1 to 4, wherein the single domain antibody binds to calcitonin, anticalcitonin and / or N-PCT. 50 The value was higher than 2 μg / mL.

7. A procalcitonin detection kit comprising the single domain antibody according to any one of claims 1 to 6.