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CN119264254B - Anti-procalcitonin antibody and detection kit containing the antibody - Google Patents

Anti-procalcitonin antibody and detection kit containing the antibody Download PDF

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CN119264254B
CN119264254B CN202411793635.1A CN202411793635A CN119264254B CN 119264254 B CN119264254 B CN 119264254B CN 202411793635 A CN202411793635 A CN 202411793635A CN 119264254 B CN119264254 B CN 119264254B
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single domain
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CN119264254A (en
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张屹
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Yantai Jiezi Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/585—Calcitonins

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Abstract

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

Description

Anti-procalcitonin antibody and detection kit comprising same
Technical Field
The present invention relates to anti-procalcitonin antibodies. The invention also relates to a detection kit comprising the procalcitonin antibody.
Background
Procalcitonin (procalcitonin, PCT) is a hormone-free active glycoprotein of 116 amino acids and has a molecular weight of about 13KD and is encoded by the CALC-I gene located on chromosome 11. Procalcitonin (PCT) is a precursor of Calcitonin (CT), comprising the N-terminal of 57 amino acids (N-PCT), active CT of the 32 peptide and the anti-calpain of the C-terminal 21 peptide (KATACALCIN, PDN 21). Under normal conditions PCT is produced from thyroid C cells and cleaved to calcitonin. PCT content in human serum is extremely low, on the order of picograms per milliliter. However, in patients with systemic inflammatory response, sepsis or septic shock, chronic renal failure, etc. caused by bacterial infection, serum PCT and its components are significantly elevated, several hundred to several tens of thousands times the normal level can be reached, but the CT content remains unchanged or slightly increased. PCT can therefore be used as a specific indicator of bacterial infection. PCT has higher accuracy and specificity than other markers. For example, PCT can be detected 2 hours after infection, peak at 6-12 hours, concentration drops after 24 hours, half-life is about 20-24 h, and the therapeutic effect can be reflected in time, while C-reactive protein (CRP) peaks only 24-48 hours after infection, and concentration remains high after inflammatory control, which is detrimental to early diagnosis and therapeutic evaluation. In addition, PCT can distinguish between bacterial and viral infections, while also being able to assess the severity of infection based on PCT levels.
Commonly used PCT detection methods include immunochromatography, electrochemiluminescence immunoassay, enzyme-linked immunosorbent assay, latex turbidimetry and the like, and detection by using an anti-PCT antibody is often required. Since the serum of healthy people has extremely low PCT content and can be interfered by CT during detection, it is required to develop anti-PCT antibodies with high affinity and specificity.
Disclosure of Invention
The invention aims to provide an anti-procalcitonin antibody and a detection kit comprising the same.
The invention adopts the following technical scheme.
The invention provides a single domain antibody targeting human procalcitonin, wherein the sequence of a complementarity determining region of the single domain antibody is as follows:
1) The sequence of HCDR1 is shown as SEQ ID NO. 5, the sequence of HCDR2 is shown as SEQ ID NO. 6, the sequence of HCDR3 is shown as SEQ ID NO. 7, or
2) The sequence of HCDR1 is shown as SEQ ID NO. 9, the sequence of HCDR2 is shown as SEQ ID NO.10, and the sequence of HCDR3 is shown as SEQ ID NO. 11.
In some embodiments, the heavy chain variable region of the single domain antibody comprises the sequence set forth in SEQ ID NO. 4 or 8.
In some embodiments, wherein the heavy chain variable region is linked at its C-terminus to a human Fc fragment.
In some embodiments, the human Fc fragment comprises the sequence shown as SEQ ID NO. 3.
In some embodiments, the single domain antibody binds to human procalcitonin with an EC 50 value of no more than 30 ng/mL.
In some embodiments, the single domain antibody binds to calcitonin, anticalin and/or N-PCT with an EC 50 value of greater than 2 μg/mL.
In another aspect, the invention provides procalcitonin assay kits comprising the single domain antibodies described above.
The single domain antibody provided by the invention can bind procalcitonin with high affinity, but does not bind procalcitonin, and has high binding specificity.
Detailed Description
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.
The terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but not to exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, are also intended to cover the circumstance that the recited elements, integers or steps consist of them. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass antibody variable regions that consist of that particular sequence.
The term "about" generally refers to ranges that vary by 10% above or below the specified value, e.g., ranges that vary by 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 the specified value.
The term "or" refers to a single element of a list of selectable elements unless the context clearly indicates otherwise. The term "and/or" means any one, any two, any three, any more, or all of the listed selectable elements.
An "antibody" refers to an immunoglobulin or other type of molecule that includes one or more antigen binding domains that specifically bind an antigen, a protein or polypeptide that exhibits binding specificity for a particular antigen. Specific examples of antibodies can include whole antibodies (e.g., classical four-chain antibody molecules), single chain antibodies, single domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are typically tetramers composed of 2 identical heavy chains and 2 identical light chains interconnected by disulfide bonds. Heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus according to the conservative differences in amino acid sequences. The variable region is used to recognize and bind antigen and the constant region (e.g., fc fragment) is used to initiate downstream effects such as antibody dependent cell-mediated cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, the amino acid composition and arrangement order of the three partial regions, respectively, have a higher degree of variation, which is a critical position for binding of antibodies to antigens and is therefore also referred to as a Complementarity Determining Region (CDR). The amino acid sequence of the CDRs can be determined using art-recognized numbering schemes, such as Kabat, chothia, IMGT or contacts. The three complementarity determining regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3, respectively, and the three complementarity determining regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, respectively. Each of the heavy chain variable region (VH) and the light chain variable region (VL) may be composed of three CDRs and four FR regions, which may be arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxyl terminus.
Unlike classical antibody molecules, "single domain antibodies (single domain antibody, sdabs)", also referred to as "VHH antibodies", nanobodies, etc., refer to antibody molecules having antigen binding capacity, including heavy chain variable regions without light chains. Structurally, single domain antibodies can also be considered fragments of classical four-chain antibody molecules. Single domain antibodies were first found in camelids, and then researchers screened through antibody libraries (e.g., phage display libraries) to find more single domain antibodies with antigen binding capacity. Single domain antibodies have several advantages over common antibody molecules (e.g., classical antibody molecules), including, but not limited to, smaller molecular weights, ease of access to tissues or sites in humans that are difficult to reach by common antibody molecules, or the ability to access epitopes in proteins or polypeptides that are difficult to reach by common antibody molecules, more stability, resistance to changes in, for example, temperature and pH, and the action of denaturants and proteases.
"Fc fragment" refers to the handle region of a classical "Y" shaped antibody molecule, i.e., a crystallizable fragment, comprising the second and third constant domains (CH 2 and CH3 domains) of the heavy chain. The antibody Fc region can be obtained by hydrolyzing an antibody molecule with a proteolytic enzyme such as papain. The effector functions of an "Fc fragment" may include binding to Fc receptors, clq binding and Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), mediated phagocytosis, and the like.
For an antibody or antigen-binding fragment thereof, "targeted" or "specifically binds" means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (e.g., an antigen) than the other molecules that are concurrently present in the environment. The binding affinity of an antibody for an antigen can be measured by a number of parameters, such as the EC 50 value or KD value of the binding of the antibody to the antigen.
"EC 50 value" refers to the concentration that causes 50% of the maximum effect. When used in an enzyme-linked immunosorbent assay (ELISA) to indicate the binding capacity of an antibody molecule to a corresponding antigen, it may refer to the concentration of the antibody molecule that produces half of the maximum detection signal (e.g., colorimetric or fluorescent intensity). The lower the EC50 value, the greater the binding affinity to the antigen.
Provided herein are single domain antibodies that bind PCT with high affinity, and are useful in a variety of detection methods and reagents for detecting PCT content in a sample based on an antigen-antibody reaction. For example, a single domain antibody provided herein can be present in a PCT assay kit with a secondary antibody (targeting the Fc fragment of the single domain antibody) conjugated to a protease that facilitates detection, such as horseradish peroxidase. Alternatively, the single domain antibodies provided herein may be self-conjugated to a protease as described above or other detectable label (e.g., radioisotope, fluorophore, etc.), and PCT detected by direct ELISA.
The invention is further illustrated by the following specific examples.
Example 1 phage antibody library preparation and affinity panning of specific clones
Fusion proteins PCT-huFc and CT-huFc linked to human IgG1 Fc fragments were expressed in 293T cells using eukaryotic expression vectors. PCT sequence is shown as SEQ ID NO. 1, CT sequence is shown as SEQ ID NO. 2, huFc sequence is shown as SEQ ID NO. 3. Separating and purifying by using Protein A chromatographic column.
PCT sequence:
APFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRSKRCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPGKKRDMSSDLERDHRPHVSMPQNAN (SEQ ID NO：1)
CT sequence:
CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO:2)
IgG1 Fc sequences
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO：3)
Alpaca was immunized with purified PCT-huFc by back-neck multiple injections 4 times at 3 week intervals. One week after the last immunization, 100 mL was collected, peripheral Blood Mononuclear Cells (PBMCs) were isolated, total RNA was extracted using an RNA extraction kit, and reverse transcribed into cDNA. A phage antibody display library was constructed by PCR amplification of a nucleic acid fragment encoding a heavy chain single domain antibody (VHH) using degenerate primers, ligation with phage display plasmid pComb3XSS followed by transformation of competent E.coli TG1 cells in the presence of helper phage M13K 07.
Streptavidin-conjugated magnetic beads were coated with biotinylated PCT-huFc for positive panning of phages. Streptavidin-coupled magnetic beads were coated with biotinylated PC-huFc for negative panning of phages. Four rounds of liquid phase panning were performed together, each round of negative panning followed by positive panning. After the last round of panning, monoclonal antibodies were randomly selected from the enriched phage pool, packaged into phage, and the binding of the monoclonal phage to PCT-huFc and PC-huFc was detected by ELISA, clones that bound to PCT-huFc but not PC-huFc were found, and the coding nucleic acid sequences were obtained by sequencing, followed by antibody amino acid sequences. A total of 23 monoclonal phages were obtained, and antibodies corresponding to two clones were found in subsequent studies to have a relatively high affinity and specificity for PCT, and the VHH sequences of the two antibodies (numbered 13# and 20 #) were as follows.
Antibody 13# VHH:
EVQLAESGGGLVQAGGALRLSCAASGRTFSLNASKWFRQAPGKERDFVAVSDASASYTYSAVAGQFAISRDNSKNTVYLQMSSLKPEDTAVYYCAADPSLMGGKYSLLREVDYWGRGTQVTVSS (SEQ ID NO：4)
Antibody 13# HCDR1: LNASK (SEQ ID NO: 5)
Antibody 13# HCDR2: VSDASASYTYSAVAG (SEQ ID NO: 6)
Antibody 13# HCDR3: DPSLMGGKYSLLREVDY (SEQ ID NO: 7)
Antibody 20# VHH:
DVQLVESGGGLVQAGGALRLSCAASGRTFSSYAMALWFRQAPGKERDFVATYSLSWSGSRIFAISRDNSKNTVYLQMNSLKPEDTAVYYCAAAADVVETEWYARLDEWGRGTQVTVSS (SEQ ID NO：8)
antibody 20# HCDR1: SYAMAL (SEQ ID NO: 9)
Antibody 20# HCDR2: TYSLSWSGSRI (SEQ ID NO: 10)
Antibody 20# HCDR3: AADVVETEWYARLDE (SEQ ID NO: 11).
EXAMPLE 2 expression and purification of Single-Domain antibodies in eukaryotic cells
The coding sequences of the heavy chain variable regions of antibodies 13# and 20# obtained in example 1 were cloned into the vector pcdna3.4 together with the human Fc gene sequence after codon optimization, and the correctness of the cloning was identified by cleavage. 293T cells were transfected, cultured for 72 hours, and the supernatant was collected by centrifugation, purified by Protein A chromatography column, and molecular weight and purity of the purified antibody were identified by SDS-PAGE. The molecular weight of the two antibodies is about 38kD, and the purity is about 95%.
Example 3 determination of EC 50 value of antibody binding to PCT and fragments thereof by ELISA
Antibodies 13# and 20# prepared in example 2 were evaluated for their ability to bind to PCT, CT, katacalcin (amino acid sequence shown in SEQ ID NO: 12) and N-PCT (amino acid sequence shown in SEQ ID NO: 13) by indirect ELISA (PCT, CT, katacalcin was obtained by commercial purchase and N-PCT was obtained by a delegated synthesis). ELISA plates were coated overnight at 4℃with PCT, CT, katacalcin. Mu.g/mL or N-PCT, respectively. Plates were washed with PBS-T (0.05% Tween), blocked with PBS-T containing 1% BSA for 2 hours at 37 ℃. The blocking solution was discarded, 100. Mu.l of antibody 13# or 20# at 2. Mu.g/mL was added to the first well, and antibody was added to each subsequent well at 3-fold gradient dilution for a total of 11 wells. After 1 hour incubation at room temperature, the plate was washed three times with PBS-T, goat anti-human Fc antibody conjugated with horseradish peroxidase was added, and incubated at 37℃for 1 hour. Plates were washed 4 times with PBS-T, then TMB color development was added and incubated in the dark for 20 minutes at room temperature. The reaction was stopped by adding 50. Mu.L of 1M HCl stop solution. EC 50 values were obtained by plotting curves based on OD450 values using a microplate reader at 450 nm. The results are shown in Table 1.
KATACALCIN sequence:
DMSSDLERDHRPHVSMPQNAN (SEQ ID NO:12)
N-PCT sequence:
APFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRS (SEQ ID NO:13)
table 1 EC50 value (ng/mL) for binding of antibodies to each antigen
Antibodies to PCT CT Katacalcin N-PCT
13# 28.3 > 2000 > 2000 > 2000
20# 12.7 > 2000 > 2000 518.4
Antibody # 13 has high affinity for PCT but does not substantially bind to each fragment after PCT degradation, and its binding epitope is presumed to involve amino acids within at least two of the fragments. Antibody 20# has high affinity for PCT, substantially does not bind to CT and KATACALCIN, has a lower binding affinity for N-PCT, and is presumed to bind to the N-PCT moiety, but also to amino acids in other parts of PCT.

Claims (7)

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 as SEQ ID NO. 5, the sequence of HCDR2 is shown as SEQ ID NO. 6, the sequence of HCDR3 is shown as SEQ ID NO. 7, or
2) The sequence of HCDR1 is shown as SEQ ID NO. 9, the sequence of HCDR2 is shown as SEQ ID NO.10, and the sequence of HCDR3 is shown as SEQ ID NO. 11.
2. The single domain antibody of claim 1, wherein the heavy chain variable region of the single domain antibody comprises the sequence set forth in SEQ ID No. 4 or 8.
3. The single domain antibody of claim 2, wherein the heavy chain variable region has a human Fc fragment attached at its C-terminus.
4. The single domain antibody of claim 3, wherein the human Fc fragment comprises the sequence set forth in SEQ ID No. 3.
5. The single domain antibody of claim 1, wherein the single domain antibody binds to human procalcitonin with an EC 50 value of no more than 30 ng/mL.
6. The single domain antibody of any one of claims 1-4, wherein the single domain antibody binds to calcitonin, an anti-calcitonin and/or N-PCT with an EC 50 value of greater than 2 μg/mL.
7. A procalcitonin assay kit comprising the single domain antibody of any one of claims 1-6.
CN202411793635.1A 2024-12-09 2024-12-09 Anti-procalcitonin antibody and detection kit containing the antibody Active CN119264254B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181308A (en) * 2021-12-28 2022-03-15 广州市雷德生物科技有限公司 Procalcitonin antibody and application thereof
CN116948027A (en) * 2023-07-28 2023-10-27 上海翎因生物科技有限公司 Nanometer antibody for resisting procalcitonin, kit and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181308A (en) * 2021-12-28 2022-03-15 广州市雷德生物科技有限公司 Procalcitonin antibody and application thereof
CN116948027A (en) * 2023-07-28 2023-10-27 上海翎因生物科技有限公司 Nanometer antibody for resisting procalcitonin, kit and application

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