Tagged Fab antibodies

Abstract

The invention discloses a labeled Fab antibody and application thereof in preparation of a diagnostic reagent / kit. The Fab antibody is used for preparing the labeled Fab antibody, and compared with preparation of the labeled antibody by using a full-length IgG antibody and other preparation processes, the labeled Fab antibody prepared by the method has the obvious advantage of high detection signal-to-noise ratio, and a new way is provided for development and application of diagnostic reagents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese Patent Application No. 202310729490.8, filed on June 19, 2023, entitled "Labeled Fab Antibody", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to immunodiagnostics, specifically to labeled Fab antibodies. Background Art

[0004] Immunodiagnostics is a common method for clinical assays, and currently, full-length IgG antibodies with a molecular weight of 150 kDa are commonly used as labeled antibodies. However, using full-length antibodies as diagnostic antibodies presents several challenges. First, their large molecular weight and wide distribution of conjugable groups lead to random conjugation positions and heterogeneous conjugates. Second, the Fc fragment of full-length antibodies may increase non-specific adsorption during detection. Furthermore, the steric hindrance effect of full-length antibodies may affect detection sensitivity.

[0005] Some researchers use fragmented antibodies as labeled antibodies. Fragmented antibodies retain residual fragments of the antigen-binding region of the full-length antibody, such as Fab, Fab', F(ab')2, and Fv. However, fragmented antibodies lack the Fc fragment, limiting the groups that can be used for chemical conjugation, thus requiring the development of suitable labeling processes. Summary of the Invention

[0006] A first aspect of the present invention provides a Fab-based labeled Fab antibody, comprising the following structure:

[0007]

[0008] From left to right: signal element, connector (i.e.) ) and Fab antibodies, where n and m are integers and are independently selected from 0 to 24.

[0009] Preferably, the amplification carrier is selected from at least one of polysaccharides, polylysine, protein carriers, polyethylene glycol, polyethyleneimine, and dendritic polymers.

[0010] Preferably, the amplification carrier is selected from at least one of bovine serum albumin, human serum albumin, hemocyanin, or ovalbumin.

[0011] A second aspect of the present invention provides a detection reagent / kit comprising the labeled Fab antibody described in the first aspect of the present invention.

[0012] Preferably, the detection reagent further includes an auxiliary agent, optionally wherein the auxiliary agent is selected from at least one of chelating agents, surfactants, preservatives, additives, buffers, and stabilizers.

[0013] Preferably, the kit further includes an instruction manual, which optionally includes step-by-step instructions for the user to perform detection operations using the reagents in the kit.

[0014] A third aspect of the present invention provides the application of the above-described labeled Fab antibody / detection reagent in the preparation of a detection kit.

[0015] The present invention has the following beneficial effects:

[0016] This invention reconstructs adjacent free thiol groups of Fab-type antibodies (or reduces the antibody's disulfide bonds to obtain adjacent free thiol groups if none exist) to add a "linker" structure. This linker is then used to connect an amplification vector, which in turn connects to a signaling agent, thus obtaining the labeled Fab antibody of this invention. This labeled Fab antibody improves the detection signal-to-noise ratio compared to labeled antibodies prepared from intact IgG. DETAILED DESCRIPTION

[0017] Explanation of terms:

[0018] The term Fab as used in this invention means, in addition to Fab, also includes antibody fragments with similar structures, such as Fab', F(ab')2, and Fv.

[0019] The chemical function of the "reconstruction crosslinking agent" (or disulfide bond reconstruction agent) described in this invention is to reconstruct sulfur-carbon bridge bonds from the disulfide bonds that have been reduced and opened.

[0020] The schematic structure of the labeled Fab antibody described in this invention includes a defined chemical structure (e.g., a linker) and an undefined chemical structure (e.g., a signaling agent, amplifying support, Fab, etc.). The schematic structure is a simplified representation of the inventive point, omitting the "linking groups" that may exist between the defined and undefined chemical structures, or between undefined chemical structures. Therefore, the schematic structure of the labeled Fab antibody of this invention may implicitly contain "linking groups." For example, the "-" between the oxygen atom in the amplifying support and the linker does not limit the connection method; the amplifying support may be directly coupled to the oxygen atom using certain inherent groups, or it may be indirectly coupled to the oxygen atom using a linking group. Whether direct or indirect coupling is used, it is for the purpose of connection. The "-" between the signaling agent and the amplifying support does not limit the connection method; the amplifying support may be directly coupled to the signaling agent using certain inherent groups, or it may be indirectly coupled to the signaling agent using a linking group.

[0021] The "linking group" as used in this invention refers to a chemical moity that may contain about 2 to about 50 atoms or 4 to about 30 atoms (excluding hydrogen), and may contain a chain of 2 to about 30 atoms or 3 to about 20 atoms, each of which is independently selected from the group generally composed of carbon, oxygen, sulfur, nitrogen, and phosphorus. In some instances, some or all of the linking group may be part of a linked molecule, such as, but not limited to, amino acid residues on a poly(amino acid). The number of heteroatoms in the linking group may be 0 to about 20, or 1 to about 15, or about 2 to about 10. The linking group may be aliphatic or aromatic. When heteroatoms are present, oxygen is generally present as an oxo or oxygen group bonded to carbon, sulfur, nitrogen, or phosphorus; nitrogen is generally present as a nitro, nitroso, or amino group generally bonded to carbon, oxygen, sulfur, or phosphorus; sulfur is similar to oxygen; and phosphorus is generally bonded to carbon, sulfur, oxygen, or nitrogen as a phosphonate mono- or diester and a phosphate mono- or diester. Common functional groups that form covalent bonds between the linking group and the molecule to be combined include alkylamines, amidines, thioamides, ethers, ureas, thioureas, guanidines, azo compounds, thioethers and carboxylic acid esters, sulfonates and phosphate esters, amides and thioesters.

[0022] In most cases, the linking group further has a linking functional group (functional group for reacting with the group), including nonoxocarbonyl groups of nitrogen and sulfur analogs, phosphate ester groups, amino groups, alkylating agents such as halo- or toluenesulfonyl alkyl groups, oxy (hydroxyl or sulfur analogs, mercapto) oxocarbonyl groups (e.g., aldehydes or ketones), or reactive olefins such as vinyl sulfones or α-, β-unsaturated esters. These functional groups can be linked to amine groups, carboxyl groups, reactive olefins, alkylating agents, such as bromoacetyl groups. When an amine is linked to a carboxylic acid or its nitrogen derivative or phosphoric acid, amides, amidines, and phosphoramides can be formed. When a thiol is linked to an activated olefin, a thioether is formed. When a thiol is linked to an alkylating agent, a thioether is formed. When an aldehyde and an amine are linked under reducing conditions, an alkylamine is formed. When a ketone or aldehyde and a hydroxylamine (including its derivatives, wherein a substituent replaces the hydrogen of the hydroxyl group) are linked, an oxime functional group (=NO-) is formed. When a carboxylic acid or phosphoric acid and an alcohol are linked, an ester is formed. When the connecting functional group is, for example, a click chemical group, including methyltetrazine, trans-cyclooctene, azide, dibenzocyclooctene, tetrazine, alkyne, cyclopropanecyclooctene, and cyclopropene, it can be connected to a pair of orthogonal click chemical groups.

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] Some embodiments of the present invention provide labeled Fab antibodies according to the first aspect of the present invention, which include the following structures:

[0025]

[0026] From left to right: signal element, connector (i.e.) ) and Fab antibodies;

[0027] Wherein, n and m are both integers and are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. Preferably, m + n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. Preferably, m is not zero. Preferably, n is not zero. Preferably, neither m nor n is zero. Preferably, m + n ≥ 4.

[0028] In some embodiments, the amplification carrier is selected from at least one of polysaccharides, polylysine, protein carriers, PEG (polyethylene glycol), polyethyleneimine, and dendritic polymers.

[0029] In some embodiments, the polysaccharide is selected from at least one of cross-linked sucrose and dextran.

[0030] In some embodiments, the protein carrier is selected from at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin.

[0031] In some embodiments, the dendritic polymer is selected from at least one of polyethylene glycol and polypropyleneimide.

[0032] Specifically, the polysaccharide includes cross-linked sucrose and dextran; the protein carrier includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin; and the dendritic polymer includes polyethylene glycol and / or polypropyleneimide. In some embodiments, the amplification carrier includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin.

[0033] In some embodiments, the signaling agent is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based markers; preferably, the fluorescent dye is selected from at least one of fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives; preferably, the enzyme is selected from any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase; preferably, the radioisotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 4 The chemiluminescent reagent is selected from at least one of 7Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F; preferably, the chemiluminescent reagent is selected from at least one of luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, rofenoxuron and its derivatives, and peroxazone and its derivatives; preferably, the nanoparticle label is selected from any one of colloids, organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles; preferably, the colloid is selected from at least one of colloidal metals, dispersed dyes, dye-labeled microspheres, and latex; preferably, the colloidal metal is selected from at least one of colloidal gold, colloidal silver, and colloidal selenium.

[0034] This invention provides a method for preparing labeled Fab antibodies as described in any of the foregoing embodiments, comprising: coupling a linker reagent 2-(toluenesulfonyl)methacrylamide-(PEG)m-amide-(PEG)n (Phipeng Biotechnology catalog number TSMA) with disulfide bond remodeling agent function to a Fab with adjacent free thiol groups to obtain a Fab-linker; and then coupling the Fab-linker to a signaling agent (or an amplification carrier carrying the signaling agent) to obtain the labeled Fab antibody of this invention; the linker reagent TSMA has the following structure:

[0035]

[0036] The Fab-connector has the following structure:

[0037]

[0038] Feipeng Bio has conducted in-depth research on the application process of TSMA antibody disulfide bond reconstruction. They have previously tested the detection performance of labeled Fab antibodies obtained by reconstructing TSMA antibodies with different n+m values ​​(i.e., different numbers of PEG units). Compared to conventional processes where antibodies directly pass through surface amino-coupled signaling molecules without reconstruction, antibodies reconstructed through disulfide bonds and coupled with linker-coupled signaling molecules exhibit superior detection performance. When n+m values ​​are between 0 and 24, they outperform labeled Fab antibodies obtained through conventional processes; when n+m values ​​are between 4 and 8, they significantly outperform labeled Fab antibodies obtained through conventional processes; and an n+m value of 4 achieves the optimal result.

[0039] In some embodiments, the linker reagent has a click chemical group (first click chemical group R1), and the coupling reaction of the linker reagent with Fab (i.e., the disulfide bond reconstruction process) is illustrated below:

[0040]

[0041] In some embodiments, the amplification carrier is chemically derivatized to have a click chemical group (second click chemical group R2), and a signal molecule is coupled to the surface of the amplification carrier. Based on the orthogonal coupling reaction of the first click chemical group R1 and the second click chemical group R2, the amplification carrier can react with the Fab-linker to form a Fab-amplification carrier-signal molecule.

[0042] In some implementations, the first click chemical group and / or the second click chemical group are selected from any one of: methyltetraazine (MTz), trans-cyclooctene (TCO), azide (N3), dibenzocyclooctene (DBCO), tetraazine (Tz), alkynes, cyclopropanecyclooctene (BCN), and cyclopropene.

[0043] In some embodiments, a kit according to a second aspect of the present invention is provided, comprising a labeled Fab antibody according to a first aspect of the present invention, and instructions for use.

[0044] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0045] Example 1: Preparation of Fab-linker-methyltetraazine (Fab-MTz)

[0046] Take anti-CTNI Fab antibody (3 mg / mL, available from Feipeng Biotechnology) and use Zeba TMThe desalting column (7K MWCO) was replaced with PBS (10mM, pH 7.4, 150mM NaCl) buffer. TCEP (Sigma) was prepared into a 20mM solution using PBS buffer. 8 eq of TCEP was added to the Fab antibody solution, and the reaction was carried out at 25°C with gentle shaking (400 rpm) for 2 hours, followed by replacement with PB (50mM, pH 7.8) buffer. TSMA-MTz (i.e., 2-(toluenesulfonyl)methacrylamide-PEG4-amide-methyltetraazine, which can be customized from Xi'an Kangfuno Biotechnology Co., Ltd. or purchased from Feipeng Biotechnology) was prepared into a 60mM solution using DMSO under dry conditions. 10 eq of TSMA-MTz was added to the antibody solution after the above reaction, and the reaction was carried out at 4°C for 16 hours. Excess chemical reagents in the antibody solution were removed using Zeba. TM The desalting column (7K MWCO) was replaced with PBS buffer. The prepared Fab antibody-linker-methyltetrazine ((Fab-MTz)) was stored at 4°C for later use.

[0047] Example 2: Coupling of BSA with acridine ester and Fab

[0048] BSA (4 mg / mL) was treated with Zeba TM A desalting column (7K MWCO) was used to replace the PBS (10mM, pH 7.4, 150mM NaCl) buffer. Based on the coupling reaction between BSA surface amino groups and NHS, 5 eq of NHS-TCO was added to the BSA solution, and the reaction was carried out at 25°C for 60 minutes. Excess chemicals in the BSA solution were removed using Zeba. TM The desalting column (7K MWCO) was replaced with PBS buffer. 20 eq of NHS-AE was added, and the reaction was carried out at 25°C for 30 minutes. Then, 250 eq of glycine solution (100 mM) was added, and the reaction was continued for another 30 minutes. The solution was then purified using Zeba. TM The desalting column (7K MWCO) was replaced with PBS buffer. Trans-cyclooctene-BSA-acridinium ester (TCO-BSA-AE) was prepared and stored at 4°C for later use.

[0049] The above-mentioned BSA-TCO-AE solution was crosslinked with Fab-MTz from Example 1 at a molar ratio of 1.5:1 and reacted at 25°C for one hour to obtain the Fab-BSA-acrididine ester conjugate of the present invention (denoted as Fab-BSA-AE), which was stored at 4°C for later use.

[0050] Comparative Example 1: Direct coupling of Fab with acridine ester

[0051] Take anti-CTNI Fab antibody (3 mg / mL) and use Zeba TMThe desalting column (7MWCO) was replaced with PBS buffer (10mM PB, 50mM NaCl, pH 7.4, 5mM EDTA). 10mM NHS-AE (10 eq, dissolved in DMSO) was added to 30µM Fab antibody solution, and the reaction was carried out at 25°C for 1 hour. After the reaction, excess reagents were removed by desalting to obtain the Fab-acridone ester conjugate (denoted as Fab-AE), which was stored at 4°C for later use.

[0052] Comparative Example 2: IgG directly coupled with acridine ester

[0053] Take full-length anti-CTNI IgG antibody (4 mg / mL, available from Feipeng Biotechnology) and use Zeba TM The desalting column (7MWCO) was replaced with PBS buffer (10mM PB, 50mM NaCl, pH 7.4, 5mM EDTA). 10mM NHS-AE (10 eq, dissolved in DMSO) was added to 30μM antibody solution, and the reaction was carried out at 25°C for 1 hour. After the reaction, excess reagents were removed by desalting to obtain the antibody-acrididium ester conjugate (denoted as IgG-AE), which was stored at 4°C for later use.

[0054] Comparative Example 3: IgG indirectly coupled with acridine ester via BSA

[0055] Take the full-length cTnI antibody (4 mg / mL) and use Zeba TM The desalting column (7K MWCO) was replaced with PBS (10 mM, pH 7.4, 150 mM NaCl) buffer. TCEP (Sigma) was prepared into a 20 mM solution using PBS buffer. 10 eq of TCEP was added to the antibody solution, and the reaction was carried out at 25°C with gentle shaking (400 rpm) for 2 hours, after which the solution was replaced with PB (50 mM, pH 7.8) buffer. TSMA-MTz was prepared into a 60 mM solution using DMSO under dry conditions. 50 eq of TSMA-MTz was added to the antibody solution after the above reactions, and the reaction was carried out at 4°C for 16 hours. Excess chemicals in the antibody solution were removed using Zeba. TM The desalting column (7KMWCO) was replaced with PBS buffer. The prepared antibody-linker-methyltetrazine (IgG-MTz) was stored at 4°C for later use.

[0056] The TCO-BSA-AE from Example 2 was crosslinked with the above-mentioned IgG-MTz at a molar ratio of 1.5:1 and reacted at 25°C for one hour to obtain the antibody-BSA-acrididine ester conjugate (denoted as IgG-BSA-AE), which was stored at 4°C for later use.

[0057] Validation Example 1: Sensitivity Assessment of Antibody Conjugate Detection

[0058] Using the Fab-BSA-AE of this invention and the comparative Fab-AE, IgG-AE, and IgG-BSA-AE, respectively, in the Shine i2000 of Phypeng Biotechnology... T The M fully automated chemiluminescence immunoassay system was used to test CTNI samples. The test results are shown in Table 1 below.

[0059] Table 1 Comparison Test

[0060]

[0061]

[0062] For the low-value sample (43.4 pg / mL), the signal-to-noise ratio (SNR) of Fab-BSA-AE was 4.00, significantly higher than that of Fab-AE, IgG-AE, and IgG-BSA-AE. For the high-value sample (693.8 pg / mL), the SNR of Fab-BSA-AE was also higher.

[0063] Comparing the detection data of IgG-AE and Fab-AE, it can be found that if an amplification carrier is not used in the preparation process of labeled Fab antibodies, the effect of using Fab is significantly worse than that of IgG-AE. This may be because Fab lacks Fc, limiting the number of AEs that can be coupled, resulting in a signal much lower than that of IgG. However, if an amplification carrier (e.g., BSA) is used in the preparation process of labeled Fab antibodies, the effect of using Fab is significantly stronger than that of IgG. This is an unexpected technical effect, because theoretically, IgG-BSA can provide more coupling sites than Fab-BSA, and the number of AEs that IgG-BSA can load should also be greater than that of Fab-BSA, resulting in a higher detection signal intensity. However, with the reconstruction cross-linking agent modification process of this invention, the antibody is site-specifically coupled to BSA via disulfide bonds (BSA carries AE molecules), and the Fab-based reagent actually has an advantage in signal intensity compared to the IgG-based reagent. The underlying mechanism needs further exploration, but this invention undoubtedly provides a holistic compound structure for chemiluminescent reagents with a signal-to-noise ratio advantage.

[0064] In other detection tests such as CA153, the labeled Fab antibody of this invention also exhibits superior performance.

Claims

1. A labeled Fab antibody, characterized in that: Includes the following structure: Wherein, n and m are both integers and are independently selected from 0 to 24.

2. The labeled Fab antibody according to claim 1, wherein the amplification carrier is selected from at least one of polysaccharides, polylysine, protein carriers, polyethylene glycol, polyethyleneimine and dendrimers.

3. The labeled Fab antibody according to claim 1, wherein the amplification carrier is selected from at least one of bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.

4. The labeled Fab antibody according to claim 1, wherein the signal substance is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers; preferably, the fluorescent dye is selected from at least one of fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives; preferably, the enzyme is selected from any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxygenase; preferably, the radioactive isotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68 At least one of Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F; preferably, the chemiluminescent agent is selected from at least one of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, lophanite and its derivatives and peroxyoxalate and its derivatives; preferably, the nanoparticle marker is selected from any one of colloids, organic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles; preferably, the colloid is selected from at least one of colloidal metals, disperse dyes, dye-labeled microspheres and latex; preferably, the colloidal metal is selected from at least one of colloidal gold, colloidal silver and colloidal selenium. The labeled Fab antibody according to claim 1 , wherein the value of n+m is 4 to 8.

6. A detection reagent, characterized in that: The method comprises the labeled Fab antibody according to any one of claims 1 to 5.

7. The detection reagent according to claim 6, further comprising an auxiliary agent, optionally, wherein the auxiliary agent is selected from at least one of a chelating agent, a surfactant, a preservative, an additive, a buffer and a stabilizer.

8. A kit comprising the labeled Fab antibody according to any one of claims 1 to 5, or the detection reagent according to any one of claims 6 to 7.

9. The kit according to claim 8, wherein the kit further comprises an instruction manual, and optionally, the instruction manual comprises step-by-step instructions for a user to perform a detection operation using the reagents in the kit.

10. Use of the labeled Fab antibody according to any one of claims 1 to 5 or the detection reagent according to any one of claims 6 to 7 in the preparation of a detection kit.

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