RU2763178C2 - Antibodies for laboratory diagnostics of the concentration of interleukin-11 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular, to an antibody specific to human IL-11 and IL-11 of model animals, such as the crab-eating macaque, or to an antigen-binding fragment thereof, as well as to a conjugate containing said antibody.

EFFECT: invention is effective for determining the concentration of IL-11 in human samples and samples of model animals, such as the crab-eating macaque.

3 cl, 2 dwg, 3 ex

Description

The field of technology to which the invention belongs

The present invention relates to the field of biotechnology, immunology and medicine and can be used for laboratory diagnosis of the concentration of interleukin-11 in biological fluids.

State of the art

Interleukin-11 (IL-11) is a pleiotropic cytokine belonging to the interleukin-6 (IL-6) family. The mature protein consists of 178 amino acid residues and has a small molecular weight (about 19 kDa). The amino acid sequence homology of human IL 11 and cynomolgus macaque (Macacafascicularis) is 93.8% (Sokolov A.S. et al., Molecules 2016, 21, 1632). IL-11 receptors are multisubunit membrane complexes consisting of a- (IL-11RA) and β-subunits (glycoprotein 130, gp 130). Specific binding of the ligand to the IL-11RA subunit causes a conformational change in the latter, leading to an increase in its affinity for gp 130. Homodimerization of gp 130 molecules in the multimeric complex triggers signal transduction into the cell via activation of the signaling cascades JAKs/STAT 1,3, ERK/RAS, and mTor/PI3K (Negahdaripour M. et al., Cytokine Growth Factor Rev. 2016;32:41-61).

IL-11 directly stimulates the proliferation of megakaryocyte progenitor cells and induces the maturation of megakaryocytes, resulting in increased platelet production. In addition, it stimulates erythropoiesis, myelopoiesis, lymphopoiesis and osteoclast development, activates the synthesis of acute phase proteins by hepatocytes and inhibits adipogenesis.

In addition to the participation of IL-11 in physiologically important processes, it is involved in the pathogenesis of a number of diseases, including cancer. The need to search for new markers for diagnosing diseases as early as possible, especially for oncological diseases, is beyond doubt.

It has been shown that an elevated level of IL-11 is significantly observed in the plasma of patients with pancreatic cancer (Ren C.L. et al., Tumor Biol. 2014,35:11467-11472).

IL-11 appears in bronchoalveolar lavage fluid in patients with lung cancer, including chronic obstructive pulmonary disease (Pastor M.D. et al., J Thorac Oncol. 2016 Dec; 11(12):2183-2192).

Spontaneous intracerebral hemorrhages are accompanied by an increase in the content of IL-11 in blood plasma and cerebrospinal fluid, moreover, the level of this cytokine correlates with mortality from this disease (Fang HY et al., Surg Neurol. 2005;64(6):511-7; Tu CJ et al., J Int Med Res. 2011;39(4): 1265-74).

The concentration of IL-11 in the blood serum increases in patients with attacks of acute pancreatitis and is directly dependent on their severity (Chen C.C. et al., Gut 1999;45:895-899).

Based on the foregoing, IL-11 can be considered as a new independent marker, monitoring the level of which in biological fluids will allow diagnosing and monitoring the course of diseases associated with the expression of this cytokine.

An increased level of IL-11 in the seminal fluid increases the fertilizing ability of spermatozoa, so the content of IL-11 can be one of the control parameters for the quality of the ejaculate during assisted reproductive technologies (Seshadri S. et al., Andrologia. 2011 Dec;43(6):378 -86).

From RF patent RU 2318829 C2, neutralizing chimeric or humanized anti-IL-6 antibodies containing at least one hypervariable region of the heavy or light chain of the mouse monoclonal antibody CLB-8 are known. Preferred antibodies according to the invention are antibodies that bind to the region of the IL-6 molecule responsible for binding to gp 130 (site 1). DNA sequences encoding hypervariable regions 1-3 of the light and heavy chains of the mouse monoclonal antibody CLB-8, as well as their amino acid sequences, are described. The composition of drugs based on these antibodies and their use for the treatment of oncological or other diseases mediated by IL-6 is disclosed. However, the antibodies described in this patent are not specific for interleukin-11, and therefore cannot be used to diagnose or treat diseases associated with IL-11.

US Pat. No. 6,998,123 (B1) describes a method for treating or alleviating the symptoms of pathological conditions in which there is a decrease in bone density by administering to the patient an effective amount of anti-IL-11 antibodies that block the formation of a multimeric ligand-receptor complex on the surface of cells. The term "anti-IL-11 antibody" is used in this patent to define neutralizing chimeric, humanized or hybrid monoclonal antibodies, as well as antibody fragments capable of binding to an antigen. The patent reports the results of using only one type of anti-IL-11 antibody molecule in a mouse model: in mice with both ovaries removed, after intraperitoneal injection of affinity-purified polyclonal goat anti-mouse IL-11 antibodies, a greater amount of cancellous bone tissue in the femur is observed compared to control group of animals. The document does not provide a description of the structure of the antibodies used, including the amino acid sequence, as well as the details of their production. This method of using anti-IL-11 antibodies refers to the therapy of diseases, while there is no information about the potential use of these anti-IL-11 antibodies in the diagnosis of pathological conditions in which there is a decrease in bone density.

Disclosure of invention

The aim of the present invention is to overcome the above disadvantages of anti-IL-11 antibody analogs by providing the present invention.

The invention provides antibodies specific for human IL-11 and model animals such as the cynomolgus monkey, or derivatives thereof, or antigen-binding fragments of antibodies, in which the variable domains of the heavy and light chains contain one of the following combinations of amino acid sequences of hypervariable regions:

Antibodies of the invention may be full length or may contain only an antigen-binding fragment (eg Fab fragment, F(ab')2).

Antibodies can be monoclonal chimeric, humanized or hybrid, of various classes and subclasses, most preferably IgG1 or IgG4.

The term "antibody" as used herein is intended to refer to full length immunoglobulin molecules consisting of four polypeptide chains (two heavy (H) chains and two light (L) chains) linked by disulfide bonds. Each heavy chain contains a variable region (abbreviated here as VH) and a constant region containing the three domains CH1, CH2 and CH3. Each light chain contains a variable region (abbreviated here as VL) and a constant region containing a single CL domain. The VH and VL regions consist of three hypervariable regions surrounded by four more conserved framework regions. Hypervariable regions are also called complementarity determining regions and are referred to as CDRs (complementarity determining regions), since they form the antigen-binding centers of an antibody molecule. Framework regions (FRs) are usually not involved in antigen binding, but are essential for the V-domain folding, which provides an adequate conformation of the antigen-binding center.

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment") as used herein refers to one or more antibody fragments that retain the ability to specifically bind human and model animal IL-11 antigen. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the loop region. Further, an antibody or antigen-binding fragment thereof may be part of larger immunoadhesion molecules formed by covalent or non-covalent bonding of an antibody or antibody fragment to one or more proteins or peptides.

In one embodiment, the invention is a full-length antibody of the IgG1 subclass, designated U13, in which the amino acid sequences of the hypervariable regions of the heavy and light chain variable domains correspond to combination #1.

In another embodiment, the invention is a full-length antibody of the IgG1 subclass, designated L1, in which the amino acid sequences of the hypervariable regions of the heavy and light chain variable domains correspond to combination #2.

In yet another embodiment, the invention is a Fab fragment, designated Fab3, in which the amino acid sequences of the hypervariable regions of the heavy and light chain variable domains correspond to combination #1.

In another embodiment, the invention is a Fab fragment, designated Fab11, in which the amino acid sequences of the hypervariable regions of the heavy and light chain variable domains correspond to combination #2.

Another object of the present invention are derivatives of antibodies or their fragments, for example, hybrid proteins. Fusion molecules can be other polypeptides, such as biotin and proteins with enzymatic activity, such as horseradish peroxidase and alkaline phosphatase, as well as organic or inorganic molecules, such as fluorescent labels (for example, fluorescein, fluorescein isothiocyanate, rhodamine, chloride 5- dimethylamine-1-naphthalenesulfonyl, phycoerythrin, etc.), radioactive labels (eg isotopes P-32 and I-125) or chemiluminescent labels (eg luminol and its derivatives).

In yet another embodiment, the invention is a U13 antibody conjugated to a biotin molecule, referred to as U13-bio.

In yet another embodiment, the invention is an L1 antibody conjugated to a biotin molecule, referred to as L1-bio.

Antibodies, or antigen-binding fragments thereof, or derivatives thereof, according to the invention, have at least one of the following properties:

1) are able to specifically bind to human IL-11 and model animals such as the cynomolgus monkey;

2) able to bind to IL-11 with high affinity: the equilibrium dissociation constant (K _d ) is 1×10 ^-8 M or less, and the kinetic dissociation constant (K _off ) 1×10 ^-4 s ^-1 or less, determined, for example, using the method of surface plasmon resonance;

3) the value of the equilibrium dissociation constant when binding human IL-11 differs from the value of K _d binding IL-11 model animals, such as cynomolgus macaque, no more than 10 times;

4) molecules with a different set of hypervariable regions do not compete with each other for binding to IL-11

U13 and L1 antibodies have the following human IL-11 binding parameters:

1) K _d equal to 1.0×10 ^-11 M and 5.0×10 ^-10 M, respectively;

2) K _off equal to 2.0×10 ^-6 s ^-1 and 8.0×10 ^-5 s ^-1 , respectively.

Antibodies U13 and L1 have the following binding parameters for cynomolgus macaque IL-11:

3) K _d equal to 7.0×10 ^-11 M and 2.0×10 ^-10 M, respectively;

4) K _off equal to 7.0×10 ^-6 s ^-1 and 2.0×10 ^-5 s ^-1 , respectively.

Antibodies can be obtained by conventional methods, for example, immunization with the appropriate antigen of animals, or hybridoma technology, or using genetic engineering technology. Methods for obtaining antibodies are described in the literature: Zola, H. (CRC Press, 1987). Monoclonal Antibodies: A Manual of Techniques; John G.R. Hurrell (CRC Press, 1982). Monoclonal Hybridoma Antibodies: Techniques and Applications.

Antibody fragments are obtained by cleavage of antibodies with appropriate proteolytic enzymes, such as papain and pepsin, as well as using recombinant DNA technology.

Techniques such as phage display are used to select recombinant antibodies of the required specificity. Most often, filamentous phages of the Ff family (M13, fd, f1) are used for phage display. Nucleotide sequences encoding fragments of antibodies, for example, Fab or scFv, are combined as part of the phage genome or phagemid with a part of the gene of one of the five bacteriophage envelope proteins; pIII and pVIII are most often used. As a result, phage particles express in the capsid copies of the core protein with the polypeptide sequence of the antibody fragment attached to them. The selection is usually carried out in the form of several successive rounds, each of which includes the adsorption of phage particles expressing the corresponding antibody fragments on carriers with immobilized antigen, washing off unbound particles, elution of the remaining bound ones, and propagation of the resulting phages in bacterial cells. Phage DNA or phagemids are isolated and sequenced. Phage display is widely described in the literature: Bradbury A.R., Marks J.D. Antibodies from phage antibody libraries. J. Immunol. Methods 2004, 290, 29-49; Ledsgaard, L. et al. Basics of Antibody Phage Display Technology. Toxins 2018, 10, 236; John M. Walker. The Protein Protocols Handbook. Springer, 2007.

Recombinant DNA technology involves the production of expression genetic constructs encoding antibody chains under the control of appropriate regulatory elements in one or different carrier vectors. Regulatory elements are promoter sequences and signal sequences, such as polyadenylation signals, that control transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Gene Expression Technology: Methods in Enzymology, vol. 185, Edited by David V. Goeddel, Academic Press, San Diego, CA (1990).

One or more expression constructs are transferred by conventional methods into cells suitable for expression and preferably secretion of antibodies into the culture medium. Cells producing full-length antibodies are preferably mammalian cell lines CHO (Chinese hamster ovary cells), SP2/0 (mouse myeloma cells) or HEK293 (human embryonic kidney cells). These cell lines are suitable both for obtaining lines that stably express the target antibody, and for producing antibodies only for a short time (transient expression). Antibody fragments such as Fab are produced in bacteria, yeast, filamentous fungi and insect cell lines. Transformed cells are cultivated by conventional methods and antibodies and their fragments are isolated from the culture medium. Standard recombinant DNA techniques are used to obtain heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells as described by Sambrook, J., Fritsch, E. F., and T. Maniatis. Molecular cloning: A laboratory manual, Second Edition, Cold Spring Harbor, N.Y. (1989); Ausubel F., Brent R. et al. Current Protocols in Molecular Biology, John Wiley & Sons (2003).

Derivatives of antibodies and their fragments are also obtained as a result of chemical modification using activated derivatives of fusion molecules. For example, the most common and versatile biotinylation method is chemical modification using activated biotin derivatives, such as biotinyl-N-hydroxysuccinimide (BNHS), while biotin is attached to the ε-amino groups of lysine residues in the antibody molecule and their fragments. A detailed description of biotinylation is given in the literature, for example, Diamandis E. P and Christopoulos T K (1991) The biotin-(strept)avidin system principles and applications in biotechnology Chn Chem 37, 625-636.

Another aspect of the invention relates to a method for determining the presence and concentration of human IL-11 and model animals in samples, such as the cynomolgus macaque, which consists in the fact that 1) the test sample containing IL-11 is brought into contact with antibodies, or fragments thereof, or derivatives that are the objects of the present invention, and 2) assess the presence and concentration of immune complexes of antibodies, or fragments or derivatives with antigen (IL-11).

Samples in which it is necessary to determine the presence or concentration of IL-11 can be samples of biological fluids (for example, blood, plasma, serum) and pieces of tissue obtained as a result of biopsy, human body and model animals, for example, cynomolgus macaque, and also samples of cell culture supernatants, solutions of purified recombinant human and animal IL-11, for example, cynomolgus macaque.

For laboratory diagnosis of the presence and concentration of IL-11 in samples of biological fluids, cell culture supernatants and solutions of purified IL-11, a wide range of immunoassay methods can be used, including immunoblotting, enzyme immunoassay, radioimmunoassay and immunofluorescence analysis, immunoturbodimetry. In tissue samples, immunohistochemistry is used to determine the presence of IL-11.

Depending on the design of the experiment, the antibodies, their derivatives or fragments of the present invention can be used alone or in pairs with each other.

In yet another embodiment, the invention is a sandwich immunoassay using pairs of antibodies U13 and L1, as well as their conjugates, for example, U13-bio or L1-bio. Antibodies U13 and L1 and their conjugates specifically recognize different non-overlapping regions of the IL-11 molecule (epitopes), due to which there is no competition between them for binding to the antigen. This method of analysis makes it possible to achieve high sensitivity, accuracy and specificity in the determination of antigen even in heterogeneous samples, such as biological fluids and tissues, cell culture supernatants.

The biological sample may be pretreated or directly contacted with at least one capture antibody under conditions conducive to contact with the epitope.

In accordance with the preferred method of implementation of the capture antibodies are subjected to immobilization on the solid phase. As non-limiting examples of the solid phase, microplates can be used, especially polystyrene microplates such as those available from Thermo scientific NUNC (Denmark) and Corning-Costar (CIIIA). The use of a solid phase makes it possible to increase the sensitivity of the method due to the removal of substances that do not participate in the reaction due to the immobilization of one of the components of the reaction mixture on the solid phase.

As a detecting antibody, as a rule, conjugated antibodies directed against another epitope of IL-11 are used.

The proposed method of ELISA includes fixing on the surface of the wells of a plastic tablet capturing antibodies against IL-11; blocking non-specific binding sites; adding test material to the wells and incubating the plate; washing with a buffer solution from antigen molecules that have not bound to the capturing antibodies; adding detection antibodies to the wells and incubating the plate; washing with a buffer solution from molecules of detecting antibodies that have not bound to the antigen-antibody complex on the surface of the solid phase; determination of the concentration of immune complexes.

If the detecting antibodies are a conjugate with an enzyme, then the concentration of immune complexes is determined by the activity of the enzyme label: an appropriate chromogenic substrate is added to the reaction mixture and the content of colored products of enzymatic reactions is determined by changing the optical density of the solution at a certain wavelength. Due to the ability of one enzyme molecule to catalyze the conversion of a large number of substrate molecules, a high sensitivity of the method is achieved.

If the detection antibodies are a biotin conjugate, then the immune complexes are detected, for example, using biotin-binding proteins, for example egg white avidin or streptavidin, linked to a fluorescent, chemiluminescent or enzymatic label.

The experimental data of the sandwich-type immunoassay are preferably presented as a plot of signal magnitude versus antigen (IL-11) concentration in a sample constructed using calibration solutions with a known antigen concentration. For the manufacture of calibration solutions, a solution of highly purified recombinant human or animal IL-11, or a reference preparation of IL-11 NIBSC 92/788, recommended by WHO as a standard, can be used. In the absence of limiting factors in the working range of the method, there should be a direct linear relationship between the signal magnitude and the antigen concentration. However, real sandwich analysis curves can be distorted to varying degrees, so linearizing transformations are carried out, for example, a logarithmic scale of values along the abscissa and/or ordinate axis is used.

The concentration of the antigen in the test sample is determined according to the calibration curve, based on the measured signal value.

The following drawings and examples disclose the invention without limiting it.

Brief description of the drawings

On Fig. Figure 1A is a plot of mean optical density at 492 nm versus log base 2 of human IL-11 concentration in calibration solutions (in ng/mL) for determining human IL-11 concentration in samples by sandwich-pair ELISA. anti-IL-11: capture antibodies U13 adsorbed on plastic in the wells of a 96-well microplate, and detecting antibodies L1-bio conjugated with biotin.

On Fig. Figure 1B shows a plot of the average optical density at a wavelength of 492 nm versus the base 2 logarithm of the concentration of human IL-11 in calibration solutions (in ng/ml) for determining the concentration of human IL-11 in samples by ELISA using a sandwich pair. anti-IL-11: capturing antibodies L1 adsorbed on plastic in the wells of a 96-well microplate, and detecting antibodies U13-bio conjugated with biotin.

On Fig. 2A shows sensorograms and theoretical curves of the interaction of antibody U13 with human IL-11 (upper panel) and cynomolgus macaque (lower panel) according to surface plasmon resonance (SPR) data. The ligands are covalently attached to the surface of the sensor chip by amino groups. Application concentrations of antibodies in working buffer: 15, 12.9, 8.57 nM. Data analysis was carried out in the ProteOn Manager 3.0 program within the framework of the bivalent analyte scheme.

On Fig. 2B shows sensorograms and theoretical curves of the interaction of antibody L1 with human IL-11 (upper panel) and cynomolgus macaque (lower panel), according to the surface plasmon resonance (SPR) method. The ligands are covalently attached to the surface of the sensor chip by amino groups. Application concentrations of antibodies in working buffer: 10, 8.57, 2.58 nM. Data analysis was carried out in the ProteOn Manager 3.0 program within the framework of the bivalent analyte scheme.

Example 1

Determination of the concentration of human IL-11 by enzyme immunoassay using a sandwich pair of antibodies L1 and U13-bio, or U13 and L1-bio

I. Sorption of capture antibodies on the solid phase Into the wells of a 96-well polystyrene plate, 100 µl of phosphate-buffered saline pH 7.2-7.4 (1xPBS) containing 1-2 µg of L1 or U13 antibodies are placed. The tablet closed with a lid is kept for 16-18 hours at a temperature of +(5±3)°C or for 2-6 hours at a temperature of +(37±2)°C with constant shaking/mixing. The contents of the wells are removed without washing or the wells are washed from unbound antibody molecules 1-3 times with 1xPBS buffer containing 0.05-0.1% polysorbate 20 (1xPBST), 250-300 μl.

II. Blocking non-specific binding sites on the solid phase 250 μl of 1xPBST buffer containing 0.5-1.0% bovine serum albumin or egg ovalbumin, or skim milk is placed in the wells of the tablet. The tablet, closed with a lid, is kept for 1-2 hours at a temperature of + (37 ± 2) ° C or at room temperature with constant shaking / stirring.

The contents of the wells are removed and the wells are washed 1-3 times with 1xPBST buffer, 250-300 µl.

III. Incubation with antigen

100 μl samples of biological fluids or two-fold dilutions of the human IL-11 standard are placed in the wells of the tablet. Antigen dilutions should be prepared with 1xPBST containing 0.1% bovine serum albumin or ovalbumin, or non-fat milk, since polysorbate 20 reduces non-specific binding of protein molecules to each other and to the surface of the plate, and the protein interferes with antigen proteolysis. The test solution and standard dilutions of the antigen are added in 2-3 replications, using two (three) wells for each dilution of the protein. The tablet closed with a lid is kept for 0.5-2 hours at a temperature of +(37±2)°C or at room temperature with constant shaking/mixing. The contents of the wells are removed and the wells are washed 3-5 times with 1xPBST buffer, 250-300 µl.

IV. Incubation with detection antibodies

100 μl of 1xPBST buffer containing 0.1% bovine serum albumin or egg ovalbumin, or non-fat milk, and at least 2 μg / ml of U13-bio or L1-bio antibodies conjugated with biotin (paired with adsorbed with L1 antibodies, U13-bio antibodies are used, and with U13 antibodies, L1-bio antibodies). The tablet, closed with a lid, is kept for 30-60 minutes at a temperature of +(37±2)°C or at room temperature with constant shaking/mixing. The contents of the wells are removed and the wells are washed 3-5 times with 1xPBST buffer, 250-300 µl.

V. Detection of immune complexes on the solid phase

Place 100 µl of 1xPBST buffer containing streptavidin-horseradish peroxidase conjugate (for example, Thermo Fisher, cat. No. N100) into the wells of the tablet. The optimal concentration of conjugated streptavidin is usually specified by the manufacturer of this reagent (usually at least 1/4,000). The tablet, closed with a lid, is kept for 30-45 minutes at a temperature of + (37 ± 2) ° C or at room temperature with constant shaking / stirring. The contents of the wells are removed and the wells are washed 3-5 times with 1xPBST buffer, 250-300 µl.

100 μl of horseradish peroxidase substrate solution are added to the wells and incubated at room temperature with constant stirring for 5-15 minutes in a place protected from light. As a substrate for horseradish peroxidase, OPD (o-phenylenediamine dihydrochloride) can be used, for example, Thermo Fisher, cat. No. 34006, in a buffer containing hydrogen peroxide, such as Thermo Fisher, cat. No. 34062.

The reaction is stopped by adding 50 µl of 10% sulfuric acid solution to each well. After that, immediately proceed to the measurement of the optical density of the solution at the appropriate wavelength (for the substrate OPD 492 nm) using a plate spectrophotometer.

The interpretation of the results is carried out as follows. A graph is constructed of the dependence of the average values of the optical density of the calibration solutions, calculated from several repeated measurements, on the logarithm of the concentration of IL-11. The formula for the area of the linear section (trend line formula) is determined, with the help of which the numerical value of the concentration of IL-11 in the samples under study is determined depending on the values of their optical density.

The results obtained are shown in Fig. 1, where in Fig. 1A shows the results obtained using a sandwich pair of antibodies U13 and L1-bio, and Fig. 1B - using a sandwich pair of antibodies L1 and U13-bio.

The “correctness” of the technique is understood as the degree of closeness of the average value of the concentration of IL-11 in the studied samples, obtained on the basis of a series of measurement results, to the accepted reference value. As a reference value, the actual value of the concentration of the analyte (IL-11) in the sample is used, calculated taking into account the concentration of the analyte in the initial solution, measured by one of the pharmacopoeial methods for determining the protein concentration (for example, spectrophotometric analysis) or indicated in the passport, and the degree of dilution of the initial solution during sample preparation.

рассчитывают по формуле:Mean value of analyte concentration

calculated by the formula:

where X _i is the result of the i-th concentration measurement,

n is the number of analyte concentration measurements in samples of the same homogeneous sample.

Correctness (T) is expressed as a percentage of the average concentration of IL-11 in samples from the reference value, the formula is used to calculate:

where X _t is the actual value of the concentration of the analyte (IL-11) in samples of the same homogeneous sample.

The true value of the accuracy value of the ELISA method described in Example 1, with a probability of 95%, lies in the range from 80 to 115%) using a sandwich pair of antibodies U13 and L1-bio, and in the range from 90 to 110%. using a sandwich pair of antibodies L1 and U13-bio.

To calculate the confidence interval, which includes the correctness values calculated for analyte concentrations in the operating range of a particular immunoassay technique, methods of mathematical statistics are used, for example, through the critical value of Student's t-test.

Example 2

Measurement of the kinetic and equilibrium association/dissociation constants of the human or model animal IL-11 complex with antibodies U13 and L1 by surface plasmon resonance

The measurement is carried out at +(25±1)°C using a Bio-Rad ProteOn™ XPR36 spectrometer. To do this, the ligand (human IL-11 or cynomolgus macaque) is immobilized on the surface of the ProteOn™ GLH #176-5013 chip through amino groups. The degree of completion of the ligand immobilization phase on the chip is controlled by the kinetics of the SPR signal.

In a perpendicular direction, a solution of the analyte (antibodies U13 or L1) is applied to the chip in working buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4) in five different protein concentrations from 2.0 nM to 20 nM, and on one track - a working buffer as a control. Control of the degree of completeness of the dissociation phase of the complex is carried out by the magnitude of the decrease in the PPR signal.

The chip surface is regenerated after the analyte binding cycle by passing 10 mM glycine pH 2.0 through the chip for 300 sec.

Calculation of the constants of interaction of the ligand with the analyte is carried out in the standard program Bio-Rad ProteOn Manager 3.0 using the simplest scheme of the bivalent analyte for at least three concentrations of the analyte.

The results obtained are shown in Fig. 2.

Example 3

Obtaining full-length antibodies

Obtaining Fab-fragments of antibodies was carried out using the technology based on three phage libraries of LLC "Anteriks", Pushchino: Fab2h-library, Fab5h-library, Fab7h-library.

Phage display technology is described in the literature, for example, Bazan, J., I. Calkosinski, and A. Gamian, Phage display-a powerful technique for immunotherapy: 1. Introduction and potential of therapeutic applications. Hum Vaccin Immunother, 2012. 8(12): p. 1817-28.

From the initial variety of phage libraries during three rounds of selection, phage particles containing on their surface Fab-fragments of antibodies that specifically bind to human and macaque IL-11 were selected. Two selection schemes were used, differing in the order of antigen presentation (human IL-11 - macaque IL-11 - human IL-11 and macaque IL-11 - human IL-11 - human IL-11).

Phagemids from bacterial cells infected with the selected virions were isolated using the QIAprep Spin Miniprep kit (Qiagen, #27106) and sequenced.

The sequences encoding the VL, VH, CL, and CH1 domains of antibodies from the selected phagemids were recloned into the pLL4 vector (Addgene plasmid #107208) for expression in E. coli strain BL-21(DE3) of Fab fragments bearing the 8XHis tag on C -end. Isolation and purification of antibody fragments from the cell lysate was performed using metal chelate chromatography. Screening of Fab fragments for their specificity to human and macaque IL-11 was performed using ELISA.

To create full-length antibodies, the sequence encoding the VH domain was cloned into the pTT5 expression vector containing sequences encoding human IgG1 constant regions (CH1, CH2, CH3), while the sequence encoding the VL domain was cloned into the pTT5 expression vector containing sequences encoding human k-light chain (CL) constant regions.

The production of antibodies was carried out in a system of transient expression in eukaryotic cells of the CHO-3E7 line (RRID: CVCL_JY74) adapted for the production of mammalian proteins. CHO-3E7 cells were co-transfected with the obtained plasmids using polyethylenimine (PEI). Isolation and purification of antibodies from the culture medium was performed using affinity chromatography using a sorbent with immobilized protein A (HiTrap rProtein A FF, GE Healthcare Life Sciences), which has a high affinity for class G immunoglobulins. The obtained antibodies were dialyzed and sterilized by filtration through 0.22 micron filter. The affinity of the obtained antibodies to IL-11 was tested using ELISA and surface plasmon resonance spectroscopy.

The routine methods used in this work are widely described in the literature (Barbas CF, Wagner J (1995). Synthetic Human Antibodies: Selecting and Evolving Functional Proteins. Methods. 8 (2): 94-103. doi: 1.0.1006/meth. 1995.9997; Barbas CF (1995) Synthetic human antibodies Nat Med 1 (8): 837-839 doi:10.1038/nm0895-837 PMID 7585190 Wild D The Immunoassay Handbook (4th edition) Theory and applications of ligand binding, ELISA and related techniques 2013; Greenfield EA, Antibodies A laboratory manual Second edition 2014).

Heavy chain sequence of HCDR1

SEQ ID NO 1: Phe Thr Phe Thr Gly Tyr Trp Met Asn Trp Val Arg;

HCDR2 heavy chain sequence

SEQ ID No. 2: Val Ser Ser Ile Ser Ser Tyr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser;

HCDR3 heavy chain sequence

SEQ ID NO: 3: Cys Ala Arg Glu Ser Leu His Gln Leu Phe Asp Tyr Trp Gly Gln;

LCDR1 light chain sequence

SEQ ID No. 4: Gln Ser Val Ser Ser Ser;

LCDR2 light chain sequence

SEQ ID NO: 5: Gly Ala Ser Ser Arg Ala Thr;

LCDR3 light chain sequence

SEQ ID NO: 6: Cys Gln Gln Ser Tyr Ser Tyr Ala Pro Ile Thr Phe Gly Gln;

Heavy chain sequence of HCDR1

SEQ ID NO: 7: Phe Thr Phe Thr Asp Tyr Trp Met Asn Trp Val Arg;

HCDR2 heavy chain sequence

SEQ ID NO: 8: Val Ser Thr Ile Ala Ser Ser Asn Ser Tyr Thr Asp Tyr Ala Asp Ser;

HCDR3 heavy chain sequence

SEQ ID NO:Cys Ala Arg Glu Ser Trp Ser Glu Gly Trp Trp Pro Ser Gly Phe Asp Tyr Trp Gly Gln;

LCDR3 light chain sequence

SEQ ID NO: 10: Cys Gln Gln Tyr Glu Thr Ser Pro Ile Thr Phe Gly Gln.

Claims (20)

1. An antibody specific for human and animal model IL-11 such as the cynomolgus monkey, or an antigen-binding fragment thereof, wherein the heavy and light chain variable domains contain one of the following combinations of hypervariable region amino acid sequences: # one: HCDR1: SEQ ID #1, HCDR2: SEQ ID #2 HCDR3: SEQ ID #3 LCDR1: SEQ ID #4 LCDR2: SEQ ID #5 LCDR3: SEQ ID #6 and/or #2: HCDR1: SEQ ID No. 7, HCDR2: SEQ ID #8 HCDR3: SEQ ID #9 LCDR1: SEQ ID #4 LCDR2: SEQ ID No. 5, LCDR3: SEQ ID No. 10. 2. A conjugate for determining the concentration of human IL-11 in samples and model animals, such as the cynomolgus macaque, containing the antibody or its antigen-binding fragment according to claim 1, conjugated with biotin. 3. A method for determining the concentration of IL-11 in human samples and model animals, such as the Javan macaque, which consists in the fact that: 1) the test sample containing IL-11 is brought into contact with the antibody or its antigen-binding fragment according to claim 1, 2) evaluate the concentration of immune complexes of the antibody or its antigen-binding fragment according to claim 1 with the IL-11 antigen.

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