WO2019031978A1

WO2019031978A1 - Diagnosing cancer using the analysis of blood antibodies on a microchip

Info

Publication number: WO2019031978A1
Application number: PCT/RU2017/000578
Authority: WO
Inventors: Николай Владимирович БОВИН; Е. Маргарет ХУФЛЕЙТ; Марко ВУСКОВИЧ
Original assignee: Общество С Ограниченной Ответственностью "Семиотик"
Priority date: 2017-08-07
Filing date: 2017-08-07
Publication date: 2019-02-14

Abstract

The invention concerns the development and creation of novel microarrays for identifying signatures for the diagnosis of cancers, based on the interaction of carbohydrate ligands with antibodies of human serum. Carbohydrate ligands that differ from the natural configurations of a glycosidic bond by virtue of the arrangement of the monosaccharide residues, the presence of a furanose form or the presence of an additional (non-carbohydrate) substituent have proven significant as cancer markers.

Description

DIAGNOSTIC CANCER USING ANALYSIS OF BLOOD ANTIBODIES ON

Microchip

TECHNICAL FIELD

The invention relates to medicine and immunology, and concerns the development and creation of new microarray for the diagnosis of diseases, based on the interaction of carbohydrate ligands with antibodies of human serum or blood plasma, as well as to a method of searching for such microarray. This invention can find application in immunology and medicine for the study and diagnosis of cancer. BACKGROUND

Carbohydrates are known to play an important role in numerous biological processes, such as the immune response, viral membrane fusion, and glycoprotein homeostasis. Carbohydrates play a role in inflammatory processes, cell-cell interaction, signal transduction, fertilization, and tumor transformation. Numerous oligosaccharides in the composition of glycoproteins and glycolipids are known as tumor-associated carbohydrate antigens (TASA) [1]; The level of human serum antibodies directed against some TASA, for example, the TF and Tp antigens, is an indicator when monitoring carcinomas.

The success of the immune system largely depends on the availability of a competent and well-stocked repertoire of carbohydrate-binding antibodies, which are the first line of defense against pathogens and remove cells or malignant cells or their fragments that have lost their function [1, 2]. These immunoglobulins are IgG, IgM, IgA, and IgD subtypes and are called natural antibodies (eAT), as they are present in the serum of all individuals in the absence of deliberate immunization [3], thus being part of the innate immunity system [4]. Natural antibodies (eAT), recognizing carbohydrates, including antibodies against blood group antigens, are not detected in the first few weeks of a person’s life. G.F. The springer proposed a hypothesis about the so-called “bacterial paradigm”, in which it is postulated that anti-carbohydrate antibodies appear in response to stimulation of the immune system by O-antigens and lipopolysaccharides of bacteria in the gastrointestinal tract [5, 6].

Despite the fact that a high level of natural lg remains almost unchanged throughout life and their repertoire is conservative [7], there are factors that influence the regulation of their biosynthesis. It has now been established that eAT is encoded by genes in a germline configuration in B cells that have not undergone somatic hypermutation and affinity maturation [8]. Many eat directed against carbohydrate antigens found in normal human tissues, and can be called autoantibodies [3]. The most well-known and studied eAT are antibodies against antigens of blood groups A and B and xenoantigens (heterophilic), such as the epitope of Galili Gala1-3Gal 1-4GlcNAc [9-11], Forssman glycolipid antigen (Fs) GalNAca1-3GalNAc31-3Gala1-4Gaipi- 4Glc [12, 13], and the Khanganutsu-Deicher (HD) antigen Neu5Gca2-3Gal 1-4Glc [14]. Anti-carbohydrate eAT appear in a number of pathologies, the group of special attention includes antibodies to TASA, for example, to Gal 1-3GalNAca (TF, Thomsen-Friedenreich antigen), GalNAca1-OSer / Thr (Tn) [5], and several related antigens [ 15]. Antibodies to ganglioside GT3 have been found in patients with type 1 diabetes [16]; antibodies of circulating islet cells recognize pancreatic glycopeptides, including sulfatides [17]. A number of neuropathies are associated with characteristic combinations of antibodies to gangliosides, sulfated glycolipids, and in some cases neutral glycosphingolipids, such as the Fs antigen [18]. Antibodies against Glca1-4Glca (glycogen fragment) turned out to be specific for patients with multiple sclerosis, which made it possible to differentiate patients with various neurological diseases [19]. Patients with peptic ulcer have an anti-1_e ^x 1_e and ^anti-y [20]. Antibodies against polylactosamines (system of blood groups I) [21], and in some cases against antigen H (fucosylated I or i) [22] are known as factors of the pathological disease of cold agglutination, and antibodies against globozide were detected during cold paroxysmal hemoglobinuria; several cases of other autoimmune diseases have been described in [23]. Antibodies against a limited glycolipid repertoire were found in patients with hemolytic anemia [24], antibodies against GlcNAc are characteristic of rheumatoid arthritis [25], antibodies against oligomannosic sequences are markers of Crohn's disease [26], it was shown that antibodies to the SARS vaccine have undesirable autoimmune reactivity to the glycan fragment of humans [27].

Although the above observations indicate the importance of the repertoire and properties of eAT, only limited data on anti-carbohydrate antibodies in humans have been available to date, largely due to the time-consuming ELISA procedure in the study of large cohorts. The development of glycan chips for several hundred positions printed on the chip platform [9, 28–30] makes it possible to conduct systematic studies based on large populations and use for prediction and diagnosis of various diseases, including oncological ones. However, due to the fact that the differences between antibodies to glycans in normal and cancer conditions are quantitative rather than qualitative, practical diagnosis based on natural antibodies to glycans is considered difficult.

At the same time, abrupt qualitative changes occur at the level of the supramolecular organization of biomolecules in oncological diseases and in case of other damaged cells. In other words, normal, regular molecules, including glycol molecules, can form stable, well-organized complexes (hazard-related molecular structures, DAMP) that are extremely different from the normal membrane combination of these molecules. The immune system is able to recognize DAMP; specific epitopes recognized by the generated antibodies include a spatial combination of atoms that cannot be accurately reproduced in vitro. Thus, the detection of these specific antibodies with the help of separate natural molecules (antigens) is impossible. Imitation (mimicking) of the relevant structures is also considered impossible, since it is not known what exactly to imitate, the structural organization of DAMP is unknown.

Therefore, there remains a need to develop approaches to the detection of antibodies to DAMP and, thus, to new methods of diagnosing cancer. DISCLOSURE OF INVENTION

The objective of this invention is to create a micro-aray to find diagnostic signatures used later for oncodiagnosis based on the interaction of carbohydrate and carbohydrate-like ("glycochints") ligands with human serum antibodies, as well as the development and creation of an effective diagnostic method.

The technical result of the invention is to develop and create a microerray based on carbohydrate and carbohydrate-like markers-antigens, capable of detecting new tumor marker antibodies and their signatures. The technical result of the present invention is also to develop a method for determining markers-antigens of this class for the effective diagnosis of cancer.

This technical result is achieved by creating a microerray containing many ligands, in particular, about ten (for practical analysis, that is, for diagnosing a disease) or several hundred (for searching signatures) of carbohydrate ligands, where one or more ligands are glycan having a combination of monosaccharides that is not characteristic of human glycans (for example, the disaccharide sequence Gal-GalNAc with a 1-4 bond, that is, the sequence Gal1-4GalNAc, whereas in human glycol molecules there is Gal1-3GalNAc), and / or glycan, which has the combination of monosaccharides characteristic of human glycans with an additional non-carbohydrate substituent, and / or glycan, which has a combination of monosaccharides unusual for human glycans and an additional non-carbohydrate substituent, and the ligands bind specifically to human serum or plasma antibodies (for example, with IgG, IgM, but usually the sum of all types of lg is detected), allowing you to diagnose cancer. In particular embodiments of the invention, the microrray according to the invention may contain from 10 to 600 ligands, in some other particular variants from 100 to 600 ligands (for conducting a signature search), in some other particular variants from 10 to 100 (for practical analysis, i.e. diagnosis of the disease).

In some embodiments, the microrray is characterized in that the carbohydrate ligand is a glycan having a combination of monosaccharides with an additional non-carbohydrate substituent. In particular embodiments of the invention, said non-carbohydrate substituent is alkyl, acyl,-glycolyl, unsubstituted or substituted aryl, or benzyl. In some embodiments, the aryl (for example, phenyl) or benzyl group may be substituted, for example, in the ortho, meta or para position with at least one substituent, in some embodiments with two substituents.

In some embodiments, the microrray is characterized in that one or more ligands are glycan, the combination of monosaccharides of which is characterized by the glycosidic bond position unusual for human glycans, such as Gai i-4GalNAc instead of Gal 1-3GalNAc.

In particular embodiments of the invention, said combination of monosaccharides is characterized by an unusual for human glycans, that is, opposite to the natural, anomeric configuration of the glycosidic bond, for example, 3Neu5Ac instead of aNeu5Ac.

In other particular embodiments of the invention, the combination of monosaccharides is characterized by an unusual for human glycans, that is, opposite to the natural, anomeric configuration of the glycosidic bond between the reducing end of the glycan and the aglyconin residue. In some embodiments of the invention, the aglyconin residue (the aglycon moiety) is a linker between a carbohydrate-containing molecule and a solid carrier. In the most preferred embodiments of the invention, the linker is an aromatic residue, in particular, o-, m- or p-substituted phenyl or o-, m- or p-substituted benzyl, or o-, m- or p-substituted 2-phenylethyl .

In some embodiments, the microrray is characterized in that the glycan is immobilized directly on a solid carrier using a covalent bond or through an intermediate polymer (for example, polyacrylamide, polyacrylic acid or polyethylene glycol) with a covalent or non-covalent bond.

In particular embodiments of the invention, the microrray is characterized in that one or more ligands are glycan having an unusual combination of monosaccharides for human glycans, which contains a monosaccharide fragment of Neu5Ac _p . In particular embodiments of the invention, the microrray is characterized in that one or more ligands are glycan having a combination of monosaccharides characteristic of human glycans with an additional non-carbohydrate substituent, which is a disaccharide fragment of Gal _p a1-3GalNAc _p aOC ₆ H ₄ .

In particular embodiments of the invention, a microrray is characterized in that one or more ligands are glycan having a combination of monosaccharides characteristic of human glycans with an additional non-carbohydrate substituent which is a tetrasaccharide (3-0-Su) Gal _p pi -4GlcNAc _P 1-3Gal _P 1 -4GlcNAc _P -.

In particular embodiments of the invention, the microrray is characterized in that one or more ligands are glycan having a combination of monosaccharides characteristic of human glycans with an additional non-carbohydrate substituent, in particular the glycopeptide GlcNAc _p i-4Mur-L-Ala-Di-Gln.

In particular embodiments of the invention, a microrray is characterized in that one or more ligands are glycan having an unusual combination of monosaccharides for humans, which is a GalNAC _f PI-4GlcNAc _p - disaccharide.

In particular embodiments, a microarray harakterizuyuetsya in that one or more ligand is a glycan having peculiar to human glycan monosaccharides combination with additional non-carbohydrate substituent such as _p r1-461s disaccharide Ca1 ^ 1-MH-amino acid Ca1 E1-4S1s _P 1 _P -1MH-amino acid-amino acid, trisaccharide Neu5Aca2-3Gal _P 31-4Glc _p pi-NH-aMHHOKH ^ OTa or Neu5Aca2-ЗСа1 _Р 1-4С1с _р p1-MH-amino acid-amino acid. In some preferred embodiments of the invention, the amino acid is glycine, i.e. Galp 1-4Glc _P 1 -NHCOCH ₂ NH- or Neu5Aca2-3Gal _P 1-4Glc _p p1-NHCOCH ₂ NH-.

In addition, the invention relates to the use of a microerray according to the invention for: 1) searching for diagnostic signatures; 2) for the diagnosis of cancer, in particular breast cancer, ovarian cancer, colorectal cancer, lung cancer, mesothelioma.

The technical result is also achieved by implementing a method for finding signatures for detecting and / or monitoring (diagnosing) an oncological disease, comprising the following successive stages:

- contact of serum with microerray according to the invention;

detection of a molecular complex formed by microerray ligands and serum antibodies using fluorescently-labeled antibodies to human immunoglobulins;

- processing of the obtained results as described in [34];

- the choice of ligands for inclusion in the signature, based on the processed data. In some particular embodiments of the invention, this method is used to detect and / or monitor breast cancer, ovarian cancer, colorectal cancer, lung cancer, mesothelioma.

The invention also relates to the diagnosis and / or monitoring of cancer using the microarray of the invention.

The invention also includes the production of microarrays of the invention.

DETAILED DISCLOSURE OF THE INVENTION

Definitions and abbreviations

The term "glycochint" as used herein means a glycan, the structure of which is an unnatural combination of monosaccharides typical of the human body (such as, for example, glucose, galactose, fructose, mannose, N-acetylglucosamine, α-acetylgalactosamine or α-acetylneuraminic acid), or natural human glycan, modified by a non-natural residue (or natural residue in an unnatural position). In terms of immunology, glycochint mimics a glycan-containing spatial epitope (DAMP, damage associated molecular pattern) - a supramolecular structure consisting of two or more adjacent molecules (or their fragments), such as glycolipids, glycoproteins, lipids, proteins etc. Glycohint should not be understood as a glycomimetic (see below); glycomimetics are analogues of glycans of a known structure, while glycochints mimic unknown (currently unknown or unknown in principle) combinations of molecules or fragments, such as those defined above as DAMP.

The term "glycomimetic" (literally: "carbohydrate simulator") in this document means a molecule (usually a peptide), the reproducing ability of a carbohydrate to bind to a specific receptor (usually a protein), for example, lectin.

The term "combination of monosaccharides" as used herein means a complex compound, where the monosaccharides are linked by a glycosidic bond, i.e. an oligosaccharide.

"GID" in this document means the identification number of the glycan in this document.

The term “solid carrier” as used herein means the surface of a chip, latex, gold particles, quantum dots, and the like.

The term "diagnostic signature" as used herein means a combination of two or more markers of a given disease.

"RFU" in this document means relative fluorescence units.

The term "glycan" in this document means a carbohydrate chain that is included in natural glycoproteins (for example, Neu5Aca2-4Gal 1-4GlcNAc) or glycolipids (for example, Neu5Aca2-4Ga ^ 1-4Glcp), in some embodiments of the invention, the carbohydrate chain of a glycan may include from 1 to 6 monomers, in the most preferred embodiments of the invention from 1 to 4. According to the invention, in a microerray, it can be used as natural glycans i.e. those whose carbohydrate chain is a combination of monosaccharides peculiar to the human body, connected by O-glycosidic bonds (such as, for example, Neu5Ac 2-3Gaipi-3GalNAca, Galp1-3 (Fuca1-4) GlcNAcp, (Neu5Aca2-8) ₃ , 3η 1-40ΙοΝΑοβ, but not limited to), and non-natural glycans, i.e. glycohint (see above).

The term "chip (microchip)" in this document means a device in the form of a glass or plastic plate, on one side of which a set of chemicals is immobilized (ligands), usually in the form of round or square discrete areas.

The term "(micro) herrey" (array) in this document means a combination of chemicals (ligands) immobilized on a glass or plastic plate (see above) or other solid carrier. That is, an arrey is a concept, not a device.

The term “PGA” printed glycan array (glycoerray) in this document means a collection of carbohydrate ligands immobilized on a solid carrier.

“Alkyl”, by itself or as part of another substituent, refers to straight or branched saturated hydrocarbon groups, including hydrocarbon groups having a specified number of carbon atoms (for example, Landfill l means from one to eight carbon atoms). Examples of alkyls include methyl, ethyl, n-propyl, iso-propyl, and others.

The term “acyl” refers to the group —C (0) R (in which R has the meaning, in particular, H, alkyl, aryl). Examples of acyl groups include formyl, acetyl, benzoyl, phenylacetyl, and similar groups.

The term "aryl", unless otherwise specified, refers to an aromatic hydrocarbon group, which may be a single cyclic structure or several rings that are condensed together, preferably, is a 5-6-membered monocyclic ring. An example of aryl cyclic groups is phenyl.

The term "benzyl", unless otherwise specified, means a hydrocarbon residue -

“Su” - in this document means sulfate. The lowercase letter " _p " denotes the pyranose cycle of the corresponding monosaccharide, and the lowercase letter "f" denotes the furanous cycle of the corresponding monosaccharide.

In the structure of GlcNAc _p 1-4Mur-L-Ala-Di-Gln, the letter "i" denotes the prefix "iso", that is, side carboxyl instead of canonical is involved in the formation of an amide bond with alanine.

Brief Description of the Drawings

Figure 1. The intensity of the binding of human serum antibodies to natural glycans (dark histogram columns) and the corresponding related non-natural, that is, glycochints (light histogram columns). PGA data (see below) on a scale of 0 - 65 RFU LLC:

a) data for a pair of glycans Gala1-4GlcNAcp1-3Gaipi-4GlcNAcP-sp / Gai i - 4GlcNAcpi-3Gaipi-4GlcNAc -sp, differing only in the configuration of one of the four glycosidic bonds; The study was conducted on a cohort of 80 individuals, including healthy subjects in the control group (n = 20), duct carcinoma in situ (DCIS; n = 20), infiltration duct carcinoma (IDC; n = 20), and metastatic breast cancer (MVS; n = 20);

b) data for a pair of glycans Neu5Aca2-8Neu5Aca-OCH ₂ CeH ₄ -p-NHCOCH ₂ NH ₂ / Neu5Aca2-8Neu5Aca-sp, the first of which contains an aromatic (benzyl) aglycon residue; The study was conducted on a cohort of 127 individuals, including healthy subjects in the control group (n = 106), early aggressive ovarian cancer (n = 21).

Detailed disclosure of the invention

Existing methods of molecular diagnostics of oncological diseases have low diagnostic specificity and sensitivity (they give unacceptably many false-positive and false-negative results), since they are based on the definition of a single marker (sometimes two or three). At the same time, the tumor is not only heterogeneous, not only biochemically individual for each patient, but also rapidly evolving throughout the course of the disease. To avoid the influence of these three factors, it is necessary to use many markers simultaneously (the so-called diagnostic signatures).

The role of natural human antibodies is to supervise the wrong structures and the wrong combinations of the right molecules in the cell membrane. Some of the naturally-occurring antibodies are directed against carbohydrates (glycans, oligosaccharides) or glyco-containing molecules, such as aberrant (biosynthetically "irregular", absent in normal cells) glycopeptides and glycolipids. Since the antigenic determinants for these antibodies are unknown complex structures, there are no algorithms and methods for their determination. Thus, to identify these determinants using diagnostic and prognostic methods, it is necessary to find samples capable of specifically binding to antibodies, despite the absence of complete structural identity with real motives.

In this document, such samples are called glycoquinta. Glycohints contain a portion of the complete antigenic determinant (the unchanged portion of the glycan), that is, carbohydrate motifs, known as targets for natural antibodies [31], in combination with a non-natural motive or a non-natural (or unusual for humans) combination of natural monosaccharides. Unexpectedly, it was found that some glycans containing an unnatural structural fragment (i.e. glycochints) bind to human blood antibodies, and their frequency of entry into diagnostic signatures is much higher than for natural glycans typical of human glycoproteins and glycolipids. Unexpectedly, it was found that glycochins provide an opportunity to distinguish between cancer patients and healthy donors by comparing the antibody binding profile on the chip. To do this, there are many ligands on the chip, among which must be present both natural glycans and glycochints. Glycans and glycochints can be attached to a solid support through covalent or non-covalent interactions. The method of attachment is limited by the need to ensure that the bond of the glycan (or glycochint) with the solid phase is strong and does not interfere with the analysis, for example, does not affect the binding affinity in one direction or another.

In other words, for the diagnosis of cancer, these glycohints are critical components in detecting certain types of cancer.

Obviously, glycohint can only be found empirically. Despite this, glycochints can be used for effective and accurate oncodiagnosis, as shown in this document. To prove the applicability of glycochints for the differentiation of healthy people and cancer patients, a comparative experiment was conducted in which one molecule of glycohint in the diagnostic signature was replaced with a very close natural glycan. The data below (Examples 1–6) demonstrate a significant difference between some signatures based on glycohint from similar signatures, where the glycochint is replaced with its closest natural prototype. The implementation of the invention

Obtaining ligands according to the invention

Glycoligands (representing both glycans and glycochints) with a purity of 95-98% are chemically synthesized. The synthesis of some of them is known from the prior art, in Specifically, the synthesis of the Ga ^ 1-4GlcNAc 1-3Gal 1-4GlcNAc3-sp glycan is known from [35], the synthesis of the Neu5Aca2-8Neu5Aca-OCH2C ₆ H4-p-NHCOCH ₂ NH2 glycans and Neu5Aca2-8Neu5Aca-sp is known from [36], the glycan GlcNAc31-3 (GlcNAc 1-6) GalNAca-sp from [37], the glycan Neu5Aca2-3Gal 1-4 (Fuca1-3) (6-0-Su-) GlcNAc from [38], the glycan Neu5Aca2-3Ga ^ 1-4 (Fuca1-3) GlcNAc from [39], glycan Ga ^ 1-4GlcpO-sp from [40]. Examples of the synthesis of some other glycans according to the invention are described below.

Examples of the synthesis of glycans

GlcNAc l -4 (GlcNAc 1 -6) GalNAca-0 (CH ₂ ) ₃ NH ₂

Glycosylation of the 3-OAc-GalNAca-0 (CH ₂ ) ₃ NH-COCF ₃ glycosyl acceptor using the a-Ac0 ₄ -GlcN (Troc) Br glycosyl donor during the synthesis of the GlcNAc i-6GalNAca-0 (CH ₂ ) ₃ disaccharide NH ₂ [37], after removing the protective groups as a by-product, produced the GlcNAc 1-4 trisaccharide (GlcNAc 1-6) GalNAca-0 (CH ₂ ) ₃ NH ₂ (-10%). ¹ H-NMR (500 MHz, D ₂ 0): 1.890-2.000 (m, 2H, CH ₂ sp); 2.023, 2.037 and 2.051 (3s, 3 x 3H, NCOCH ₃ ); 3.123 (m ^« t, 2H, NCH ₂ sp 7.76); 3.384-3.588 (m, 7H); 3.690-3.812 (m, 6H); 3.912-3.973 (m, 2H); 4.002 ^ .050 (m, 2H); 4.095-4.183 (m, 3H); 4.508 (d, 1 H, H-1 GlcNAc, J _{1 2} 8.5); 4.703 (d, 1 H, H-1 GlcNAc, J _{1 i2} 8.4); 4.870 (d, 1 H, H-1 GalNAc, J _1i2 3.6).

Gala1-4GlcNAcp1-3Galp1-4GlcNAcp-0 (CH ₂ ) ₃ NH ₂

Upon receipt of Ga ^ 1-4GlcNAcpi-3Gaipi-4GlcNAc -0 (CH ₂ ) ₃ NH ₂ , the synthesis of which is described in [35], its α-anomer Gala1-4GlcNAc 1-3Gal 1-4GlcNAc-0 (CH ₂ ) is also formed ₃ NH ₂ ; H NMR (500 MHz, D ₂ 0): 1.910-2.002 (m, 2H, CH ₂ sp); 2.050 and 2.058 (3s, DxZN, NCOCH ₃ ); 3.097 (m ^« t, 2H, NCH ₂ sp 6.86); 3.542-3.653 (m, 3H); 3.650 ^ .073 (m, 20H); 4.168 (dd * d, 1 H, H-4, J 3.1); 4.474 (d, 1 H, H-1, J _1i2 7.9); 4.527 (d, 1 H, H-1, J _1i2 7.9); 4.715 (d, 1 H, H-1, Ji _{, 2} 8.4); 5.452 (d, 1 H, H-1d, J _{1 2} 3.6).

Chip printing

The chips were printed as described previously [30], at a concentration of 50 μM, each in eight repetitions. The quality control of the chips included the analysis of the quality of repetitions inside the chip and the analysis of PGA reproducibility between the chips. The first one included the calculation of variation coefficients and general correlation coefficients of compliance [32] in order to monitor and control the precision of measurements using PGA, and the latter included the calculation of correlation coefficients of Lin's correspondence [33, 34] for PGA printed on different series of slides and incubated with the same serum, but with different development schemes. Quality control also included variational analysis, leading to the obtaining of correlation coefficients between classes (ICC), which made it possible to finally determine whether a particular cohort is suitable for further statistical analysis. The analysis of serum.

The chips were incubated with human serum (a 1: 15 dilution in phosphate-buffered saline containing 3% BSA and 1% Tween 20) with gentle rocking for 2 hours at 37 ° C. Antibodies binding to printed ligands were visualized using a combination of biotinylated secondary antibodies against the sum of human IgG, IgM and IgA (Pierce, Rockford, IL) followed by treatment with streptavidin (streptavidin-Alexa555, Invitrogen / Molecular Probes, Carlsbad, CA), that is, Separately, the level of IgG, IgM, IgA binding was not measured. The intensity of the fluorescence signals was read at the power of the laser reader and processed using the BioDiscovery / lmaGene software (El Segundo, CA). The range of all values of the relative intensity of the fluorescence signal of bound antibodies was 0 ~ 65 x 10 ³ RFU [31]. Statistical analysis.

To analyze the statistical correlation between the initial values of the signal intensity (measured on a laser scanner as RFU, relative fluorescence units) for all pairs of ligands, the Pearson cross-correlation coefficient was calculated. The result was a cross-correlation matrix of 205 x 205 (there were 205 ligands on a chip), with which ligands with a statistical correlation were determined. An example of a diagnostic signature according to the invention is presented in table 1.

Table 1. Diagnostic signature for the diagnosis of breast cancer.

After analyzing a cohort of 20 patients with breast cancer and 20 healthy controls, the following parameters of this signature were found: AUC 0.945; sensitivity of 85%; specificity is 80%. A comparative analysis was also carried out in which two series of chips were used, one of which used the glycochints of the invention, and the other, the most similar natural glycan in structure. However, as shown by the results of the analysis, in the case of using natural glycans, the diagnostic signature did not work for the diagnosis of tumor diseases. The results of this study are presented in table 2.

Table 2. Comparison of diagnostic results by using a signature with a glycochint according to the invention and the same signature, where the glycochint is replaced by the most structurally close natural glycan.

Table 2 shows examples of the comparison of six pairs of glycohint substitution with natural glycans: the upper glycan in the “glycan structure” column is the glycochint included in the signature for this analysis, the lower glycan is the closest natural structurally related glycan. Pearson's correlation coefficient (p) is calculated for each example. The Rank column indicates the position of the glycan in the signature in the corresponding Wilcoxon rank table. WITH - Invasive Ductal Carcinoma (invasive ductal carcinoma), AIM - Metaplastic Breast Cancer (metaplastic breast cancer).

In the event that p (Wilcoxon) has very low values, this means that the signature works well as a diagnostic tool, that is, patients with it reliably differ from healthy ones. If this value lies in the range of 0.01-0.9, then this indicates that the diagnosis is ineffective.

The results obtained in the table show that the replacement of glycohints for the nearest structural analogues - natural glycans lead to a dramatic loss of diagnostic information. Thus, the use of synthetic close unnatural analogues of natural glycans can improve the diagnostic value of information obtained using microchip technology, that is, increase the accuracy of diagnosis.

Although the invention has been described with reference to the disclosed embodiments, it should be obvious to those skilled in the art that the specific experiments described in detail are given only for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be clear that it is possible to implement various modifications without departing from the gist of the present invention.

References, which are included in the present description of the invention as references:

1. S.Hakomori. Tumor-associated carbohydrate antigens. Annu. Rev. Immunol., 2, 103-126 (1984).

2. K.O.Lloyd. Humoral immune responses to tumor-associated carbohydrate antigens. Semin. Cancer Biol., 2, 421-431 (1991).

3. J. Milland, M.S. Sandrin. ABO blood group and related antigens, natural antibodies and transplantation. Tissue antigens, 68, 459-466 (2006).

4. G.Dighiero, N.R.Rose. Critical self-epitopes are key to understanding self-tolerance and autoimmunity. Immunology today, 20, 423-428 (1999).

5. R.A.C.Hughes, R.D.M.Hadden, N.A.Gregson, K.J.Smith. Pathogenesis of Guillain-Barre syndrome. J. Neuroimmunol., 100, 74-97 (1999).

6. AFPreece, KMStrahan, J.Devitt, F.Yamamoto, K.Gustaffson. Expression of ABOs is associated with specific natural antibodies and complement. Blood, 99, 2477-2482 (2008). 7. S.Henry, R.Oriol, B.Samuelsson. Lewis blood group system and associated secretory phenotypes. Vox Sanquinis, 69, 166-182 (1995).

8. Y.Shoenfeld. The mosaic of autoimmunity. Immunol. Today, 10, 13-126 (1989).

9. W. McKane, J. Lee, R. Preston, A. Hasking, P. Simpson, S. Lynds, L. Goldberg, T. Carns, D. Taube. Polymorphism in the human anti-pig natural antibody repertoire.

Transplantation, 66, 626-633 (1998).

10. I.V.Lekakh, N.V.Bovin, G.P.Bezyaeva, A.M.Poverenny. Naturally hidden autoantibodies react with negatively charged carbohydrates and xenoantigen Bdi. Biochemistry (Moscow), 66, 205-210 (2001).

11. R.A.Alvarez. O.BIixt. Identification of ligand specificities for glycan-binding proteins using glycan arrays. Methods Enzymol., 415, 292-310 (2006).

12. J.Wieslander, O.Mansson, E.Kallin, A.Gabrielli, H.Nowack, R.Timple. Specificity of human antibodies against Galct1-3Gal carbohydrate; Epitope and Gala1-2Gal or Gala1-4Gal. Glycocon. J., 7, 85-100 (1990).

13. a) Huflait, M.E., Blixt, O., Vuskovic, M., Xu, H., Shaw, L, Reuben, J.M., Kuerer,

H., and Cristofanilli, M. 28th Annual San Antonio Breast Cancer Symposium, San Antonio, TX.

(2005) Br. Cancer Res. Treat. 94, Suppl.S85.

b) Arun, B., Vuskovic, M., Vasiliu, D., Xu, H., Atchley, D., Chambers, J., Bovin, N.V.,

Sneige, N., Hortobagyi, G.N., and Hufleit, M.E. 30th San Antonio Breast Cancer Symposium, San Antonio, TX (2007) Br. Cancer Res. Treatment 106, Suppl.1: S180.

14. G.F.Springer. T and Tn, general carcinoma autoantigens. Science, 224, 1198-1206 (1984).

15. E. Cabrera-Rode, O.Diaz-Horta, L.E. Fernandez, A.Carr, G.Marquina, O.Valente, R.M.Gonzalez-Suarez, A.Uriarte. Glycolipids as the major autoantigens of cytoplasmatic islet cell antibodies. Autoimmunity, 20, 145-151 (1995).

16. D.Wang, J.Lu. Glycan arrays lead to auto discovery of autoimmunogenic activity of SARS-CoV. Physiol. Genomics, 18, 245-248 (2004).

17. N.Dotan, R.T.AItstock, M.Schwarz, A.Dukler. Anti-glycan antibodies for biomaterials for diagnosis and prognosis. Lupus, 15, 1-10 (2006).

18. S.Avrameas. Natural autoantibodies: from "horror autotoxicus" to "gnothi seauton". Immunology Today, 12, 154-159 (1991).

19. K.Kamiya, T.Arisawa, H.Goto, K.Shibayama, T.Horii, T.Hayakawa, M.Ohta. Are autoantibodies against Lewis antigens involved in the pathogenesis of Helicobacter pylori-induced peptic ulcers? Microbiol. Immunol., 43, 403-408 (1999).

20. B.A.Gaillard, J.M.Thomas, L.J.Nell, D.M.Marcus. Antibodies against ganglioside

GT3 in sera of patients with type 1 diabetes mellitus. J. Immunol., 142, 3826-3832 (1989).

21. T. Feizi. Antibodies to defined carbohydrate sequences in immunological disorders of man. Med. Biol., 59, 131-133 (1981). 22. ECIVeerman, JGMBolscher, BJAppelmelk, E.Biemena, TKvan den Berg, AVNAmerongen. A monoclonal antibody directed against high salivary mucins recognizes the s03-3Gal beta 1-3GlcNAc moiety of sulfo-Lewis (a): a) Glycobiology, 7, 37-43 (1997).

23. U.Nydegger, T.Hardegger, A.Tobler, H.Rieder, A.Cherniak, B.Lammle. Cold agglutinin syndrome and liver transplantation. Vox Sang, 67, 85 (1994).

24. E.Y.Korchagina, T.V.Pochechueva, P.S.Obukhova, A.A.Formanovsky, A.lmberty, R.Rieben, N.V.Bovin. Design of the blood group AB glycotope. Glycoconj. J., 22, 127-133 (2005).

25. N.I.Briko, N.V.Bovin, B.I.Shevelev, L.O.Dynga, Y.I.BIinnikova, P.P.Kuksyuk,

S.E.Myasoyedova, I.V.Ambrosov, N.N.Filatov. Streptococcus on the base of conjugated N-acetylglucosamine and its medical application. Klin. Lab. Diagnost, N 9, 43-46 (1997).

26. K.Furukawa, K.Yokoayma, T.Sato, J.Wiels, Y.Hirayama, M.Ohta, K.Furukawa. Expression of the Gb3 / CD77 synthase gene in megakaryoblastic leukemia cells: J. Biol. Chem., 277, 11247-11254 (2002).

27. T. Feizi, R.A.Childs. Blood-group-li-active gangliosides of human erythrocyte membranes. Biochem. J., 173, 245-254 (1978).

28. A.Coutinho, M.D.Kazatchkine, S.Avrameas. Natural autoantibodies. Curr. Opin. Immunol., 7, 812-818 (1995).

29. P.Bailly, J.-F.Bouhours. P blood group and related antigens. In: Blood Cell Biochemistry, Vol. 6: Antigens Molecular Basis of Major Human Blood Group, J.-P.Cartron and P.Rouger, Eds., Plenum Press, New York, 1995, pp.299-329.

30. O.BIixt, S.Head, T.Mondala, C.Scanlan, MEHuflate, R.Alvarez, MCBryan, F.Fazio, D.Calarese, J.Stevens, N.Razi, DJStevens, JJSkehel, I. van Die, DRBurton,

I.A.Wilson, R.Cummings, N.Bovin, C.-H.Wong, J.C.Paulson. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA, 101, 17033-17038 (2004).

31. Hufleit, ME, Vuskovic, MI, Vasiliu, D., Xu, H., Obukhova, P., Shilova, N., Tuzikov, A., Galanina, O., Arun, V., Lu, K., and NV Bovin. Anti-carbohydrate antibodies of normal sera: findings, surprises and challenges. Mol. Immunol., 46, 3037-3049 (2009) ..

32. R.B. Parekh, R.A.Dwek, B.J.Sutton, D.L.Fernandes, A.Leung, D.Snanworth, T.W.Rademacher, T.Mizuochi, T.Taniguchi, K.Matsuta. Association of rheumatoid arthritis and primary osteoarthritis with changes in serum IgG. Nature, 316, 452-457 (1985).

33. LILin. A concordance correlation coefficient to evaluate reproducibility. Biometrics, 45, 255-268, (1989). 34. Vuskovic, Ml., Xu, H., Bovin, NV., Pass, HI, and Huflate, ME. (2011). Printed Glycan Arrays for diagnostic and prognostic applications. Int. J. of Bioinformatics Research and Applications, 7, 402-426 (2011).

35. V.V.Severov, I.M.Belyanchikov, G.V.Pazynina, N.V.Bovin. Synthesis of N-acetyllactosamine-containing oligosaccharides, galectin ligands. Rus. J. Bioorgan. Chem., 33,

122-138 (2007).

36. G.Pazynina, V.Nasonov, I.Belyanchikov, R.Brossmer, M.Maisel, A.Tuzikov, N. Bovin. Koenigs-Knorr glycosylation with neuraminic acid derivatives. Int. J. Carbohydr. Chem., 2010, 8 pages (2010). Doi: 10.1155 / 2010/594247.

37. G.V.Pazynina, N.V.Bovin. Synthesis of GlcNAc-terminated oligosaccharides - fragments of glycoprotein O-chains. Mendeleev Communs., 4, 132-133 (2000).

38. G.Pazynina, M.Sablina, M.Mayzel, V.Nasonov, A.Tuzikov, N. Bovin. Chemical synthesis of 6 ^(GlcNAc) - and 6 ^(Gal) -0-sulfated Sial_e ^x tetrasaccharides in space-armed form.

Glycobiology, 19, 1078-1081 (2009).

39. G.V.Pazynina, M.A.Sablina, A.B.Tuzikov, A.A.Chinarev, N.V.Bovin. Synthesis of complex Π2-3 sialooligosaccharides, including sulfated and fucosylated ones, using Neu5AcD2-

3Gal as a building block. Mendeleev Communs., 13, 245-248 (2003).

40. M.Duk, G.Kuznierz-Alejska, E.Y.Korchagina, N.V.Bovin, S..Bochenek, E.Lisowska.

Anti-a-galactosyl antibodies recognizing epitopes terminating with a1, 4-linked galactose: human "natural" and mouse monoclonal anti-NOR and anti-P1 antibodies. Glycobiology, 15, 109-118

(2005).

Claims

Claim

1. A microarray containing a variety of carbohydrate ligands, where one or more ligand is a glycan having an unusual combination of monosaccharides for human glycans, and / or a glycan having a combination of monosaccharides characteristic of human glycans with an additional non-carbohydrate substituent, and / or a glycan having an unusual for human glycans, the combination of monosaccharides with an additional non-carbohydrate substituent.

2. Microerray according to claim 1, characterized in that the microerray contains 10-600 ligands.

3. Microerray according to claim 2, characterized in that the microerray contains 100-600 ligands.

4. Microerray according to claim 2, characterized in that the microerray contains 10-100 ligands.

5. A microarray according to claim 1, characterized in that said one or more carbohydrate ligand is a human glycan, in which a non-carbohydrate substituent is additionally introduced.

6. The microarray of claim 5, wherein the non-carbohydrate substituent is an alkyl, acyl, unsubstituted or substituted aryl, or benzyl.

7. A microarray according to claim 1, characterized in that one or more ligand is a glycan, the combination of monosaccharides of which is characterized by the glycosidic bond position unusual for human glycans.

8. A microarray according to claim 1, characterized in that one or more ligand is a glycan, the combination of monosaccharides of which is characterized by an unusual for human glycans, that is, opposite to the natural, anomeric configuration of the glycosidic bond.

9. A microarray according to claim 1, characterized in that one or more ligand is a glycan, the combination of monosaccharides of which is characterized by an unusual for human glycans, that is, opposite to the natural, anomeric configuration of the glycosidic bond between the reducing end of the glycan and the aglycon residue.

10. Microerray according to claim 9, characterized in that the aglycone residue is a linker between a carbohydrate-containing molecule and a solid carrier.

11. The microarray of claim 10, characterized in that the aglyconin residue is an aromatic residue.

12. The microarray according to claim 11, characterized in that the aromatic residue is substituted benzyl or substituted phenyl.

13. The microarray under item 12, characterized in that the benzyl or phenyl is substituted in the para-position.

14. Microerrey according to claim 1, characterized in that the ligands are attached to a solid carrier by covalent bond.

15. A microarray according to claim 1, characterized in that one or more ligands are glycan having a combination of monosaccharides characteristic of human glycans with an additional non-carbohydrate substituent, and is tetrasaccharide.

16. The microarray according to claim 15, wherein the glycan is (3-0-Su) Gal _p 1 - 4GlcNAc _p p1 -3Ga ^ 1 -4GlcNAc _p p-.

17. The microarray according to claim 1, characterized in that one or more ligand is a glycan having peculiar to human glycan monosaccharides combination with additional non-sugar substituent which is a disaccharide Ca1 _p _p 1-4S1s P1-NH-amino acid 6a1 _p p1-4C1c _p 1-MH-amino acid-amino acid, Neu5Aca2-3Gal _P 1-4Glc _P 1-NH-aMMHOKHOioTa trisaccharide or Neu5Aca2-3Gal _P 1 -4Glc _p i -ΝΗ-amino acid-amino acid.

18. Microerrey on 17, characterized in that the amino acid is glycine.

19. The microarray of claim 18, characterized in that the glycan is Galppi-4Glcpp1 -NHCOCH ₂ NH- or Neu5Aca2-3Galpp1 -4Glcpp1 -NHCOCH ₂ NH-.

20. A method for finding a signature for detecting cancer, comprising the following successive stages:

- the contact of the patient's serum with a microerray according to any one of claims 1 to 19;

detection of a molecular complex formed by microerray ligands and serum antibodies using fluorescently labeled antibodies to human immunoglobulins;

- processing of the data;

- the choice of ligands for inclusion in the signature, based on the processed data.

21. The method according to claim 20, characterized in that the search for signatures of such cancers as breast cancer, ovarian cancer, colorectal cancer, and lung cancer is carried out.