WO2024117938A1 - Methods for producing bioconjugates for liver-specific targeted proteome modulation - Google Patents

Abstract

The invention relates to the field of biotechnology and medicine, and more particularly to the production of bioconjugates for liver-specific targeted proteome modulation, which can be used for treating a broad spectrum of diseases stemming from an imbalance in protein activity or concentration. For this purpose, a method is proposed for producing conjugates that include an antibody against a target of interest, wherein said antibody is conjugated with ASGPR ligands. The proposed method includes a step of activating a ligand by reacting same with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide, and a step of bioconjugating activated ligands with an antibody. The proposed method makes it possible to produce a high yield of bioconjugates which do not require additional purification, while preserving the specificity of the antibodies therein.

Description

METHODS FOR OBTAINING BIOCONJUGATES FOR LIVER-SPECIFIC TARGETED PROTEOME MODULATION

Field of technology

The invention relates to the field of biotechnology and medicine, namely, to the production of bioconjugates for liver-specific targeted modulation of the proteome, which can be used for the treatment of a wide range of diseases based on an imbalance in the activity or concentration of proteins. The resulting bioconjugates cause liver-specific targeted degradation of undesirable components of the proteome. Due to this, they can be aimed, in particular, at increasing the effectiveness of antitumor therapy and solving the problem of drug resistance of tumor cells.

Prior Art

Modulation of the functions of most proteins is ensured by targeted proteolysis and further degradation in proteasomes and lysosomes.

In 2020, a group of scientists led by K. Bertozzi described a variant of a way to direct this natural process to solve medical problems, namely the method of targeted degradation of membrane proteins using glycoconjugates, called LYTAC [Banik SM, Pedram K, Wisnovsky S, Ahn G, Riley NM, Bertozzi CR. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature. 2020 Aug;584(7820):291 -297. doi:10.1038/S41586-020-2545-9. Epub 2020 Jul 29. PMID: 32728216]

The therapeutic value of molecules of the antibody-linker-ligand type, where the antibody is capable of specifically binding to a target located on the surface of cells (for example, a cell receptor), and the ligand is a fragment that mediates transport to lysosomes (in particular, the ligand to the mannose-6-phosphate receptor ), potentially very high. With the help of such molecules, it is possible to degrade both membrane (surface) and extracellular proteins responsible for the development of various diseases.

In 2021, the LYTAC method was improved: a method for liver-specific targeted degradation of target proteins using antibodies conjugated to ASGPR1 receptor ligands was described [Ahn G, Banik SM, Miller CL, Riley NM, Cochran JR, Bertozzi CR. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat Chem Biol. 2021 Sep;17(9):937-946. doi: 10.1038/s41589-021 -00770- 1. Epub 2021 Mar 25. PMID: 33767387; PMCID: PMC8387313.]. Both variations of LYTAC are reflected in the international application of Lycia Therapeutics WO 2021/142377.

However, the bioconjugates and their synthesis scheme proposed by Bertozzi’s group are appropriate for proving the concept of targeted protein degradation via lysosomes, but reveal their unsuitability upon transition to the synthesis of minimal industrially significant quantities of bioconjugates.

Thus, it is known that a common problem in the synthesis of medicinal bioconjugates is residual amounts of toxic organic solvents [https://www.sciencedirect.com/science/article/abs/pii/S0003267014010927]. It is known that the bioconjugation described by Bertozzi's group involves the presence of dimethyl sulfoxide (DMSO), the toxicity of which is well known.

Accordingly, a purification step for the resulting bioconjugate is required. However, even this does not solve the problem of protein denaturation that has already occurred upon contact with the solvent. Indeed, while carrying out a synthesis similar to that proposed by Bertocci's group, using organic solvents, we encountered low yields of bioconjugates, presumably due to the fact that antibodies aggregate and are retained on the filter membrane.

The article by Zhou Y. et al. Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins // ACS Cent. Sci. 2021, 7, 3, 499-506 describes the preparation of trimeric GalNAc-antibody conjugates to form bifunctional molecules that can selectively target extracellular proteins to the lysosomes of liver cells for degradation. The synthesis proposed by this research group requires a separate activation step of the ligand with the carboxy form of the (PEC)4 linker using an N-hydroxysuccinimide (NHS)/1,3-dicyclohexylcarbodiimide (DCC) mixture in dimethylformamide (DMF). In this case, the activated ligand is not released from unreacted components. Accordingly, the target antibody during conjugation is mixed with a solution of the ligand in an organic solvent, which, as in the case of the synthesis proposed by the Bertozzi group, can affect the conformation and functionality of the target antibody during the conjugation process, especially at a high molar ratio of antibody to linker (in publication use a ratio of 1:25, indicating that the higher the ratio, the greater the effect on the target protein). In addition, unused excess carbodiimide component, 1,3-dicyclohexylcarbodiimide (DCC), in this mixture is capable of activating random carboxyl groups in the target antibody and causing intramolecular or intermolecular cross-linking, which is known to lead to the formation of non-functional forms of conjugates and their aggregation as a result of cross-linking (https://www.sciencedirect.com/topics/medicine-and-dentistry/dicyclohexylcarbodiimide).

Disclosure of the Invention

где п - целое число, выбранное из диапазона 3-12,

где Ri представляет собой The technical objective of the invention is to develop a method for producing bioconjugate molecules capable of causing targeted degradation of proteins involved in pathological processes, ensuring a high yield of the resulting bioconjugates while maintaining the specificity of the antibody in the resulting bioconjugate. To this end, a method is proposed for producing antibody-ligand conjugates for liver-specific modulation of the proteome, in which the ligand is a molecule capable of binding to the ASGPR receptor, selected from the group consisting of

where n is an integer selected from the range 3-12,

where Ri represents

R2 and R3 are independently selected from C3-Ciu-alkyl or C3-Ciu-alkyloxide having from 1 to 5 oxygen atoms, m is an integer selected from the range of 1-3; including stages:

- activation of the ligand in the process of sequential interaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide in phosphate or phosphate-saline buffer at pH 5.0-7.0 with the formation of N-hydroxysulfosuccinimide ester of the ligand;

- bioconjugation, during which the N-hydroxysulfosuccinimide ester of the ligand interacts with the primary amino group of the antibody in a phosphate or phosphate-saline buffer at pH 7.2-7.4.

In some embodiments, the antibody is a monospecific or bispecific antibody or antibody fragment comprising at least one heavy chain and light chain variable region that binds to a target selected from the group consisting of EGFR, VEGF-A, VEGFR2, CTLA-4, PD-1, PD-L1, PDGFRa, HER2, LOXL2 and PCSK9. In some particular embodiments of the invention, the antibody is cetuximab.

In some embodiments, the ligand is a ligand of formula (I), wherein n is an integer selected from the range of 5-9. In some particular embodiments of the invention, the ligand is a ligand of formula (I), in which n=5 (36-[[2-(acetylamino)-2-deoxy-p-O-galactopyranosyl]oxy]-21,21-bis[[ 3-[[3-[[5-[[2-(acetylamino)-2-deoxy-p-E-galactopyranosyl]oxy]-1-ocopentyl]amino]propyl]amino]-3-oxopropoxy]methyl]-19 ,26,32-trioxo-4,7,10,13,16,23-hexaoxa-20,27,31 - triazahexatriacontanoic acid). This ligand variant is hereafter referred to as triGalNac.

In some embodiments, the molar ratio of antibody to ligand is from 1:7.5 to 1:45.

As a result of the invention, the following results are achieved:

- a new method has been developed for producing conjugate molecules capable of causing targeted degradation of proteins involved in pathological processes;

- the developed method eliminates the use of organic solvents, which significantly affects the chemical purity and quality of the resulting conjugates, since the use of organic solvents, on the one hand, can negatively affect the conformation and functionality of the target antibody during the conjugation process, and secondly, requires additional purification of the resulting conjugates , which, however, cannot completely exclude forms of antibodies and impurities damaged by organic solvents;

- the developed method involves the use of reagents (such as solvents; reagents used to activate the ligand and to stop synthesis, etc.) that minimally cause denaturation of protein molecules, which minimizes serious damage to the antibody sites included in the conjugates, responsible for the specific binding of target proteins, to the nonspecific binding of such damaged antibodies and, accordingly, to the associated side effects;

- the developed method provides a high yield of bioconjugate molecules while maintaining the specificity of the antibody in the resulting bioconjugate;

- the resulting bioconjugates have high solubility due to the structure of the linker (high content of hydrophilic groups) formed as a result of synthesis, connecting the antibody and ligand(s) included in the bioconjugate.

Definitions and terms

The following terms and definitions apply throughout this document unless otherwise expressly stated. References to techniques used in the description of this invention refer to well known techniques, including modifications of these techniques and replacement thereof with equivalent techniques known to those skilled in the art.

As used herein, the terms “includes” and “including” are interpreted to mean “including, but not limited to.” These terms are not intended to be construed as “consisting only of.”

The term “and/or” means one, more, or all of the above.

The term “treatment” means to cure, slow, stop, or reverse the progression of a disease or disorder. As used herein, “treating” also means alleviating symptoms associated with a disease or disorder.

As used herein, the term “therapeutically effective amount” refers to an amount of a medicinal (biologically active) substance that is non-toxic but sufficient to provide the desired therapeutic (prophylactic) effect. The amount that is “effective” may vary depending on the particular drug substance or combination of substances, disease, severity of the condition, and the like, as known to those skilled in the art. Such an effective amount may also vary from subject to subject depending on the age, body weight, general condition of the individual, etc. When carrying out treatment and/or prophylaxis, the administration can be carried out either once or several times a day, more often in the form of a course of administration for over a period of time sufficient to achieve a therapeutic effect (from several days to a week, several weeks and up to months), while courses of drug administration can be repeated. In particular, in moderate forms of the disease, the single dose, frequency and/or duration of administration of the conjugates according to the invention can be increased. Preferably, the conjugate of the invention is administered to a patient as part of a pharmaceutical composition comprising, in addition to the active ingredient, pharmaceutically acceptable carriers and/or excipients. The terms "subject" and "patient" include any mammalian species, and more preferably include humans.

The term "antibody", as used herein, refers to a monospecific or bispecific antibody or antibody fragment containing at least one heavy chain and light chain variable region. In specific embodiments, the term "antibody" refers to a monoclonal antibody (mAb) containing two heavy chains and two light chains, or an antigen binding fragment thereof, in which at least one interchain disulfide bond is present, for example, a Fab, Fab' or F fragment (ab')2 . The antibodies of the invention may be of any isotype, such as IgG, IgA or IgM antibodies. Preferably, the antibody is an IgG antibody, more preferably an IgG1 or IgG2 antibody. Antibodies can be chimeric, humanized or human. Preferably, the antibodies are humanized. Even more preferably, the antibody is a humanized or human IgG antibody, most preferably a humanized or human IgG1 mAb. The antibody may have K (kappa) or L (lambda) light chains, preferably K (kappa) light chains, i.e., a humanized or human IgG1-K antibody.

In humanized antibodies, the antigen-binding complementarity determining regions (CDRs) in the HC and LC variable regions are derived from antibodies from a non-human species, typically mouse, rat or rabbit. These non-human CDRs can be placed among the human framework residues (FR1, FR2, FR3 and FR4) of the HC and LC variable regions. Selected amino acids in human FRs can be replaced with corresponding parent amino acids from a non-human species to improve binding affinity while maintaining low immunogenicity. Alternatively, selected amino acids from the original non-human FRs are replaced with their corresponding human amino acids to reduce immunogenicity while maintaining antibody binding affinity. The variable regions thus humanized are combined with human constant regions. The terms “monoclonal antibody” and “mAb”, as used herein, refer to an antibody derived from a population of essentially homogeneous antibodies, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may present in small quantities. Antibodies can be produced by immunizing animals with a mixture of peptides representing the desired antigen. B lymphocytes are isolated and fused with myeloma cells, or individual B lymphocytes are cultured for several days in the presence of conditioned medium and feeder cells. Myeloma or B-lymphocyte supernatants containing The antibodies produced are tested to select suitable B lymphocytes or hybridomas. Monoclonal antibodies can be produced from suitable hybridomas by the hybridoma method first described in Kohler et al., Nature, 1975, 256, 495-497. Alternatively, RNA from suitable B cells or lymphoma can be lysed, the RNA can be isolated, reverse transcribed and sequenced. Antibodies can be produced by recombinant DNA in bacterial, eukaryotic animal or plant cells (see, for example, US Pat. No. 4816567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using methods described in the art, for example, in Clackson et al., Nature 199, 1352, 624-628 and Marks et al., J. Mol. Biol. 1991, 222, 581-597.

The term "ligand" as used herein is an asialoglycoprotein receptor (ASGPR)-specific binding molecule.

The term "antibody-ligand conjugate" ("conjugate", "bioconjugate"), as used herein, refers to an antibody, as defined herein above, to which one or more ligands are conjugated. The number of ligands conjugated to the antibody is generally expressed as the ratio ligand (drug) to antibody (DAR). By conjugation methods, as a rule, a heterogeneous mixture of various conjugate molecules is obtained, i.e. various variants of conjugates having different ligand to antibody ratios. Thus, the term “conjugate” also refers to such mixtures of conjugate variants. The term "average DAR" refers to the average DAR of a population of such DAR variants. As is well known in the art, the distribution of DAR and ligand loading can be determined, for example, using hydrophobic interaction chromatography (HIC) or reverse phase high performance liquid chromatography (RPHPLC). HIC is particularly useful for determining average DAR. In some embodiments, the antibody-ligand conjugate has an average ligand to antibody ratio (DAR) of at least 1.0. In some other embodiments, the antibody-ligand conjugate has an average ligand to antibody ratio (DAR) of at least 25.0.

Functional parts of polyfunctional molecules (fragments) can also be used as parts of mono- and bifunctional molecules, as well as their isoforms and chemically modified variants.

The terminology is used only to describe the functional parts of the molecule, to understand the mechanism of targeting target proteins and is not limiting; other names for the molecules described in this patent are possible.

Unless otherwise defined, technical and scientific terms in this application have their standard meanings commonly accepted in the scientific and technical literature. Brief description of the drawings

Figure 1 is a simplified diagram of the stages of synthesis of the active form of the ligand (1) and conjugation of the ligand with the target antibody (2).

Figure 2 is a detailed diagram of the activation of the triGalNAC ligand.

Fig.3 - polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS PAGE) of conjugates obtained as a result of bioconjugation of an antibody (cetuximab) and a ligand (triGalNac) in a reaction with various molar ratios and buffer systems: A - antibody:ligand ratio = 1: 45, B - antibody:ligand ratio = 1:15, C - antibody:ligand ratio = 1:7.5. Samples in lanes: 1) Original Erbitux (cetuximab antibody), 2) Ultrafiltration/Diafiltration (UF/DF) in PBS, 3) Bioconjugation in PBS at pH 7.4 for 1 hour, 4) Bioconjugation in PBS at pH 7.4 for 3 hours, 5) Bioconjugation in PBS at pH 7.4 overnight, M) PageRullerPlus molecular weight marker, 6) Preparation after LIF/DF in PBS (repeat sample 2), 7) Bioconjugation in PBS at pH 8.1 for 1 hours, 8) Bioconjugation in PBS at pH 8.1 for 3 hours, 9) Bioconjugation in PBS at pH 8.1 overnight

Figure 4 - Western blot: degradation of EGFR after treatment of HepG2 cells with the resulting bifunctional molecule containing cetuximab and triGalNac in the structure for 48 hours. On the left is a lysate of control cells, on the right is cells after treatment with a bioconjugate.

Figure 5 - proliferative test (MTT test): reduction in proliferation of HepG2 hepatocellular carcinoma cells after treatment with cetuximab-triGalNac bioconjugate.

Figure 6 - proliferative test (assessment of cell density): reduction in proliferation of hepatocellular carcinoma cells HepG2 after treatment with cetuximab-triGalNac bioconjugate.

Figure 7 - HPLC results: 1 (red) - HPLC profile of the antibody before modification; 2-4 HPLC profiles of antibodies after modification: 2 (black) - AT ratio: tri-GalNAc-COOH 1:3; 3 (blue) - ratio AT: tri-GalNAc-COOH 1:12; 4 (blue) - ratio AT: tri-GalNAc-COOH 1:25.

Detailed Description of the Invention

The present invention relates to the production of bioconjugates for liver-specific targeted modulation of the proteome, which can be used for the treatment of a wide range of diseases based on an imbalance in the activity or concentration of proteins. To this end, a method has been proposed for producing conjugates comprising an antibody against a target of interest conjugated to ASGPR receptor ligands.

On the surface of hepatocytes there are about 0.5-1 million asialoglycoprotein receptors (ASGPR). ASGPR ability to transport glycosylated proteins across the membrane, high expression level, rapid cycle regeneration and preferential location on the surface of hepatocytes and hepatocellular carcinoma cells make it a promising target for targeted delivery of biologically active substances in the treatment of diseases. ASGPR recognizes glycoproteins bearing D-galactose (Gal) and M-acetyl-O-galactosamine (GalNAc) ligands and internalizes them through clathrin-mediated endocytosis. Following internalization and endosomal acidification, ASGPR releases GalNAc (or Gal) and is recycled back to the plasma membrane, while glycoproteins are directed to lysosomes for further degradation under acidic conditions. This mechanism makes it possible to use ASGPR as a target for the transport of target compounds into hepatocytes and their further degradation in lysosomes, including the degradation of extracellular proteins, including membrane proteins.

Thus, the present invention relates to a method for specific proteolysis of a target protein using bifunctional bioconjugates that have a specific binding site for the target protein and binding sites for receptors and/or coreceptors that mediate the involvement of the target protein in the process of proteolysis within the intracellular organelle.

The present invention can be used to obtain bioconjugates for the purpose of their use for the correction of various pathological processes associated with qualitative and quantitative protein imbalances, since the proteolytic process can be directed to target proteins associated with the cell membrane, intracellular proteins, as well as extracellular proteins . The patent also describes the structures and types of bifunctional molecules, possible target proteins for therapy and nosological forms for which targeting and proteolysis are relevant, while it is possible to use molecules and target proteins similar in structure (isoforms and chemically modified molecules).

More specifically, the present invention is directed to the production of bispecific conjugates containing an antibody against a target of interest conjugated to ASGPR receptor ligands. The result of the binding of such a conjugate to the target of interest and its interaction with the ASGPR receptor is the targeted degradation of the specified target; in this case, the target is selected depending on the purpose of therapy.

In particular, the diseases listed in Table 1 are offered for treatment, because It is known that in these cases, cells of the organ region associated with the development of pathology, or its microenvironment, carry on the surface ASGPR, which can be used to direct the target protein involved in the pathogenesis of the disease into lysosomes.

Particularly recommended for treatment using synthesized molecules are those diseases in which ASGPR is carried on the surface directly by the cells of that part of the organ that is associated with the development of pathology. Such diseases include for example, hepatocellular carcinoma, because In this type of cancer, malignant transformation is directly experienced by hepatocytes, and, as is known, they are characterized by a high level of ASGPR expression.

In the hepatocellular carcinoma embodiment, the antibody included in the conjugate of the invention is directed against a target protein responsible for the growth, invasion and migration of hepatocellular carcinoma cells. Examples of such target proteins include the epidermal growth factor receptor EGFR; G protein-coupled receptors (GPCRs), including prostaglandin E2 receptors (EP1, EP2, EP3, and EP4); C-C chemokine receptors (CCR1, CCR2, CCR5, CCR6, CCR7, CCR9); CC chemokines (eg CCL2, CCL21); nucleolin; C-X-C chemokine receptor (CXCR2, CXCR4, CXCR6, CXCR7, XCR1); chemokine receptor ligands (CXCL5, CXCL9, CXCL12, CXCL16, CX3CL1/CX3CR1); metalloproteases (MMP2; MMP9; MMP10); transforming growth factor beta (TGF-P), but not limited to. In the case of other diseases, the antibody included in the conjugate according to the invention is directed against a target protein responsible for the development of such a disease.

Table 1 provides examples of known antibodies that are approved for the treatment of these diseases, are in late-stage clinical trials, or have promising studies showing their effectiveness in the case of these pathologies. Accordingly, it is expected that conjugates of these antibodies (or biosimilars of these antibodies, or antibodies matching the antigen specificity with antibodies according to Table 1) with ASGPR receptor ligands (ASGPR1) may have therapeutic efficacy significantly superior to that of antibodies not conjugated to these ligands .

It is obvious that in the future more effective therapeutic antibodies against the same antigens can be developed and tested, which can also be converted into the conjugates proposed according to the invention according to the proposed synthesis scheme.

Table 1. Diseases and molecular targets for promising use of the proposed conjugates

According to the invention, a method is proposed for producing bifunctional bioconjugates that have a specific binding site for the target protein and binding sites for receptors and/or coreceptors that mediate the involvement of the target protein in the process of proteolysis inside the intracellular organelle.

In more specific embodiments, the present invention relates to a method for producing an antibody-ligand conjugate, comprising the steps (see Figure 1):

1) Stage No. 1 - activation of the ligand.

At this stage, the carboxyl group of ligand (1) is converted to the sulfo-NHS-activated form.

To do this, the ligand first reacts with the carbodiimide agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form the ligand ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The resulting ester then reacts with the sulfo form of the N-hydroxysuccinimide agent (N-hydroxysulfosuccinimide, sulfo-NHS) to form an ester of the ligand and N-hydroxysulfosuccinimide, which is the activated ligand. The activation reaction with EDC and sulfo-NHS is most effective at pH 4.5-7.2 and room temperature (about 15-25°C). It is preferable to carry out the reaction in phosphate or phosphate-buffered saline with a concentration of 10 to 100 mM and a pH of 5.0 to 7.0, because this ensures compatibility with the subsequent bioconjugation reaction of the sulfo-NHS-activated ligand to the target protein. In particular embodiments, the activation reaction occurs in a phosphate buffer at pH 6.0

Stage No. 2 - bioconjugation.

At the second stage, the sulfo-INZ-activated form of the ligand (1) is attached to the target antibody. The binding of the activated ligand occurs at primary amine residues in the protein sequence of the antibody, predominantly N-terminal amines and lysine amines. In preferred embodiments of the invention, the reaction takes place in phosphate or phosphate-buffered saline at pH 7.2-7.4 and room temperature (about 15-25°C). To carry out the bioconjugation reaction, the target activated ligand ester and the target antibody must first be dissolved or transferred to the reaction buffer, as described above. The solution of the target ligand and the solution of the target antibody are mixed based on different molar ratios of antibody: ligand. The optimal range of molar ratios is from 1:7.5 to 1:45, and with increasing proportion of the ligand in the reaction mixture the number of ligands introduced into the target antibody molecule increases. The chemistry of the bioconjugation reaction is depicted in Figure 1 .

As a buffer system for carrying out the bioconjugation reaction, buffer solutions that do not contain components with carboxyl and amino groups can be used to avoid inactivation of the reaction components. Preferred buffer systems for carrying out the bioconjugation reaction are phosphate or phosphate-saline buffer in the concentration range from 10 mM to 100 mM and in the pH range from 7.0 to 7.5, or carbonate buffer in the concentration range from 10 mM to 100 mM and in the pH range from 7.5 up to 8.0.

Thus, in the proposed method, the activation of the ligand and the bioconjugation reaction are carried out in aqueous solutions, which allows minimizing the contact of the target antibody with organic solvents that can affect its functionality. Often, ligand preparation or activation is carried out in organic solvents such as dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), which can affect protein conformation (On the Structural Stability and Solvent Denaturation of Proteins III. DENATURATION BY THE AMIDES, https://www .jbc.org/article/S0021 -9258(18)62563-3/pdf). To reduce this effect, the bioconjugation process was carried out in aqueous solutions in the buffer systems described above.

The proposed optimized process for the synthesis of bioconjugates allows one to stop one of the stages of synthesis using the proteinogenic amino acid glycine, which is normally present in tissues and cells, is not toxic and does not damage proteins.

Таким образом, предлагаемые условия синтеза биоконъюгатов решают задачу сохранения специфичности антитела в составе получаемого биоконъюгата. Thus, the proposed method for the synthesis of bioconjugates is carried out in only two stages, which leads to the least loss of antibody molecules at the conjugation stage. In this case, the proposed synthesis method uses reagents (solvents; reagents used to activate the ligand and to stop the synthesis, etc.) that minimally cause denaturation of protein molecules (antibodies), which avoids serious damage to their areas responsible for specific binding target proteins, as well as to avoid nonspecific binding of such damaged antibodies and, accordingly, possible side effects (which are difficult to assess in advance, at the development stage; and which can vary greatly from batch to batch of the produced bioconjugate, which is incompatible with modern requirements for medicinal products molecules) [On the Structural Stability and Solvent Denaturation of Proteins III. DENATURATION BY THE AMIDES https://www.ibc.org/article/S0021 -9258 62563-3/pdf.

Thus, the proposed conditions for the synthesis of bioconjugates solve the problem of preserving the specificity of the antibody in the resulting bioconjugate.

An additional advantage of the proposed synthesis method and the resulting conjugates is that no DBCO- remains in the linker after bioconjugation. grouping. Instead, the linker is represented by a small fragment of polyethylene glycol, bounded on both sides by amide bonds, similar in nature and in expected stability to peptide bonds already present in living systems, including in the molecule of the conjugated antibody. It is known that polyethylene glycol is well compatible with living systems, has exceptional hydrophilicity and is therefore widely used to solve the problem of drug bioavailability - see, for example,

[https://pubs.rsc.org/en/content/articlelanding/2021/nr/d1 nr02065j].

The possibility of objective manifestation of the technical result in the implementation of the invention is confirmed by reliable data given below in the examples containing experimental information obtained in the process of conducting research using methods accepted in this field.

It should be understood that the examples given in the application materials are not limiting and are provided only to illustrate the present invention.

Example 1. Preparation of cetuximab-triGalNac bioconjugate by the method according to the invention

The antibody cetuximab was isolated from the finished drug product Erbitux. For this purpose, the drug was purified from excipients that could interfere with the bioconjugation reaction. The procedure for transferring the target antibody from the storage buffer system to the reaction (suitable for conjugation) buffer system was carried out using ultrafiltration. For this purpose, centrifugal concentrators UFC905024 Amicon Ultra-15 Centrifugal Filters Ultracel - 50K (MerkMillipore) were used. The buffer was replaced with phosphate buffered saline, prepared from P4417-100TAB Phosphate buffered saline tablets (Sigma) and deionized water at the rate of 1 tablet per 200 ml of water.

2 ml of the Erbitux preparation were distributed into 1 ml of 2 Amicon 50K tubes, the ultrafiltration/diafiltration process was carried out as follows: 9 ml of buffer was added to 1 ml of sample, centrifugation was carried out on a Tegto Scientific SL16R centrifuge, in a swing-bucket rotor with inserts for 50 ml tubes at 4C and 4000g (RCF) for 10 min, repeating the concentration-dilution procedure three times. Thus, when the content of the initial buffer is reduced by at least 1000 times, the content of interfering substances becomes negligible. The resulting concentrate in phosphate-buffered saline was combined in 1 test tube and adjusted with the same buffer to a volume of 1.9-2 ml. Next, spectrophotometric concentration measurements were carried out using a microvolume cuvette pCuvette G1.0 with an optical path length of 1 mm for absorption at a wavelength of 280 nm on an Eppendorf Biospectrometer Kinetic spectrophotometer. Loss of target protein (cetuximab) due to aggregation and no precipitation was observed during ultrafiltration into the reaction buffer system. The retentate remains transparent.

To obtain an active ester of a ligand capable of forming amide bonds with the primary amines of the target protein (cetuximab antibody), an activation reaction of the carboxyl group was carried out using the carbodiimide agent EDC (, Thermo Scientific, Product No. 22980) and the sulfo form of the N-hydroxysuccinimide agent (Thermo Scientific , Product No. 24510). The reaction scheme is shown in Figure 2. The conjugation reaction of cetuximab and the sulfo-NH3-ester of the triGalNac ligand was carried out at different pH values, molar ratios of antibody:ligand, and incubation durations. The options are below:

Table 2. Options for applied conjugation parameters

ON - overnight, incubation overnight (~17 hours)

The change in the molecular weight of the antibody chains was assessed using reducing denaturing SDS-PAGE (gradient 4-15%) electrophoresis with gel staining using a reagent based on Coomassie blue dye. Gel documentation was carried out on a ThermoFisher Invitrogen iBright 1500 device in the Coomassie blue mode. The measurement results are shown in Fig.3.

Confirmation of the formation of a covalent bond between the protein and the ligand is the presence of a shift to a region of higher molecular weight of the heavy and light chains of the bioconjugate relative to the unmodified form of the antibody. In lanes 3-5, where cetuximab-triGalNac bioconjugates were applied, a band shift upward relative to control lanes 1-2, where the original drug cetuximab was applied, is visible, which confirms the success of the conjugation (increase in molecular weight due to conjugation of the antibody with triGalNac). Similar results were obtained for drugs conjugated in a buffer solution with pH 8.1.

Example 2. Evaluation of the functional activity of the resulting cetuximab-triGalNac bioconjugate

The EGFR protein is expressed in 60% of cases on the surface of hepatocellular carcinoma cells and causes their proliferation, and the membrane was incubated with Pierce™ Clear Milk buffer (10X) with primary antibodies against EGFR, clone C4. The Western blot shown in Figure 4 has two lanes, the first is treatment control (DMSO), the second is treatment of cells with a bifunctional molecule, 30 nM. Actin was used as a control for housekeeping protein. As follows from the presented data, the cetuximab-triGalNac conjugate causes effective targeted degradation of EGFR.

2. Assessment of the functional activity of the bioconjugate using the proliferative test (MTT test and cell density test).

Assessment of proliferative activity 96 hours after treatment with cetuximab and triGalNac bioconjugate molecules (molarity ratio 1:15) was carried out using a proliferative MTT test and a cell density test.

Hepatocellular carcinoma cells (HepG2) were divided into several groups: 1. control group (cells treated with DMSO); 2. cells treated with Cetuximab (Ctx) at concentrations of 15 nM; 3. cells treated with cetuximab-triGalNac bioconjugate at a concentration of 5 nM; 4. cells treated with cetuximab-triGalNac bioconjugate at a concentration of 12.5 nM.

Performing the MTT test: Cells were seeded in a 96-well plate at a density of 175,000 cells/well and incubated in DMEM for 24 hours. Then the medium was changed to OptiMeM and incubated for 24 hours. Next, DMSO and cetuximab-triGalNac bioconjugate molecules of various concentrations (5 nM and 12.5 nM) were added to the corresponding wells and incubated for another 24 hours. After this, 100 μl of medium was taken from each well and 10 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and incubated overnight. In the morning, SDS was added, pre-dissolved in 1 ml of HCI (0.01 M), at the rate of 100 μl per well and incubated for 4 hours at 37°C. Absorbance measurement (A) was read at 570 nm using a Spark YuM tablet (spectro)photometer. In this method, it was assumed that the absorbance at 570 nm at the end of the test allows us to talk about the number of living cells in the well of the plate.

The MTT test showed a decrease in the proliferation of HepG2 hepatocellular carcinoma cells after treatment with the cetuximab-triGalNac bioconjugate at a concentration of 12.5 nM (group 4) compared to the control. Treatment of cells with cetuximab (2) and treatment of cells with cetuximab-triGalNac at a concentration of 5 nM (group 3) did not reduce cell proliferation.

Cell Density Test: Cells were seeded in a 96-well plate at a density of 175,000 cells/well and incubated in DMEM for 24 hours. Then we changed the environment to OptiMeM. Next, DMSO and cetuximab-triGalNac bioconjugate molecules of varying concentrations (5 nM and 12.5 nM) were added to the corresponding wells and incubated for 48 hours. Automatic assessment of confluency (cell density) was carried out on a Spark YuM tablet spectrophotometer. The result showed a decrease in the proliferation of HepG2 hepatocellular carcinoma cells after treatment cetuximab-triGalNac bioconjugate at a concentration of 12.5 nM (group 4) compared to the control (group 1). Treatment of cells with cetuximab (group 2) and treatment of cells with cetuximab-triGalNac at a concentration of 5 nM (group 3) did not affect cell proliferation.

In both proliferation tests, the resulting bioconjugate showed a significant advantage over cetuximab without a conjugated ligand, which indicates effective specific binding and functional activity of the conjugates obtained by the proposed method.

Example 3. Preparation of cetuximab-triGalNac bioconjugate by the method described in Zhou Y. et al. Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins // ACS Cent. Sci. 2021, 7, 3, 499-506

The experimental antibody (cetuximab) was dissolved in 1 ml sodium phosphate buffer (PBS), and 200 μl was collected. The concentration was checked by absorbance on a Nabi spectrophotometer in a 5 μl drop. Absorption 0.152 O.U. at 280 nm was taken to correspond to a concentration of 1 mg/ml. 4 ml PBS was added and desalted on a 10 kDa Amicon centrifuge filter, concentrating to a volume of 200 µl. The concentration according to the spectrophotometer did not change. From 200 μl of the experimental antibody solution, after washing, 60 μl were taken in three reactions (0.35 nmol each). The remaining 20 μl was used for analysis (SEC-HPLC).

Ether synthesis:

1 mg tr-GalNAc was dissolved in 40 μl of M,M-Dimethylformamide (DMF), and 4 μl (58 nmol) was taken.

1,3-Dicyclohexylcarbodiimide (DCC) (0.88 mg) was dissolved in 100 μl of DMF, 2 μl (0.018 mg, 87 nmol) was taken.

N-hydroxysuccinimide (NHS) (0.67 mg) was dissolved in 100 μl of DMF, 3 μl (0.020 mg, 174 nmol) was taken.

The DCC solution was added to the triGalNAc solution, followed by the NHS solution. The total reaction volume was 9 μl. The reaction was kept for 1 hour at room temperature.

Antibody conjugation:

The reaction mixture was diluted 10 times by adding 81 μl of DMF and added to Eppendorfs with antibody: 2 μl (1:3, 0.35 nmol: 1.29 nmol), 7 μl (1:12 0.35 nmol: 4.51 nmol), 14 μl (1: 250.35 nmol:9.02 nmol). Incubated overnight on a rotator at a maintained temperature of 20°C.

The reaction mixtures were washed and concentrated to 60 µl on centrifuge filters 10 kDa Amicon Ultra Ultracel-10 regenerated cellulose membrane, 0.5 ml (5 times 400 µl PBS). Absorbance on a Nabi spectrophotometer showed that the latter reaction contained little substance (see Table 3). Table 3. Absorption measurement results

мг/мл Concentration,

mg/ml

There was no substance in the wash. It is possible that the modified experimental antibody remains on the membrane due to its structure; or too much DMF in the reaction negatively affects the membrane.

For additional analysis of the presence of the conjugate in solutions, HPLC was performed.

HPLC conditions:

Column BioSep™ 5 µm SEC-S2000 145 A, LC Column 300 x 4.6 mm.

Speed 0.5 ml/min.

Peaks with Rt 6.8 and Rt 8.2 - PBS buffer.

The HPLC results are shown in Figure 7 (for clarity, all 4 obtained chromatograms are shown in one figure). As can be seen from the figure, the HPLC data correlate with the data obtained on the spectrophotometer.

Thus, as the experimental results showed, the yield of the conjugate as a result of the reaction was extremely low. Most likely, this is due to the fact that antibodies aggregate during conjugation and are retained on the filter membrane. In this regard, the known method for producing conjugates of antibodies and ASGPR receptor ligands has significant limitations for practical use.

Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments detailed are provided for purposes of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the spirit of the present invention.

Claims

Claim 1 . A method for producing an antibody-ligand conjugate, wherein the ligand is a molecule capable of binding to an ASGPR receptor selected from the group consisting of

where n is an integer selected from the range 3-12,

where Ri represents

R2 and R3 are independently selected from C3-Ciu-alkyl or C3-Ciu-alkyloxide having from 1 to 5 oxygen atoms, m is an integer selected from the range of 1-3; including stages: - activation of the ligand in the process of sequential interaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide in phosphate or phosphate-saline buffer at pH 5.0-7.0 with the formation of N-hydroxysulfosuccinimide ester of the ligand; - bioconjugation, during which the N-hydroxysulfosuccinimide ester of the ligand interacts with the primary amino group of the antibody in a phosphate or phosphate-saline buffer at pH 7.2-7.4. 2. The method of claim 1, wherein the antibody is a monospecific or bispecific antibody or antibody fragment containing at least one heavy chain and light chain variable region that binds to a target selected from the group consisting of EGFR, VEGF-A, VEGFR2, CTLA-4, PD-1, PD-L1, PDGFRa, HER2, LOXL2 and PCSK9. 3. The method according to claim 1, in which the ligand is 36-[[2-(acetylamino)-2-deoxy-p-O-galactopyranosyl]oxy]-21,21-bis[[3-[[3-[ [5-[[2-(acetylamino)-2-deoxy-p-O-galactopyranosyl]oxy]-1-ocopentyl]amino]propyl]amino]-3-oxopropoxy]methyl]- 19,26,32-trioxo-4,7, 10,13,16,23-hexaoxa-20,27,31-triazahexatriacontanoic acid. 4. The method according to claim 3, in which the antibody is cetuximab. 5. The method according to claim 1, in which the molar ratio of antibody and ligand is from 1:7.5 to 1:45.