Anti-transferrin receptor antibodies and use thereof

Abstract

The invention relates to the production of novel low-affinity antibodies to the transferrin receptor that are capable of transporting therapeutic agents across the blood-brain barrier, and to therapeutic agent conjugates based thereon. The antibodies produced exhibit optimal affinity to the receptor and high stability.

Description

[0001] ANTIBODIES AGAINST TRANSFERRIN RECEPTOR AND THEIR APPLICATION

[0002] Field of technology to which the invention relates

[0003] The invention relates to biotechnology, immunology, medicine and concerns the design of antibodies that penetrate the blood-brain barrier (BBB) ​​and contain a domain with affinity to the transferrin receptor, which are intended for conjugation with a therapeutic agent, methods for their production and use.

[0004] State of the art

[0005] Neurological disorders are a major cause of death and disability worldwide. Despite significant progress, current treatment options have a number of limitations. One of the main reasons for this limitation is the blood-brain barrier (BBB). The BBB is an anatomical and molecular barrier that separates the brain from the blood. This barrier has evolved to prevent the transport of peripheral neurotransmitters, cytokines, and microorganisms into the brain that may have a negative impact on the central nervous system (CNS).

[0006] Unlike capillaries in other tissues such as muscles, capillaries that supply blood to most brain tissues except for a few areas (pineal gland, pituitary gland, etc.) are distinguished by the fact that the endothelial cells that form their endothelium are interconnected by intercellular junctions. This limits the passive transfer of substances from the capillaries to the brain. This system, which limits the exchange of substances between the blood and the tissue fluid of the brain through the endothelium of the capillaries of the brain, is called the blood-brain barrier (BBB). Thus, the BBB limits the exchange of substances not only between the blood and the brain, but also between the blood and the tissue fluid of the central nervous system, including the brain and spinal cord. Thanks to the BBB, most cells of the central nervous system avoid the effects of fluctuating concentrations of substances such as hormones and cytokines in the blood, and thus their biochemical homeostasis is maintained.Although this is a useful protective mechanism, its presence results in many potentially useful molecules not having access to the CNS and thus not being able to exert therapeutic effects in neurological diseases or other CNS disorders that require delivery of the active component across the BBB.

[0007] Medicines based on recombinant therapeutic proteins are increasingly used in the treatment of autoimmune, oncological, cardiovascular diseases, in hormone replacement therapy, etc. High affinity to the target with a minimum of side effects and, in some cases, duration of action determine the ever-increasing popularity of this type of drugs. The use of therapeutic proteins, such as monoclonal antibodies, enzymes, however, has one important limitation - the inability to penetrate the BBB, which significantly limits their use.

[0008] Currently, one of the approaches to solving this problem is the construction of recombinant proteins containing transport and therapeutic domains, respectively. The transport domain is a monoclonal antibody or its fragment, targeted at the BBB-specific receptor, which induces receptor-mediated transcytosis in the brain. The therapeutic domain of the protein fused with IgG provides the pharmacological effect. Due to active transport through the BBB with the participation of receptors, large molecules of proteins and polypeptides can be transferred, such as transferrin, insulin, etc. The use of antibodies that bind to the specified receptors-carriers allows for the transport of the protein through the BBB and ensures the biological activity of the conjugated protein in the central nervous system (CNS).

[0009] The transferrin receptor (TfR) currently appears to be the most promising. TfR is involved in intracellular iron delivery by internalizing the Fe3+-transferrin complex. It is characterized by broad tissue expression, including the endothelium of brain capillaries (BBB), cardiomyocytes, and skeletal muscle cells, while rapid internalization followed by recycling make TfR a good target for the development of intracellular delivery systems. TfR is characterized by the highest expression level in the epithelium of brain capillaries and the choroid plexus (blood / CSN barrier) compared to other receptors studied for delivery beyond the BBB (insulin receptor InsR, LRP1). However, it has recently become clear that obtaining an antibody to TfR is not enough to ensure an effective therapeutic effect of the protein fused with it.It was found that high-affinity TfR antibodies exhibit reduced persistent effects in both the brain and CNS periphery due to rapid clearance and degradation of the TfR receptor. Low-affinity TfR antibodies are characterized by reduced BBB passage; however, TfR receptor expression levels do not change, and antibody concentrations in both plasma and intra-CNS are maintained due to slow clearance (Atwale et al., Sci. Transl. Med. 3 (2011) 84ra43). Antibodies with lower affinity for the receptor are not cleared from the system as efficiently as antibodies with higher affinity, and thus may provide higher circulating concentrations (Yu YJ, Zhang Y, Kenrick M, et al., SciTranslMed. 2011;3(84):84ra44, doi: 10.1126 / scitranslmed.3002230).This may reduce the frequency of drug administration, which will have advantages both in terms of patient quality of life and in terms of reducing potential side effects of the drug. Also importantly, the lower affinity reduces the risk of TfR receptor degradation in patients during treatment with the drug.

[0010] On the other hand, it has been shown that the primary response to the administration of anti-TfR antibodies leads to reticulocyte depletion and the occurrence of acute clinical side effects (Sci. Transl. Med. 2013, 5, 183ga57), which is associated with antibody-dependent cell-mediated cytotoxicity (ADCC) of the antibody, while the residual effect of reticulocyte depletion is mediated by the complement pathway. This problem can be solved by disabling or reducing the effector function (WO2014189973). At the same time, in some cases, disabling the effector function led to an increase in antibody clearance (Mol. Pharm. 2019, 16, 3534-3543), therefore, the effect of eliminating the effector function on clearance is not obvious and should be studied for each antibody separately.

[0011] Antibodies to TfR and conjugates with therapeutic proteins based on them, for which successful penetration through the BBB has been demonstrated, are disclosed, for example, in publications WO2021092482, EA38367, EA39366, US10722576, etc., however, at present, the only drug that has shown clinical efficacy is PabinafuspAlfa (JR-141), developed by JCR Pharmaceuticals, which consists of human iduronate-2-sulfatase (IDS), an enzyme deficient in patients with mucopolysaccharidosis type II (MPS-II), fused to the C-terminus of the heavy chain of an antibody to the human transferrin receptor (TfR). Its successful delivery across the BBB into the CNS via TfR-mediated transcytosis has been demonstrated in animal models, along with the resulting effect of reducing heparan sulfate (HS) accumulation in the brain. A first-in-human phase I / II study in Japan involving MPS-II patients also yielded encouraging results (Molecular Therapy Vol. 27 No. 2 February 2019).A subsequent phase II study in Brazil and a phase II trial in Japan (Giuglianietal. Iduronate-2-sulfatase fused with anti-hTfR antibody, pabinafusp alfa, for MPS-II: A phase 2 trial in Brazil. MolTher. 2021;29(7):2378–2386.) further confirmed the somatic / peripheral and central efficacy of PabinafuspAlfa, leading to its approval by Japanese regulatory authorities in March 2002–211 as the first BBB-crossing enzyme replacement therapy. Thus, PabinafuspAlfa confirmed the real possibility of obtaining drugs based on anti-TfR antibodies.

[0012] However, the task of obtaining new anti-TfR antibodies that combine optimal affinity, safety and stability for the creation of drugs on their basis that can penetrate the BBB, including for enzyme replacement therapy, remains an urgent problem, especially for Russia in the context of import substitution of essential drugs. Such antibodies will expand the arsenal of means for treating diseases that require the introduction of drugs that can penetrate the BBB.

[0013] The closest technical solution to the claimed invention is the antibody H6L7, disclosed in the publication WO2021092482. This antibody has good thermal stability, but still has insufficiently low affinity.

[0014] Thus, taking into account the above, the main task of the present invention is to obtain an antibody having such an affinity for the receptor that, on the one hand, would ensure binding to the receptor, and on the other hand, would be low enough that a relatively slow clearance would allow achieving stable therapeutic values ​​of concentrations of the corresponding conjugates in the brain. That is, the ultimate result of the present invention is the achievement of a relatively low affinity of the antibody with respect to the TfR receptor, which ensures the advantages of the present invention. With such optimal affinity of TfR, a moderate clearance of antibodies to TfR is observed, as was shown in the given examples of the invention. Another technical result is the expansion of the arsenal of means for treating diseases requiring the administration of drugs capable of penetrating the BBB.

[0015] The specified technical results are achieved by introducing mutations into the amino acid sequences of the hypervariable regions of CDRf-3 of the light and heavy chains, which made it possible to further select an antibody with the required affinity for the receptor; as well as additional structural modifications of the CHf and Fc regions, ensuring the shutdown or significant weakening of effector functions and reducing clearance, which has an advantage in therapeutic use.

[0016] Thus, the task was solved by creating a line of antibodies with the desired characteristics, i.e., relatively low affinity for TfR and high stability, as well as conjugates based on them, which demonstrate slow clearance.

[0017] Evaluation of the obtained antibodies also showed that they are characterized by the absence of high molecular weight aggregates, high solubility and improved storage stability compared to the prototype. It is known from the prior art (Gilliland et al., Methods Mol Biol, 841: 321-349 (2012)) that high thermal stability leads to better expression, less aggregation and better long-term stability. Therefore, it is expected that the improved properties of the claimed antibodies will lead to the production of conjugates with therapeutic molecules based on them, more suitable for the development of drugs than existing antibodies to TfR.

[0018] The essence of the invention

[0019] The present invention relates to new anti-TfR antibodies that have low affinity for the receptor, in particular, lower affinity for TfR compared to PabinafuspAlfa and the parent antibody H6L7. Additional mutations provide for the switching off or significant reduction of the effector function. It was also unexpectedly found that the obtained antibody has increased stability compared to the parent antibody. The proposed antibodies can be used for conjugation with various therapeutic agents, i.e. used as "shuttles" for the transfer of active drugs across the BBB.

[0020] In particular, the invention relates to a humanized monoclonal antibody to transferrin receptor 1 (TfR1) having the ability to penetrate the BBB and having an affinity for TfR1 in the Kd range from 10 to 500 nM, comprising sets of sequences of the CDR1-3 regions of the heavy chain and CDR1-3 of the light chain selected from a) SEQIDNO: 19, 29, 36, 45, 57, 70; b) SEQIDNO: 19, 27, 34, 43, 59, 106; c) SEQIDNO: 21, 31, 35, 54, 105, 73; d) SEQIDNO: 19, 102, 39, 45, 57, 106; d) SEQIDNO: 22, 28, 36, 46, 57, 73; in this case, the effector functions of the antibody are absent or significantly reduced by introducing amino acid mutations in the Ec domain.

[0021] In this case, mutations in the Fc domain can be selected from L234A and / or L235A and / or G236A and / or P237S and / or H268A and / or N297A and / or K322A and / or P329S, and also additionally from: M252Y and / or T256D and / or T307Q and / or T307W and / or S254T and / or E380A.

[0022] In a specific embodiment, the antibody comprises: a) a heavy chain of SEQIDNO: 79 and a light chain of SEQIDNO: 89; b) a heavy chain of SEQIDNO: 80 and a light chain of SEQIDNO: 90; c) a heavy chain of SEQIDNO: 81 and a light chain of SEQIDNO: 91; d) a heavy chain of SEQIDNO: 86 and a light chain of SEQIDNO: 96; e) a heavy chain of SEQIDNO: 88 and a light chain of SEQIDNO: 98.

[0023] In another aspect, the invention relates to the use of the obtained antibody for the production of conjugates with therapeutic proteins intended for the treatment of diseases requiring the delivery of a drug across the BBB, in particular diseases associated with the CNS. Such diseases include, but are not limited to, Gaucher disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type VII, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, galactosylceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, alpha-mannosidosis, beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, mucoollippidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

[0024] The invention also relates to a nucleic acid encoding an antibody and a nucleic acid encoding a conjugate with a therapeutic protein based thereon.

[0025] In a further aspect, the invention relates to an expression vector comprising a nucleic acid encoding an antibody or a nucleic acid encoding a conjugate with a therapeutic protein based thereon.

[0026] In a further aspect, the invention relates to a host cell containing an expression vector encoding an antibody or antibody conjugate, which is intended for the production of an antibody or conjugate based on it.

[0027] In a further aspect, the invention relates to a method for producing an antibody or a conjugate based thereon, comprising culturing host cells, isolating and purifying the target protein.

[0028] In a further aspect, the invention relates to a pharmaceutical composition comprising a conjugate of an antibody with a therapeutic protein in an effective amount and pharmaceutically acceptable excipients.

[0029] The said composition can be used to treat CNS diseases, in particular, such as Gaucher disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type VII, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, galactose-ceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, Alpha-mannosidosis, Beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, mucolipidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

[0030] The invention also relates to a method for treating CNS diseases, comprising administering to a patient a conjugate of an antibody with a therapeutic protein in a therapeutically effective amount or a pharmaceutical composition containing it. The said method can be used, in particular, for the treatment of diseases such as Gaucher disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type VII, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, galactosylceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, Alpha-mannosidosis, Beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, mucolipidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

[0031] Brief description of the figures

[0032] Fig. 1a and 1b show schematic images of the antibody of the invention and its conjugate with a protein.

[0033] Fig. 2 shows a scheme for introducing mutations in the CDR1-3 region of an antibody.

[0034] Fig. 3 shows a scheme for introducing mutations into the constant regions of the light and heavy chains.

[0035] Fig. 4a and 4b show maps of the obtained expression vectors. 4a is a plasmid encoding the heavy chain of the antibody to the transferrin receptor, 4b is a plasmid encoding the light chain of the antibody to the transferrin receptor.

[0036] Fig. 5 shows the amplification scheme for conjugating the heavy chain gene to a therapeutic protein.

[0037] Fig. 6 shows a map of the pOptivecTfrMabHC expression vector with conjugated protein.

[0038] Fig. 7 shows a typical electropherogram of the obtained antibodies and conjugates using the example of variants of I-Shi antibodies and conjugates based on them.

[0039] Fig. 8a shows comparative data regarding the ADCC and ADCP values ​​for the anti-TfR antibody Variant I according to the invention and the control sample.

[0040] Fig. 86 shows comparative data regarding in vivo clearance in mice for the anti-TfR antibody Variant I of the invention and a control sample.

[0041] Figures 9a and 9b show the results of a comparative study of the stability of antibody preparations during storage at a temperature of 25±2 °C. 9a - capillary gel electrophoresis (non-reducing conditions); 9b - size-exclusion chromatography.

[0042] Fig. 10a-m show spectra obtained for samples of PabinafuspAlfa, H6L7, and variant 1-X antibodies.

[0043] Detailed description of the invention

[0044] As used herein, the term "transferrin receptor" (TfR) refers to any naturally occurring TfR of a vertebrate, including mammals, including primates, such as humans, and rodents, such as mice and krryyyss. Transferrin receptor includes both the full-length unprocessed TfR and any form of TfR that is produced by processing in the body, including spliced ​​or allelic variants of such receptors. TfR is a transmembrane glycoprotein (with a molecular weight of about 180,000 Da) consisting of two disulfide-linked subunits (each with an apparent molecular weight of about 90,000 Da) involved in iron uptake in vertebrates. In a preferred embodiment, TfR is a human TfR comprising an amino acid sequence as set forth, for example, in Schneider et al. Nature 311:675–678 (1984).

[0045] The term "anti-transferrin receptor antibody" (or anti-TfR antibody) refers to a recombinant antibody produced using genetic engineering techniques, for example, a humanized IgG1 antibody or an antigen-binding fragment thereof, which is expressed by a recombinant host cell containing a nucleic acid encoding the antibody. The recombinant antibody may include modifications designed to change its characteristics, such as effector functions, target affinity, stability, clearance, etc. Such modifications may include the introduction of mutations in the antibody sequence that are amino acid insertions, substitutions, or deletions, as well as changes in the glycosylation profile.

[0046] "Efficiency functions" of an antibody are typically associated with an Fc domain sequence, which may be a native Fc domain sequence or variants thereof containing mutations. Examples of effector functions of an antibody include, for example, Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP). The antibody of the present invention preferably has a reduced or absent effector function. Preferably, such elimination of function is accomplished by mutations of the Fc domain including, but not limited to, substitutions at positions L234A, L235A, G236A, P237S, H268A, N297A, K322A, and P329S.

[0047] By antibody clearance, as used herein, is meant the rate of elimination of a drug (e.g., an antibody or antibody conjugate) from the body. Decreased clearance of a particular antibody can be achieved either by changing its affinity for the receptor or by introducing mutations, preferably Fc domain mutations including substitutions at positions M252Y, S254T, T256E, T307Q, T307W, and E380A. Unless otherwise indicated, amino acid residue numbering in an antibody sequence is performed in accordance with the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Mutations in Medicine, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0048] "Conjugate" is an antibody fused to one or more therapeutic molecules, optionally via a linker. Schematic representations of the resulting antibodies and their conjugates with a protein are shown in Fig. EB. The therapeutic molecules may include the drugs indicated herein for the treatment of diseases of the central nervous system, as well as any other suitable therapeutic agents that can be fused to an antibody for targeted delivery across the BBB. Methods for conjugating antibodies to proteins and small molecules are described in detail in the prior art and are well known to those skilled in the art. As a rule, they are carried out using special protein and non-protein linkers, the choice of which depends on the therapeutic agent.

[0049] "A therapeutic agent for the treatment of diseases requiring delivery of a drug across the BBB" includes drugs or therapeutic agents intended for the treatment of neurological disorders and enzyme replacement therapy. These agents include therapeutic antibodies, peptides, proteins, nucleic acids and small molecules or active fragments thereof.Preferably, the therapeutic agent can be selected from the following enzymes: alpha-L-iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase, N-acetyl-alpha-glucosaminidase, N-acetylglucosamine-6-sulfate sulfatase, beta-galactosidase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, hyaluronidase, β-beettaa galactosidase, beta-hexosaminidase, beta-glucocerebrosidase, imiglucerase, arylsulfatase A, alpha galactosidase A, N-acylsphingosin amidohydrolase, acid sphingomyelinase, alpha mannosidase, beta mannosidase, alpha-fucosidase, aspartyl glucosaminidase, a-N-acetylgalactosaminidase, a-neuraminidase, a-1,4-glucosidase, alglucosidase alpha, avalglucosidase alpha, lysosomal acid lipase, cathepsin K, palmitoyl protein thioesterase, tripeptidyl peptidase I.

[0050] As exemplary proteins, the present invention uses lysosomal therapeutic proteins such as iduronate-2 sulfatase (IDS) (Gene ID: 3423) and acid 1,4-alpha-glucosidase (GAA) (Gene ID: 2548) (designations according to the database of the US National Library of Medicine). Naturally occurring human IDS is a lysosomal enzyme with the sequence SEQ ID NO: 1

[0051] Natural human GAA is a lysosomal enzyme with the sequence SEQ ID NO: 2.

[0052] Any natural proteins intended for conjugation with an antibody to TfR may contain modifications, including substitutions, insertions and deletions of amino acids, that do not affect biological function and safety. The amino acid sequence of the modified therapeutic proteins should preferably have at least 90% homology to the amino acid sequence of the natural protein, such as natural IDS and natural GAA.

[0053] "Linker" in the context of this document is a peptide of 1-50 amino acids in size, designed to connect the domains of an antibody, as well as conjugate the antibody to a therapeutic moiety. Preferred variants of amino acid linkers that can be used to conjugate an antibody to a protein are given in Table 1.

[0054] Table 1. Sequences of amino acid linkers

[0055] The term "expression vector" is used herein interchangeably with the term "plasmid" and refers to a nucleic acid molecule into which a nucleic acid encoding a light and / or heavy chain of an antibody, alone or conjugated to a therapeutic agent, is cloned for expression. The term includes both the self-replicating nucleic acid structure and the vector incorporated into the genome of the host cell into which it is introduced.

[0056] "Host cell" is a cell into which the expression vector is introduced, as well as the progeny of such cells. Examples of "host cells" for the production of recombinant antibodies and conjugates based on them preferably include mammalian cells, including Chinese hamster ovary cells (CHO), monkey kidney cells (COS), myeloma cells (including Y0 and NS0 cells), baby hamster kidney cells (BHK), human embryonic kidney cells (HEK3), Hela cells, Vero cells and other cell lines suitable for the production of recombinant proteins, but are not limited to them.

[0057] "Neurological disorders" or diseases of the central nervous system are disorders or diseases that manifest in the central nervous system. Illustrative CNS diseases or disorders include, but are not limited to, Gaucher disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type VII, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, galactosylceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, alpha-mannosidosis, beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, mucolipidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

[0058] Preferred diseases for which the therapeutic protein conjugate with the claimed antibody may be used are lysosomal storage diseases, which are a class of over 50 different inherited metabolic diseases caused by a deficiency of a specific lysosomal protein. In particular, these include Hunter disease and Pompe disease. Hunter syndrome (mucopolysaccharidosis type II, MPS-II) is caused by a deficiency of iduronate-2-sulfatase (IDS), which is an important enzyme for the degradation of lysosomal glycosaminoglycans (GAGs), including heparan sulfate and dermatan sulfate. Loss of IDS activity results in the abnormal accumulation of GAGs in most cells in all tissues and organs, resulting in a wide range of symptoms, including coarse facial features, skeletal deformities, joint stiffness, recurrent respiratory infections, heart failure, renal degeneration and deafness.Severe forms of the disease are characterized by progressive deterioration of central nervous system function, manifested by cognitive impairment. Pompe disease or glycogen storage disease type II is associated with a deficiency of the enzyme acid 1,4-alpha-glucosidase (GAA).

[0059] In the case of the above diseases, enzyme replacement therapy (ERT) is not effective, so there remains a high unmet need for ERT with improved bioavailability for the brain and CNS.

[0060] The pharmaceutical compositions of the invention comprise a conjugate of an anti-TfR antibody with a therapeutic agent in an effective amount, as well as pharmaceutically acceptable ingredients.The therapeutic agent may be a special agent for the treatment of diseases requiring delivery of the drug across the BBB (including those listed above), in particular the following diseases: Fabry disease, BBB hepatitis, Wolman disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type IV, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type IX, GM1 gangliosidosis, Pompe disease, Tay-Sachs disease, Sandhoff disease, galactosylceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, Alpha-mannosidosis, Beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler's disease, mucolipidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, pycnodysostosis, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

[0061] "Pharmaceutically acceptable ingredient" means any safe component of the composition that is not an active therapeutic agent, including a buffer, filler, diluent, stabilizer, preservative, pH-influencing agent, isotonic agent, antioxidant, and other components commonly used in antibody and therapeutic protein compositions, as described in detail, for example, in Remington's Pharmaceutical Sciences, (16th ed., Osol A., 1980).

[0062] Typically, the pharmaceutical compositions according to the present invention are prepared in liquid or lyophilized forms. In this case, in a liquid preparation or after dilution of a lyophilized preparation, the concentration of the conjugate with an anti-TfR antibody in the composition is typically, for example, 1-50 mg / ml, preferably 5-30 mg / ml.

[0063] The buffer preferably selected is a citrate, phosphate or sodium phosphate buffer. Nonionic surfactants such as polysorbate and poloxamers, examples of which include polysorbate 20 and polysorbate 80, poloxamer 188 and others, can be used as stabilizers. Amino acids such as glycine, histidine, arginine and methionine can be used as stabilizers. Mannitol, sorbitol or sodium chloride can be used as an isotonic agent.

[0064] The anti-TfR antibody conjugates of the invention may be administered by any suitable route, including intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0065] Compositions intended for injection are prepared sterile. Sterility is generally ensured by filtration through sterile filter membranes.

[0066] "Therapeutically effective amount" means an amount of the active component of the composition sufficient to provide a beneficial effect in relation to the treatment of a disease or disorder when administered to a patient. It usually depends on the type of disease to be treated, the severity and progression of the disease, whether the conjugate is administered for prophylactic or therapeutic purposes, previous or combination therapy, clinical history and other characteristics of the patient, based on which the attending physician determines an appropriate dosage. As a rule, a therapeutically effective amount of the conjugate of the invention is about 1-100 μg / kg, preferably 1-30 mg / kg.

[0067] EXAMPLES Example 1. Design and modification of light and heavy chains of antibodies.

[0068] In the first stage, the design of hypervariable regions for new antibodies was developed.

[0069] The variable region of the heavy chain of the anti-TfR parent antibody is represented by the sequence SEQIDNO18 (CDRs are highlighted):

[0070] EVQLVQSGAEVKKPGESLKISCKGSGYSFTGYTMNWVRQMPGKGLEWMGRINP

[0071] HNGGTDYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYYSLDYW

[0072] GQGTLVTVSS, for which hypervariable regions (CDR1-

[0073] CDR3) listed in Table 2:

[0074] Table 2, Sequences of hypervariable regions of the heavy chain.

[0075] The variable region of the light chain of the anti-TfR antibody is represented by the sequence SEQIDNO:42 (hypervariable regions are highlighted):

[0076] EIVLTQSPATLSLSPGERATLSCSASSSIRYIHWYQQKPGQAPRRWIYDTSNLASGTPA

[0077] RFSGSGSGTSYSLTISSLEPEDFAVYYCHQRNSYPWTFGQGTKVEIK, for which the hypervariable regions (CDR1-CDR3) shown in Table 3 were constructed:

[0078] Table 3. Sequences of hypervariable regions of the light chain

[0079] The introduction of mutations in the CDRs of the heavy (SEQIDNOs 19-26, 27-31, 32-41) and light chains (SEQIDNOs 43-56, 57-65, 66-74) for some antibody variants made it possible to reduce the affinity of the antibody to TfR by 10-100 times (Example 6), while it was unexpectedly discovered that certain combinations of CDRs make it possible to obtain an antibody or a conjugate based on it with improved stability (Example 7).

[0080] The constant portion of the light chain of the anti-TfR antibody is represented by the human light chain sequence SEQIDNO: 75:

[0081] PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

[0082] SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR.

[0083] In the next step, the effect of mutations in the Fc region of the antibody on effector properties and clearance was studied using methods described below in the examples. The CH1 domain, hinge region and Ec fragment of the heavy chain of the original antibody are represented by the sequence SEQIDNO:76

[0084] GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0085] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL

[0086] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0087] REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, in which the positions into which mutations were introduced to select antibodies with the desired characteristics are highlighted. The effect of mutations on the effector properties and clearance of the antibody was assessed using methods similar to those described in Examples 8 and 9. The parental anti-TfR antibody H6L7 was used as a control against which the effect of mutation was assessed.

[0088] The results of the study of the influence of various mutations on the properties of antibodies are shown in Table 4:

[0089] Table 4. Mutations and their impact on antibody properties

[0090] * Numbering according to the Kabat system.

[0091] Based on combinations of CDR sequences and other regions that result in reduced affinity for TIR and reduced or eliminated effector properties, the 1-X antibody variants shown in Table 5 were constructed.

[0092] Table 5. Antibody variants.

[0093] The heavy chain sequence of the parental anti-transferrin receptor antibody is given in SEQIDNO:77 (heavy chain variable region highlighted):

[0094] SEQIDNO:77

[0095] EVQLVQSGAEVKKPGESLKISCKGSGYSFTGYTMNWVRQMPGKGLEWMGR

[0096] INPHNGGTDYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYYS

[0097] LDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

[0098] GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0099] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0100] WYVDGVEVHNAI <TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CKVSNKALPAP

[0101] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

[0102] NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0103] TO

[0104] The sequence of the light chain of the parent antibody is given in

[0105] SEQIDNO:78(light chain variable region highlighted):

[0106] SEQIDNO:78

[0107] EIVLTQSPATLSLSPGERATLSCSASSSIRYIHWYQQKPGQAPRRWIYDTSNLA

[0108] SGTPARFSGSGSGTSYSLTISSLEPEDFAVYYCHQRNSYPWTFGQGTKVEIKRTVA

[0109] APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK

[0110] DSTYSLSSTLTSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0111] A series of affinity-altered antibodies, designated variants I-X, were constructed from this antibody. These antibodies contained mutations in the heavy (SEQIDNO:77) and light (SEQIDNO:78) chains corresponding to those listed in Tables 2-5 above. The new heavy chain sequences were designated SEQIDNO:79-88 and the new light chain sequences were designated SEQIDNO:89-98, respectively. All of these sequences are listed in the Sequence Listing at the end of this specification.

[0112] Example 2. Synthesis of anti-TfR antibody genes and conjugates based on it

[0113] At the first stage, mutations were introduced into the hypervariable regions CDR1-3 of the light and heavy chains of the original antibody according to the scheme shown in Fig. 2.

[0114] Using primer pairs 1, 2, 3 and 4 for the light chain and 1H, 2H, 3H, 4H for the heavy chain, overlapping fragments with the required nucleotide sequence of hypervariable regions were obtained by polymerase chain reaction using DNA polymerase with proofreading activity (e.g. Pfu polymerase). The reaction products were purified from the reaction mixture. The PCR products from the light and heavy chains were mixed in a molar ratio of 1:1:1:1 and amplified in the Overlap extension PCR reaction using Pfu polymerase and flanking primers - IF, 4R - for the light chain and 1HF and 4HR - for the heavy chain. The resulting product was cloned into a vector for molecular biological operations (for example: pUCC19, pUC57, pAE2T, etc.) and the vector with the correctly assembled fragment was amplified with end primers, obtaining ready VL and VH fragments.

[0115] In the second step, the CL region of the light chain was amplified using primers CEP containing the VL fragment and CLR (Fig. 3). Depending on the number of mutations that need to be introduced into the heavy chain, fragments of the heavy chain were amplified using primers and no more than four adjacent fragments were ligated using the Overlap extension PCR method, the resulting fragments were cloned into a vector for molecular biological operations, amplified with end primers and then the resulting large fragments were ligated into one fragment of the heavy chain containing the CH1 domain and the Fc fragment (Fig. 3). The resulting fragment of the heavy chain was cloned into the vector and amplified using flanking primers.

[0116] In the third step, the VH and CH1+CH2+CH3 fragments were ligated using Overlap extension PCR and flanking primers into a single gene, thus obtaining a full-length heavy chain with the necessary mutations. The VL fragments were ligated in a similar manner, obtaining a full-length light chain. The light chain was cloned into the pcDNA 3.3 vector, and the heavy chain (if there was no need for further operations with the gene) into pOptiVec, obtaining the vectors shown in Fig. 4. To obtain a conjugate of the heavy chain with another protein or enzyme, the heavy chain and the conjugated protein were amplified using primers, while the Reverse primer of the heavy chain at the 5 '-end should contain the nucleotide sequence of the linker that is supposed to be used. Similarly, a forward primer for amplification of another protein must contain a reverse complementary linker sequence at the 5-terminus. The scheme is shown in Fig. 5.

[0117] The resulting PCR products were cross-linked in an Overlap extension PCR reaction with flanking primers and cloned into the pOptiVec vector. Figure 6 shows a map of the pOptivecTfrMabHC expression vector with conjugated protein.

[0118] To obtain conjugates, the sequences of the obtained heavy chains of antibodies of Variants I - X to the transferrin receptor were fused via linkers (SEQIDNO: 6, 7 and 15) with the therapeutic protein - iduronate-2-sulfatase (SEQIDNO: 1). Also, the albumin leader peptide SEQIDNO: 26: MKWVTFISLLFLFSSAYS was added to the N-termini of the light and heavy chains.

[0119] The sequence of the heavy chain of the Variant I antibody, conjugated at the C-terminus to iduronate 2-sulfatase, is given in SEQIDNO: 99:

[0120] MKWVTFISLLFLFSSAYSEVQLVQSGAEVKKPGESLKISCKGSGYSMTGYTMNW

[0121] VRQMPGKGLEWMGRINPHSGGTDTNQLFKDQVTISADKSISTAYLQWSSLKASDTA

[0122] MYYCARGYYYASLEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK

[0123] DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0124] PSNTKVDKRVEPKSCDKTHTCPPCPAPEALGASSVFLFPPKPKDTLMISRDPEVTCVVV

[0125] DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLQVLHQDWLNGI<EY

[0126] KCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA

[0127] VAWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

[0128] HYTQKSLSLSPGKGGGGSGGGSETQANSTTDALNVLLIIVDDLRPSLGCYGDKLVRSP

[0129] NIDQLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGNFSTIP

[0130] QYFKENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGEL

[0131] HANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPK

[0132] EFQKLYPLENITLAPDPEVPDGLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRK

[0133] IRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAFTSDHGWALGEHGEWAKYSNFD

[0134] VATHVPLIFYVPGRTASLPEAGEKLFPYLDPFDSASQLMEPGRQSMDLVELVSLFPTLA

[0135] GLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEEDPYLPGNPRELIAYSQYPRPS DIPQWNSDKPSLKDIKIMGYSIRTIDYRYTVWVGFNPDEFLANFSDIHAGELYFVDSDP

[0136] LQDHNMYNDSQGGDLFQLLMP

[0137] The final version of the light chain of antibody I is shown in SEQIDNO: 100: MKWVTFISLLFLFSSAYSEIVLTQSPATLSLSPGERATLSCSASSSLRYIHWYQQKPGQ

[0138] APRRWIYDTSNLSSGTPARFSGSGSGTSYSLTISSLEPEDFAVYYCHQKNSYPWTFGQ

[0139] GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0140] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0141] In a similar manner, i.e., by adding the albumin leader peptide with SEQIDNO: 26 to the N-terminus of the heavy chain, and the iduronate-2-sulfatase sequence fused via one of the linkers indicated in Table 6 to the C-terminus of the heavy chain, conjugates of antibodies of variants II - X were obtained.

[0142] Table 6 lists the linkers used to create the antibody conjugates.

[0143] Table 6. Linker sequences

[0144] The final light chains of Variant II - X antibodies, as in the case of Variant I antibody, included a leader pentyl with SEQIDNO: 26 at the N-terminus of the light chain. A schematic representation of the antibodies and their protein conjugates is shown in Fig. 1a and 1b, respectively.

[0145] Example 3. Obtaining expression vectors and preparing them for transfection. The obtained sequences of the heavy chain of antibodies 1-X and the conjugate of the heavy chain of antibodies 1-X were cloned bluntly into the pOptiVec vector (Invitrogen, USA), with the gene of dihydrofolate reductase (resistance to high concentrations of methotrexate). The light chain of antibodies 1-X was cloned into the pcDNA 3.3 vector. (Invitrogen, USA)

[0146] To produce the above antibody variants and antibody conjugates, gene fragments were amplified using Pfu DNA polymerase. The reaction product was purified using a spin column DNA purification protocol. pOptiVec and pcDNA vectors were linearized using EcoRV restriction enzyme (previously introduced between the CMV promoter and polyadenylation signal), the product was purified by agarose gel electrophoresis and isolated from the gel using a spin column DNA purification protocol. The ligation reaction was performed using T4 DNA ligase at an insert / vector ratio of 1 / 10 using 5% PEG-4000 in the ligation reaction. The resulting plasmid DNA was purified using a spin column DNA purification protocol. The ligase mixture was transformed into chemically competent E. coli cells; after transformation, the cells were plated in LB agar on a Petri dish containing 100 μg / ml ampicillin.After incubation at 37 °C for 18 h, colonies were picked, resuspended in 20 µl of sterile water, and checked for the presence of the insert and correct gene orientation.

[0147] After the PCR reaction, the reaction product was analyzed by agarose gel electrophoresis (1% agarose). Clones whose PCR products had the PCR product size appropriate for the two reactions were transferred to 50 ml of EB medium containing 100 μg / ml ampicillin and cultured with stirring for 24 h. The resulting suspension was centrifuged and the supernatant was removed without affecting the cell pellet. Plasmid DNA was isolated from the cell pellet using a spin column purification protocol. 0.1 V sodium acetate 3M and 2.7 V ethanol 95% were added to the resulting eluate (IV), mixed and centrifuged at 10,000 g for 30 min. The supernatant was removed under aseptic conditions, the pellet was washed with plasmid DNA with 70% ethanol, the supernatant was removed and the pellet was dried. 10 µl of sterile water were added and mixed until completely dissolved. 2 µl were collected to assess the DNA concentration and determine the sequence of the introduced genes.

[0148] Example 4. Transfection and selection of transfectants. Creation of a producer strain. For transfection of CHO cells deficient in the dihydrofolate reductase gene, pure (“transfectiongrade”) plasmid DNA with a concentration of at least 2 mg / ml was used: for the production of antibody No. I (hereinafter Mabl)

[0149] - pcDNA 3.3. TfRLCMab 1;

[0150] - pOptiVecTfRHCMab 1 for the production of antibody conjugate No. I (hereinafter Mabl-IDS) pcDNA 3.3. TfRLCMab 1;

[0151] - pOptiVecTfRHCMab 1 + IDS for the production of antibody No. II (hereinafter referred to as MaЬ2)

[0152] - pcDNA 3.3. TfRLCMab 2;

[0153] - pOptiVecTfRHCMab 2 for the production of antibody conjugate No. II (hereinafter Mab2-IDS) pcDNA 3.3. TfRLCMab 2;

[0154] - pOptiVecTfRHCMab 2 + IDS for the production of antibody No. III (hereinafter referred to as MAb3)

[0155] - pcDNA 3.3. TfRLCMab 3;

[0156] - pOptiVecTfRHCMab 3 for the production of antibody conjugate No. III (hereinafter referred to as MAb3-IDS) pcDNA 3.3. TfRLCMab 3;

[0157] - pOptiVecTfRHCMab 3 + IDS, etc.

[0158] As a result of cotransfection with expression vectors, one pool of cells was obtained for each set of plasmids, in which the viability of the cell culture 48 h after transfection was at least 85%. Control transfection with a vector containing the gene of the fluorescent protein GFP demonstrated transfection efficiency at the level of 30±10%. After 72 h, transfectant selection was started by culturing with the addition of 400 μg / ml geneticin and 1 μM MTX. Within 10 days under selective pressure, the viability reached a minimum of 35±15%. Viability in all cultures in the pool was restored to 90±5% on the 16th and 18th days, after which some of the cells in the pool were frozen.

[0159] The remaining part was used to produce protein and clone a cell pool. In this way, 20 amplified pools of cells producing 10 variants of a monoclonal antibody to the transferrin receptor were created, as well as 10 variants of conjugates of these antibodies with IDS. The proteins produced in preparative (100 mg - 1 g) quantities were used to evaluate their properties and select the most promising of them.

[0160] Limiting dilutions were performed at a density of 0.5 cells per well. The total number of plates for each protein was 100 pcs.

[0161] Producer clones

[0162] A total of 389 clones were selected from the 96-well plates from the limiting dilution cloning stage and transferred to 24-well plates. 96 clones grown in 24-well plates were transferred to 6-well plates with an increased content of Anti Clumping Agent B to 1.4% to destroy cell aggregates.

[0163] After scaling for adaptation to shaking flask cultivation, 5 subclones for each protein producer were selected that had the best characteristics according to the parameters: specific productivity (ped) > 10 pg / cell, viable cell content > 0.9 million / ml, protein content in the culture fluid > 30 μg / ml.

[0164] Subclone reseeding was carried out over 2 passages (1 passage - 4 days), after the second passage the cells were frozen with a cell content of 5±1.5 million cells / cryotube in the amount of 63 tubes for each producer clone.

[0165] Example 5. Method for obtaining recombinant protein from producer strain cells.

[0166] The developed method for obtaining the conjugate includes the following stages:

[0167] 1) cultivation of a strain producing recombinant protein;

[0168] 2) selection and clarifying filtration of the culture liquid;

[0169] 3) chromatographic purification of recombinant protein;

[0170] 4) confirmation of the authenticity of the obtained recombinant protein.

[0171] Cultivation

[0172] For cultivation in a bioreactor, the inoculum was grown in the required quantity for initial seeding in a reactor with a cell density of 0.5x10 6 cells / ml. Cultivation was carried out in ActiPro medium (Hyclone, USA) containing 4 mM L-alanine-L-glutamine (Gibco, USA) and 0.4% anti-cell aggregation reagent, and incubated for 24 hours in an incubator at 37 °C and a humidity of at least 70% in T-25 flasks.

[0173] The producer strain cells were cultured in a BioFlo 320 bioreactor with a working volume of 1 liter (Eppendorf, Germany) on ActiPro medium (Нуclone, USA) containing 4 mM L-alanine-L-glutamine (Gibco, USA) and 0.4% anti-cell aggregation reagent (Lonza, Switzerland). A complex of additives was added daily from the third day of cultivation: CellBoost 7 A (Cytiva, USA) 1.5-3.5% of the cultivation volume and a mixture of amino acids E-cystine (152 mM), L-tryptophan (90 mM), L-tyrosine (250 mM) (AppliChem, USA for three amino acids). The amount of amino acid mixture used was 10 times less relative to the volume of CellBoost 7 A. During the entire process, a concentrated glucose solution of 200 g / l (Panreas, USA) was added daily to maintain the glucose concentration in the culture liquid at about 5 g / l.

[0174] The gas mixture was fed through a macrobubbler at a rate of 0.2 to 1.5 l / min. Cultivation was carried out at a marine propeller stirrer speed of 60 to 100 rpm (cascade control) and a temperature of 37 °C, followed by a decrease to 35 °C in the exponential growth phase and to 33 °C on the following day of cultivation.

[0175] The cultivation was stopped when the viability dropped below 80%. The culture fluid was clarified by centrifugation in two stages: 1000 g for 30 minutes and 3270 g for 15 minutes. A filter with a rating of 0.22 μm was used to sterilize the culture fluid. The resulting volume of sterile culture fluid was 1.05 l. From the resulting culture fluid, 565 mg of the conjugate with the variant 1 antibody and 352 mg of the conjugate with the variant II antibody were obtained by purification according to the method described below. Conjugates with the III-X antibodies were obtained in a similar manner.

[0176] Protein purification.

[0177] Chromatographic purification of protein from clarified culture liquid is carried out on a preparative chromatograph. The first stage of purification is the affinity sorbent MabSelectShure LX (Cytiva). After equilibration with a neutral buffer, the culture liquid was applied to the column, then washed with a Wash buffer of 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 and eluted with a buffer of 20 mM glycine, pH 3.0. The pH in the semi-finished product was adjusted to 7.0 with a 1 M Tris solution.

[0178] The second stage of purification was the hydrophobic sorbent Butyl Sepharose HP (Cytiva). The sorbent was equilibrated with a buffer of 20 mM sodium phosphate, 2 M sodium chloride, pH 6.0, the semi-product obtained from the first stage of purification was applied to the column, washed with a buffer of 20 mM sodium phosphate, 2 M sodium chloride, pH 6.0, and then elution was carried out with a buffer of 20 mM sodium phosphate, pH 6.0.

[0179] The third stage of purification is transfer to the GLF buffer using 53 ml of Sephadex G25 sorbent. Before application, the column was equilibrated with a buffer of 20 mM sodium phosphate, pH 7.0,

[0180] The fourth stage is normalization of protein concentration. The concentration is adjusted with a 20 mM sodium phosphate buffer, pH 6.3 to a value of 20 mg / ml or sterile filtered through a Steritop 0.22 mkm bottle filter (Merck - Millipore, Germany). A solution of sterile purified antibody or antibody conjugate is obtained.

[0181] Confirmation of protein authenticity.

[0182] The authenticity of the obtained recombinant proteins was confirmed by electrophoresis in polyacrylamide gel followed by immunoblotting. Gradient polyacrylamide gel 4-20% (Bio-Rad, cat. No. 4561093) was used for electrophoresis. The samples were analyzed by electrophoresis under reducing conditions. 100 ng of protein solution were added to the gel wells. Separation was carried out at a voltage of 200 V for 50 min. Staining was performed using Coomassie R250. The electropherogram for antibody variants I-III and the corresponding conjugates is shown in Fig. 7. The sizes of other antibodies and conjugates based on them were estimated in a similar manner.

[0183] Example 6. Study of the properties of the obtained antibodies and their conjugates with IDS using the surface plasmon resonance method

[0184] The dissociation constants of the antibody (or conjugate) with the transferrin receptor were measured by the surface plasmon resonance method on a Bio-Rad ProteOn™ XPR36 spectrometer at 25 °C. Buffer conditions: 10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4.

[0185] The ligand (transferrin receptor) was immobilized onto the ProteOn™ GLH chip (Bio-Rad #176-5013) via amino groups using the Bio-Rad Surface Ligand Immobilization Reagent Kit at a ligand concentration of 0.03 mg / mL.

[0186] In the perpendicular direction, a solution of the antibody preparation in the working buffer was applied to the chip in five different protein concentrations from 5 nM to 40 nM, and the working buffer was applied to one lane as a control. The flow rate through the chip was 30 μl / min, the duration of the association phase was 300 sec. During the dissociation phase of the ligand-analyte complex, the working buffer was passed through the chip at the same speed, the duration of the dissociation phase was 3600 sec. The degree of completion of the dissociation phase of the complex was monitored by the magnitude of the decrease in the SPR signal.

[0187] The chip was regenerated after the ligand-analyte binding cycle by passing a 10 mM glycine solution, pH 2.0, through the chip.

[0188] The calculation of the dissociation constants of the ligand with the analyte was performed in the standard Bio-Rad ProteOn Manager program using the bivalent analyte scheme.

[0189] The results of the study of dissociation constants are presented in Table 7.

[0190] Table 7. Dissociation constant values ​​for the obtained antibodies.

[0191] Fig. 10a-m show the spectra obtained from evaluating 12 samples.

[0192] Based on the data obtained, it can be concluded that the most preferred are antibody variants I, II, III, VIII and X. The affinity of these proteins is 5-30 times lower than that of Pabinafusp Alfa and antibody H6L7, which is more preferable from the point of view of subsequent use as a drug, as discussed above, which has a positive effect on the safety of therapy in patients during the course of treatment with the drug.

[0193] Example 7. The obtained antibodies have high stability.

[0194] The stability of antibodies during storage was assessed for antibody solutions with a concentration of 10.0 ± 3.0 mg / ml, which were stored in glass vials made of hydrolytic class 1 glass at a temperature of 25 ± 2 °C for 336 days. At points 0, 7, 14, 28, 84, 168, 336 days from the beginning of the experiment, samples were taken and analyzed by capillary gel electrophoresis and size-exclusion chromatography. The stability assessment results are shown in Figs. 9a and 996b. AA - capillary gel electrophoresis (non-reducing conditions); B - size-exclusion chromatography.

[0195] Example 8. The obtained antibodies do not have an effector function.

[0196] Unexpectedly, it was found that the effector function shutdown provides a decrease in the level of not only antibody-dependent cellular cytotoxicity (ADCC), but also a decrease in antibody-dependent cellular phagocytosis (ADCP). Ramos cell culture was used to study the obtained antibodies to TfR, since these cells are the standard for studying the effector properties of a large number of therapeutic monoclonal antibodies and they express TfR1 and CD20 receptors.

[0197] The study design was as follows: two cell cultures were used in the study - Ramos and J774.2 (mouse monocytes / macrophages). J774.2 cells are a subclonal cell line of reticular cell sarcoma and have macrophage-like properties.

[0198] The study was designed to simultaneously evaluate antibody-dependent cellular phagocytosis (ADCP) and ADCC activity induced by anti-TfR antibody in a single experiment. For this purpose, flow cytometry with three different dyes was used. The studied anti-TfR antibody was added to the cells at a concentration of 5 μg / ml, the H6L7 antibody served as a control.

[0199] Ramos cells were labeled with the fluorescent molecule CFSE (this dye binds to both membrane and cytoplasmic proteins, easily penetrating into the cell and having low cytotoxicity). To stain the effector cells J774.2, a PE-conjugate of the antibody to F4 / 80 (mouse epidermal growth factor expressed on these cells) was used. In addition, the experiment involved the dye DAPI, which specifically binds to cellular DNA.

[0200] Thus, it was possible to distinguish between cell populations:

[0201] — cells positive for CFSE and DAPI staining served as a marker for ADCC activity (since J774.2 effector cells activated by anti-TfR antibody triggered lysis of target Ramos cells);

[0202] — cells positive for staining with F4 / 80 and CFSE labels were an indicator of the phagocytosis that had occurred (since the J774.2 effector cells activated by the anti-TfR antibody phagocytized the target Ramos cells).

[0203] It was found (see Fig. 8a) that the total number of killed Ramos tumor cells in the control (H6L7) was 75%, in the presence of anti-TfR antibody Variant I - 20%. Similar values ​​(in the range from 17 to 46%) were obtained for antibody variants II, III, VIII and X.

[0204] A similar technique was also used to assess the impact of mutations on the antibody effector functions. The results of the corresponding studies are presented in Table 4.

[0205] Example 9. The obtained antibodies have reduced clearance.

[0206] A clearance study was performed using the anti-TfR Variant I antibody with the L234A / G236A / P237S / T256E / T307Q / K322A / E380A Fc domain mutations and the H6L7 antibody (control). Female SCID-Beige mice (8-12 weeks old) were injected intravenously with 100 mg of antibody. Blood was collected from the tail vein at 2, 24, 48 and 168 hours after injection. Serum from each time point was pooled (n=4-5) and added to an ELISA plate coated with goat anti-human IgG. Binding was detected using AR-conjugated goat anti-human kappa chain antibody and AR substrate.

[0207] It was found (see Fig. 86) that the clearance of the anti-TfR Variant I antibody was almost two times less than the clearance of the control anti-TfR antibody H6L7.

[0208] A similar technique was used to assess the effect of mutations on antibody clearance. The results are shown in Table 4. Example 10. Preparation of a Lyophilized Pharmaceutical Composition

[0209] The conjugate obtained in Example 5, in a buffer containing 20 mM sodium phosphate with a pH of 6.3, was subjected to sterilizing filtration and then concentrated using tangential filtration to a concentration of 50 mg / ml, simultaneously changing the buffer to sodium phosphate 17 mM, pH 6.3. A stock solution of polysorbate 20 - 100 mg / ml was added to the resulting solution to a concentration of 0.05 mg / ml, the volume was brought up to water for injection. A composition of the following composition was obtained:

[0210] Conjugate of antibody variant I with iduronate-2-sulfatase - 30 mg / ml;

[0211] Sodium hydrogen phosphate dihydrate - 2.0 mg / ml;

[0212] Sodium dihydrogen phosphate monohydrate 0.8 mg / ml;

[0213] Sorbitol - 200 mg / ml;

[0214] Polysorbate 20 - 0.05 mg / ml;

[0215] Water for injection - up to 1 ml

[0216] 5 ml of the resulting liquid composition were filtered through a sterilizing filter, placed in vials made of class 1 hydrolytic glass and lyophilized.

[0217] After transfer to 20 mM sodium phosphate buffer pH 6.3 and sterilizing filtration (Example 5), the antibody sample was concentrated using tangential filtration to a concentration of 50 mg / mL by transferring the protein to a buffer of the following composition:

[0218] Sodium hydrogen phosphate dihydrate - 2.0 mg / ml;

[0219] Sodium dihydrogen phosphate monohydrate 0.8 mg / ml.

[0220] A stock solution of sorbitol 1000 mg / ml was added to a concentration of 200 mg / ml and a stock solution of polysorbate 20 - 100 mg / ml to a concentration of 0.4 mg / ml.

[0221] 5 ml of the solution were filtered through a sterilizing filter, placed in vials made of class 1 hydrolytic glass and lyophilized.

[0222] Example H. Obtaining a liquid pharmaceutical composition

[0223] After transfer to 20 mM sodium phosphate buffer pH 6.3 and sterilizing filtration (Example 5), the antibody sample is transferred using tangential filtration to a concentration of 20 mg / ml, in a buffer of the following composition:

[0224] Sodium hydrogen phosphate dihydrate - 4.6 mg;

[0225] Sodium dihydrogen phosphate monohydrate 1.8 mg;

[0226] Histidine - 2.05 mg; Polysorbate 20 - 0.1 mg;

[0227] Water for injection - up to 1 ml

[0228] 5 ml of the solution are filtered through a sterilizing filter and placed in vials made of class 1 hydrolytic glass.

[0229] List of sequences

[0230] SEQ ID NO:1 (iduronate-2 sulfatase):

[0231] MPPPRTGRGLLWLGLVLSSVCVALGSETQANSTTDALNVLLIIVDDLRPSLGCYGDKL

[0232] VRSPNIDQLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGN

[0233] FSTIPQYFKENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGP

[0234] DGELHANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPF

[0235] RYPKEFQKLYPLENITLAPDPEVPDGLPPVAYNPWMDIRQREDVQ ALNIS VP YGPIPVD

[0236] FQRKIRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAFTSDHGWALGEHGEWAKYS

[0237] NFDVATHVPLIFYVPGRTASLPEAGEKLFPYLDPFDSASQLMEPGRQSMDLVELVSLF

[0238] PTLAGLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEEDPYLPGNPRELIAYSQY

[0239] PRPSDIPQWNSDKPSLKDIKIMGYSIRTIDYRYTVWVGFNPDEFLANFSDIHAGELYFV

[0240] DSDPLQDHNMYNDSQGGDLFQLLMP.

[0241] SEQ ID NO: 2 (кислая 1,4-альфа-глюкозидаза):

[0242] MGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPRELSGSSPVLEETHPAHQQ

[0243] GASRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQ

[0244] GLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMM

[0245] ETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLL

[0246] NTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANL

[0247] YGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKS

[0248] VVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWND

[0249] LDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEG

[0250] LRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWI

[0251] DMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLY

[0252] GLTEAIASHRALVKARGTRPF VISRSTF AGHGRYAGHWTGD VWS SWEQLAS S VPEIL

[0253] QFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEP

[0254] AQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLW

[0255] GEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQW

[0256] VTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDG

[0257] ESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSN

[0258] GVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC.

[0259] SEQ ID NO: 3 (linker): SAAS SEQ ID NO: 4 (linker): YYYY

[0260] SEQ ID NO: 5 (linker): GGGGGSG

[0261] SEQ ID NO: 6 (linker): YYYYYYYY

[0262] SEQ ID NO: 7 (linker): GGSGGSGGSG

[0263] SEQ ID NO: 8 (linker): GGSGGSGGSGGSGG

[0264] SEQ ID NO: 9 (linker): GGS GGSGGSGGSGG

[0265] SEQ ID NO: 10 (linker): GGS GGS GGSGGSGGSGG

[0266] SEQ ID NO: 11 (linker): GSGGSGGG

[0267] SEQ ID NO: 12 (linker): GSGGSGGSGGG

[0268] SEQ ID NO: 13 (linker): GSGGSGGSGGSGGG

[0269] SEQ ID NO: 14 (linker): GSGGSGGSGGSGGSGGG

[0270] SEQ ID NO: 15 (linker): SGGGGIG

[0271] SEQ ID NO: 16 (linker): SGGGGSGGGGSGGGGIG

[0272] SEQ ID NO: 17 (linker): SGGGGSGGGGSGGGGSGGGGIG

[0273] SEQ ID NO: 18 (anti-TfR heavy chain variable region of parental antibody H6L7):

[0274] EVQLVQSGAEVI <I<PGESLKISCI<GSGYSFTGYTMNWVRQMPGI<GLEWMGRJNPHNG

[0275] GTDYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYYSLDYWGQGT LVTVSS

[0276] SEQ ID NO:19 (CDR1 heavy chain): GYSMTGYTMN

[0277] SEQ ID NO:20 (CDR1 heavy chain): GYSFATGYTMN

[0278] SEQ ID NO:21 (heavy chain CDR1): GYSFTGYTHN

[0279] SEQ ID NO:22 (CDR1 heavy chain): GYSFTAYTMN

[0280] SEQ ID NO:23 (CDR1 heavy chain): AYSFTGYTMN

[0281] SEQ ID NO:24 (CDR1 heavy chain): GNSFTAYSMN

[0282] SEQ ID NO:25 (CDR1 heavy chain): GYSFTGYMN

[0283] SEQ ID NO:26(albumin leader peptide): MKWVTFISLLFLFSSAYS

[0284] SEQ ID NO:27 (heavy chain CDR2): RINPHNGATDYNNKFKD

[0285] SEQ ID NO:28 (CDR2 heavy chain): RINFPHNGATDYSQKFKD

[0286] SEQ ID NO:29 (CDR2 heavy chain): RINPHSGGTDTNQLFKD

[0287] SEQ ID NO:30 (CDR2 heavy chain): LINPHNAGTDYNFKFKD

[0288] SEQ ID NO:31 (CDR2 heavy chain): RVNPHNTGTDFNQLFKD

[0289] SEQ ID NO:32 (CDR3 heavy chain): GYAYYSLDY SEQ ID NO:33 (CDR3 heavy chain): GATYYYSLDY

[0290] SEQ ID NO:34 (CDR3 heavy chain): GYYYFSLDY

[0291] SEQ ID NO:35 (CDR3 heavy chain): GYYEYSLDY

[0292] SEQ ID NO:36 (CDR3 heavy chain): GYYYASLEY

[0293] SEQ ID NO:37 (CDR3 heavy chain): GYIYYSLDY

[0294] SEQ ID NO:38 (CDR3 heavy chain): SYYYYSLDY

[0295] SEQ ID NO:39 (CDR3 heavy chain): SYYYYSKDY

[0296] SEQ ID NO:40 (CDR3 heavy chain): GYYSSTLDY

[0297] SEQ ID NO:41 (CDR3 heavy chain): GYYYYSLDYS

[0298] SEQ ID NO: 42 (parental anti-TfR antibody light chain variable region

[0299] H6L7):

[0300] EIVLTQSPATLSLSPGERATLSCSASSSIRYIHWYQQKPGQAPRRWIYDTSNLASGTPA

[0301] RFSGSGSGTSYSLTISSLEPEDFAVYYCHQRNSYPWTFGQGTKVEIK

[0302] SEQ ID NO:43 (CDR1 light chain): SASSAIRYIH

[0303] SEQ ID NO: 44 (light chain CDR1): SASAAIRYIH

[0304] SEQ ID NO:45 (CDR1 light chain): SASSSLRYIH

[0305] SEQ ID NO:46 (CDR1 light chain): SASASLRYIH

[0306] SEQ ID NO:47 (CDR1 light chain): SASSAIRYIF

[0307] SEQ ID NO:48 (CDR1 light chain): SASSSIRYIF

[0308] SEQ ID NO:49 (CDR1 light chain): SASSAIRYLH

[0309] SEQ ID NO:50 (CDR1 light chain): SASSSIRYIW

[0310] SEQ ID NO:51 (CDR1 light chain): SASSAIRYIW

[0311] SEQ ID NO:52 (CDR1 light chain): SASSSAIRYIH

[0312] SEQ ID NO:53 (CDR1 light chain): SASSSAIRYIF

[0313] SEQ ID NO:54 (CDR1 light chain): SASSSAIRYLH

[0314] SEQ ID NO:55 (CDR1 light chain): SASSSAIRYLF

[0315] SEQ ID NO:56 (CDR1 light chain): SASSAIRYLAH

[0316] SEQ ID NO:57 (CDR2 light chain): DTSNLSSGTPA

[0317] SEQ ID NO:58 (CDR2 light chain): DTSQLASGSPA

[0318] SEQ ID NO:59 (CDR2 light chain): DTSQLSSGTPA

[0319] SEQ ID NO:60 (CDR2 light chain): DTSQLASATPA

[0320] SEQ ID NO:61 (CDR2 light chain): DTSNLASATPA

[0321] SEQ ID NO: 62 (CDR2 light chain): DTSNLASASPA SEQ ID NO: 63 (CDR2 light chain): DSSNLASGTPA

[0322] SEQ ID NO:64 (CDR2 light chain): DSSNLASATPA

[0323] SEQ ID NO:65 (CDR2 light chain): DSSNLSSGTPA

[0324] SEQ ID NO:66 (CDR3 light chain): HNRNSYPW

[0325] SEQ ID NO:67 (CDR3 light chain): HQRNSYAPW

[0326] SEQ ID NO:68 (CDR3 light chain): HQRNSYGPW

[0327] SEQ ID NO:69 (CDR3 light chain): HQRNSYSPW

[0328] SEQ ID NO:70 (CDR3 light chain): HQKNSYPW

[0329] SEQ ID NO:71 (CDR3 light chain): HQKNSYAPW

[0330] SEQ ID NO:72 (CDR3 light chain): HQKNSYSPW

[0331] SEQ ID NO:73 (CDR3 light chain):HQKNSYGPW

[0332] SEQ ID NO:74 (CDR3 light chain): HQRQSYPW

[0333] SEQ IIDD NO:75 (constant portion of anti-TfR antibody sequence):

[0334] PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

[0335] STYSLSSTLTSKADYEKHKVYACEVTHQGLSSPVTKSFNR

[0336] SEQ ID NO: 76 (CHI domain, hinge region and Fc portion of the heavy chain of anti-TfR antibody):

[0337] GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL

[0338] YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP

[0339] SVFLFPKPKDTLMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0340] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

[0341] PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

[0342] TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0343] SEQIDNO:77 (chain link of anti-TfR antibodies H6L7):

[0344] EVQLVQSGAEVI <I<PGESLKISCI<GSGYSFTGYTMNWVRQMPGI<GLEWMGRINPHNG

[0345] GTDYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYYSLDYWGQGT

[0346] LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

[0347] FP AVLQS SGLYSLS S VVTVPS S SLGTQT YICVNNHKPSNTKVDKRVEPKSCDKTHTCPP

[0348] CPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0349] NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

[0350] PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0351] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0352] SEQIDNO:78 (light chain of parent anti-TfR antibody H6L7): EIVLTQSPATLSLSPGERATLSCSASSSIRYIHWYQQKPGQAPRRWIYDTSNLASGTPA

[0353] RFSGSGSGTSYSLTISSLEPEDFAVYCHQRNSYPWTFGQGTKVEIKRTVAAPSVFIFPP

[0354] SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0355] TLTLSKADYEKKVYACEVTHQGLSSPVTKSFNRGEC

[0356] SEQIDNO:79 (Heavy Chain Antibody Option I):

[0357] EVQLVQSGAEVKKPGESLKISCKGSGYSMTGYTMNWVRQMPGKGLEWMGRINPHS

[0358] GGTDTNQLFKDQVTISADKSISTYLQWSSLKASDTAMYYCARGYYASLEYWGQ

[0359] GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGV

[0360] HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHT

[0361] CPPCPAPEALGASSVFLFPKPKDTLMISRDPEVTVCVVDVSHEDPEVKFNWYVDGVE

[0362] VHNAKTKPREEQYNSTYRVVSVLQVLHQDWLNGKEYKCAVSNKALPAPIEKTISKA

[0363] KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVAWESNGQPENNYKTTPP

[0364] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0365] SEQIDNO:80 (Тяжелая цепь антибоды Variant II):

[0366] EVQLVQSGAEVKKPGESLKISCKGSGYSMTGYTMNWVRQMPGKGLEWMGRINPH

[0367] NGATDYNNKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYFSLDYWG

[0368] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG

[0369] VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH

[0370] TCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGV

[0371] EVHNAKTKPREEQYNSTYRVVSVLWVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0372] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

[0373] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0374] SEQIDNO:81 (Heavy Chain Antibody Option III):

[0375] EVQLVQSGAEVKKPGESLKISCKGSGYSFTGYTHNWVRQMPGKGLEWMGRVNPHN

[0376] TGTDFNQLFKDQVTISADKSISTYLQWSSLKASDTAMYYCARGYYESLDYWGQ

[0377] GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGV

[0378] HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHT

[0379] CPPCPAPELLGGSSVFLFPPKPKDTLMISRDPEVTCVVVDVSAEDPEVKFNWYVDGVE

[0380] VHNACTKPREEQYNSTYRVVSVLQVLHQDWLNGKEYKCKVSNCALGAPIEKTISKA

[0381] KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0382] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0383] SEQIDNO:82 (Heavy Chain Antibody Option IV): EVQLVQSGAEVKKPGESLKISCKGSGYSMTGYTMNWVRQMPGKGLEWMGRINPHS

[0384] GGTDTNQLFKDQVTISADKSISTYLQWSSLKASDTAMYYCARGYYYYSLDYWGQ

[0385] GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGV

[0386] HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICVNNHKPSNTKVDKRVEPKSCDKTHT

[0387] CPPCPAPELLGASSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE

[0388] VHNAKTKPREEQYNSTYRVVSVLWVLHQDWLNGKEYKCAVSNKALPAPIEKTISKA

[0389] KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVAWESNGQPENNYKTTPP

[0390] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0391] SEQIDNO:83 (Тяжелая цепь антибодите Variant V):

[0392] EVQLVQSGAEVKKPGESLKISCKGSGYSFATGYTMNWVRQMPGKGLEWMGRINPH

[0393] SGGTDTNQLFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYYSLDYWG

[0394] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG

[0395] VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH

[0396] TCPPCPAPEALGGPSVFLFPKPKDTLYISRDPEVTVCVVDVSHEDPEVKFNWYVDGV

[0397] EVHNAKTKPREEQYNSTYRVVSVLQVLHQDWLNGKEYKCAVSNKALGAPIEKTISK

[0398] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVAWESNGQPENNYKTTP

[0399] PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0400] SEQIDNO:84 (Тяжелая цепь антибоды Variant VI):

[0401] EVQLVQSGAEVKKPGESLKISCKGSGYSMTGYTMNWVRQMPGKGLEWMGRINPH

[0402] NGGTDYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYAYYSLDYWG

[0403] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG

[0404] VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH

[0405] TCPPCPAPEALGGSSVFLFPKPKDTLYISRTPEVTCVVVDVSAEDPEVKFNWYVDGV

[0406] EVHNAKTKPREEQYNSTYRVVSVLWVLHQDWLNGKEYKCAVSNKALGAPIEKTISK

[0407] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVAWESNGQPENNYKTTP

[0408] PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0409] SEQIDNO:85 (Heavy Chain Antibody Option VII):

[0410] EVQLVQSGAEVKKPGESLKISCKGSGNSFTAYSMNWVRQMPGKGLEWMGLINPHN

[0411] AGTDYNFKFKDQVTISADKSISTYLQWSSLKASDTAMYYCARGYYYFSLDYWGQ

[0412] GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGV

[0413] HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHT

[0414] CPPCPAPELLGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVE

[0415] VHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0416] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0417] SEQIDNO:86 (Heavy Chain Antibody Option VIII):

[0418] EVQLVQSGAEVKCPGESLKISCKGSGYSMTGYTMNWVRQMPGKGLEWMGRINPH

[0419] NGGTDYNQKFKDQVTISADKSISTYLQWSSLKASDTAMYYCARSYYYSKDYWG

[0420] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG

[0421] VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTH

[0422] TCPPCPAPELLGGSSVFLFPKPKDTLYITRTTPEVTCVVVDVSHEDPEVKFNWYVDGV

[0423] EVHNAKTKPREEQYNSTYRVVSVLWVLHQDWLNGKEYKCAVSNKALPAPIEKTISK

[0424] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

[0425] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0426] SEQIDNO:87 (Тяжелая цепь антибоды Variant IX):

[0427] EVQLVQSGAEVKKPGESLKISCKGSGYSFTAYTMNWVRQMPGKGLEWMGRVNPH

[0428] NTGTDFNQLFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYFSLDYWG

[0429] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG

[0430] VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH

[0431] TCPPCPAPEAAGGPSVFLFPKPKDTLMISRDPEVTCVVVDVSAEDPEVKFNWYVDGV

[0432] EVHNAKTKPREEQYNSTYRVVSVLQVLHQDWLNGKEYKCKVSNKALGAPIEKTISK

[0433] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

[0434] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0435] SEQIDNO:88 (Тяжелая цепь антибодите Variant X):

[0436] EVQLVQSGAEVKKPGESLKISCKGSGYSFTAYTMNWVRQMPGKGLEWMGRINFPH

[0437] NGATDYSQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGYYYASLEYWG

[0438] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG

[0439] VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH

[0440] TCPPCPAPELLGGSSVFLFPKPKDTLYITRDPEVTVCVVDVSHEDPEVKFNWYVDGV

[0441] EVHNAKTKPREEQYNSTYRVVSVLWVLHQDWLNGKEYKCKVSNKALGAPIEKTISK

[0442] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVAWESNGQPENNYKTTP

[0443] PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0444] SEQIDNO:89 (Antibody Light Chain Variant I):

[0445] EIVLTQSPATLSLSPGERATLSCSASSSLRYIHWYQQKPGQAPRRWIYDTSNLSSGTPA

[0446] RFSGSGSGTSYSLTISSLEPEDFAVYYCHQKNSYPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0447] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0448] SEQIDNO:90 (Antibody Light Chain Variant II):

[0449] EIVLTQSPATLSLSPGERATLSCSASSAIRYIHWYQQKPGQAPRRWIYDTSQLSSGTPA

[0450] RFSGSGSGTSYSLTISSLEPEDFAVYYCHQRNSYPWTFGQGTKVEIKRTVAAPSVFIFP

[0451] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0452] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0453] SEQIDNO:91 (Antibody Light Chain Variant III):

[0454] EIVLTQSPATLSLSPGERATLSCSASSSAIRYLHWYQQKPGQAPRRWIYDTSNLASGT

[0455] PARFSGSGSGTSYSLTISSLEPEDFAVYYCHQKNSYGPWTFGQGTKVEIKRTVAAPSV

[0456] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

[0457] YSLS STLTLSKADYEKHKVYACEVTHQGLS SP VTKSFNRGEC

[0458] SEQIDNO:92 (Antibody Light Chain Variant IV):

[0459] EIVLTQSPATLSLSPGERATLSCSASSSIRYIHWYQQKPGQAPRRWIYDTSQLSSGTPA

[0460] RFSGSGSGTSYSLTISSLEPEDFAVYYCHQKNSYPWTFGQGTKVEIKRTVAAPSVFIFP

[0461] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0462] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0463] SEQIDNO:93 (Antibody Light Chain Variant V):

[0464] EIVLTQSPATLSLSPGERATLSCSASSSIRYIFWYQQKPGQAPRRWIYDTSNLASASPA

[0465] RFSGSGSGTSYSLTISSLEPEDFAVYYCHQKNSYSPWTFGQGTKVEIKRTVAAPSVFIF

[0466] PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0467] S STLTLSKADYEKHKVYACEVTHQGLS SP VTKSFNRGEC

[0468] SEQIDNO:94 (Antibody Light Chain Variant VI):

[0469] EIVLTQSPATLSLSPGERATLSSCSASSSAIRYIFWYQQKPGQAPRRWIYDTSQLASATP

[0470] ARFSGSGSGTSYSLTISSLEPEDFAVYCHQRNSYPWTFGQGTKVEIKRTVAAPSVFIF

[0471] PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0472] S STLTLSKADYEKKVYACEVTHQGLS SP VTKSFNRGEC

[0473] SEQIDNO:95 (Light chain antibody Option VII):

[0474] EIVLTQSPATLSLSPGERATLSCSSSSSIRYIHWYQQKPGQAPRRWIYDTSNLASGTPA

[0475] RFSGSGSGTSYSLTISSLEPEDFAVYCHQRNSYPWFGQGTKVEIKRTVAAPSVFIFPP

[0476] SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0477] TLTLSKADYEKKVYACEVTHQGLSSPVTKSFNRGEC

[0478] SEQIDNO:96 (Light Chain Antibody Option VIII): EIVLTQSPATLSLSPGERATLSCSASSSLRYIHWYQQKPGQAPRRWIYDTSNLSSGTPA

[0479] RFSGSGSGTSYSLTISSLEPEDFAVYCHQRNSYPWTFGQGTKVEIKRTVAAPSVFIFP

[0480] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0481] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0482] SEQIDNO:97 (Antibody Light Chain Variant IX):

[0483] EIVLTQSPATLSLSPGERATLSCSASSAIRYIHWYQQKPGQAPRRWIYDTSNLASGTP

[0484] ARFSGSGSGTSYSLTISSLEPEDFAVYYCHQRNSYPWTFGQGTKVEIKRTVAAPSVFIF

[0485] PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0486] S STLTLSKADYEKHKVYACEVTHQGLS SP VTKSFNRGEC

[0487] SEQIDNO:98 (Antibody Light Chain Variant X):

[0488] EIVLTQSPATLSLSPGERATLSCSASSSLRYIHWYQQKPGQAPRRWIYDTSNLSSGTPA

[0489] RFSGSGSGTSYSLTISSLEPEDFAVYYCHQKNSYGPWTFGQGTKVEIKRTVAAPSVFIF

[0490] PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0491] S STLTLSKADYEKHKVYACEVTHQGLS SP VTKSFNRGEC

[0492] SEQIDNO: 99(antibody heavy chain variant I conjugated to IDS):

[0493] MKWVTFISLLFLFSSAYSEVQLVQSGAEVKKPGESLKISCKGSGYSMTGYTMNW

[0494] VRQMPGKGLEWMGRINPHSGGTDTNQLFKDQVTISADKSISTAYLQWSSLKASDTA

[0495] MYYCARGYYYASLEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK

[0496] DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0497] PSNTKVDKRVEPKSCDKTHTCPPCPAPEALGASSVFLFPPKPKDTLMISRDPEVTCVVV

[0498] DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLQVLHQDWLNGKEY

[0499] KCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA

[0500] VAWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

[0501] HYTQKSLSLSPGKGGGGSGGGSETQANSTTDALNVLLIIVDDLRPSLGCYGDKLVRSP

[0502] NIDQLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGNFSTIP

[0503] QYFKENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGEL

[0504] HANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPK

[0505] EFQCLYPLENITLAPDPEWPDGLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRK

[0506] IRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAFTSDHGWALGEHGEWAKYSNFD

[0507] VATHVPLIFYVPGRTASLPEAGEKLFPYLDPFDSASQLMEPGRQSMDLVELVSLFPTLA

[0508] GLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEEDPYLPGNPRELIAYSQYPRPS

[0509] DIPQWNSDKPSLCDIKIMGYSIRTIDYRYTVWVGFNPPDEFLANFSDIHAGELYFVDSDP

[0510] LQDHNMYNDSQGGDLFQLLMP SEQIDNO: 100 (light chain antibody option I):

[0511] MKWVTFISLLFLFSSAYSEIVLTQSPATLSLSPGERATLSCSASSSLRYIHWYQQKPGQ

[0512] APRRWIYDTSNLSSGTPARFSGSGSGTSYSLTISSLEPEDFAVYYCHQKNSYPWTFGQ

[0513] GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0514] NSQESVTEQDSKDSTYSLSSTLLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0515] SEQIDNO: 101 (CDR1 heavy chain of parental anti-TfR antibody H6L7):

[0516] GYSFTGYTMN

[0517] SEQIDNO:102 (CDR2 of heavy chain of parental anti-TfR antibody H6L7): RINPHNGGTDYNQKFKD

[0518] SEQIDNO:103 (CDR3 of heavy chain of parental anti-TfR antibody H6L7): GYYYYSLDY

[0519] SEQIDNO:104 (light chain CDR1 of parental anti-TfR antibody H6L7):

[0520] SASSSIRYIH

[0521] SEQIDNO:105 (light chain CDR2 of parental anti-TfR antibody H6L7):

[0522] DTSNLASGTPA

[0523] SEQIDNO:106 (light chain CDR3 of parental anti-TfR antibody H6L7): HQRNSYPW

Claims

CLAUSE OF INVENTION 1. A humanized monoclonal antibody to transferrin receptor 1 (TfR1) that has the ability to penetrate the BBB and has an affinity for TfR1 of at least a Kd < 10 dB < 500 nM, comprising sets of heavy chain CDR1-3 and light chain CDR1-3 sequences selected from a) SEQIDNOs: 19, 29, 36, 45, 57, 70; b) SEQIDNOs: 19, 27, 34, 43, 59, 106; c) SEQIDNOs: 21, 31, 35, 54, 105, 73; d) SEQIDNOs: 19, 102, 39, 45, 57, 106; d) SEQIDNO: 22, 28, 36, 46, 57, 73; in this case, the effector functions of the antibody are absent or significantly reduced by introducing amino acid mutations in the Ec domain.

2. The antibody according to item 1, wherein the mutations in the Fc domain are selected from L234A and / or L235A and / or G236A and / or P237S and / or H268A and / or N297A and / or K322A and / or P329S.

3. The antibody according to claim 1 or 2, additionally containing mutations in the Fc domain M252Y and / or T256D and / or T307Q and / or T307W and / or S254T and / or E380A.

4. The antibody according to I.1, comprising a) a heavy chain with SEQIDNO: 79 and a light chain with SEQIDNO: 89; b) a heavy chain with SEQIDNO: 80 and a light chain with SEQIDNO: 90; c) a heavy chain with SEQIDNO: 81 and a light chain with SEQIDNO: 91; d) a heavy chain with SEQIDNO: 86 and a light chain with SEQIDNO: 96; d) a heavy chain with SEQIDNO: 88 and a light chain with SEQIDNO:

98.

5. A nucleic acid encoding an antibody according to any one of claims 1-4.

6. An expression vector containing the nucleic acid according to I.

5.

7. A host cell comprising an expression vector according to claim 6 for expressing an antibody according to any one of claims 1-4.

8. The host cell according to I.7, which is a CHO or HEK3 cell.

9. A method for producing an antibody according to any of paragraphs 1-4, by culturing host cells according to paragraph 7 or 8, isolating and purifying the antibody.

10. A conjugate of an antibody according to any of paragraphs 1-4 with a therapeutic agent for the treatment of diseases requiring delivery of a drug across the BBB.

11. An antibody conjugate according to i.10, wherein the therapeutic agent is linked to the C-terminus of the heavy chain of the antibody.

12. The conjugate according to paragraph 10-11, wherein the antibody is linked to the therapeutic agent via a peptide linker selected from: GG, GGG, GGGG, GGGGGSG, GGGGSGGG, GGS(GGS) 2 G, (GGS) 3 GG, (GGS) 4 GG, (GGS) 5 GG, (GSG) 2 GG, (GSG) 3 GG, (GSG) 4 GG, (GSG) 5 GG,SGGGGIG,(SGGGG) 3 IG OR (SGGGG) 4 IG.

13. The conjugate according to nn.10-12, wherein the therapeutic agent is an enzyme replacement therapy agent.

14. The conjugate of claim 13, wherein said therapeutic agent is selected from the following enzymes: alpha-L-iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase, N-acetyl glucosamine-6-sulfatase, rgaallactoosamine-4-sulfatase, beta glucuronidase, hyaluronidase, beta galactosidase, beta-hexosaminidase, beta-glucocerebrosidase, imiglucerase, arylsulfatase A, alpha Galactosidase A, N-acylsphingosin amidohydrolase, acid sphingomyelinase, alpha mannosidase, beta mannosidase, alpha-fucosidase, aspartyl glucosaminidase, aN-acetylgalactosaminidase, α - neuraminidase, a-1,4-glucosidase, alglucosidase alpha, avalglucosidase alpha, lysosomal acid lipase, Cathepsin K, palmitoyl protein thioesterase, tripeptidyl peptidase I.

15. The conjugate according to paragraph 14, wherein the therapeutic agent is selected from iduronate-2-sulfatase or acidic alpha-1,4-glucosidase.

16. The conjugate according to paragraph 15, wherein the therapeutic agent is iduronate-2-sulfatase fused to the C-terminus of the heavy chain of the antibody via a GGGGSGGG or GGSGGSGGSG linker.

17. The conjugate of claim 15 or 16, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 89-91, 96 or 98, and the heavy chain of the antibody is linked via a linker to human iduronate-2- sulfatase and has the amino acid sequence of SEQ ID NO: 79-81, 86 or 88.

18. The conjugate according to I.17, comprising a light chain with SEQ ID NO: 100 and a heavy chain conjugated to human iduronate-2-sulfatase with SEQ ID NO:

99.

19. The conjugate of any one of paragraphs 10-18, wherein the disease is selected from the following: Fabry disease, Gaucher disease, Wolman disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type IV, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type IX, GM1 gangliosidosis, Pompe disease, Tay-Sachs disease, Sandhoff disease, galactosylceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, Alpha-mannosidosis, Beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, mucolipidosis 31, mucolipidosis II, mucolipidosis III, mucolipidosis IV, pycnodysostosis, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

20. The conjugate according to paragraph 10-12, wherein the therapeutic agent is a small molecule or small interfering RNA used to reduce the expression of target genes.

21. The conjugate according to I.19, wherein the genes are selected from the following: GYSI, DMD, FBX032, TRIM63, FOXOI, FOXO3, MSTN, DM1, DM4, SCN9A, CFB, TMPRSS6, MYOC, APOE, MART, TTR, C9orf72, ANGPTL3, APOC3, KNK, LRRK2.

22. A pharmaceutical composition for the treatment of diseases requiring delivery of a medicinal product across the BBB, containing a conjugate according to p. 10-18 in an effective amount and pharmaceutically acceptable ingredients.

23. Use of the antibody according to paragraphs 1-4 to obtain conjugates with drugs intended for the delivery of said drugs across the BBB.

24. Use of a conjugate according to paragraphs 10-18 or a composition according to paragraph 22 for the treatment of neurological diseases.

25. The use according to item 24, wherein the disease is selected from the following: Fabry disease, Gaucher disease, Wolman disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type IV, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type IX, GM1 gangliosidosis, Pompe disease, Tay-Sachs disease, Sandhoff disease, galactosylceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, Alpha-mannosidosis, Beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, mucolipidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, pycnodysostosis, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

26. A method for treating diseases requiring delivery of a medicinal product across the BBB, comprising administering an effective amount of a conjugate according to paragraph 10-18 or a composition according to paragraph 22 to a patient in need thereof.

27. The method according to item 26, wherein the disease is selected from the following: Fabry disease, Gaucher disease, Wolman disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type IV, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type IX, GM1 gangliosidosis, Pompe disease, Tay-Sachs disease, Sandhoff disease, galactosylceramide lipidosis, metachromatic leukodystrophy, Farber syndrome, Niemann-Pick disease, Alpha-mannosidosis, Beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, mucolipidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, pycnodysostosis, ceroid lipofuscinosis type I, ceroid lipofuscinosis type II.

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