UA75318C2 - Antibodies against protein being affined to parathyreoid human hormone - Google Patents

Abstract

The invention relates to chimeric and humanized antibodies against albumen, being affined to parathyreoid human hormone, which DNA encodes indicated antibodies, recombinant vector which contains mentioned DNA, transformant, regenerated by means of mentioned recombinant vector, method for obtaining the mentioned antibodies and their use.

Description

Description of the invention

This invention relates to a human/mouse chimeric antibody containing the variable region (M-region) of the 2 Mmoclonal mouse antibody against the parathyroid hormone-related protein and the constant region (C-region) of the human antibody; to a humanized antibody in which the hypervariable regions, M-regions of the lungs of the lung (I-chain) and heavy chain (H-chain) of a mouse monoclonal antibody against the parathyroid hormone-related protein (PTNHgR) are transplanted into a human antibody chain; to the I - and H-chains of the specified antibody, as well as to the polypeptide, which has an M-region consisting of the I - or H-chain of the specified antibody. 70 This invention also relates to DNA containing the sequence of bases encoding the above-mentioned antibody, in particular, its M-region, and to DNA encoding the I- or H-chain creating the M-region. This invention further relates to a recombinant vector containing this DNA and to a host transformed by said vector.

In addition, this invention relates to methods of obtaining chimeric and humanized antibodies against RTNHR. This 72 invention further relates to a pharmaceutical composition and medicinal product for suppressing hypercalcemia or improving hypophosphatemia, which contains an antibody against RTNH as an active ingredient.

Hypercalcemia due to malignancy is a serious complicating symptom found in 5-2090 of all subjects with malignancy. This is considered a terminal symptom of a malignant tumor and inevitably leads to the death of the subject in the absence of appropriate treatment. Controlling hypercalcemia can significantly affect the prognosis and quality of life of the subject, so its purpose is very important.

As a rule, hypercalcemia in subjects with a malignant tumor can be roughly classified as humoral hypercalcemia of malignancy (HMM), arising under the influence of tumor-forming humoral factors of bone tissue resorption, and as local osteolytic hypercalcemia (ION), arising as a result of the local influence of the tumor transferred or infiltrated into bone tissue. It is believed that in the case of humoral hypercalcemia of a malignant disease, the content of the flow of calcium increases due to the resorption of He) bone tissue or due to osteolization, leading to hypercalcemia in combination with the deterioration of the ability of the kidneys to excrete calcium | Z.U/ada apa M. Madaya, Ipiegpa ! Medisipe, 69,644-6481.

Symptoms of hypercalcemia usually appear when the concentration of calcium in the serum is more than 12 mg/ml; although its non-specific symptoms, such as anorexia (lack of appetite), nausea and vomiting, are observed already at the early stage of a malignant disease. As hypercalcemia develops, the subject's ability to concentrate water decreases due to damage to the renal peripheral tubules, which leads to increased urine output (polyuria), while anorexia and nausea are accompanied by dehydration of the body due to insufficient water absorption. yu

Moseley J.M. and others found that one of the humoral factors causing humoral hypercalcemia of malignant disease is a protein related to parathyroid hormone (PTH) (hereinafter referred to as "PTHgR") in

I (Rhos. Mai). Asad Zsi., OBA (1987) 84, 5048-5052.

Following this, the gene encoding RTNHR |Zima I... ei was isolated. A.I., Zsiepse (1987) 237, 893). The analysis of this gene showed the presence of three types of human RTNHR, having 139, 141 and 173 amino acids, as a result of alternate splicing of this gene, and the presence of various fragments in the blood as a result of limited cleavage of RTNHR C 50 (1-139) with the complete structure |Vara, N., Siipisai SaiIsisht (1995) 5, 229-223). In RTNHR, 8 amino acids from the M-terminal 13 amino acids are identical to the indicated amino acids of parathyroid hormone. It follows that

The "Iz" amino acid site corresponding to positions 14-34 has a spatial structure that is also similar to parathyroid hormone. Thus, PTNHR and PTN bind to the common receptor PTN/PTNgR at least in the M-terminal region Yuerrpeg, N. ei aiYi., Zsiepse (1991) 254, 1024-1026; Aroi-bZatga, A-V. eh oh

Rkos May). Asad Zsi., OZA (1992) 89, 2732-2736). and As is known, RTNHGR is produced by various tumor tissues, while it has been established that RTNHGR sl is produced not only in the tumor, but also in various normal tissues starting from the fetus and ending in adult subjects. Such tissues include the skin, central nervous system, uterus, placenta, mammary glands during lactation, thyroid gland, parathyroid gland, adrenal gland, liver, kidneys, and urinary bladder IVigpv, - 70 MU.3., Sp. Spent (1992) 38, 2171-2183; Byemzh-ap. A.R. 5 Vgoadiv, A.E., U. Sp. Epdosghtoi (1991) 71, 1410-1414). In addition, it is believed that RTNGR plays an important role in the metabolic regulation of calcium, the content of which is higher in the fetus and newborn than in the mother.

It is known that RTN/RTNGR receptors are located mainly in bones and kidneys |S. ZPidepo, Siipisa! SaiYisit (1995) 5, 353-359) and activate several systems of intracellular signal transmission by binding RTNHR with 29 receptors. One of them is allenylate cyclase, and the other is phospholipase C. Activation of allenylate cyclase increases

HF) concentration of intracellular cyclic adenosine monophosphate (in AMP), which activates protein kinase A.

Phospholipase C cleaves phosphatidylinositol-4,5-bisphosphonate with the formation of inositol-1,4,5-triphosphonate and diacylglycerol. In the specified signal transmission systems (z-protein |Soietap, O.T.

Viospetisa! tespapiztev oi ragaiyugoid pogtope asip. Ip: "Te ragaiyugoiidv" (Viegikiap, O.R. esa!Y), Kamep 60 Rgevv, Mem/ Hogk (1994) 2391.

Under the influence of these signaling systems, RTHGR causes hypercalcemia and hypophosphothemia, reduces the ability of phosphate resorption by the kidneys, increases the excretion of UAMF by the kidneys, and determines other similar processes that occur in the case of hypercalcemia of malignant disease (NM).

Thus, it was established that RTNgR is directly related to hypercalcemia caused by a malignant tumor. Calcitonin, steroids, indomethacin, inorganic phosphate salts, bisphosphonates, and similar agents, as well as fluid repletion agents, are used to treat hypercalcemia associated with malignant tumors. However, these drugs are characterized by a decrease in effectiveness with long-term use, the occurrence of serious side effects and a slow manifestation of their pharmacological action, therefore, new drugs with a stronger therapeutic effect and less pronounced side effects are needed.

Kukreya S.S. and others (Kikgeda, 5.S. ei. a. reported that for the treatment of hypercalcemia caused by a malignant tumor, they used a neutralizing antiserum against RTNgGR. When this antiserum was administered to nonstimulated mice that were transplanted with human lung or larynx cancer cells, which 7/0 It causes hypercalcemia, a decrease in the calcium content in the blood was observed. Her level of UAMF in the urine.

Ipmevi (1988) 82, 1798-1802). Kanya Sato and others. reported that with the introduction of antibodies against RTNgGR. (1-34) "naked" mice, which were implanted with an RTNgR-producing human tumor, the symptoms of hypercalcemia became less pronounced and the lifespan of the mice significantly increased (). Vope 5 Mipe. Caves. (1993) 8, 849-860). Further, in the open publication of Japanese patent application Mo4-228089, mouse/human chimeric antibodies against human RTNgGR 7/5 (1-34) are described.

Mouse monoclonal antibodies have a strong immunogenic effect (sometimes defined as "antigenic") in the human body, which limits the use of monoclonal antibodies for human treatment. For example, a mouse antibody injected into a human may be metabolized as a foreign substance; therefore, the half-life of the mouse antibody from the human body is relatively short, and it does not provide the necessary effect. In addition, an increase in the level of anti-mouse antibodies (MAA) in the human body when mouse antibodies are administered can cause immune reactions that are unacceptable or dangerous to the patient, such as serum sickness and other allergic reactions. Therefore, mouse monoclonal antibodies are often unsuitable for administration to humans.

To solve these problems, methods were developed aimed at reducing the immunogenicity of superhuman antibodies, in particular, mouse monoclonal antibodies. One of these methods is the creation of a chimeric antibody in which the variable region (M-region) is isolated from a mouse monoclonal antibody and the constant region (C-region) is isolated from the corresponding human antibody.

Since the resulting chimeric antibody has an intact variable region of the original mouse antibody, it can be expected that this chimeric antibody will bind to the antigen with the same specificity as the original mouse antibody. In addition, such a chimeric antibody has a much smaller part of the amino acid sequence (Fuzo obtained from an animal; from the above it follows that the immunogenicity of this antibody should be lower compared to the original mouse antibody. Although the chimeric antibody binds to its antigen like the original - - a mouse monoclonal antibody, characterized at the same time by lower immunogenicity, some immune reactions to the variable region of the mouse may still occur (I oVidiyir, A.R. ei ai., Rgos. Mai). Asad. Zsi. O5A, 86 , 4220-4224, 1989). at

The second method of reducing the immunogenicity of mouse antibodies is more complex, but, as it is read, allows to further reduce the potential immunogenicity of mouse antibodies.

In accordance with this method, only the hypervariable region (HIV) of the variable region of the mouse antibody is transplanted into the human variable region in order to create a reconstructed human variable region. If necessary, a partial amino acid sequence of the framework region (EC) containing hypervariable regions in the variable region of a mouse antibody can be transplanted into the human variable region, in order to create a structure of hypervariable regions in the reconstructed human variable region, which is closer to the structure of the original mouse antibody. ;" These humanized reconstructed human variable regions are then connected to human constant regions.

In the finally reconstructed humanized antibody, foreign amino acid sequences are only hypervariable regions and a very small part of the framework region. Hypervariable sites consist of -I hypervariable amino acid sequences and do not have any species-specific sequences. Therefore, a humanized antibody containing mouse hypervariable regions has no stronger immunogenicity than a natural human antibody containing human hypervariable regions. 2) The use of humanized antibodies is discussed in the following references: Kiesptapp, GI. Eisai., Mayige, 332, 323-327, 1988; Megpoeue, M. Eya!Y., Zsiepse, 239, 1534-1536, 1988; KeSherogotsudp, S.A. hey). Rgoivip Epopa, - 4, 773-783, 1991; Maeyeeda, N. eiaI,, Nytap Apiiirodiez apa NuBgidota, 2, 124-134, 1991; bootup, 5.0. eyai., from Rgos. Property Asad si. OBA, 88, 4181-4185, 1991; Tetrezi, R.K. eh AI., BioLesppoiodu, 9, 266-271, 1991; So,

M.5. eh aiYi., Rhos. May Asai. 5 O5BA, 88, 2869-2873, 1991; Sapeg, R. ei. aiYi.,, Rhos. May). Asai. 5

OBA, 89, 4285-4289, 1992; So, M.5. eh AI., U. Ittipoi, 148, 1149-1154, 1992; apa ayu, K. eyi. AIY., Sapseg vv Kev., 53, 851-856, 1993).

Although it is believed that humanized antibodies should be useful for therapeutic purposes, in the aforementioned

F) the references do not contain any mention of a humanized antibody against RTRGN. In addition, a standardized method that could be applied to any antibody to obtain humanized antibodies has not yet been developed; therefore, there is a need to create effective methods of obtaining a humanized antibody, which is characterized by sufficient activity of binding and neutralizing a specific antigen | see, for example, Za, To. eh ai.,

Sapseg Kezv., 53, 851-856, 1993).

The object of the present invention is a human/mouse chimeric antibody containing the variable region (M-region) of a mouse monoclonal antibody against RTRgHN and the constant region (C-region) of a human antibody; a humanized antibody in which the hypervariable regions of the M-regions of the lung chain (I-chain) and heavy chain 65 (H-chain) of the mouse monoclonal antibody against RTRGN are transplanted into a human antibody; I- and H-chains of the specified antibody, as well as a polypeptide containing the M-region consisting of the I- or H-chain of the specified antibody.

Another object of the present invention is to obtain DNA containing the base sequence encoding the above-mentioned antibody, in particular, its M-region, and DNA encoding the I- or H-chain. holding polypeptide containing

M-region. Another object of the present invention is the creation of a recombinant vector containing the specified DNA and a host transformed by the specified vector. Another object of the present invention are methods of obtaining chimeric and humanized antibodies against RTRGN. Another object of the present invention is to obtain antibodies against

RTRGN, which has strong neutralizing activity. Another object of the present invention is the creation of a pharmaceutical composition and means for suppressing hypercalcemia, or improving hypophosphatemia and/or alkalosis containing an antibody or a humanized antibody against RTRGN as an active ingredient.

As a result of a comprehensive study aimed at achieving the above-mentioned goals, the authors of the present invention obtained an antibody with low immunogenicity of mouse monoclonal antibodies against RTRGN in the human body; thus the present invention was completed.

The object of the present invention is a chimeric I-chain containing the C-region of the I-chain of a human antibody and the M-region of the 7/5 I-chain of a mouse monoclonal antibody against RTRGN. The M-region of the I-chain contains one amino acid sequence expressed by the sequence with identifying Mo45, and the C region of the I chain includes the CX region.

The object of the present invention is also a chimeric H-chain containing the C-region of the H-chain of a human antibody and

M-region of the H-chain of a mouse monoclonal antibody against RTRGN. The M-region of the H-chain contains the amino acid sequence expressed by the sequence with the identification Mo46, and the C-region includes the C)-region.

In addition, the object of the present invention is a chimeric monoclonal antibody against RTRGN containing the specified chimeric I-chain and the specified chimeric H-chain.

The object of the present invention is further a polypeptide having the M-region I-chain of a humanized antibody, which contains the framework regions 1-4 of the M-region I-chain of a human antibody and the hypervariable regions 1-3 of the M-region I-chain of a mouse monoclonal antibody against RTRGN. Hypervariable sites 1-3 contain amino acid sequences, p

Expressed respectively by sequences with identification MoMo 59-61; framework regions 1-3 are obtained from framework regions 1-3 of human antibody NZIOOZ868 and framework region 4 is obtained from framework region 4 of human antibody o 525755; or framework regions 1-3 are essentially identical to framework regions 1-3 of human antibody NZIOZ3868, and framework region 4 is essentially identical to framework region 4 of human antibody 525755.

The term "substantially identical" means that the framework regions of the human antibody used in the humanized antibody may have deletions, substitutions, and/or additions of amino acids necessary to form the hypervariable regions of the mouse monoclonal antibody, as a result of which the humanized antibody should possess an activity that equivalent to the activity of a mouse monoclonal antibody. co

Thus, this invention relates to a polypeptide having the M-region of the I-chain of a humanized antibody, in which the 36th and 49th amino acids in the framework regions in accordance with the recommendations of Kabat |Kabai, E.A. ehaI,, 05 juyu

Oeri Neak apa Nytap zZegmisez, OZ Somegptepi Rgipipuo Oyisez, 1991) are, respectively, tyrosine and y-aspartic acid.

This invention also relates to a polypeptide having the M-region of the I-chain of a humanized antibody, which contains an amino acid sequence expressed by any sequence with the identification MoMo 48-51.

This invention further relates to a polypeptide having the M-region of the I-chain of a humanized antibody, where the 45th and 87th amino acids in the framework regions, in accordance with Kabat's recommendations, are respectively lysine and isoleucine. and This invention further relates to a polypeptide having an M-region of the I-chain of a humanized antibody, which contains the "" amino acid sequence expressed by any sequence with the identification MoMo 52-55.

This invention further relates to a polypeptide having an M-region of the H-chain of a humanized antibody that contains

Framework regions 1-4 of the M-region of the H-chain of the M-region of the human antibody and hypervariable regions 1-3 of the M-region and the H-chain of the M-region of the mouse monoclonal antibody against human RTRGN. Hypervariable regions 1-3 contain amino acid sequences, expressed respectively by sequences with identification MoMo 62-64, framework regions 1-4 are isolated from framework regions 1-4 of human antibodies belonging to the human subgroup PI (HO

Ge) 111, Kabrai, E.A. eh ai., OB Oeri. Neay apa Nytap Zegmisevz, O5 bomegptepi Rgipiipd Oiisev. 1991), in particular, 5o from framework regions 1-4 of human antibody 531679, respectively, or they are essentially identical to framework regions 1-4 of human antibody 531679, respectively. In addition, this invention relates to a polypeptide having the M-region of the H-chain of a humanized antibody, which contains the amino acid sequence expressed by the sequence with the identification Mo56.

This invention also relates to the I-chain of a humanized antibody against human PTNgR, which contains a polypeptide having the M-region of the I|-chain of said humanized antibody and a polypeptide having a C-region

Y-chain of human antibodies. The C-region includes the C;X-region, framework regions 1-3 are essentially identical to framework regions

F, regions 1-3 of the human antibody NZIOZ868, the framework region 4 is essentially identical to the framework region 4 of the anti-human antibody 525755, and the amino acid sequences of the hypervariable sites 1-3 are expressed, respectively, by sequences with the identification MoMo 59-61. 60 This invention further relates to the H-chain of a humanized antibody against human PTNgR, which contains polypeptides having the C-region of the H-chain and the M-region of the H-chain of the indicated human antibody. The C-region includes C), the 1-region, the framework regions 1-4 are isolated from the framework regions 1-4 of the human antibody related to H5OSh, and the hypervariable regions 1-3 contain amino acid sequences expressed, respectively, by sequences with the identification MoMo 62-64 . 65 This invention further relates to an antibody against RTNGR with weak antigenicity and high neutralizing activity. The anti-RTHgR antibody includes a human antibody, a humanized antibody, a chimeric antibody, and a primed antibody that can be used to treat human diseases. This antibody is characterized by a low dissociation constant. In addition, the antibody according to the present invention has high neutralizing activity due to the low dissociation constant, so it can be used for the treatment of human diseases.

The antibody of the present invention has a dissociation constant of 1.86x1077 MI or less, a dissociation rate constant of 1.22x107 |1/sec| or less, and the association rate constant equal to 6b,55x107 |1/M.sec| or more These constants can be measured by Scatchard analysis using radiolabeled ligands or a surface plasmon resonance sensor.

The present invention further relates to DNA containing a base sequence encoding the M region of the I chain or

The M-region of the H-chain of a mouse monoclonal antibody against human RTNGR. M-region of the I-chain and M-region

The H-chain contains amino acid sequences, expressed respectively by sequences with identification MoMo 45-46; The DNA encoding the M-region of the I-chain contains, for example, the sequence of bases expressed by the sequence with the identification Mob5, and the DNA encoding the M-region of the H-chain contains the sequence of bases expressed by the sequence with the identification Mo57.

This invention further relates to DNA encoding said chimeric I- or H-chain. The DNA encoding the I chain contains, for example, the sequence of bases expressed by the sequence with the identifying Mob5, and the DNA encoding

H-chain, contains the sequence of bases, expressed by the sequence with identification Mo57.

This invention also relates to DNA containing a base sequence encoding the M region of the I chain or

The M-region of the H-chain of the specified humanized antibody. DNA containing the base sequence encoding the M region

Y-chain, expressed by any sequence with identification MoMo 66-74, and DNA containing the base sequence encoding the M-region of the H-chain, expressed by sequence with identification Mo58.

This invention also relates to DNA for the M-region |-chain of a humanized antibody, which contains a sequence of bases encoding an amino acid sequence expressed by any sequence with the sem identification MoMo 47-55. The indicated DNA contains a base sequence expressed by one of the sequences with the MoMo 66-74 identification range.

This invention further relates to DNA for the M-region of the H-chain of a humanized antibody, which encodes the amino acid sequence expressed by the sequence with the identification Mo56b6. The specified DNA contains a sequence of bases expressed by the sequence with identification Mo58. with

This invention further relates to a recombinant vector containing any of the specified DNAs. "-

This invention also relates to a transformant transformed using the specified recombinant vector. (ze)

In addition, this invention relates to a method of obtaining a chimeric or humanized antibody against a protein related to the human parathyroid hormone, which includes cultivating the specified transformant and isolating from the obtained culture a chimeric or humanized antibody against a protein related to the human paracytoid hormone.

This invention further relates to a pharmaceutical composition or means intended to suppress hypercalcemia or improve hypophosphatemia, which contain the specified antibody as an active "ingredient." Calciumemia occurs as a result of malignant disease, and hypophosphatemia is often observed in subjects suffering from hypercalcemia due to a malignant tumor. Thus, the antibody according to this invention can be used for the treatment of a malignant tumor or for alleviating the symptoms of hypercalcemia or hypophosphatemia. Malignant tissue may include, but is not limited to, at least one selected from the group consisting of cancer of the pancreas, lung, pharynx, larynx, tongue, gums, esophagus, stomach, bile ducts, breast, kidney, bladder, uterus, and prostate, as well as malignant lymphoma. Means for

This invention, designed to suppress hypercalcemia, can be used in the case of any malignant disease that causes hypercalcemia. This invention will be described in detail below. 1. Preparation of mouse monoclonal antibodies against human RTNHRI (95) Mouse monoclonal antibodies against human RTNHRI can be obtained by creating a hybridoma as a result of the fusion of myeloma cells and antibody-forming cells isolated from animals immunized with the antigen, and selection of antibody-producing clones obtained by the hybrid , which specifically inhibit the activity of RTNGR. and42) (1) Obtaining antigens

RTHGR used for animal immunization includes peptides containing all or part of the amino acid sequence of RTHGR obtained by recombinant DNA technology or chemical synthesis, and RTHGR isolated from the supernatants of cancer cells that cause hypercalcemia. o For example, as an antigen, you can use the peptide IRTNgR(1-34)3), which contains amino acids 1-34 of the known

RTNGR IKetr, V.E. eh aiYi Zsiepse (1987) 238, 1568-1570). RTNHGR (1-34) of a person has an amino acid sequence expressed by the sequence with identification Mo75.

The obtained RTNHGR is attached to a carrier protein, such as thyroglobulin, followed by the addition of an adjuvant. 60 Any adjuvant can be mixed, including complete and incomplete Freund's adjuvants. (2) Immunization and isolation of antibody-forming cells.

The antigen obtained above is injected into a mammal, such as a mouse, rat, horse, monkey, rabbit; goat or sheep.

Immunization can be carried out by any known methods, including intravenous, subcutaneous and intra-abdominal injections. Time intervals between injections for the purpose of immunization do not have any 65 specific limitations and can range from several days to several weeks, preferably from 4 days to 21 days.

Two or three days after the last immunization, antibody-forming cells are released. Antibody-forming cells are cells of the spleen, lymph node, and peripheral blood; and spleen cells are usually used. A single dose of the antigen intended for immunization is 10O0μg/mouse. (3) Determination of antibody titers

To determine the levels of immune response of immunized animals and to select those of interest hybridomas from cells subjected to fusion, measure the titer of antibodies in the blood of the immunized animal or the titer of antibodies in the supernatant of antibody-forming cells.

Antibody detection methods are well known and include enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and solid-phase enzyme-linked immunosorbent assay (ELISA). 70 (4) Fusion of cells

Myeloma cells used for fusion with antibody-forming cells include cell lines obtained from various animals, such as mice, rats, and humans, and generally can be readily obtained by those skilled in the art. Acceptable cell lines are characterized by drug resistance, the inability to survive in a selective environment such as NAT medium in the unattached state, and the ability to survive in the specified /5 habitat only in the attached state. Commonly uses cell lines resistant to 8-azaguanine, which lack hypoxanthine-guanine-phosphoribosyltransferase and cannot grow in hypoxanthine-aminopterin-thymidine (HAT) medium.

Suitable myeloma cells are various known cell lines such as RZ (RZ3ХбЗА98.653)

AND). Ittipoi/(1979) 123:1548-15501; RZhbZA9d8И0.1 |Sygttepi Torisv ip Misgobioyudu apa Ittipoidu (1978) 811) 2o (1-7y; M5-1 (Compieg, b apa Miiviei, S, E. U. Ittipoi. (1976) 6:511-519); MRO -11 |Magoshev, O.N.

Hsieh (1976) 8:405-415)1; 5R2/O |ZpcItap, M. ei. AI., Maishge (1978) 276:269-1701; RO (de 5 Ogo, 5.R. ei. aI., U. IttypoYi. Meodz (1980) 35:1-211; 5194 Ptomrgidde, I.5, U. Ehr. Mea. (1978) 148:313-323 | and k210

ISaite, 0. ei. AI., Makighe (1979) 277:131-1331.

Antibody-forming cells can be obtained from spleen, lymph node, or similar cells. For this purpose, the spleen, lymph node, or other organ is removed from any of the above-mentioned animals and the tissue is shredded. The resulting crushed material is suspended in a medium or buffer, such as i) phosphate-buffered saline (PB5), Dulbecco's modified Eagle's medium (OMEM) or KRMI1640, filtered through a stainless steel filter or similar and centrifuged to obtain the necessary antibody-forming cells. Then the specified myeloma cells and antibody-forming cells are subjected to fusion.

Cell fusion can be achieved by combining myeloma and antibody-forming cells at a ratio of -171 to 1:10 in an animal cell culture medium such as minimal support medium (MEM), with

OMEM or KRME-1640, in the presence of a fusion accelerator at a temperature of 30-372C for 1-15 minutes. Any fusion accelerator or virus such as polyethylene glycol with an average molecular weight of 1000-6000, polyvinyl alcohol or Sendai virus can be used to accelerate cell fusion. Fusion of antibody-forming and myeloma cells can also be performed in a commercially available cell fusion device using electrical stimulation, such as electroporation. (5) Selection and cloning of hybridomas

The hybridomas of interest are selected from the cells after the fusion of the cells, for example, by the method of "selective growth of cells in selective media." 8 s For this purpose, the cell suspension is diluted in an acceptable medium and inoculated on a titration microplate. A selective medium, such as NAT-medium, is added to each well and incubated, periodically replacing the selected medium with fresh medium.

Grown cells are collected and used as hybridomas.

These hybridomas are then examined using limited dilution, fluorescence-activated cell sorter, or other methods. And finally, hybridomas producing monoclonal antibody are obtained. (6) Isolation of monoclonal antibodies i-th To obtain monoclonal antibodies from the obtained hybrid, methods d) cultivation of cells and formation of ascites are usually used.

According to the cell culture method, the hybridomas are cultured in animal cell culture medium, such as KRMI-1640, containing 10-2095 fetal bovine serum, minimally supportive medium or serum-free medium, under acceptable conditions (e.g., 372C, 5956 ») for 2-14 days, after which antibodies are isolated from the supernatant.

In accordance with the method of formation of ascites, hybridomas are intraperitoneally inoculated into the same mammal

The type that is used as a source of myeloma cells to achieve abundant hybridoma growth. Ascites or serum is obtained after 1-4 weeks. i) To obtain purified antibodies, such well-known methods as co-precipitation with ammonium sulfate, ion exchange chromatography and affinity chromatography are used separately or in combination. 2. Construction of chimeric antibodies 60 (1) Cloning of DNA containing the sequence of bases encoding the M-region of a mouse monoclonal antibody against human RTNGR. () Receipt of MENK

In order to clone the DNA containing the sequence of bases encoding the M-region of the mouse monoclonal antibody against the human RTNgGR, the resulting hybridomas are processed by a known method, for example, by 65 guanidine ultracentrifugation (SpPigomip, U.M. ei ai., Viospetivighu (1979) 18, 5294 -5299) or by the ASRS method

ISpotsgupki, R. ei ai., Apaiuisa! Viospetivigh (1987) 162, 156-159), with obtaining complete RNA, from which mRNA is isolated, for example, in a separation column with oligo (thymidine) and cellulose filler, which are included in the kit for purification of mRNA (RIagtasia). A purification kit can also be used to obtain mRNA

MRNA OcyskK Rger (Rpagtasia AV), while it is not necessary to isolate the complete RNA. (ii) Obtaining and amplifying cDNA

cDNA is synthesized from the mRNA obtained above in the M-regions of the I- and H-chains using reverse transcriptase. In the process of cDNA synthesis, a primer such as oligothymidine or any other suitable primer that hybridizes to the C-region of the I- or H-chain can be used, for example, the MHC2 primer having the sequence of bases expressed by the sequence with the identification Mo1. 70 For cDNA synthesis, the specified mRNA and seed are mixed and subjected to interaction in the presence of reverse transcriptase, for example, at a temperature of 522C for 30 minutes.

cDNA amplification of both the I-chain and the H-chain can be carried out by polymerase chain reaction (PCR) using the 5-KACE method | Egoptap, M.A. eh aiYi., Rhos. May Asad All together. OBA, 85, 8998-9002, 1988;

VeihuahuzkKu, A. eyi. ai., Mysivis Asids Kevz., 17, 2919-2932, 1989) using the set of 5-Atrii RIMOEK KASE 75 "SGOMTESN Ips.). Thus, the binding fragment of Atria RIMOEK (sequence with identification Mo42) is attached to the 5' end of the above-synthesized cDNA and the polymerase chain reaction is carried out in relation to

DNA containing sequences of bases encoding the M-regions of the I- and H-chains. (DNA containing the sequence of bases encoding the M region of the I chain is sometimes referred to as "DNA for the M region of the I chain" or "DNA encoding the M region of the I chain". The same applies to M-regions of the H-chain, C-regions, etc.).

An acceptable primer for the amplification of DNA encoding the M-region of the I-chain may be, for example, a binding primer (a sequence with an identifying Mo2) and primers derived from conservative sequences in the constant region of the I-chain (C;-region) of mouse antibodies , such as MI C seeds having a sequence of bases expressed by the sequence with the identification Mo4. Acceptable seed for amplification of coding DNA

The M-region of the H-chain, possibly, for example, binding seeds (the sequence with the identification Mo2) and the seeds of su

МНО-О1 (sequence with identification Мо3) |5. T. dopezv, eh. AI., Viotesppoiodu, 9, 88, 19911. (iii) DNA purification and determination of the sequence of bases i)

The products of the polymerase chain reaction (PCR) are electrophoresed in agarose gel by known methods in order to cut out the DNA fragments of interest, which are then isolated, purified and ligated from the vector DNA. eh

DNA purification can be carried out using commercially available kits, such as ZEMESI EAM 1;

VIO101. Vector DNA carrying DNA fragments is known; for this purpose, you can, for example, use РИОСС19 or --

Vinesoghiri. co

The specified DNA and vector DNA are ligated using a known kit for ligation (Takaga Zpi2o) to obtain a recombinant vector. i am

The obtained recombinant vector is introduced into EzspPegispia soii )M109 and into ampicillin-resistant colonies; in this way, vector DNA is obtained by a known method |U. Hold on, hey. aI., "Moiestsshag Siopipa", Soyd Zrgipd Nagrog

And aroghaiyuogu Rgezz, 1989). After cleavage of the vector DNA by one or more restriction enzymes, the sequence of the bases of the required DNA is determined by a known method, such as the dideoxy method (U. Zatrbgook, ei. « aI., "MoiIesciag Siopipd", Soiy Zrgipd Nagrog I arogaiogu Rgezz, 1989). When implementing this invention, you can use an automatic device for determining the sequence of bases (DNA sequencer 373ZA; AVI Ips.). - c (im) The hypervariable site of the M-region of the H- and Y-chains will form an antigen-binding site, and their complete structures have some similarity. "» That is, four sections of the frame area (EC) are connected by three hypervariable sections (SOK).

The amino acid sequence in the framework region is quite conservative, while the amino acid sequence of the hypervariable region is characterized by high variability IKabai, E.A. eiaII.,, "Zedoepse -I oi Rgoieipv oi Ittipoiodisa! Ipiegesis!", O5 Oeri. Neaky apa Nytap z5eguisez, 1983).

Many sections of the specified four framework regions have a p-fold structure, as a result of which the three gshypervariable sections will form a loop. A hypervariable region can sometimes be (95) part of a p-fold structure. Therefore, the three hypervariable sites are spatially located very close to each other due to the specified structure of the framework regions, which will form an antigen-binding site together with the three hypervariable sites in the paired regions. taking into account these factors, hypervariable sites can be detected by comparing the amino acid sequence in the variable region of the mouse monoclonal antibody against human RTNgR with the database on the structure of amino acid sequences for antibodies obtained by the method of Kabat et al. ("Zedcepse oi Rgoieip5 22 ОИ |Ittipoodisai! Ipieges!", O5 Yueri. Neaky apa Nytap Zeguisez, 1983), with the aim of researching their homology.

Gee! (2) Construction of a chimeric antibody expressing vector.

After cloning the DNA fragments encoding the M-regions of the I- and H-chains of the mouse monoclonal antibody (hereinafter, the 1-ko or H-chain of the antibody is sometimes defined as "mouse I-chain", etc. for mouse antibodies and "H- human chain" etc. for human antibodies), DNA encoding the M-regions of the mouse and DNA encoding the constant regions of the human antibody 60 are ligated and expressed to obtain chimeric antibodies against human RTNHR.

The standard method of obtaining chimeric antibodies is ligation of the mouse leader sequence and the sequence

of the M-region present in the cloned cDNA with the sequence encoding the C-region of the human antibody, which is already present in the mammalian cell expression vector. Alternatively, the leader sequence of the mouse and the sequence of the M region present in the cloned cDNA are ligated with the sequence encoding the C region of the human antibody and then ligated with a b5 expressing vector of a mammalian cell.

A polypeptide containing the C-region of a human antibody can be any C-region H- or I-chain,

including, for example, Sut, Sug2, SuZ or Sud for human H-chains or C), or Sk for I-chains.

To obtain a chimeric antibody, two expression vectors are first constructed; that is, an expression vector containing DNA encoding the M region of the mouse I chain and the C region of the human I chain under the control of an expression regulatory region such as an enhancer/promoter system, and an expression vector containing DNA encoding The M-region of the mouse H-chain and the C-region of the human H-chain, under the control of an expression-regulating region, such as an enhancer/promoter system. Host cells, such as mammalian cells, are then cotransformed with these expression vectors, after which the transformed cells are cultured with ip migo or ip mimo to obtain a chimeric antibody | see, for example, M/091/16928I. 70 Alternatively, the mouse leader sequence present in the cloned cDNA and DNA encoding the M-region of the mouse I-chain and the C-region of the human I-chain, as well as the mouse leader sequence and DNA encoding the M-region of the mouse H-chain and The C-region of the human H-chain is introduced into one expressing vector (see, for example, U/094/115231) and the indicated vector is used to transform the host cell; after which the transformed host cells are cultured by IP mimo or IP migo in order to obtain the necessary chimeric antibody. () Obtaining the H-chain of a chimeric antibody

A chimeric antibody H-chain expression vector can be obtained by introducing a cDNA containing the base sequence encoding the M-region of the mouse H-chain (hereinafter also referred to as the "M-region cDNA

H chain"), into an acceptable expression vector containing genomic DNA with a base sequence encoding

The C-region of the H-chain of a human antibody (hereinafter also referred to as "genomic DNA for the C-region of the H-chain"), 2o or cDNA encoding the specified region (hereinafter also referred to as "cDNA for the C-region of the H-chain") C-region

H-chain includes, for example, Sut, Su2, SuZ or Sud oblast. (i-a) Construction of a chimeric H-chain expression vector containing genomic DNA encoding

C-region of the H-chain.

Expression vectors containing genomic DNA encoding the C-region of the H-chain, in particular, encoding the HaSu1-region, include, for example, НЕР-РМН-ду1 (М/091/19759) and ONEK-АЕ-КМп-РМИ (M/092/19759). at

After introducing the cDNA encoding the M-region of the H-chain of the mouse into these expression vectors, the corresponding sequence of bases can be introduced into the specified cDNA by the polymerase chain reaction (PCR). For example, in order to introduce the corresponding base sequences into the expression vector, the polymerase chain reaction can be carried out with the help of a primer for RSC, which is designed to obtain a cDNA containing the recognition (he) sequence for an acceptable restriction enzyme at the 5-end and the agreed Kozak sequence, - located immediately before the initiation codon, in order to increase the efficiency of transcription, and a primer for RSC, which is designed to obtain a cDNA containing a recognition sequence for an acceptable (ze) restriction enzyme at the 3-end and a donor region, which is necessary for the correct splicing of primary products transcription of genomic DNA in order to obtain mRNA, for the introduction of the specified base sequences into the expressing vector. -

After treatment of the thus constructed cDNA encoding the M-region of the mouse H-chain with one or more suitable restriction enzymes, it is inserted into the indicated expression vector in order to create a chimeric H-chain expression vector containing the genomic DNA encoding the C-region "

H-chain (Su1-region). (I-5) Construction of a chimeric H-chain expression vector containing a cDNA with a base sequence encoding the H-chain h» Expression vectors containing a cDNA encoding the C-region of the H-chain, such as the C y1-region , can be "constructed as follows: mRNA is obtained from Chinese hamster ovary cells, into which the expressing vector ONEK-AE-KMP-RMI is introduced, see UU091/19759|, containing DNA encoding the M-region of the H-chain of the humanized RMI antibodies, and genomic DNA of the C-region of the H-chain (Su!) of human antibodies (|M. Takanazii, eyai!., Seiyi, 29, and 671-679 (1982)), and the expressing vector KM1-RMTa |see UM/091/19759), containing the genomic DNA encoding the 4! M-region of the I-chain of the humanized RMI antibody, and the genomic DNA of the C-region of the I-chain of the human antibody; using the CT-RSK method, cDNA encoding the M- the region of the H-chain of the humanized antibody RMI, and the cDNA encoding the C-region of the H-chain (Su!) of the human antibody, and ligated with the expressing vector of an animal cell, which is treated with an acceptable restriction enzyme m, to construct the desired expressing vector. co When the cDNA encoding the M-region of the mouse H-chain is ligated directly to the cDNA encoding the C-region

H-chain (SU1) of human antibodies, in the fragment containing the cDNA encoding the M-region of the H-chain, it is possible to introduce the appropriate sequence of bases by the RSK method. For example, the polymerase chain reaction can be carried out using a special primer for PCR, which is designed to obtain a cDNA containing the recognition sequence for an acceptable restriction enzyme at the 5'-end and the agreed sequence of Kozak, (F) located immediately before the initiation codon, with in order to increase the efficiency of transcription, and

He is a primer for RSK, which is intended for obtaining a cDNA containing a recognition sequence for an acceptable restriction enzyme at the 3'-end, which is necessary for direct ligation with the C-region of the H-chain (C y1) for the purpose of introducing the specified base sequences into the specified cDNA.

The thus constructed cDNA encoding the M-region of the mouse H-chain is treated with one or more suitable restriction enzymes, ligated with the cDNA encoding the C-region of the H-chain (Si1) and inserted into an expression vector such as PCO51 or pCHO. in order to create an expression vector containing a cDNA encoding a chimeric H-chain. 65 (her) Obtaining the I-chain of a chimeric antibody

Expression vector | -chain of a chimeric antibody can be obtained by ligation of the cDNA encoding the M-region

Y-chain of the mouse, from genomic DNA or cDNA encoding the C-region of the I-chain of the human antibody, and introduction into an acceptable expressing vector. The C-region of the I-chain includes, for example, the k-chain and the x-chain. (i-a) Construction of an expression vector containing a cDNA encoding a chimeric 15-chain

Having constructed an expressing vector containing the cDNA encoding the M-region of the I-chain of the mouse, the corresponding base sequences can be introduced into the indicated expressing vector by the PCR method. For example, the polymerase chain reaction can be carried out with a special primer for RSC, which is designed to obtain a cDNA containing a sequence that is recognized by an acceptable restriction enzyme at the 5-end and an agreed Kozak sequence, in order to increase the efficiency of transcription, and the primer for ROK, 70 which is designed to obtain a cDNA containing a recognition sequence for an acceptable restriction enzyme at the 3-end, which is necessary for the introduction of the indicated sequences of bases into the indicated cDNA.

The entire sequence of cDNA bases encoding the C-region of the I)-chain of a person can be synthesized using a DNA synthesizer and constructed by the ROK method. It is known that the C region of the human X chain has at least 4 different isotypes, and each isotype can be used to create an expression vector. For example, an isotype can be selected based on 75 homology data with the C-regions and the A-chain of cloned mouse monoclonal antibodies

MsodKenO2-fragment of the C-region of human IX-chain (MokhX57819) | R. Oagiamasy ea., Rgos. May Asad All together. O5A, 84, 9074-9078, 1987) and use it to create an expressing vector. To construct cDNA for the known human C-region and X-chain, such as MsdaiKetO2-, the following four primers can be used, expressed by sequences with identification MoMo 11-44: primers MVSINORI (sequence with identification Mo11) and MVSINORZ (sequence with identification Mo13) contain semantic sequences

DNA, and primers MVSINOR2 (sequence with identification Mo12) and MVSINORA4 (sequence with identification Mo14) contain antisense DNA sequences, and each primer has a complementary sequence of 20 to 23 base pairs at each end.

MVSTNORZ (sequence with identification Mo15) and MVSINORE (sequence with identification Moib) are M 259 external primers, have sequences homologous to MVSINORI and MVSINORA, respectively, and a recognition sequence for an acceptable restriction enzyme. After collecting four primers by the ESC method, synthesize a complete cDNA chain and add an external primer for cDNA amplification.

Assembly by the RSK method is carried out as follows: in MVSTNOR1 and MVSTNOR2 or MVSTNORZ and MVSTNO RA, complementary sequences are hybridized, synthesizing a fragment MVSITNORI-MVSINOR2 and a fragment with MVSInOoRZ-MVSINORA, after which complementary sequences are hybridized again in each fragment, "- synthesizing cDNA encoding the unprocessed C-region of the human X-chain.

The cDNA thus constructed encoding the human C-region 1 75-chain and the above-constructed cDNA encoding the mouse M-region I-chain can be ligated between acceptable restriction enzyme sites by inserting a Kk expression vector such as PCO51 or pCHO, in order to create an expression vector containing cDNA,

What does the X-chain of the chimeric antibody encode? - (i-b) Construction of an expression vector containing a cDNA encoding a chimeric I k-chain

Having constructed an expressing vector containing cDNA encoding the M-region of the I-chain, the corresponding sequence of bases can be introduced into the indicated cDNA using the PCR method. For example, polymerase chain reaction can be performed using a special primer for PCR, which is designed to obtain a cDNA that contains a recognition sequence for an acceptable restriction enzyme at the 5'-end and a coordinated sequence with Kozak, in order to increase the efficiency of transcription, and a primer for RSC, which is designed to obtain :z" cDNA, containing a recognition sequence for an acceptable restriction enzyme at the 3-end, which is necessary for the introduction of the indicated sequences of bases into the indicated cDNA.

DNA encoding the C-region of the human I k-chain, which is designed to be ligated with the DNA encoding the M-region of the mouse Y-chain, can be constructed, for example, from HER-RMIK-YAK containing genomic DNA |see UUO 92/19759)I.

Recognition sequences for acceptable restriction enzymes can be introduced by the ROK method in the 5-- and 1-3-end of the DNA encoding the C-region of the I k-chain, and the DNA encoding the M-region of the mouse I-chain, and the DNA that encodes the C-region of the I k-chain, can be ligated with each other and inserted into an expression vector such as pCO51 or p OSNO, in order to create an expression vector containing cDNA encoding the I k-chain of a chimeric antibody. - Z. Obtaining humanized antibodies o (1) Detection of homology with human antibodies

In order to obtain a humanized antibody in which the hypervariable region of a mouse monoclonal antibody is transplanted into a human antibody, it is desirable that there is significant homology between the framework regions of the mouse monoclonal antibody dv and the human antibody. Therefore, it is necessary to make a comparison of the M-regions of the H- and I-chains of the mouse monoclonal antibody against human RTNGR with the M-regions of all known antibodies, the structure of which

F) determined on the basis of a data bank on protein structure. In addition, at the same time, a comparison is made with subgroups of human antibodies (HO: human subgroup), classified by Kabat et al., taking into account the length of the framework region of the antibody, homology of amino acids, and the like IKarai, E.A. egai, 05 OYUOer. Neay apa Nytap bo Zemisev, 05 bomegptepi Rgipipu OpPisev, 19911.

The M-region of the human H-chain can be attributed to one of the No II-I subgroups based on the classification of Kabat et al., while the M-region of the H-chain of the mouse monoclonal antibody against human RTNHR is characterized by 82,795 homology with the agreed sequence of the No PI subgroup. On the other hand, ??M-regions??! (human -chain can be attributed to one of the subgroups of NZO1-MI based on the classification of Kabat et al., while the M-region I X-chain 65 of the mouse monoclonal antibody against human RTNGR does not have a sufficiently high homology with the agreed sequences of the M-regions I X - a chain of people included in these subgroups.

If it is necessary to humanize a mouse monoclonal antibody against human RTNgR, it is preferable to use it

The M-region of the human H-chain, which belongs to the HO PI subgroup and has the highest homology, or

The M-region of the human H-chain, in which the framework region has the corresponding standard structure (Spoipia C, ей.ай.,

U. Moi. Viol., 196, 901-917, 19871. In addition, since there is no consistent sequence with high homology in subgroups of the M-regions of the human X-chain, it is desirable to use the M-region of the human X-chain antibody with the highest homology to construct a humanized antibody homology registered in the protein structure data bank. (2) Construction of DNA encoding the M region of the humanized antibody.

At the first stage of the construction of the DNA encoding the M-region of the humanized antibody, it is necessary to select the M-region of the human antibody as the basis for the specified construction.

In the implementation of the present invention, the framework region of the M-region of a human antibody, which is homologous to the framework region of the M-region of a mouse antibody by more than 8095, can be used in a humanized antibody.

The M-region of the H-chain, which is used as a fragment of an essentially identical framework region, may include a 75 K framework region belonging to subgroup I, such as 531679 (MVKE-ROV, Scivipieg A.M. eyai., Echshg. 9.

Ittipoi, 23, 110-118, 1993). In addition, the framework region of the M-region of the I chain, which is used as a fragment of an essentially identical framework region, can include, for example, EKI, EK2 and ECZ isolated from the human antibody nzZIOOozZVb68 |(SEM-VAMK, Oepoz M. eai., 5sapa. 9. Ittipoi, 39, 95-103, 1994), and EC4 isolated from human antibody 525755 (MVKE-ROV).

The human antibody 5311679 was cloned from a human fetal liver cDNA library, and the human antibody NZOOZ868 was cloned as a novel gene for the human M-region I x-chain. (3) Preparation of polypeptides containing the M-region of a humanized antibody.

In the humanized antibody according to the present invention, the C-region and framework regions (EC) of the M-region of the specified antibody are taken from a human, and the hypervariable sections of the M-region are taken from a mouse (Fig. 1). The polypeptide containing the M-region of the humanized antibody according to the present invention can be obtained by transplanting hypervariable regions by the RSFK method using a DNA fragment of a human antibody as a matrix. The method of transplanting hypervariable regions consists in taking a DNA fragment encoding a mouse hypervariable region and replacing it with a human antibody used as a template.

If the DNA fragment of the human antibody intended for use as a template is not available, the (ee) base sequence registered in the database can be synthesized in a DNA synthesizer, and the DNA for the M region of the humanized antibody can be obtained by polymerase chain reaction (PCR). In addition, if only the amino acid sequence is registered in the database, the entire sequence of bases can be deduced from "9 of the indicated amino acid sequence, taking into account the data on the use of codons in antibodies by the method

Kabata and others. IKarai, E.A. eyai)., ip 05 YuOer. Neay apa Nytap Zegmise5, O5 (zomegptepi Rgipipd Opyisev, that 1991). This sequence of bases is synthesized in a DNA synthesizer, then DNA of the M-region of the humanized antibody is obtained by the ROK method and injected into an acceptable host with subsequent expression necessary to obtain the required polypeptide.

The following describes general procedures for transplantation of hypervariable regions using the RSC method, which are performed in the presence of a human antibody DNA fragment used as a matrix. (Y) Transplantation of hypervariable sites. - c Assume that the DNA encoding the M-region is the DNA encoding EC7!, SOCI, EC2, SOC2, and ECZ, SOCZ, and EKA, which are linked to each other in the indicated order, as shown in Fig. 2. "» First, fragments of mouse DNA corresponding to the necessary hypervariable regions are synthesized.

Hypervariable sites 1-3 are synthesized on the basis of the sequences of the bases of previously cloned M-regions H- and

I|-chain of the mouse. Transplanting seeds B and E are synthesized with such a calculation that the seed B has -| the sequence hybridizing with SOKI1 mouse and human EK2 antibodies in the direction of the sense sequence, and the primer E sl had a sequence hybridizing with SOKI1 and human EK1 antibodies in the direction of the antisense sequence (Fig. 2 (1)). Similarly, transplanting seeds C and E and seeds O and b are synthesized. In addition, the so-called "external seeds" corresponding to A and H in Fig. 2 (1) are synthesized (95), which can respectively hybridize with the upper shu 20 regions from EKI and the lower regions from EK4. Transplanting seeds can be isolated and extracted by known methods (IZatrbgookK, ei.aiYi., MoYiIescshiag Siopipd: A I arogagagu Mapia!i, Soii Zrgipd Nagbrog I arogagugu so Rgezv, 1989).

Then the first polymerase chain synthesis reaction is performed, using transplanting primer E and external primer A, transplanting primers B and E, transplanting primers C and b, as well as transplanting Z-prime 0 and external primer H, as a result of which fragments E, B-ER, C-0 and OB-H (Fig. 2 (2)).

Since the upper region of the transplant seed B and part of the lower region of the transplant seed E o have been designed to overlap each other (the same is true for the transplant seeds C and EP, and also have) OO and C), these fragments can be hybridized with the corresponding complementary sequences during the reaction under acceptable temperature conditions and assembled as a result of the polymerase chain reaction 60 with the formation of DNA length from A to H. Having obtained a DNA fragment encoding the M-region, you can add external primers A and H and perform a second polymerase chain synthesis reaction , which gives the coding DNA

M-region of a humanized antibody, in which framework regions 1-4 are isolated from humans and hypervariable regions 1-3 are isolated from mice. This DNA can then be introduced into a suitable host for expression to produce the required polypeptide (Fig. 2 (3)). 6E (i) Construction of DNA and expression vector encoding the humanized M-region of the H-chain.

In the implementation of this invention, the entire sequence of DNA bases encoding the M-region of the H-chain of a human antibody, which is intended for use as a matrix of a humanized antibody, can be synthesized in a synthesizer

DNA and construct according to the RSK method, although the indicated DNA cannot be obtained from natural sources.

The M-region of the H-chain of the mouse monoclonal antibody against the human RTNgR is characterized by high homology with 531679, which is included in the human subgroup Ii. For the use of this human antibody as a template for the construction of DNA encoding the humanized M-region of the H-chain, four primers are required, expressed, for example, by sequences with identification MoMo 23-26. The seeds of MVSYAINORI (sequence with identification Mo23) Her MVSITNORZ (sequence with identification Mo24) contain meaningful sequences

DNA, and primers MVSINOR2 (sequence with identifying Mo25) and MVSINORA4 (sequence with 70 identifying Mo26) contain antisense DNA sequences. Each of them must have a complementary sequence of 15 to 21 base pairs at either end.

External primers MVSYAINMU5Z1 (sequence with identification Mo27) and MVSTNMK1 (sequence with identification Mo28) have a sequence homologous to MVSYATNORI and MVSINORA, respectively, and each of them contains a recognition sequence for the corresponding acceptable restriction enzyme. Four primers are collected by PCR and unprocessed cDNA is synthesized, after which external primers are added for DNA amplification. "Collection by the RSK method" means the hybridization of MVSYATNORI and MVSITNOR2 or MVSINORZ and

MVSINORA by their complementary sequences to obtain the MVST1NORI1-MVSTNORZ fragment and the MVSTNOR2-MVSYAINORA fragment with subsequent hybridization of these fragments by their complementary sequences to obtain unprocessed DNA for the humanized M-region of the H-chain.

The C-region of the H-chain of a human antibody can be any C-region of the human H-chain, for example, Sut,

Sug2, SuZ or Su4 region of the human H-chain.

DNA for the M-region of the H-chain of a humanized antibody, which is obtained according to the above description, can be ligated with DNA for any C-region of the H-chain of a human antibody, for example. C in the 1-region of the human H-chain. As indicated in the section "Preparation of the H chain of a chimeric antibody", the DNA for the M region of the He H chain can be treated with an acceptable restriction enzyme and ligated with DNA encoding the C region of the human H chain under the control of an expression regulatory region such as an enhancer/promoter system to obtain an expression vector containing DNA for the humanized M-region of the H-chain and the C-region of the human H-chain. (ii) Construction of DNA and an expression vector encoding the humanized M region of the I chain.

In the implementation of the present invention, the entire sequence of DNA bases encoding the M-region of the I-chain of a human antibody, (ee) used as a matrix, can be synthesized in a DNA synthesizer and constructed by the RSK method, although

The DNA for the M region of the I chain is not available in the same way as the DNA encoding the M region of the H chain. -

To construct DNA for the humanized M-region of the I-chain, using as a template a human antibody with

ZYCOZ3868, which is characterized by the greatest homology with the M-region of the I-chain of the mouse monoclonal antibody against the human RTNgGR, requires four primers, expressed, for example, by sequences with identification that

MoMo 29-32. Primers MVSTLISRI (sequence with identification Mo29) and MVSTLISRZ (sequence with identification Mo30) contain sense DNA sequences, and primers MVSTIOSR2 (sequence with identification Mo31) and MVSLI ORA (sequence with identification Mo32) contain antisense sequences

DNA. All were designed to have a complementary sequence of 15 to 21" base pairs at either end.

External primers МВС1ИИМ51 (sequence with identifying Mo33) and МВСЛИМКИ1 (sequence with - with identifying Мо34) have a sequence homologous to MVSTLISRI and MVSLI OR, respectively, and contain a recognition sequence for the corresponding acceptable restriction enzyme. Four seeds are collected by the ROK method to obtain unprocessed DNA and an external primer is added for DNA amplification. "Collection by the ROK method" means the hybridization of the MVSLI ORI and MVSTI ORZ primers or the MVSLTI P2 and MVSLI RA primers according to their complementary sequences to obtain the MVSLI ORI1I fragment -MVSTI ORZ and fragment -and MVS OP2-MVSTI OP with subsequent hybridization of these fragments along their complementary sequences with sl obtaining unprocessed DNA encoding the humanized M-region of the H-chain.

The C-region of the I-chain of a human antibody can be any C-region of the I-chain of a person, for example, C X- (95) or the Sk-region of the I-chain of a person. -oUu 70 DNA for the M-region of the I-chain of a humanized antibody, which is obtained according to the above description, can be ligated with DNA for any C-region of the I-chain of a human antibody, for example, C 7-region of the I-chain of a human . The DNA for the M region of the I chain can be treated with a suitable restriction enzyme and ligated with DNA encoding the C region of the human I chain under the control of an expression regulatory region, such as an enhancer/promoter system, to produce an expression vector containing DNA, encoding the populated M-region | -chain and the C-region of the human X-chain.

GF) The fact of obtaining a polypeptide by the method described above, containing the M-region of a humanized antibody, does not mean at all that the indicated polypeptide will have activity as an antibody, for example, have binding or even neutralizing activity against its antigen. This, in particular, applies to the I-chain, since the M-region

The Y-chain of a mouse monoclonal antibody against human RTNgR is isolated from a very rare Muh-gene, therefore it is necessary to examine the indicated polypeptide for the presence or absence of this activity by combining it with a humanized H-chain and expressing it in an animal cell, such as CO5 -7.

An effective method of detecting the framework region in the M-region of a humanized antibody, possessing the binding and neutralizing activity of a humanized antibody, can be the construction of a hybrid M-region (Opioto, T.eiaII., Moiesciag Ittipiodou, 32, 407-416, 1995) and confirmation obtained results. In order to detect the amino acid in the M-region | - the chain of the humanized antibody according to the present invention, which must be mutated to obtain an amino acid with the required activity, construct DNA in which a fragment of the framework region of the humanized antibody is recombined with a fragment of the framework region isolated from a mouse, after which each region is analyzed for humanization.

As shown in Fig. 3, receive an antibody with a polypeptide containing a recombinant M-region, in which EC! and

ECZ is isolated from a human antibody and ECZ and EC4A are isolated from a mouse antibody (such an antibody having a recombinant fragment is defined as a "hybrid antibody"), a hybrid antibody in which only EC1 is isolated from human, and a hybrid antibody in which only EC2 is isolated in a person Each DNA encoding these hybrid antibodies is introduced into an expression vector and humanized antibodies are temporarily expressed to test for the presence of the necessary /o activity.

Using this method, the author of the present invention studied polypeptides containing M-regions

Y-chain, in terms of antigen-binding and neutralizing activity and found that certain amino acids that are replaced are in the framework regions of IVF and IVF.

Having discovered that the amino acids that determine this activity are located in the EK2 and IVF regions, the author of this /5 Invention established that the indicated activity is possessed by the 36th, 45th and 49th amino acids in the EKZ2 region and the 87th amino acid in the IVF (the numbering of amino acids in antibodies is determined by Kabat (|KaBbai, E.A. ei. aI., 05 Oer.

Neay apa Nytap Zegmisez, O5 bomegptepi Rgipiipud Oyisevs, 19911).

Thus, in the implementation of the present invention, a polypeptide containing an M-region in which one or more of these amino acids is mutated (for example, replaced) is obtained.

According to the method of transplantation of hypervariable sites discussed above, a polypeptide containing an M-region with an amino acid sequence as a basis for amino acid mutation is first obtained. This initial polypeptide contains the amino acid sequence expressed by the sequence with identification Mo47, and is defined as "variant a" ("a" in Table I).

Then, using "variant a" as a basis, various variant fragments are obtained, in which one or several amino acids of the framework region are mutated.

The mutation can be carried out by constructing an oligonucleotide primer (mutagenic primer) that i) encodes an amino acid introduced as a necessary mutation, which is used to perform a polymerase chain reaction using the specified primer.

This is how polypeptides containing M-regions (variants B-) are obtained, in which specific amino acids are mutated in the so-so regions of EK2 and RKZ (B-th in Table 1).

Table 1 - and scan: i)

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The DNA obtained above encoding each variant of the M-region | - a chain of a humanized antibody, can be ligated with

DNA of any C-region I -chain of a human antibody, such as C X-region I -chain of a person. To this end, it is treated with an appropriate restriction enzyme and ligated with DNA encoding the human C-region and X-chain under the control of an expression regulatory region such as an enhancer/promoter system to produce an expression vector containing DNA encoding each variant. of the humanized M-region of the I-chain, and DNA encoding the humanized c) C-region of the I-chain. c" The above-constructed DNA encoding the M-region of the H-chain of the humanized antibody and the C-region of the human H-chain, and the DNA encoding the humanized M-region of the I-chain and the C-region of the human I-chain can also be introduced into one expressing vector described in patent M/094/11523, and said vector can be used to transform a host cell and the transformed host can be cultured ip mimo or ip migo to obtain

The necessary humanized antibodies. 4. Obtaining a chimeric antibody and a humanized antibody

To obtain a chimeric or humanized antibody, it is necessary to prepare the two above-mentioned expressing vectors. Thus, in the case of a chimeric antibody, an expression vector containing DNA encoding the M-region -shsh 20 of the mouse H-chain and the C-region of the human H-chain is constructed under the control of an expression-regulating region, such as an enhancer/promoter system, and expressing a vector containing DNA encoding the M-region of the I-chain of the mouse and the C-region of the Y-chain of the human, under the control of the regulatory expression of the region, such as the enhancer/promoter system. In the case of a humanized antibody, an expression vector containing DNA encoding the humanized M-region is constructed

The H-chain and C-region of the human H-chain under the control of an expression regulatory region such as the 22 enhancer/promoter system and an expression vector containing DNA encoding the humanized M-region of the I-chain and the C-region

F! A human Y-chain under the control of a regulatory expression region, such as an enhancer/promoter system. Then host cells, such as a mammalian cell, are cotransformed with these expression vectors and the resulting transformed cell is cultured ip migo or ip mimo to produce a chimeric or humanized antibody | see, for example, UU091/169281.

Alternatively, the DNA encoding the M- and C-regions of the H-chain and the DNA encoding the M- and C-regions of the I-chain can be ligated with a single vector and transformed into an acceptable host cell to produce an antibody. Thus, for the expression of a chimeric antibody DNA encoding the mouse leader sequence present in the cloned cDNA, the M-region

The mouse H-chain and the C-region of the human H-chain, as well as the DNA encoding the mouse leader sequence, the M-region 62 | -chain of the mouse and the C-region of the human I-chain are introduced into one expressing vector described, for example, in

MU/094/11523. For the expression of a humanized antibody, DNA encoding the humanized M-region of the H-chain and the C-region

The H-chain of a person and DNA encoding the humanized M-region of the I-chain and the C-region of the I-chain of a person are introduced into a single expressing vector described, for example, in UU094/11523. Such a vector is used to transform a host cell, after which the transformed host is cultured by ip mimo or ip mygo to obtain the chimeric or humanized antibody of interest.

A chimeric or humanized antibody of interest obtained by culturing a transformant transformed with DNA encoding said chimeric or humanized antibody can be isolated from the interior or exterior of the cell and purified to homogeneity. 70 The chimeric or humanized antibody of interest of the present invention can be isolated and purified in a protein A and agarose column. For this purpose, you can also use any other methods that are used for the isolation and purification of ordinary proteins, and their choice is not limited by anything. For example, different types of chromatography, ultrafiltration, salting out, and dialysis can be used individually or in combination to isolate and purify chimeric or humanized antibodies.

Any expressing system can be used to obtain a chimeric or humanized antibody against human RTNHR according to the present invention. For example, eukaryotic cells include animal cells, in particular, isolated mammalian cell lines, mold cells, fungi and yeast cells; prokryotic cells include bacterial cells, such as EzvsPegispia soya cells. The chimeric or humanized antibody according to the present invention is preferably expressed in a mammalian cell, such as cells of SOC or CHO.

Any known promoters designed for expression in mammalian cells can be used. For example, the ShshFast-acting human cytomegalovirus (HCMV) protomore is mainly used. Examples of expression vectors containing the HCMM promoter are HCMM-MN-HC 1 and HCMM-MI-NSCs isolated from reM2peo

MU092/19759).

In addition, in the implementation of the present invention, as promoters for gene expression in mammalian cells, viral promoters such as retrovirus, polyoma virus, adenovirus and simian virus (ZM) 40 can be used, and promoters isolated from mammalian cells, for example, from the factor -1o, human polypeptide chain elongation (NERB-19). at

For example, the 5M40 promoter can be easily used according to the method of Mulligan et al. (Miiydap eyai., Mayige, 277, 108, 1979) and the NEBR-1u promoter. can be effectively used according to the method of Mizushima et al. (Migizpita, 5. ei. aiI., Mysivys Asidz Kezeagsy, 18, 5322, 1990). (uh)

The starting material for replication used in the implementation of the present invention is obtained from ZM40 virus, polyoma virus, adenovirus or bovine papillomavirus (BPM). In addition, the expressing vector may contain a gene for ARN(Z) phosphotransferase II or I! (peso), thymidine kinase (TK), co xanthine-guanine-phosphoribosyl-transferase (Esodri) of E. soybean or dihydrofolate reductase (ONEK) as a selective marker for increasing the number of gene copies in the host cell system. o 5. Assessment of antigen-binding and neutralizing activity of chimeric and humanized antibodies. (1) Determination of antibody concentration.

The concentration of the obtained purified antibody can be determined by the method of solid-phase immunoenzymatic analysis (ESIBA). "

Tablets for determining the concentration of antibodies by the EGISA method are obtained as follows: in each well of a 70 9b-well tablet for EGISA analysis (for example, Makhizogr, MOMS) immobilize 100 μl of goat - c antibodies against human immunoglobulin y o (Id) with a concentration equal to, for example , μg/ml. The contents of the well are blocked with 200 μl of diluting buffers (for example, 5 mM Tris-HCI, mM MaosI, 0.1 M Masi, 0.0595 Tween-20, 0.0295 Mam, 195 "» bovine serum albumin (BSA), pH 7, 2), gradually diluted supernatant of CO5-7 or CHO cells in which a chimeric, hybrid or humanized antibody is expressed or purified chimeric, hybrid or humanized antibody is added to each well; 100 μl conjugated from alkaline. Phosphatase, goat anti-human dO antibodies and -and 1 mg/ml substrate solutions (Zidta 104, paranitrophenylphosphoric acid, ZIOMA), after which the optical density is measured at a wavelength of 405 nm with the help of a microplate reader (Vio Kai). As a standard for determining concentrations, you can use purified human (binding site). (95) (2) Determination of antigen-binding capacity. -l 20 Tablets for the determination of antigen-binding capacity by the EGISA method are obtained as follows: in each well of a 96-well tablet for the analysis of EG IZA (for example, Makhisogr, MOIS) real estate mix 100 μl of RAKNHR 42) of a person (1-34) with a concentration of 1 μg/ml. The contents of the well are blocked with 200 μl of dilution buffers, gradually diluted supernatant of CO5-7 or CHO cells, in which a chimeric, hybrid or humanized antibody is expressed or a purified chimeric, hybrid or humanized antibody, 100 μl of alkaline phosphatase-conjugated goat anti-IDO antibody is added to each well human and img/ml substrate solutions (Zidta 104, paranitrophenylphosphoric acid, ZIOMA), after which the optical density at a wavelength of 405 nm is measured using a microplate reader (Vio Kai). eat) (3) Determination of neutralizing activity.

The neutralizing activity of a mouse antibody, a chimeric antibody, and a humanized antibody can be determined using, for example, the rat osteosarcoma cell line KOB517/2.8-5 (Za, K.etsai., Asia EpaosgipoIodu, 116, 113-120, 1987). KOB5 17/2,8-B5 cells were stimulated using 4 mM hydrocortisone to induce the PTN/PTNHR receptor. The degenerate enzyme for cyclic adenosine monophosphate (cAMP) is inhibited by MM isobutyl-1-methylxanthine (IVMH, 5ISMA). A mouse antibody, or a chimeric or humanized antibody designed to determine the neutralizing activity, is mixed with an equal amount of RTNHGR (1-34) and 65 of the resulting mixture of antibody and RTNHGR is added to each well. (1-34). The neutralizing activity of the mouse antibody, chimeric or humanized antibody can be determined by measuring the amount of CAMP produced by KO5 17/2.8-5 rat osteosarcoma cell lines as a result of RTNGhR stimulation. (4) Kinetic analysis of the interaction between RTNHgR and anti-RTNHgR antibody.

In the implementation of the present invention, the kinetics of interaction between RTNGR and anti-RTNgr can be determined by various means and methods. In particular, dissociation constants, dissociation rate constants, and association rate constants can be measured by the Scatchard method (5saispaga) and with the help of a resonance surface plasmon sensor VIASOKE (developed and sold by RNaptasia Vioyesi). The analysis using the surface plasmon resonant sensor VIOSOKE will be described below in one of the examples.

VIASOMKE consists of an optical source, a prism, a detector and a microchannel. In the process of practical application, the ligand is immobilized at the end of the cassette-type sensor and the substance being analyzed is initiated into it. If there is a relationship between them, it optically determines the degree of bonding.

The detection principle of this method is defined as surface plasmon resonance. The incident light is directed to the interface between the glass and the metal film so that total reflection occurs, while the light incident at a certain angle causes the excitation of the surface plasmon. The angle of incidence of light 7/5 Changes depending on the change in the concentration of the solvent touching the metal film (sensor). VIASOKE determines this change.

In VIASOK, this change is called a resonance signal (5PE-signal), and a change of 0.1 degree is 1000 resonance units (KI). 1000 resonance units corresponds to a binding change equal to approximately ng of protein on a thin gold sensor with a surface area of mm2. For a protein, a change equal to approximately 50 resonance units (5OpgHg) can be fully determined.

Detected signals are transformed in a computer connected to VIASOKE into a binding curve, called a sensor-gram, which is built on the computer monitor in real time |Maivite, T., ей. AI., (1995)

Ehregitepia! Meadisipe, 13, 563-569) (Kagizzop, K., ei. aI.) ((1991) 9U. Ittipoi.) (Meipoaz 145, 229-240)|.

Using the above-described VIASOKE device, it is possible to measure the kinetic parameters, i.e., the dissociation constant (KO), the dissociation rate constant (Kaizv) and the association rate constant (Kazz) of antibodies against RTNHR according to the present invention. and)

Antibodies against RTNGR according to the present invention preferably have the lowest possible dissociation constant (value

KO) from the point of view of neutralizing activity. Against RTNHR antibodies according to the present invention preferably have a value of KO equal to 1.86 x 1077 or less, more preferably 1.86 x 109 or less and most preferably c 3.58.1079 or less. -

The value of KO is determined on the basis of two parameters: dissociation rate constants (Kaizv) and association rate constants (Kazv) (KO-Kaizv/Kavzv). Therefore i) it is absolutely obvious that the values of КО should be smaller at smaller values of Kaizz and large values of ю

Kazzv

In particular, the value of Kivv antibodies against RTNHR according to the present invention can be equal to 1.22 x107 |1/sec| or - less. Kaizz values are preferably 1.22x1072 or less, more preferably 3.16x107 or less, and most preferably 2.32x1077 (1/sec| or less.

On the other hand, values of Kazv can be equal to 6.55x107 |(1/M.sec| or more. Values of Kazz are preferably equal to 6.55x10? or more, more preferably 0.883x109 or more and most preferably 1.03x109 M1/ M.sec | ShSH8Z s or more. "z In addition, antibodies against RTNGR having a Kaivv value equal to 1.22 x 107 |(1/sec) and a Kavzv value equal to 6.55 x 107 |1/M are preferred. sec) or more.

In particular, antibodies against RTNGR according to the present invention have a KO value in the range from 1.02x10-11 to 1.86x1077 - 15 IMI, preferably from 1.01x10719 to 1.86x107 |MI, more preferably from 1.34x10719 to 3.58x10719 | MI, most preferably from 2.25X1079 to 3.58X10719) MI,

Mn Values of Kaizz are in the range from 7.38x1079 to 1.22x107 P/sec), preferably from 7.38x1075 to about 1.22.102 |(1/sec), more preferably from 1.66x1077 to 3.16x1077 |1/ sec), most preferably from 1.66x107 to - 20232510 |M/sec|. Co values of Kaze are in the range from 6.55x107 to 1.24x10-7 P/M.sec), preferably from 6.55x109 to 1.24X105 P1/M.sec|, more preferably from 7.23X109 to 1.03X109 (1/M.sec |, most preferably from 0.883x105 to 1.03x1059 P1/M.sec).

These KO, Kaizz, and Kazz values can be obtained using Scatchard analysis or a resonant surface plasmon sensor such as a VIASOKE, and preferably a VIASOKE.om. i) 6. Pharmaceutical composition and medicinal product for suppressing hypercalcemia, containing an antibody against co RTNHR or a humanized antibody as an active ingredient.

The therapeutic effect of a humanized antibody against RTNGR can be tested by administering an antibody against 60 RTNGR or a humanized antibody to an animal suffering from hypercalcemia and measuring the index of hypercalcemia. In animals with hypercalcemia and in subjects suffering from hypercalcemia, hypophosphatemia is often observed; antibodies according to the present invention can also be used to improve hypophosphatemia.

In the implementation of the present invention, an anti-RTHNR antibody is used, including a human antibody, a chimeric antibody, a primed antibody or a humanized anti-RTHNR antibody, which have a value of 65 dissociation constants, dissociation rate constants, and association rate constants. The antibody that will neutralize the activity of RTNHR by binding to it preferably includes the humanized antibody Mo23-57-137-1.

The method of obtaining the humanized antibody Mo23-57-137-1 is described in examples 1-3.

The antibody of the present invention can be purified to a high degree of purification by any combination of known purification methods, such as salting out, gel filtration using high performance liquid chromatography, etc., and affinity chromatography using a protein packed column A, etc. The accuracy of recognition of RTNHR by a purified antibody can be checked by known immunological means, such as radioimmunoassay (RAI), enzyme-linked immunosorbent assay (EIIA, EGI5ZA) and immunofluorescence analysis.

As a test animal with symptoms of hypercalcemia, an animal model obtained 7/0 by transplantation of RThR-producing cancer cells into an experimental animal with reduced or absent immunological function can be used. Transplanted cancer cells, which are preferably taken from humans, include, for example, human pancreatic cancer cells RAM-7. An animal with reduced or absent immunological function to which cancer cells are transplanted is an "old" mouse and a mouse of the ZSIU line.

The degree of suppression of hypercalcemia can be determined by measuring the concentration of calcium in the blood, the weight of the body or the restoration of movement activity after a certain period of time, with a further assessment of the change in the subject's condition based on the obtained data.

The pharmaceutical composition and means for suppressing hypercalcemia, containing an antibody or a humanized antibody against RTNgR according to the present invention as an active ingredient, can be administered parenterally systemically or locally. For example, intravenous injections, including drips, 2o intramuscular injections, intra-abdominal injections or subcutaneous injections, can be made. The method of administration is chosen depending on the age of the subject and the severity of the disease. An effective single dose can range from 0.01 to 100 Ωg/kg of body weight. Alternatively, the indicated dose may be from 5 to 1000 Ωg/body weight, preferably from 50 to 1000 Ωg/body weight.

A pharmaceutical composition and a means for suppressing hypercalcemia containing an antibody or a humanized tissue antibody against RTNGR according to the present invention as an active ingredient may further include a pharmaceutically acceptable carrier and/or an additive or additives depending on the method of administration. Examples of such carriers and (8) additives are water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, poly(sodium acrylate), sodium arginate, water soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose ) , lactose and surorants, suitable for use as pharmaceutical additives. The additives can be used individually or in combination, without being limited to the above-mentioned substances.

The antibody according to the present invention can be used for the treatment of hypercalcemia caused by various types of cancer (malignant tumors). These types of cancer do not have any specific limitations and include not only any one type of cancer, but also a combination of different types of cancer. Types of cancer may include, for example, cancer of the pancreas, lung, pharynx, larynx, tongue, gums, esophagus, stomach, bile ducts, breast, kidney, bladder, uterus, and prostate, as well as malignant lymphoma. "

Figure 1 shows a schematic representation of an antibody according to the present invention. z c Fig. 2 shows a schematic representation of the transplantation of hypervariable sites.

Figure 3 shows the definition of frame regions and hypervariable sections of the M-region. ;" Figure 4 shows a graph of the results of measuring the antigen-binding activity of antibodies.

Figure 5 shows a graph of the results of measuring the antigen-binding activity of antibodies.

Figure 6 shows a graph of the results of measuring the antigen-binding activity of antibodies. -I Figure 7 shows a graph of the results of measuring the antigen-binding activity of antibodies.

Figure 8 shows a graph of the results of measuring the antigen-binding activity of antibodies. Figure 9 shows a graph of the results of measuring the antigen-binding activity of antibodies. 2) Fig. 10 shows a graph of the results of measuring the antigen-binding activity of antibodies.

Figure 11 shows a graph of the results of measuring the antigen-binding activity of antibodies. - Figure 12 shows a graph of the neutralizing activity of humanized antibodies. c Figure 13 shows a graph of the neutralizing activity of humanized antibodies.

Figure 14 shows a graph of the neutralizing activity of humanized antibodies.

Figure 15 shows graphs illustrating the effect of antibodies according to the present invention on an animal model of pipercalcemia.

Figure 1b6 shows graphs illustrating the effect of antibodies according to the present invention on an animal model

F) hypercalcemia. Figure 1/7 shows graphs illustrating the effect of antibodies according to the present invention on an animal model of hypercalcemia. in Figure 18 shows graphs illustrating the effect of antibodies of the present invention on an animal model of hypercalcemia.

Fig. 19 shows a sensorgram illustrating the immobilization of RTNGR at the end of the sensor.

Figure 20 shows a graph of the results of the kinetic analysis of the antibody according to the present invention.

Figure 21 shows a graph of the results of the kinetic analysis of the antibody according to the present invention. 65 Fig. 22 shows a graph of the results of the kinetic analysis of the antibody according to the present invention.

Figure 23 shows a graph of the results of the kinetic analysis of the antibody according to the present invention.

Figure 24 shows a graph of the results of the kinetic analysis of the antibody according to the present invention.

Figure 25 shows a graph of the results of the effect of the humanized antibody of the present invention on the fractional excretion of phosphate.

Figure 26 shows a graph of the effects of the humanized antibody of the present invention on the concentration of phosphorus in the plasma.

Fig. 27 shows a photograph of pronounced clinical symptoms in the tested mice suffering from hypercalcemia after the introduction of an antibody against RTNHR according to the present invention (morphology of a live animal).

Fig. 28 shows a photograph of pronounced clinical symptoms in tested mice suffering from 7/0 Pipercalcemia after administration of an antibody against RTNHR according to the present invention (morphology of a live animal).

Fig. 29 shows a graph of changes in movement activity over time in a test animal suffering from hypercalcemia after administration of an antibody against RTNHR according to the present invention in comparison with the same indicator in a control animal that was injected with a saline solution.

Fig. 30 shows a graph of changes over time in the body temperature of a tested animal suffering from 7/5 pipercalcemia after the administration of an antibody against RTNHR according to the present invention in comparison with the same indicator in a control animal that was injected with a saline solution.

Fig. 31 shows a graph of the change over time in the pH value of the blood in a test animal suffering from hypercalcemia after the introduction of an antibody against RTNHgR according to the present invention in comparison with the pH value in a control animal that was injected with saline.

Examples

The following is a more detailed description of the present invention with reference to the following examples, which should not limit the scope of the present invention.

Reference example 1

The method of obtaining a hybridoma producing mouse monoclonal antibody against RTNGR (1-34) ch

Hybridomas capable of producing a monoclonal antibody against human RTNgR (1-34), Mo23-57-154 and

Mo23-57-137-1, obtained in accordance with the method described by Kanji Sato et al. (Za, DO. ей. ай., 9). Vope Fr

Mipeg. Kez. 8, 849-860, 1993).

RTHGR is used as an immunogen. (1-34) (Repipztia), with which carrier protein and thyroglobulin are conjugated using carbodiimide (O|ipp). RTNGR conjugated with thyroglobulin. (1-34) are dialyzed to obtain a solution with a protein concentration of 2 μg/ml. The resulting solution is mixed with Freund's adjuvant (Oiso) in a ratio of 111 to obtain an emulsion. This emulsion is administered as a dorsal-subcutaneous or intraperitoneal injection to 16 female VAI V/S mice at a dose of 100 μg/mouse for the purpose of immunization of the specified mice. This injection will be performed 11 times. with

For the first immunization use complete Freund's adjuvant and for subsequent immunizations use incomplete Freund's adjuvant. Shit,

Antibody titers in sera are determined in immunized mice as follows: cha

All mice have blood taken from the tail vein and centrifuged to obtain serum. This serum is diluted with buffer for radioimmunoassay (RIA), mixed with 125-|I-labeled RTNHGR(1-34) and its binding activity is determined. Mice with a sufficiently high titer of antibodies are injected intraperitoneally with RTNGR (1-34) without a carrier protein in the amount of bHOmkg/mouse for final immunization.

Three days after the final immunization, mice are sacrificed and their spleens are removed. Then the fusion of spleen cells with the mouse myeloma cell line RZUKhbZAd80.1 is carried out in accordance with the known method based on the use of 5095 polyethylene glycol 4000. The resulting connected cells are inoculated into the wells of 96-well plates "" (a total of 85 plates) in the amount of 2x107/ well Screening of hybridomas of interest is performed using hypoxanthine-aminopterin-thymidium (HAT) medium as follows.

Hybridomas are examined for the presence of RTHGR-recognizing antibodies in the culture supernatant, that well, where cell growth was observed in the NAT environment, using a solid-phase radioimmunoassay. sl Hybridomas are collected from a well in which the ability of the culture to bind to the RTHgR-recognizing antibody has been reliably established. Hybridomas obtained in this way are suspended in KRMI-1640 medium containing 1595 fetal (95) calf serum (EC5) and ORI supplement (Zidta), unified by the limited dilution method, which gives two types of 20 hybridoma clones, Mo23-57- 154 and Mo23-57-137-1, each of which has a strong ability to bind to RTNGR (1-34).

IR e) The hybridoma clone Mo23-57-137-14, named "mouse-mouse hybridoma Mo23-57-137-1", was included in the collection of the National Institute of Bioscience of Human Technology, Agency for Industrial Science and Technology, Japan (1 -3, Nidazpi 1-spote, T8yKira-5PPi, Iragadi Cape, Japan) under the number of REKM VR-5631 on the basis of the Budapest Treaty of August 15, 1996.

Example 1 o Cloning of the DNA encoding the M-region of the mouse monoclonal antibody against human RTNHGR (1-34) ime) Cloning of the DNA encoding the M-region of the mouse monoclonal antibody against the human RTNHGR (1-34) obtained above Mo23-57-137 -1, perform as follows. 60 (1) Obtaining the meRnkK

The mRNA is obtained from the Mo23-57-137-1 hybridoma using a kit for the purification of Ocysk Rger (Rpagtasia vioiesi) mRNA, similar to the procedure described below.

The cells of the Mo23-57-137-1 hybridoma obtained above are completely homogenized with the extraction buffer, after which

The mRNA is extracted from this buffer on a column with oligo(thymidine) cellulose packing according to the instructions 65 of the manufacturer of this kit. mRNA is precipitated with ethanol from the extraction solution and the obtained precipitate is dissolved

mRNA in elution buffer.

(2) Obtaining and amplifying the cDNA of the gene encoding the M-region of the H-chain of the mouse antigen. (Y) Cloning of cDNA for the M-region of the H-chain antibody Mo23-57-137-1 DNA encoding the M-region of the H-chain of the mouse monoclonal antibody against human RTNGR is cloned by the 5-KACE method |Egoptap, M.A. eh ai., Rgoum.

Property Asad All together. OBA, 85, 8998-9002, 1988; VeiuamuzKu, A. ei. aiYi., Mysivis Asiav Kev. 17, 2919-2932, 1989).

This method is performed using the 5-Atria RIMOEK KASE kit (SI OMETESN) in accordance with the manufacturer's instructions. When performing this method, primers for cDNA synthesis are used, for example, MHC2 primers (sequence with identification Mo1), which hybridizes with the C region of H -mouse antibody chain. Approximately 2 μg of the mRNA obtained above, which is a template for cDNA synthesis, is mixed with 10 pmol of MNOS2 seed. The resulting 7/0 buffer is reacted with reverse transcriptase at a temperature of 5292 for 30 minutes to prepare cDNA, complementary mRNA.

The obtained substance is mixed with bn Mahon for mRNA hydrolysis (at a temperature of 6593 for 30 minutes) and the cDNA is precipitated with ethanol. The cDNA isolated in this way is ligated from Atria RIMOEK (sequence with identification Mo42) at the 5-end due to interaction with RNA ligase T4 at a temperature of 372C for 75 hours and then at room temperature for 16 hours. Apspog primers (sequence with identification Mo2) and primers МНО-С1 (sequence with identification Mo3) are used as primers for cDNA amplification by the RSK method |5.T. dopezv, eia!., Vioyesppoiodo, 9, 88, 19911.

The solution for the polymerase chain reaction (50 μl) used in the implementation of this method consists of 10 mM Tris-HCl (pH 8.3), 500 mM KSI, 0.25 mM amcTeR (adteR, aste, astre, atteR), 1.5 mM Maosi", 2.5 units of TaKaKa Tad (TaKaga 5pyg20o), 100 ml of Apsgog seeds and 1 ml of the cDNA reaction mixture, from which MNO-C1 seeds and Atria RIMOEK Apspog seeds are ligated, placing 5 omcl of mineral oil on top. In the thermal cycler model 480) (Regkip EIteg) thirty cycles of the polymerase reaction of chain synthesis will be performed according to the program of cyclic temperature changes in the following sequence: 9422 for 45 seconds; 602 for 45 seconds and 729 for 2 minutes. Ge (i) Cloning of cDNA for the M-region of the I-chain antibody Mo23-57-137-1 DNA encoding the U-region of the I-chain of a mouse monoclonal antibody against human RTNGR, cloned by the 5-KACE method Ghehoptap, M.A . eh ai., Rhos.

Property Asad All together. OBA, 85, 8998-9002, 1988; VeiuamuzKu, A. ei. aYi., Mysieis Asidv Kev. 17, 2919-2932, 1989).

This Cetode will be performed using the 5-Atrial Ripdeg KASE kit (CiopeTesi) according to the manufacturer's instructions. When implementing this method, an oligo (thymidine) (2,0) primer is used as a primer for cDNA synthesis. Approximately 2 μg of the mRNA obtained above, which is a template for cDNA synthesis, is mixed with an oligothymidine "- primer. The resulting mixture is subjected to interaction with reverse transcriptase at a temperature of 5223 for 30 minutes to obtain cDNA. The obtained substance is mixed with bn Mahon for RNA hydrolysis (at a temperature of 65920 and) for 30 minutes). The obtained mixture is precipitated with ethanol to isolate the cDNA in the form of a precipitate, the cDNA isolated in this way is ligated with the binding fragment of Atria RIMOEK. U) Apspog at the 5-end due to interaction with

With T4 RNA ligase at 372C for 6 hours and then at room temperature for 16 hours. -

MI C primers (sequence with identification Moe4) for the polymerase reaction of chain synthesis are constructed on the basis of the conservative sequence of the C-region of the y-chain of the mouse I-chain and synthesized in synthesizer 394

DNY/RNA (AVI). "

The solution for the polymerase reaction of chain synthesis (10 μl) used in the process of obtaining З7Z 70 seeds consists of 10 mM Tris-HCII (pH 8.3), 50 mM KSI, 0.25 mM amMteR (adtR, astR, aste, attRr), 1 ,0mM s Massi", 2.5 units of AtriaTad (REKKIM EI MEK), 5 Opmole of primer Apspog (sequence with identification Mo2) "with and μl of reaction mixture of cDNA, from which the primers MI C (sequence with identification Mo4) and seeds are ligated

RIMOEK Apsgog atria were ligated by placing 5Omcl of mineral oil on top. In the thermal cycler model 480) (Regkip EITeg) thirty-five cycles of the polymerase reaction of chain synthesis will be performed according to the program -And 15 cyclic temperature changes in the following sequence: 942 for 45 seconds, 602C for 45 seconds and 7226 for 2 minutes. 1 (3) Purification and fragmentation of the polymerase chain reaction product. c All DNA fragments, amplified by the RSK methods described above, are separated using electrophoresis in an agarose gel using 395 agarose of Mi Zieme STO (EMS Vio. Rhodisiv). For each M-region of the H-chain - 70 and M-region of the I-chain, a fraction of the agarose gel containing a DNA fragment with a length of about 550 base pairs is cut from the gel, respectively. DNA is isolated from all the obtained gel fractions using the ZEMESI EAM 1I kit (VIO 101) according to the manufacturer's instructions. Purified DNA is precipitated from the solution with ethanol and dissolved in 20 μl of a solution containing TOMM tris-HCI (pH 7.4) and 1 mM EOTA. One μl of the DNA solution obtained in this way is cleaved with the restriction enzyme Khtai (Mem Epdiapa Vioyarv) at a temperature of 37 °C for 1 hour and with the restriction enzyme EsokiI (Takaga 5p!i20o) at a temperature of 372 °C for another 1 hour. The hydrolyzed (F) mixture is extracted with phenol and chloroform, and the DNA is precipitated with ethanol.

DNA encoding the M-region of the mouse H-chain is obtained in a similar way, DNA encoding the M-region

Mouse Y chain, which has an EsoKiI recognition sequence at the 5-end and an Xta/!-recognition sequence at the 3'-end. All DNA fragments of EsokiI-Chtai, containing, respectively, DNA encoding the M region of the mouse H-chain and DNA encoding the M-region of the I chain of the mouse, are subjected to interaction with the vector rusSt19, which is previously cleaved by Esoki and

Htai, at a temperature of 1623 for 1 hour using a DNA ligation kit option 2 (Takaga

Zpyg2o) according to the manufacturer's instructions. The ligated mixture obtained in this way (1 μl) is added to 100 μl of a solution containing competent cells of E. soya, UM 109 (Mirrop Sepe). The mixture of cells is left to stand on ice at 65 for 15 minutes, then at a temperature of 422 C for 1 minute and again on ice for another 1 minute. The resulting product is mixed with ZbOOmkl cultural medium 5OS | Moiesciag Siopipu: A I arogaigu

Mapaiai, Zatgook her. ai., Soya Zrgipo-Nagbog I aroghaiogu Rgezv, 1989) and incubated at a temperature of 372C for 30 minutes. The resulting solution of cells is placed on an agar medium IV or 2xUuT

Mapia), ZatrgbooK, ey. aiY., Soy Zrgipd Nagro Iarogaiogu Rgezz, 1989), containing 100 or 5BOmkg/ml ampicillin, O0.1mM IRTO 2Omkg/ml X-day, and incubated at a temperature of 372С during the night. Similarly transformants of E. soybean are obtained in this way.

Transformants are cultured overnight in 2 ml of IV medium or 2xHuUT containing 100 or 5 Ωkg/ml of ampicillin at a temperature of 372C, after which plasmid DNA is obtained from the cellular fraction using the P1-1007 plasmid extractor (Kygarots) or the OIArger plasmid isolation kit (OIASEM ). DNA sequences are determined in the 70 plasmid DNAs obtained in this way. (4) Sequencing of the cDNA encoding the mouse antibody M region.

The sequence of the cDNA encoding the plasmid region is determined using the DNA sequester 37ZA (AVI;

Regkip-EIteg), using a kit of cyclic sequencing with terminator staining (RegkKkip-EIteg). To determine this DNA sequence, base sequences are examined in both orientations using such 75 primers as primers M13 Rgiteg MA (Takaga Zp!yi20) (sequence with identification Mo5) and primers M1Z

Rgiteg KM (TaKkaga Zpy20) (sequence with identification Mob).

Thus obtained plasmids containing the cDNA encoding the M-region of the mouse H-chain and the cDNA encoding

M-region of mouse I-chain, isolated from hybridoma Mo23-57-137-1, named "MVS1TNO4" and "MVS 24", respectively.

The sequences (including the corresponding amino acid sequences) of the DNA encoding the M-region of the H-chain and the DNA encoding the M-region of the I-chain of the mouse antibody Mo23-57-137-1 (related to the plasmids МВС1НОЯА and

МВС1НЗ24), respectively, are represented by the sequences with identification MoMo 57 and 65. Both polypeptides for the fragment of the M-region of the H-chain and the fragment of the M-region of the I-chain are translated starting from the 58-0th base (which codes for glutamine) in the DNA sequences expressed by the sequences with identification MoMo 57 and 65.

The amino acid sequences for the M-region of the H-chain and the M-region of the I-chain are expressed respectively by He sequences with identification MoMo -6 and 45. o

The strain of E. soybean having the plasmid МВС1ТНО4 and the strain of E. soybean having the plasmid МВС11И 24 are respectively named "Evepegispia soii /"М109 (МВС1ТНОЗ)" and "Evepegispia soii UM109 (МВС1И 2439). These strains of E. soybean are included in the collection

National Institute of Bioscience of Human Technology, Agency of Industrial Science and Technology, Japan (1-3,

Nidazni 1-spote, TwiKkibwa-vpyi, Iragadi-kep, Japan), under the numbers RGEKM VR-5628 for Espegispia soya dHM109 (ee) (MVS1NO4) and REKM VR-5627 for Espegispia soya UM109 (MVS1I 24) on the basis of the Budapest Treaty from August 15, 1996 - (5) Determination of the hypervariable region of the mouse monoclonal antibody Mo23-57-137-1 to undergo human RTnGr.

General structures of the M-region of the H-chain and the M-region | - chain have significant similarities. That is, both o structures have four frame regions subsidized with the help of three hypervariable sites (that is,

JUICE). The amino acid sequences of the framework regions are quite conservative, while the amino acid sequences of the hypervariable regions are characterized by strong mutagenicity (Karai, E.A. ei. 1983). "

On the basis of the above-mentioned factors, hypervariable regions (HIVs) are determined by searching for the homology of 470 amino acid sequences of the M-region of a mouse monoclonal antibody as a result of referring to the database of the structure of amino acid sequences for antibodies created by Kabat et al. a Amino acid sequences of hypervariable sites 1-3 of the M-region of the I-chain are expressed respectively by "" sequences with identification MoMo59-61, and amino acid sequences of hypervariable sites 1-3

The M-regions of the H-chain are respectively expressed by sequences with identification MoMob2-64. -

F g - Example 2 c Construction of a chimeric antibody (1) Construction of the H-chain of a chimeric antibody (Y) Construction of the M-region of the H-chain 5B The cloned cDNA encoding the M-region of the mouse H-chain is modified by the RSC method and ligated with the expressing a vector containing the DNA genome for C in the C region of the human H chain. The lower seed of MVS1-51

Ф) (the sequence with identification Mo7) is constructed in such a way that it hybridizes with DNA encoding the 5-terminal region of the leader sequence of the M-region and has the agreed sequence of Kozak |(Kogak, M. ei. ai,, U. Moi .Vioi, 196, 947-950, 1987| and the Nipai recognition sequence. The upper primer of MVS1-abo (identification sequence Mo8) is designed to hybridize with DNA encoding the 33-terminal region of the yU region, and a small donor sequence of the spliced fragment and a VatNi recognition sequence. Polymerase chain synthesis reaction is carried out using TaKaKKa Ex Tad (TaKaKa 5pyg20) and the appropriate buffer. The solution for polymerase chain synthesis reaction (5Oml) contains 0.07μg of plasmid

MVSTNOYA as a template DNA, KhoOpmoley MVS1-a and Hopmoley MVS1-51 as seeds, 2.5 units of TaKaKa Ex 65 Tad and 0.25 MM amtR in a buffer over which 50 μl of mineral oil is placed. Perform thirty cycles of the polymerase chain reaction according to the temperature cycling program in the following sequence: 94927 for 1 minute, 5592 for 1 minute, 722 for 2 minutes. DNA fragments, amplified as a result of the polymerase chain reaction, are separated by electrophoresis in an agarose gel using 390 agarose of Mi Zieme STO (EMO Vio. Rhodisiv).

Then cut out a section of the agarose gel containing a DNA fragment with a length of 437 base pairs, and purify the specified DNA fragment using the SEMESI EAM II kit (VIO 101) according to the instructions attached to it. Purified DNA is precipitated with ethanol and dissolved in 20 μl of a solution containing 10 mM tris-HCl (pH 7.4) and 1 mM ethylenediaminetetraacetic acid (EDTA). One μl of the obtained DNA solution is cleaved with the restriction enzymes Watney and Nipaiy ("Ta-kaga Zypi2o") at a temperature of 372C for 1 hour. 70 The hydrolyzed mixture is extracted with phenol and chloroform and the DNA is precipitated with ethanol.

The resulting Nipap-WatNi DNA fragment, which contains DNA encoding the M-region of the H-chain of the mouse, is subcloned into the pOS19 vector, which is previously cleaved with Nipai and Watni enzymes. The resulting plasmid is sequenced with a 37ZA DNA sequencer (Regkip-EIteg), using M1Z Rgiteg M4 and M13Z Rgiteg EM primers and a kit for cyclic sequencing with terminator staining (Regkip-EIteg). A plasmid containing 75 DNA with the required base sequence encoding the M-region of the mouse H-chain isolated from the hybridoma is indicated

Mo23-57-137-1, and has a NipaP recognition sequence, a Kozak sequence in the 5-terminal region, and

Watni-recognition sequence in the 3'-terminal region, named "IMVSTN/rist19. (i) Construction of the M-region of the H-chain used to obtain the cDNA of the chimeric H-chain of the "mouse-human" type

The DNA obtained at the previous stage, encoding the M-region of the H-chain of the mouse, is modified by the ROK method and ligated with the cDNA for the C and 1 C-region of the human H-chain. The reverse primer MKSITRMUZ2 (sequence with identification Me9) used to modify the M-region of the H-chain is designed to replace the second amino acid (i.e., asparagine) of the sequence encoding the front of the M-region leader sequence with glycine to obtain a consistent sequence Kozaka | Kolak, M. ekaiY., U. Moi. Viol., 196, Ge 947-950, 1987) and Nipap-y EesokI-recognition sequences The forward primer MVSITRMK2 (sequence with the identification number Mo10) used to modify the M-region of the H-chain is designed in such a way that it hybridizes with a DNA sequence encoding the 3'-terminal region of the -region, and with a DNA sequence encoding the 5-terminal region of the C-region, and had Ara!1- and Ztai!-recognition sequences.

Polymerase chain synthesis reaction is carried out using TaKaKa Ex Tad (Takaga Zpyzo) and (ee) the appropriate buffer. The solution for the polymerase chain reaction (5 μm) contains 0.bmcg of plasmid

MVSTN/rist19 as a template DNA, Khopmoley MVSTNMU52 and ZHopmoley MVSYANMK2 as seeds, 2.5 units -

TaKakKa Ex Tad and 0.25mM amtR in a buffer over which 5µl of mineral oil is placed. Thirty hundred cycles of the polymerase chain synthesis reaction will be performed in accordance with the program of cyclic temperature changes in the following sequence: 942; within 1 minute; 55923 for 1 minute; 722C for 1 minute. DNA fragments amplified as a result of the polymerase synthesis reaction are separated by electrophoresis in - agarose gel using 1950 agarose Zea Kegpp STO (EMO Vio. Rgodisiv). Then cut out the section of the agarose gel containing the DNA fragment with a length of 456 base pairs, and purify the specified DNA fragment using the SEMESI EAM HER kit (VIO 101) according to the manufacturer's instructions. Purified DNA fragments are precipitated with ethanol and dissolved in 20 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EOTA.

One μl of the obtained DNA solution is cleaved with restriction enzymes Esoki and Ztai! (Takaga Zpy2o) - s at a temperature of 372C for 1 hour. The hydrolyzed mixture is extracted with phenol and chloroform and the DNA is precipitated with ethanol. The resulting EsokiI-Ztai DNA fragments, which contain DNA encoding the M-region of the mouse H-chain, are subcloned into the pOS19 vector, and the plasmid is cleaved in advance with Esoki and Ztai enzymes. The resulting plasmid is sequenced with a DNA sequencer 37ZA (Regkip-EIteg), using M13Z Rgiteg MA and M13Z Rgiteg primers

PM and a kit for cyclic sequencing with terminator staining (Regkeep-EItag). The indicated plasmid contains DNA with the required base sequence encoding the M-region of the mouse H-chain, isolated from the hybridoma sl Mo23-57-137-1, and has a NipapP recognition sequence, a Kozak sequence in the 5-terminal region and Arai! - and

Ztai!-recognizing sequences in the 3'-terminal region, called "ZhMVS1Nm/ryC19", o (iii) Construction of the expressing vector for the H-chain of the chimeric antibody -oUu 70 cDNA containing Su! The C-region of the H-chain of human antibodies is obtained as follows.

From the ovary cell of a Chinese hamster, into which the expressing vector ONEK-LE-KMA-RM-1 is introduced in advance. UU092/19759), encoding the genomic DNA of the M-region of the H-chain of the humanized antibody RMI and IDSI of the C-region

H-chain of human antibodies, and expressing vector KMI-RMIa | see UU092/19759|), coding DNA genokhai

M-area | - chain of the humanized antibody RMI and the C-region of the k-chain of the I-chain of the human antibody, 22 mRNAs are obtained. The obtained mRNA is used to clone cDNA containing the M-region of the H-chain using the CT-RSK method

GF) of humanized antibody RMI and Su! C-regions of human antibodies, which are then subcloned into the Nipap-Watnyi site of plasmid risC19. The DNA sequence is determined in the subcloned plasmid. Plasmid with the required base sequence is named "rAMP-RM-CDNA".

To construct an expression vector for cDNA containing the M-region of the H-chain of the humanized antibody 60 RMII Su! C-regions of human antibodies receive the ONEK-DE-KMP-RM-1 expressing vector with deletions in the NipaiI site between the 5M40 promoter and the ONEK gene and in the EsokKI site between the EBE-19u promoter and the M-region of the H-chain of the humanized antibody PM1 .

The resulting RKMA-RMI1 cDNA plasmid is cleaved by Watni, dephosphorylated with the Klenova fragment and again cleaved by Nipay! with obtaining the dephosphorylated Nipap-VatnNi fragment. This dephosphorylated Nipap-WatNi fragment is ligated with the expression vector UNEK-DE-KMA-RM-1, which has deletions in the sites

Nipayi and EEsokiI, which are split in advance by Nipayi and Vatni, in order to construct an expressing vector

KMp-RMIEKDNA, containing cDNA encoding, respectively, the M-region of the H-chain of the humanized antibody RMI1 and SuU1

C-regions of human antibodies.

The expression vector KMP-RMII-cDNA, containing cDNA encoding the M-region of the H-chain of the humanized antibody of RMI and SuU1 of the C-region of the human antibody, is cleaved by Arai and Watni, after which a DNA fragment containing the C-region of H is obtained - a chain The resulting DNA fragment is introduced into the above-mentioned plasmid MVS1Nm/pOst19, which is cleaved in advance by Aha! and Watni. The plasma obtained in this way was named "Zhmyvysi1NnKDNnNK/rOsSt19". This plasmid contains cDNAs that encode, respectively, the M-region of the H-chain antibody of the mouse and the Sui C-region of the human antibody 70 and have Esoki- and NipaPI-recognizing sequences at the 5-end and WatnHi-recognizing sequence at the 3'-end.

Plasmid МВС1НСДНКУК/рысСт19 was digested by EsokI and Watni to obtain DNA encoding the H-chain of the chimeric antibody. The resulting DNA fragment is introduced into the pCO51 expressing vector, which is cleaved beforehand by Esoki and VatNi. The expressing vector for the chimeric antibody obtained in this way is called "MVSTNkDNA/pCO51". The pSOBI expressing vector is constructed using HER-PMY-9 y1 (see 75.M092/19759), removing the antibody gene by cleavage with Esoki and Ztai and ligating it with an adapter

EsoKI-Moi-Watni (TakKagi Zpyg20).

In order to obtain a plasmid for expression in a Chinese hamster ovary cell, the MVSYANnNKDNKyurOS19 plasmid is cleaved by Esoki and Watney to obtain DNA encoding the Hn chain of a chimeric antibody, which is then introduced into the pSNOI expressing plasmid, which is cleaved in advance by EsoK! and Watni. The expressing plasmid for the chimeric antibody obtained in this way is called "YUZhMVSTNKDNnNK/pSnHOi1. The pSNoO!1 expressing vector is constructed using ONEK-DE-gmN-PM1-i, see UMU092/19759)|), removing the antibody gene by cleavage with the help of Esoki and Ztai and ligating it with the adapter EsoKI-Moi-Watni (iakaga Zpiz20).(2) Construction of the C-region of the human β-chain () Obtaining the cloning vector c

To construct the vector рыС19 containing the C-region | -chain of a person, receive the pOsC19 vector with a deletion of the Nipai site. Two micrograms of pOS19 vectors are cleaved in 20 microliters of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MoCl, 1 mM dithiothreitol (OT), 100 mM KCl, 8 units of Nipai (Takaga Zpy20), at

The temperature is 372C for 1 hour. The resulting hydrolyzed mixture is extracted with phenol and chloroform and the necessary DNA is precipitated with ethanol. co

The resulting DNA is subjected to interaction in 500 μl of a reaction solution containing 50 mM tris-HCIII (pH 7.5), 10 mM -d-

MaSi", 1 mM dithiothreitol (01), 100 mM Masi, 0.5 mM amMte and 6 units of the Klenov fragment (SIVSO VK), at room temperature for 20 minutes, as a result of which DNA dephosphorylation occurs. The resulting i) reaction mixture is extracted with phenol and chloroform and the vector DNA is precipitated with ethanol. yu

The obtained vector DNA is subjected to interaction in 1 μl of a reaction solution containing 5 μM Tris-HCl (pH 7.6),

With 10 mm of Mosi, 1 mm of adenosine-5'-triphosphate (ATP), 1 mm of thiothreitol (OT), 595 (by volume) - polyethylene glycol-80O0O and 0.5 units of T4 DNA ligase (SBIVSO VK), at at a temperature of 169C for 2 hours, as a result of which autoligation of vector DNA occurs. The reaction mixture (bmcl) is added to 100 μl of the solution containing competent cells of the strain UM109 of E. soy (Mirrop Sepe) and the resulting solution is left to stand on ice for 30 minutes, then at a temperature of 42 °C for 1 minute and again on ice 50 for 1 minute. Add 500 ml of OS culture medium to the reaction solution and incubate at a temperature of 372C for 1 hour. The resulting solution is placed on agar medium 2KHUT (containing 5 µg/ml of ampicillin), on the surface of which X-da! and IRTO | , 1989), and cultured at a temperature of 372C overnight, thereby obtaining a transformant. DNA using the 1-strand plasmid purification minikit (OIACEM) according to the instructions provided. This purified plasmid is cleaved by Nipai. This Nipap site-deleted plasmid is named "pOS19 ANipai". (i) Construction of DNA encoding C -region ;y-chain I -chain of human - The C-region of the y-chain I -chain of human antibody is known to have at least four isotypes, including o Msod'Ke"O27, MsdKeO27, MsudKe 07" and I Msd'Ke" O27 |R. Oagiamas, hey. aI., Rhos. May Asad All together. O5A, 84, 9074-9078, 1987). Based on the EMVI database. a search was made for the C-region of the x-chain and I-chain of the human antibody, which is homologous to the C-region of the x-chain and I-chain of the mouse Mo23-57-137-1. As a result, it was established that the isotype Msa 29 "Ke"O7 (Mo accession 1X57819) |P. Oagiamas, hey. ai, Rgos. Maya Asad All together. O5A, 84, 9074-9078, 1987) 5-chains

HF) Y-chain of the human antibody is most homologous to the C-region of the x-chain of the I-chain of the mouse Mo23-57-137-1, characterized by 64.495 homology with respect to the amino acid sequence and 73.495 homology with respect to the DNA sequence.

Then, DNA encoding the C-region of the y-chain of the I-chain of a human antibody is constructed using the PCR method. All 60 primers below are synthesized using the DNA/RNA synthesizer 394 (AVI). Seeds are synthesized

NIGAMVI (sequence with identification Mo11) and NI AMVZ (sequence with identification Me13), which contain a sense sequence of DNA, and primers NIAMV2 (sequence with identification Mo12) and NIH AMV4 (sequence with identification Mo14), which contain an antisense DNA sequence, while all primers have a complementary sequence of 20-23 base pairs at both ends. because External primers NIAMV5 (sequence with identification Mo15) and NIAMVK (sequence. with identification Me16) have a sequence complementary to the primers NI AMV1 and NI AMBA, respectively, and also contain, respectively, EsoKi-, Nipai- and B'pi-recognition sequences and EsoKi -recognizable sequence. During the first polymerase chain synthesis reaction, the interaction of NG AMV 1-NO AMV2 and NO AMV3Z-NO AMVA4 occurs.

After the completion of the specified reactions, both of the obtained products are mixed with each other in equivalent quantities and their collection is carried out during the second polymerase chain synthesis reaction. External seeds NIAMV5Z and NG AMVK are added to the obtained product. This reaction mixture is subjected to a third polymerase chain reaction to amplify unprocessed DNA.

Polymerase chain synthesis reactions are carried out with the help of TaKaKa Ex Tad (Takaga Zpy20o) in accordance with the manufacturer's 70 instructions. During the first polymerase chain reaction, use 100 μl of a reaction solution containing bpmoles of NIH AMV 1, 0.5 pmoles of NI AMV2 and 5 units of TaKaCa Ex Tad (Takaga

Zpy20), or a reaction solution containing 0.5 pmol NI AMV5, 5 pmol NI AMVA and 5 units of TaKaKa Ex Tad (Takaga 5p!y20), over which 5Oml of mineral oil is placed, and perform five cycles of the polymerase reaction of chain synthesis according to temperature cycle programs in the following sequence: 9422 for 1 7/5 minutes, 602C for 1 minute and 72922 for 1 minute. During the second polymerase chain synthesis reaction, use a mixture containing 5O0μl of each reaction solution, over which 5μl of mineral oil is placed, and perform three cycles of the polymerase chain synthesis reaction according to the cyclic temperature change program in the following sequence: 9422 for 1 minute, 602С for 1 minute and 729bb for 1 minute. During the implementation of the third polymerase chain synthesis reaction, 20 reaction solution will be used, to which 5HOpmol of external primers NI AMV5 and NIH AMVE are added, and thirty cycles of the polymerase chain synthesis reaction will be performed according to the program of cyclic temperature changes in the following sequence: 9422 for 1 minute, 602 for 1 minute and 722C for 1 minute.

The DNA fragment obtained as a result of the third polymerase chain synthesis reaction is subjected to electrophoresis in a 396 low-melting agarose gel (MybZieme STO Adagoze, EMC), after which it is isolated and purified from a 25-s gel using the SEMESI EAM II kit (VIO 101) in accordance with the instructions attached to he instructions. at

The thus obtained DNA fragment is cleaved in 2 μl of a reaction solution containing 50 mM Tris-HCl (pH 7.5), 10 mM Moais, 1 mM dithiothreitol (OT), 100 mM Mass, and 8 units of EsokI (Takaga Zpy20), at a temperature of 372C for 1 hours The hydrolyzed solution is extracted with phenol and chloroform and precipitated

DNA with ethanol. The obtained DNA is collected and dissolved in μl of a solution containing 100 mM tris-HCl (pH 7.4) and 30 mM ethylenediaminetetraacetic acid (EOTA). "-

Plasmid ryC19 ANipai (0.μg) is cleaved by EsokKiI similarly to the procedure described above. The hydrolyzed solution is extracted with phenol and chloroform and precipitated with ethanol, which gives the cleaved plasmid She pOist19 ANipai. The cleaved plasmid is subjected to interaction in a reaction solution (5 µl) containing 56 mM Tris-HCl (pH 9.0), 1 mM Maosi" and alkaline phosphatase (E. soya C75; TaKkaga 5pi20) at a temperature of 372 for 30-35 minutes, as a result of which dephosphorylation of the indicated plasmid occurs (that is, it is treated with bacterial alkaline phosphatase). The reaction solution is extracted with phenol and chloroform and the DNA is precipitated with ethanol.

The DNA obtained in this way is dissolved in 1 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and mM EOTA.

One μl of bacterial phosphatase-treated plasmid pOS19 AnNipaii is mixed with 4 μl of DNA obtained as a result of the above-mentioned polymerase chain reaction to ligate them with each other using the DNA ligation kit, option 2 (TaKaga 5pi20). The resulting plasmid is introduced into a competent cell of E. soybean, with UM109, to form a transformant. The transformant is cultured overnight in 2 ml of 2KHUT medium containing 3 mg/ml of ampicillin. The plasmid obtained from this cell is purified using a plasmid purification kit

OPIArger (OIASEM).

The sequence of the cloned DNA is confirmed in the plasmid described above. DNA sequencer Z37ZA (AVI) and primers "M13 Rgiteg M4" and "M13 Rgiteg KM" (TaKkaga 5p!yg20) are used to determine the sequence of DNA-1. This allows us to establish that the cloned DNA has a deletion of 12 base pairs. The specified plasmid containing this 1 DNA is named "shl/pOS19". Then, to obtain the recombinant part, the primers NSI M5 s (sequence with identification Mo17) and NSIMK (sequence with identification Mo18) are synthesized again and the necessary DNA is reconstructed using the polymerase chain reaction (PCR) method. - The first polymerase reaction of chain synthesis is carried out using a plasmid from shl/pOS19, containing deleted DNA as a template, and primers NIAMV5 and НСИМ5 or primers НСИМ5 and NI АМВ4. All products of the polymerase chain reaction are sequentially purified. When carrying out the second polymerase chain reaction, the assembly of the PCR products is carried out. External primers NI AMV:5 and 5B NIGAMVA are added to the obtained product, after which a third polymerase chain synthesis reaction is performed to amplify the unprocessed

DNA. (F) During the implementation of the first polymerase chain synthesis reaction, use 100 μl of the reaction solution containing 0.1 μg of СХА/рысС19 as a matrix, 100 mol of the seed NI AMV5 and НСИ MK or 5 000 mol of the seed

NOSI M5 and NG AMBA and 5 units of TaKakKa Ex Taz (TaKaga Zpiyi20), over which 50 μl of mineral oil are placed, and bo Perform thirty cycles of the polymerase reaction of chain synthesis according to the program of cyclic temperature changes in the following sequence: 942 for 1 minute, 602С for 1 minutes and 722 for 1 minute.

The products of the polymerase chain reaction, NI AMV5-HCI MK (236 base pairs) and NCI M5-NI AMBA (147 base pairs) are electrophoresed in a 395 low-melting point agarose gel to isolate the DNA fragment. Fragment 65 DNA is collected and purified from the gel using the SEMESI EAM II kit (VIO 101). During the second polymerase chain reaction, use 20 μl of a reaction solution containing 4 ng of purified DNA fragments and 1 unit of TaKaka Ex Tayad (TaKkaga Zp!y20), over which 25 μl of mineral oil is placed, and perform five cycles of the polymerase chain synthesis reaction, respectively to the cyclic temperature change program in the following sequence: 942 for 1 minute, 602 C for 1 minute and 722 C for 1 minute.

During the third polymerase chain reaction, use 100 μl of reaction solution containing 2 μl of the reaction solution obtained during the second polymerase chain synthesis reaction, 5 Opmol each of NI AMV5 and NIH AMVA primers and 5 units of TaKaKa EX Tai (TaKaga 5py20), above which is placed 5 µl of mineral oil, and perform thirty cycles of the polymerase reaction of chain synthesis according to the program of cyclic temperature changes in the following sequence: 942 for 1 minute, 602 for 1 70 minutes and 7292C for 1 minute, which gives a DNA fragment with a length of 357 base pairs (the product of the third polymerase chain synthesis reactions). The DNA fragment is subjected to electrophoresis in a 395 low-melting agarose gel. The resulting DNA fragment is removed and purified using the SEMESI EAM kit (VIO 101).

The thus obtained DNA fragment (0.mkg) is cleaved by EsoK!I and subcloned into the pOist19 ANipai plasmid, which is pre-treated with bacterial alkaline phosphatase (BAP). The resulting plasmid is injected into a 75-competent E. soybean cell, UM109, to form a transformant. The transformant obtained in this way is cultured overnight in 2 ml of 2KHUT medium containing 5O0μg/ml of ampicillin. The plasmid isolated from the cellular fraction is purified using the OIArger (OIASEM) plasmid purification kit.

The DNA sequence of the obtained plasmid is confirmed using M13 Rgiteg MA and MIZ Rgiteg EM (Takaga 5pig2o) primers and determined on a 37Z3A DNA sequencer (AVI). Plasmid, with confirmed correct DNA sequence without any deletion, named "СХ/rust19", (ii) Construction of DNA encoding the C-region of the k-chain of the human I-chain

The DNA fragment encoding the C-region of the k-chain and the I-chain is cloned from the HER-RMIK-akK plasmid (VDO92/19759) by the ROK method. The forward primer NKARZ (sequence with identification Me19) is designed so that it contains the EsoKi-, Nipai- and Vipi-recognition sequences, and the reverse primer NKARA (sequence with identification Mo20) is designed so that it contains the EsoKi-recognition sequence, (3 using DNA/RNA synthesizer 394 (AVI) for their synthesis.

The polymerase chain synthesis reaction is carried out using 100 μl of a reaction solution containing 0.1 μg of the NER-RMIK-OK plasmid as a matrix, 5 μmol each of NKARZ and NKARA primers and 5 units of TaKaKa Ex Tad (Takaga Zpy!y20o), over which 5 μmol of mineral oil is placed . Thirty cycles of the polymerase reaction with chain synthesis will be performed according to the program of cyclical temperature changes in the following sequence: 9422 for 1 minute, B02C for 1 minute and 7229C for 1 minute, as a result of which DNA fragment - is obtained. o co with a length of 360 base pairs. The resulting DNA fragment is isolated by electrophoresis in a 3906 low-melting agarose gel, removed and purified using the ZEMESI EAM 1I kit (VIO 101). i am

The DNA fragment obtained in this way is cleaved by EsoK!I and cloned in the pOS19 AnNipai plasmid, which is pre-treated with bacterial alkaline phosphatase (BAP). The resulting plasmid is introduced into a competent cell

E. soybean, UM109, for the formation of a transformant. The transformant obtained in this way is cultured overnight in 2 ml of 2KHUT medium containing 5 Ωkg/ml of ampicillin. The plasmid isolated from the cellular fraction is purified using the OIArger (OIASEM) plasmid purification kit. " 20 Purified plasmid DNA is sequenced using primers M13 Rgiteg M4 and M13 Rgiteg KM (TakKaga -v

Zpy20), using DNA sequencer 37ZA (AVI). Plasmid, with the confirmed correct sequence of bases, is named "Sk/rusC19" :z" (3) Construction of the chimeric antibody expressing vector I -chain

Construct an expressing vector of the I-chain of the chimeric antibody Mo23-57-137-1. DNA encoding the M region

The Y-chain of Mo23-57-137-1 is ligated to the NipaiSh and Vipi sites, located immediately before the C-region -I of the human antibody, of both plasmids CX/rist19 and Sk/rIisC19, as a result of which vectors risC19 containing DNA are obtained, which encodes the M-region of the I-chain of the chimeric antibody Mo23-57-137-1 and the C-region of the x- or k-chain of the I-chain. i-th Both vectors are then cleaved with EsoKiI to cut the DNA encoding the I-chain of the chimeric antibody, which d) is then subcloned into the HER expressing vector.

DNA encoding the M-region of the I-chain of the Mo23-57-137-1 antibody is cloned from the MVS11 24 plasmid by the ROK method. - Separate primers are synthesized using the DNA/RNA synthesizer 394 (AVI). Reverse primer MVOSSNI 1 o (sequence with identification Mo21) is constructed in such a way that it contains the NipapP recognition sequence and the Kozak sequence (Kogak, M. ei. ai., 9. Moi. Vioi. 196, 947-950, 1987), and direct seeding

MVSOSNIZ (sequence with identification Mo22) is constructed in such a way that it contains Vai- and XoKiI-u-knowing sequences.

Polymerase chain synthesis reaction is carried out using 100 μl of reaction solution containing

Ф) 10 mM Tris-NSI (pH 8.3), 50 mM KSI, 1.5 mM Masi», O0.2 mM akymtR, 0.1 μg MVS11I 24, per Zopmoley of MVOSNI seeds 1 ko Her MVOSNI Z and Imkl AtriaTad (Regkip EITeg), over which 5O0μl of mineral oil is placed. Thirty cycles of the polymerase chain synthesis reaction will be performed in accordance with the program of cyclic temperature changes in the following sequence: 9492; for 45 seconds, 602C for 45 seconds and 722C for 2 minutes.

A DNA fragment with a length of 444 base pairs is subjected to electrophoresis in a 3906 low-melting agarose gel, collected and purified using the SEMESI EAM kit (VU 101). The purified DNA fragment is dissolved in 2 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and mM EOTA. One μl of the product of the polymerase chain synthesis reaction is cleaved in 20 μl of a reaction solution containing 10 mM Tris-HCIII (pH 7.5), 10 mM Maosi, 1 mM 65 dithiothreitol, 50 mM Masi, 8 units of Nipai (TaKaga Zpy20) and 8 units of EsokI (TaKaga Zpy20), at a temperature of 372C for 1 hour. The hydrolyzed solution is extracted with phenol and chloroform and the DNA is precipitated with ethanol. The DNA obtained in this way is dissolved in μl of a solution containing 10 mM Tris-HCl (pH 74) and 1 mM

EOTA.

One μg of pOS19 plasma breaks down Nipaiyi and EsoKi! similarly to the procedure described above, it is extracted with phenol and chloroform, after which the split schiazmid is precipitated with ethanol. The resulting product is treated with bacterial alkaline phosphatase (E. soya C75; Takaga Zpy20)), extracted with phenol and chloroform, and the DNA is precipitated with ethanol. The obtained DNA is dissolved in 10 ml of a solution containing 10 mM Tris-HCl (pH 7.4) and mM EOTA.

One μl of the alkaline phosphatase-treated pOS19 plasmid is mixed with 4 μl of the above-mentioned 7/0 polymerase chain reaction product to ligate with each other using the DNA ligation kit option 2 (Takaga 5Pp!i20o). The resulting plasmid is introduced into a competent cell of E. soy, UM109 (Mirrop Sepe) similar to the procedure described above for the formation of a transformant. The resulting transformant is inoculated overnight on 2KHUT agar medium containing 50 μg/ml of ampicillin at a temperature of 372C. Then the obtained transformant is cultivated overnight in 2 ml of 2KHUT medium containing 5 Ωkg/ml of ampicillin at a temperature of 75.С72С. The plasmid isolated from the cell fraction is purified using the OiIArger plasmid purification kit (OIACEM). After determining the DNA sequence, the plasmid with a confirmed DNA regulatory sequence is named "CH/picC19".

One μg each of the plasmids CX/ryC19 and Sk/pOS19 is split into 20 μl of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM Massi", 1 mM dithiothreitol, 100 mM KSI, 8 units of Nipai (Takaga Zpy2o) and 2 units

Vipi (Takaga 5py20), at a temperature of 372C for 1 hour. The hydrolyzed solution is extracted with phenol and chloroform and the DNA is precipitated with ethanol. The obtained DNA is treated with alkaline phosphatase of bacteria at a temperature of 372C for 30 minutes, extracted with phenol and chloroform and precipitated with ethanol. The obtained product is dissolved in 1 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EOTA.

Eight micrograms of the SNI /pOsSt19 plasmid, which contains DNA encoding the M-region of the I-chain of Mo23-57-137-1, is cleaved by SM Nipayi| and VIP! as described above. The thus obtained DNA fragment with a length of 409 base pairs is subjected to electrophoresis in a 3965 low-melting agarose gel, collected and purified from the gel using a kit

SEMESI EAM (VIO 101). This DNA fragment is dissolved in 10 μl of a solution containing T0mM Tris-HCl (pH 74) and 1Mm EOTA.

Four microliters of DNA of the M-region of the I-chain are subcloned into a microliter of alkaline phosphatase-treated plasmids 99 Shuris19 or Sk/ryC19 and introduced into a competent E. soybean cell, /M109, in order to obtain a transformant. "-

The resulting transformant is cultured overnight in Zml medium 2KHUT containing 5 Ωkg/ml ampicillin.

The plasmid isolated from the cellular fraction is purified using the OIArger (OIASEM) plasmid purification kit. and,

Plasmids obtained in this way are respectively named "MVS. (5) /"pOSC19" and "MVSLI (k) /"pROS19", and

Plasmids of MIA (5) /pOS19 and MIA. (k) /pOS19 is cleaved by EsoKiI and subjected to electrophoresis at 390

From a low-melting agarose gel. A DNA fragment with a length of 743 base pairs is isolated, it is purified from the gel with the help of the ZEVESOI EMIA kit! (VIO 101) and dissolve in 1 μl of a solution containing 10 mM Tris-HCl (pH 74) and 1 mM EOTA.

The expressing vector of the plasmid HER-RM'IK-YaOK (2.7 μg) is cleaved with EsoKI, extracted with phenol and chloroform, and the DNA fragment is precipitated with ethanol. The resulting DNA fragment is treated with alkaline Z7Z70 bacterial phosphatase and subjected to electrophoresis in a 195 low-melting agarose gel. A DNA fragment with a length of 6561 base pairs is isolated and purified from the gel using the SEMESI EAMIIA kit! (VIO 101). The DNA fragment "from" is dissolved in 1 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EOTA.

The NEE vector obtained in this way is treated with bacterial alkaline phosphatase, and 2 μl of alkaline phosphatase-treated NEE vectors are mixed with 3 μl of EsoKI-fragments of MVU11G plasmids. (5) "pOS19 or MVS. (k) /"pOSSt19, - 15 to ligate them with each other. The ligation product is injected into a competent cell of E. soybean, "UM109, to form a transformant. The resulting transformant is cultured in 2 ml of 2HUuUT medium containing 500 μg/ml of ampicillin. 1 The plasmid isolated from the cellular fraction is purified using the OIRrger plasmid purification kit (OIACEM). Plasmid DNA purified in this way is cleaved in 200 μl of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM Massi, mM dithiothreitol, 100 mM KCl, 5 units Nipai! (Takaga Zpy20) and 2 units - 70 Rush ( Takaga Zp!ig20o), at a temperature of 372C for 1 hour. It is assumed that if the above-mentioned DNA fragment is introduced into a vector with the correct orientation, then as a result of hydrolysis, a cleaved fragment with a length of 5104/2195 base pairs will be obtained, and if the above-mentioned DNA fragment is introduced into a vector with the reverse orientation, then a cleaved fragment with a length of 4387/2926 base pairs will be obtained. about the Ministry of Internal Affairs. (5). against the antigen. 60 Temporary expression of a chimeric antibody is carried out using a combination of plasmids MVSYANKDNyursSob5I

Her Ministry of Internal Affairs. (5) / peo or plasmid MVSTNKDNA/rsSoZ5I and MVSTI. (k) / peo, by the method of electroporation using an electroimpulse device (zepe Riizeg (Vio Kai) for cotransfection of these combinations of plasmid DNA into cells

СО5-7. That is, 7Okg of each plasmid DNA is added to O.vml of suspension of CO5-7 cells in phosphate-buffered physiological solution (PBO(-)) with a concentration of 1x107 cells/ml. The resulting solution is exposed to 65 pulses at an electrostatic capacity of 15008 and an amperage of 2μF to cause electroporation. The solution is kept for 10 minutes at room temperature, after which the cells are suspended in Dulbecco's modified Igla's medium containing 295 fetal calf serum with an ultra-low content of dO (SIVSO), and cultured in a 10 cm dish in an incubator with CO. Cells are cultured for 72 hours, after which the culture supernatant is collected, centrifuged to remove cellular debris, and used as

Sample for solid-phase enzyme immunoassay (EGI5A).

When performing this procedure, purification of chimeric antibodies from the culture supernatant of cells СО5-7 is carried out using the AiiSbe kit! Rgoieip A MARBII (Vio Kai) in accordance with the instructions attached to it. (5) Solid-phase enzyme immunoassay (EP IZA) 70 () Determination of antibody concentration

A solid-phase enzyme-linked immunosorbent assay (ELISA) tablet designed to determine the concentration of antibodies is prepared as follows. Into all wells of a 9b-well tablet for EGISA (Makhizogr, MOMS) inject 100 μl of a solution containing a goat anti-human antibody (TASO) in a buffer for surface sensitization (0.1 M solution of ManHsSoO»z, 0.02956 MamM»z) , with a concentration of μg/ml and block 200 μl of buffers for dilution | 5 MM Tris-HCI, TMM Mosi, 0.1 M Masi, 0.0595 Tween-20, 0.0295 MamM»z, 195 bovine serum albumin (VZA); pH 7.21. In all wells, add the culture supernatant of cells CO5-7, in which chimeric antibodies were expressed or purified chimeric antibodies obtained as a result of serial dilution. The contents of the well are incubated at room temperature for 1 hour and washed with a mixture of phosphate-buffered saline and Tween-20, after which 100 μl of a solution of alkaline phosphatase-conjugated goat anti-human dO antibodies (TASO) is added to each well. The resulting mixture is incubated at room temperature for 1 hour, washed with a mixture of phosphate-buffered saline and Tween-20, and substrate (Zidta 104, paranitrophenylphosphoric acid, BISMA) is added to each well of the mg/ml solution. The optical density of the resulting solution is measured at a wavelength of 405 nm using a microplate reader (Vio Kai). Purified immunoglobulin IdoIX, human (binding site) is used as a standard for this measurement. (i) Determination of antigen-binding capacity.

A tablet for solid-phase enzyme-linked immunosorbent assay (ELISA), intended for the determination of antigen-binding capacity, is prepared as follows. Into all wells of a 9b-well plate, 100 μl of a solution containing human RTHgR (1-34) (Reriide Kezeagsp Ipzicie), in a buffer for sensitization of surfaces with a concentration of μg/ml, and block 200 μl of buffers for dilution. Cultural soil is added to all holes

From the supernatant of CO5-7 cells, in which chimeric antibodies were expressed or purified chimeric antibodies obtained as a result of serial dilution. The contents of the wells are incubated at room temperature and washed with a mixture of phosphate-buffered saline and Tween-20, after which 100 μl of a solution of alkaline phosphatase-conjugated goat anti-human (TASO) antibodies is added to each well. The resulting mixture is incubated at room temperature , washed with a mixture of phosphate-buffered saline and Tween-20 and

35 mg/ml substrate solution ("Zidta 104", paranitrophenylphosphoric acid, BISMA) is added to each well. The optical density of the resulting solution is measured at a wavelength of 405 nm using a microplate reader (Vio Cad).

The results of this analysis show that the chimeric antibody has the binding capacity against human RTNgR (1-34) and has the correct structure of the M-region of the cloned mouse antibody (Fig.4). In addition, it was established that "there is no difference between the binding capacity against RTNHGR (1-34) of a chimeric antibody in which the C-region of the Хсh c Y-chain has an хХ-chain, and a chimeric antibody in which the C-region | -chain has a k-chain. Therefore, the C-region of the I-chain of the humanized antibody is constructed using the X-chain of the I-chain of the humanized antibody. "» (6) Creation of a stably transformed Chinese hamster ovary cell line (CHO)

To create a stable transformant for the chimeric antibody, the above-mentioned expressing plasmid

INTRODUCE SNO (ОХВ11) into the cage. -I A stable transformant for a chimeric antibody is created using a combination of expressing plasmids for the CHO cell, MVSTNKEDNyurSsnO and MVS (5) / peo, or expressing plasmids for the CHO cell, and MVSTNKDNyurSNO and MVS. (k) /peo. These combinations of plasmids are separately cotransfected into CHO cells according to (95) the electroporation method using the Sepe Riizeg device (Vio Kai). All expressing vectors are cleaved by -l 20 restriction enzyme Rmuci to obtain linear DNA. The obtained DNA is extracted with phenol and chloroform and precipitated with ethanol. Plasmid DNA obtained in this way is subjected to electroporation. 1 µg of each plasmid DNA is added to 0.8 ml of suspension of cells with CHO in РВ5(-) with a concentration of 1x10 cells/ml. The resulting mixture is exposed to pulses with an electrostatic capacity of 15008 and an amperage of 2μF. Electroporated cells are kept at room temperature for 10 minutes and suspended in MEM medium (SIVSO) containing 1095 29 fetal calf serum (SIVSO). The obtained suspension is cultured on three 96-well plates (Eaisop)

GF) in an incubator with CO". The next day after the start of cultivation, this medium is replaced with a selective medium

MEM-o medium containing 1095 fetal calf serum (SIVSO) and 50O0mg/ml CEMETISIM (541851y11IHa(e; o SIVSO) without ribonucleoside or deoxyribonucleoside). Cells into which the antibody gene has been introduced are selected from the culture medium. The selective medium is replaced with fresh medium until culturing and after two weeks of culturing, after which the cells are examined under a microscope. In the cells characterized by satisfactory growth, the number of antibodies obtained as a result of performing the aforementioned solid-phase immunoassay is determined. The cells that produced the largest number of antibodies are selectively collected.

Raising the level of culture of a stable transformant for the obtained antibodies is carried out in a rotating flask using minimal support medium (MEM) supplemented with 296 fetal calf serum with an ultra-low content (DO without ribonucleoside or deoxyribonucleoside. On the 3rd and 4th day of cultivation the culture supernatant is collected and filtered through a 0.2 µm pore size filter (MiPiroge) to remove cellular debris.

Then the chimeric antibodies are purified from the culture supernatant of CHO cells in a ROKO5 column filled with protein A (RegzZeriime Viozuzietv), on Sopber I! C100 (MyProge) in accordance with the attached instructions.

Purified chimeric antibodies are used as samples to determine neutralizing activity and to study effectiveness in animal models of hypercalcemia. The concentration and binding activity of purified chimeric antibodies against the antigen are determined using a similar system for solid-phase enzyme-linked immunosorbent assay (EGIZA). 70 Example Z

Construction of a humanized antibody (1) Construction of the H-chain of a humanized antibody (Y) Construction of the humanized M-region of the H-chain

The H-chain of the humanized antibody Mo23-57-137-1 is obtained by transplanting the hypervariable site /5 by the ROK method. To obtain the H-chain of the humanized antibody Mo23-57-137-1 (variant "a"), which has a framework region isolated from the human antibody 531679 |MMKE-ROV; Siivipieg, A.M. hey!., Eig. Uittipoi!., 23, 110-118, 1993), uses the following six types of primers for the polymerase chain reaction: primers for transplantation of hypervariable sites MVSTRORI1 (sequence with identification Mo23) and MVSINOSRZ (sequence with identification Mo24) (both contain the sense sequence of DNA ) Her MVSTNOR (sequence

With identification Mo25) and MVSTNORA (sequence with identification Mo26b) (both contain an antisense DNA sequence), which have a complementary sequence 15-21 base pairs long at both ends; external primers MVSYATNMMU5I1 (sequence with identification Mo27) and MVSYATNMKT (sequence with identification

Mo28), which are, respectively, homologous to seeds for transplantation of hypervariable regions of MVS1NORI and

MvVs1NnOoga. with

Seeds for transplantation of hypervariable sites MVSTNORI, MVSITNOR2, MVSYATNORZ and MVSYA1NO RA are isolated with the help of urea-denatured polyacrylamide gel.

Mapiai, ZatrhookK, ey. aiYi., So 5rgipd Nagrog I aroghaiogu Rgezv, 1989| and extracted from the gel fraction by grinding and soaking.

Y arogayugu Rgezv, 1989), performing this procedure in this way. with the help of denatured polyacrylamide gel, one nmole of all the seeds for transplantation of the hypervariable site with obtaining DNA fragments is isolated. The obtained DNA fragments of the required length are identified on a thin plate of silica gel irradiated with ultraviolet light, and they are removed by grinding and soaking. The obtained product is dissolved in 20 μl of a solution containing 10 mM Tris-HCIII (pH 7.4) and 1 mM EOTA. Polymerase chain synthesis reaction is carried out using TaKaKa Ex o

There (Takaga 5p!y20). The reaction solution (100 μl) used for the polymerase chain reaction contains 1 μl of the above-mentioned seeds for transplantation of hypervariable sites

MVSYAINORI, MVSTNOR2, MVSYAINORZ and MVSTNORA, 0.25 mM amMtR and 2.5 units of TaKaKa Ex Tad in the buffer.

Perform five cycles of the polymerase chain reaction according to the temperature cycling program in the following sequence: 942 for 1 minute, 5593 for 1 minute, and 729C for 1 minute. To the resulting reaction mixture, add both external seeds MVSTNMU51 and MVSINMKI in the amount of Hopmoles. Using this reaction mixture, perform 30 more cycles of the polymerase chain reaction according to the same temperature cycling program. The DNA fragment amplified in this way is isolated by "" electrophoresis in an agarose gel using 4905 agarose of Mi Zieme STO (EMO Vio. Rhodisiv).

Then cut out the agarose section containing the DNA fragment with a length of 421 base pairs, and purify the specified

DNA fragment using the SEMESI EAMN kit (VIO 101) according to the instructions attached to it. The purified DNA fragment is precipitated with ethanol and dissolved in 20 μl of a solution containing 100 mM Tris-HCl (pH 7.4) and 1 mM EOTA. The reaction mixture for the polymerase chain synthesis reaction is used for subcloning this DNA fragment in the pOS19 plasmid, which is cleaved in advance by Vatni and Nipaiyi, after which it is determined

Ge) sequence of bases of the obtained plasmid. Plasmid having the correct sequence is named "yIMVvOSNm/rOsSt19", (i) Construction of the M-region of the H-chain for cDNA of the humanized H-chain - DNA of the humanized M-region of the H-chain, obtained at the previous stage, is modified by the ROK method, so to link it with the humanized Su's kKDNK! C-regions of the H-chain. For this, a reverse seed is constructed

MMSTNM51 so that it hybridizes with the sequence encoding the 5'-terminal region of the leader sequence

M-region, had a consistent Kozak sequence (Koak ei. aiYi., U. Moi. Vioi. 196, 947-950, 1987) and Nipai- and EsoKi-recognizing sequences. Direct seed MVSTNMUK2 used for DNA modification for

The M-region of the H-chain is designed so that it hybridizes with the DNA sequence encoding the 33-terminal region of the .)-region and with the DNA sequence encoding the 5-terminal region of the C-region, and has I-recognizing sequences.

Polymerase chain synthesis reaction is performed using TaKaKa Ex Tad (Takaga 5pyg2o0) and the appropriate buffer. The reaction solution, the one used for the polymerase reaction of chain synthesis, contains 04 μg of IMVSN/risC19 as a DNA matrix, 5 opmoles of MVSTNMUZ2 and MVSITNMKO primers, 2.5 units

TaKakKaeghTad and 025mM amMmTR in buffer. Thirty cycles of the polymerase chain reaction will be performed according to the temperature cycle program in the following sequence: 9492 for 1 minute, 559 for 1 minute and 722C for 1 minute. The DNA fragment amplified in this way is isolated by electrophoresis in an agarose gel using 39o agarose of Mi Zieme STO (EMO Vio. Rhodisiv).

A DNA fragment with a length of 456 base pairs is excised and purified using the ZEMESI EAMIA kit (VIO 101)

according to the instructions attached to it. The purified DNA fragment is precipitated with ethanol and dissolved in 20 μl of a solution containing 10 mM Tris-HCI (pH 7.4) and 1 mM EOTA. The reaction mixture obtained in this way will be used for subcloning of this DNA fragment in the pOsC19 plasmid, which was previously cleaved by Esoki and Ztai, after which the DNA sequence of the resulting plasmid is determined. Plasmid DNA that contains the coding DNA

The M-region of the mouse H-chain, isolated from the hybridoma Mo23-57-137-1, and has Esoki- and NipapP-recognizing sequences, a Kozak sequence at the 5' end and Arai - and Ztai!-recognizing sequences at the 3'-end, named "yIMVS1Nu/rust19", (2) Construction of an expressing vector for the H-chain of a humanized antibody

Plasmid KMP-RM'11-cDNA, containing the cDNA sequence of the H-chain of the antibody PRMI, is cleaved by AraiY and 7/0 Vatni to obtain a DNA fragment containing the sequence of the bases of the C-region of the H-chain. The resulting DNA fragment is introduced into the YAMVSTNm/pOsC19 plasmid, which is cleaved in advance by Ara! and Watni. The resulting plasmid is named "yImMVSTNKDNA/rOst19". This plasmid contains DNA encoding the M-region of the H-chain of the humanized antibody Mo23-57-137-1 and DNA encoding Su! The C-region of the H-chain of a person has Esoki- and

Nipapi-recognition sequence in the 5'-end region and WatnHi-recognition sequence in the 3'-end region. 75 This base sequence and the corresponding amino acid sequence of the humanized H-chain of the "a" variant, which is present in the plasmid НЯМВСТНКДНКриОст19, are expressed, respectively, by the sequences with identification MoMo 58 and 56.

Plasmid AYAMVSTNKDNA/pOS19 is cleaved by EsokKi!I and WatNi to obtain a DNA fragment containing the base sequence encoding the H-chain. The obtained DNA fragment is introduced into the RSOB51 expressing plasmid, which is cleaved in advance by EsokK!I and VatNi. The expression plasmid for the humanized antibody obtained in this way is called "yIMVSTnKDNnKrsSozt1".

To obtain a plasmid for expression in a CHO cell, the plasmid NIMVSYATNKDNA/pOS19 is cleaved by Esoki and

Cotton wool to obtain a DNA fragment encoding the H-chain. The resulting DNA fragment is introduced into the pRSNOI expressing vector, which is obtained by splitting the EsoK!I and VatNi plasmids. The thus obtained expressing plasmid for the humanized antibody is named "ivVS!nKkDnNK/rsnoT". c (3) Construction of the I-chain for the variable region of the hybrid antibody (Y) Obtaining the hybrid antibody EK, 2/EKZ3,4 o

DNA encoding the I-chain is constructed in which the framework regions are recombined with regions of the humanized antibody and (chimeric) mouse antibody, and these regions are evaluated for their suitability for humanization. At this stage, a hybrid antibody containing ECTi is obtained using the restriction site AYA, which is in SOC2.

IVF isolated from human antibody and IVF and RK4 isolated from mouse antibody.

In 100 μl of a reaction solution containing T0MM Tris-HCII (pH 7.5), 10MM Mass, 1MMM dithiothreitol, 5X0MMM - mass, 0.0195 (in the ratio of weight to volume) bovine serum albumin and 10 units of ALYA (TaKkaga ЗПпы20), "In cleave ten μg of plasmids MVSTI (5) / peo and AMVSII. (5) / ppeo at a temperature of 372 for 1 hour. The reaction solution is subjected to electrophoresis in a 295 low-melting agarose gel, the

DNA fragments with a length of 6282 base pairs (defined as "c1") and a length of 1022 base pairs (defined as o zcha "p17) are purified from the gel using the Semesi Eamiya kit (VIO 101).

One microgram of the obtained fragments of si! and pj1 are treated with bacterial alkaline phosphatase (BAP). The resulting DNA fragment is extracted with phenol and ethanol, precipitated with ethanol and dissolved in 1 μl of a solution containing 10 mM Tris-HCII (pH 7.4) and 1 mM EOTA.

One μl of fragments si and p1, treated with alkaline phosphatase, is mixed respectively with 4Amcl DNA fragments Y c 2 Her c2 to ligate them with each other (at a temperature of 49 overnight). The ligation product is injected into a competent cell of E. soybean, "UM109, in order to obtain a transformant. The resulting transformant is cultured in "» 2ml of 2KHUT medium containing 5Omkg/ml of ampicillin. The plasmid isolated from the cellular fraction is purified using the OIArger (OIASEM) plasmid purification kit.

Purified plasmid DNA is cleaved in 20 μl of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 10 mM Maci, 1 mM dithiothreitol, 2 units of Ara/I (Takaga Zpy2o) and 8 units of Vashni (Takaga Zpygo) or Nipap! sl (Takaga 5py20o), at a temperature of 372C for 1 hour. At this stage, the identification of the plasmid is carried out considering that, if the c1-p2 fragment is correctly ligated in the plasmid, this ligation should give (95) an AraIl-cleaved fragment containing 5560/1246/498 base pairs, or a VatNi/NipaPI-cleaved fragment, - in 20 containing 7134/269 base pairs.

The expression vector encoding the I-chain of a mouse hybrid antibody containing EC1,2 human and ECZ,4 mouse, 42) is named "p/tMVeII. (5) / peo". On the other hand, it is not possible to obtain a clone for p1-c1. Therefore, recombination is carried out on the pOS vector with subsequent cloning in the HER vector. As matrices, use the plasmid NMVSII ah/pOst19, which contains DNA encoding the M-region of the I-chain of a humanized antibody without amino acid substitutions, and the plasmid of the MVS ah/pOst19, which contains DNA encoding the M-region of the I-chain of a humanized antibody with amino acid replacement

GF) in position 91 of the framework region of IVF (amino acid Mo87 according to Kabat's definition), i.e. tyrosine, isoleucine. di In ZOmcl reaction solutions containing TMM tris-HCI (pH 7.5), 10 mM Mosi, 1 mM dithiothreitol, 50 mM mass, 0.0195 (in the ratio of weight to volume) bovine serum albumin, 16 Nipai units and 4 units of 60 ANI, cleave ten μl of MVS11 plasmids. (5) /рОС19, МВС ах/0ОС19 and NMVСиЙ 45/роС19 at a temperature of 372С for 1 hour. The reaction solution is processed by electrophoresis in a 295 low-melting agarose gel, after which the following DNA fragments are collected and purified using the ZEMESIEAMII kit (VIO 101): a DNA fragment with a length of 215 base pairs from the MVST1/ plasmid. (5) /"pOSC19 (defined as "c2") or a DNA fragment with a length of 3218 base pairs from the plasmids AMVSII ah/pOS19 and YaMveSII a45/pOSC19 (defined respectively as "Na1" and 65 "nat,

The pat and pat fragments are individually ligated with the c2' fragment and introduced into a competent cell of E. soybean in order to obtain a transformant. The obtained transformant is cultured in 2 ml of 2HUuUT medium containing 5 Ω/ml of ampicillin. The plasmid isolated from the cell fraction is purified using a plasmid purification kit

OIArger (O1LASEM). The resulting plasmid DNAs containing the Mag fragment and the Pai fragment are named "pt/pMVs1akh/pOS19" and "t/MVsI ah/pOst19", respectively. Plasmids t/pMVSsiI ah/pOS19 and t/pmMVSi ah/pOSIiV cleave

EsokII. A DNA fragment of 743 base pairs is electrophoresed in a 295 low-melting point agarose gel, removed and purified from the gel using the SEMESI EMIA kit (810101). The obtained product is dissolved in 20 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EOTA. 70 Four μl of the obtained DNA fragments are mixed with one μl of the above-mentioned HERE vector treated with bacterial alkaline phosphatase to ligate them with each other. The ligation product is introduced into a competent E. soybean cell,

UM109, in order to obtain a transformant. The resulting transformant is cultured in 2 ml of 2KHUT medium supplemented with 5 µg/ml of ampicillin. Plasmid DNA isolated from the cellular fraction is purified using the OIArger (OIASEM) plasmid purification kit.

Purified plasmid DNA is cleaved in 20 μl of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MacCl, 1 mM dithiothreitol, 100 mM KCl, 8 units of Nipai (Takaga 5piz20) and 2 units of Ruci (Takaga Zpil2o). at a temperature of 372C for 1 hour. At this stage, the plasmid DNA base is identified based on the assumption that if a DNA fragment is inserted into a plasmid with the correct orientation, hydrolysis will produce a cleaved fragment of length 5104/2195 base pairs, and if the specified DNA fragment is inserted into a plasmid with reverse orientation, then a cleaved fragment with a length of 4387/2526 base pairs will be obtained.

Plasmids obtained in this way are expression vectors encoding the I-chain of a hybrid antibody containing mouse EC1,2 and human ECZ,4, which are named, respectively, "p/pMVS HER ah/peo" and "p/pMVS I ah/peo" . (i) Preparation of EKL/EK2 hybrid antibody

The hybrid antibody ECT/EK2 is obtained as described above, using the Zpavi restriction site in the hypervariable region of the COM. at

In 20 μl of a reaction solution containing T0mM tris-HCl (pH 7.9), 10mM Mosi", 1mM dithiothreitol, 50mM mass, 0.01965 (in the ratio of weight to volume) of bovine serum albumin and 6 units of Zpavi (TaKaga 5pigo ), cleave ten μg of plasmids MVSLI u/peo and tMVS115/peo at a temperature of 372C for 1 hour.

The resulting reaction solution is further hydrolyzed in 5 mL of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 00 10 mM Mosi, 1 mM dithiothreitol, 100 mM KS, 0.0195 (weight to volume) bovine serum albumin and 6 units of Rmia, at a temperature of 372C for 1 hour.

The resulting reaction solution is processed by electrophoresis in a 1.595 low-melting point agarose gel, after which DNA fragments with a length of 4955 and 2349 base pairs are removed and purified from the gel using a set of

SEMESI EAMIYA (VIO 101). DNA fragments obtained from the MVS (5) / peo plasmid are named "t!" (4955 base pairs)

Zo and "t2" (2349 base pairs), and DNA fragments obtained from the plasmid p/t MVS (5) / peo, named "nt1" (4955 base pairs) and "pt2" (2349 base pairs). All the obtained DNA fragments are dissolved in 400 μl of a solution containing 10 mM tris-HCIII (pH 7.4) and mM EOTA.

One microliter of t1 and pt1 fragments is ligated, respectively, with 4 microliters of yt2 and t2 fragments, and then injected into a competent cell of E. soybean, /M109, in order to obtain a transformant. The resulting transformant is cultured in 2 ml of 2xUT medium containing 5 µg/ml of ampicillin. Plasmid DNA isolated from the cell fraction is purified according to C with the OIArger (OIASEM) plasmid purification kit. The purified plasmid DNA is cleaved in 20 μl of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 10 mM Massi, 1 mM dithiothreitol, and 8 units of Arai (Takaga Zpygo) or 2 units of AlyaI (Takaga Zpygo), at at a temperature of 372C for 1 hour.

Plasmids obtained in this way (t!1-pt2 and pt1-t2) are identified based on the assumption that, if the DNA fragments are ligated in the plasmid with the correct orientation, then as a result of hydrolysis of the plasmid (t1-pt2) Arai 4! and Aha! respectively, a cleaved fragment with a length of 7304 base pairs and fragments with a length of 5560/1246/498 base pairs will be obtained, and as a result of the hydrolysis of plasmids (pt1-t2) Arai and Ari! fragments with a length of 6538/766 base pairs and a fragment with a length of 3535/2025/1246/498 base pairs will be obtained, respectively. -k 70 An expression vector encoding the I-chain of a hybrid antibody containing human EC1 and mouse EC2,3,4, named "ntt MV. (5) /peo", and an expression vector encoding the I-chain of a mouse EC hybrid antibody , EK2 so human and EKZ,4 mouse, called "MVe11 type. (5) /peo". (4) Construction of the I-chain of a humanized antibody

The Y-chain of the humanized antibody Mo23-57-137-1 is obtained by transplantation of the hypervariable site using the RSC method. That is, the I-chain of the humanized antibody Mo23-57-137-1 (variant "a") is obtained, which contains

GF) regions of EC, ECZ and ECZ, isolated from the antibody of the family NZOOZ868 |(ZEM-VAMK, YuOeipoz, M. eia!., Zsapa. 9. Ittipoi, 7 39, 95-103, 19941, and EC4 region isolated from the antibody human 525755 (MVKE-ROV), using six types of primers for polymerase chain reaction: primers for transplantation of hypervariable regions of MVSLTI SR (sequence with identification Mo29) and bo MVS ORZ (sequence with identification Moz30), which contain the sense sequence of DNA, primers for transplantation of hypervariable sites, MVSTI ORI (sequence with identification Mo31) Her MVSLI RA (sequence with identification Mo32), which contain an antisense DNA sequence, while all specified seeds for transplantation of hypervariable sites have a complementary sequence 15-21 base pairs long at both ends; and external primers MVSTI M51 (sequence with identification Me33) and MVSLI MK1 bo (sequence with identification Mo34), which are respectively homologous primers for trans plantations of the hypervariable districts of the Ministry of Internal Affairs of Ukraine and the Ministry of Internal Affairs of Ukraine.

Seeds for transplantation of hypervariable sections of MVSTLI ORI, MVSTLI OR2, MVOTI ORZ and MVSLI ORA are isolated with the help of urea-denatured polyacrylamide gel.

MapiaYi, ZatbgooK ei. ai., Sboi4 5rgipd Nagrog I aroghaiogu Rgezv, 1989| and extracted from the gel fraction by the method of grinding and soaking. A I aroghaiogu Mapia), ZatrgooK ei. aiI., Soy brgipd Nagrog

I arogayugu Rgezz, 1989).

With the help of a denatured polyacrylamide gel, one nmole of all seeds for transplantation of hypervariable sites is isolated. Fragments of the obtained DNA of the required length are identified on a 70 thin plate of silica gel irradiated with ultraviolet light, and removed by grinding and soaking. The obtained product is dissolved in 20 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EOTA.

Polymerase chain synthesis reaction is carried out using TaKaKa Ex Tad (TaKaka 5pygo) with buffer.

The reaction solution (10 Ωcl) intended for the polymerase reaction of chain synthesis contains 1 µl of seeds for transplantation of hypervariable sites of MVSLI ORI, MVSLI OR2, MVSLI ORZ and MVSLI RA, 0.25 mM amtPr and /5 2.5 units of TaKaKa Ex Tayad in buffer. Perform five cycles of the polymerase chain reaction according to the temperature cycling program in the following sequence: 949 for 1 minute, 5592 for 1 minute, and 7227 for 1 minute. 5 Opmol of external seeds are added to the resulting reaction mixture

Ministry of Interior M51 and Ministry of Interior MK1. Using this reaction mixture, perform another 30 cycles of the polymerase chain reaction according to the same temperature cycling program. The amplified DNA fragment is isolated by electrophoresis in an agarose gel using 395 agarose of Mi Zieme STO (EMO Vio. Rhodisiv).

Cut out the agarose region containing the DNA fragment 421 base pairs long, and purify the specified DNA fragment from the gel using the ZEMESI EAMI!Y kit (810101) according to the instructions attached to it. The reaction mixture obtained in this way for the polymerase chain synthesis reaction is used for subcloning of this DNA fragment in the pOS19 plasmid, which is cleaved in advance by Vatni and Nipai. Then Ga r; determine the DNA sequence of the obtained plasmid. This plasmid is named "MVS/rusS19". However, it was established that in this plasmid the amino acid in the 104-position (amino acid Mo96 according to the definition of i9)

The pocket) of the hypervariable part of SOC4 of this plasmid is replaced by arginine. In order to replace this amino acid with tyrosine, primers of MVSTI SRITOK (sequence with identification Me35) are designed and synthesized. Then perform polymerase chain reaction using TaKaKa Ex Tad (Takaga 5pygo) with buffer. c The reaction solution (100 μl) used to perform the polymerase chain synthesis reaction contains

0.bmkg of YAMVSI/rOST19 plasmids as matrix DNA, 5 hopmoles of MVS1I/M51 and MVSLI SOROK seeds, 2.5 units of TaKaKa Ex Tad (TaKaga Zpygo) and 0.25 mM amte in a buffer over which a layer of mineral oil is placed. "WITH

Perform thirty cycles of the polymerase reaction of chain synthesis according to the program of cyclic temperature changes in the following sequence: 942 for 1 minute, 552C for 1 minute and 722 for 1 minute. The amplified DNA fragment is separated by electrophoresis in an agarose gel, using 390 agarose from Mi Zieme STO (EMO Vio. Rhodisiv).

A DNA fragment with a length of 421 base pairs is excised and purified from the gel using the ZEMESI EMIA kit (VIO 101) according to the instructions attached to it. The obtained reaction mixture for the polymerase chain synthesis reaction is used for subcloning of the DNA fragment into the pOS19 plasmid, which was prepared by splitting the Watni and Nipayi plasmids. - c In the obtained plasmid, the DNA sequence is determined using primers M 13 Rgiteg MA and M1Z Rgiteg KM. a The results obtained confirm that the plasmid has the correct sequence. This plasmid is cleaved by Nipai!i and ViIpiI and with the help of electrophoresis in a 190 agarose gel, a DNA fragment with a length of 416 base pairs is isolated from it. The obtained DNA fragment is purified using the ZEMESI EMIA kit (VIO 101) in accordance with the instructions attached to it and is introduced into the C;X/risC plasmid, which was prepared by splitting Nipai and Vipi plasmids. The resulting plasmid is named "MVS ah/pOS19". This plasmid is cleaved by Esoki to obtain a DNA fragment encoding a humanized I-chain. The resulting fragment DNA is introduced into the pCoO51 plasmid so that the initiation codon of the humanized I-chain is located downstream of the EE1To promoter. The resulting plasmid is named "MVS al/pCO51". The DNA sequence (including the corresponding amino acid sequence of 20) of the humanized I-chain (variant "and is expressed by the sequence with the identification Mobb.

The amino acid sequence of variant "a" is expressed by the sequence with identification Mo47. co Variant "B" is obtained by introducing a mutagen by the ROK method. The "p" variant is designed in such a way to replace the amino acid in the 43-position, i.e. glycine (amino acid Mo43 according to the Kabat definition), with proline and the amino acid in the 49-position, i.e. lysine (amino acid Mo49 according to the Kabat definition), with 22 aspartic acid. Polymerase chain synthesis reaction is carried out using a plasmid

HF) MVS ah/rOsS19 as a matrix, mutagenic seed MVMTI!SORBK (sequence with identification Mo36b) and t seed MVS1IIM51. The resulting DNA fragment is cleaved by Vatni and Nipai and the cleaved fragment is subcloned at the Vatni-Nip site!! pOS19 plasmids. The resulting plasmid DNA is sequenced and cleaved by Nipayi! and

ATII, after which the obtained cleaved fragment is ligated with the PMVSIGh x/rist19 plasmid, which is previously cleaved by Nipai!! and ANI.

The resulting plasmid is named "MVS 55/pOS19". This plasmid DNA is cleaved by EsokK!I to obtain a DNA fragment containing DNA encoding a humanized I chain. The specified DNA fragment is introduced into the pCoO51 plasmid so that the initiating codon of the humanized I-chain is located downstream of the EE1 y promoter. The resulting 65 plasmid is called "imVeSII 55/rsCo1".

Variant "c" is obtained by introducing a mutagen using the ROK method. Variant "c" is designed in such a way to replace the amino acid in the 84-position, that is, the series (amino acid Mo80 according to Kabat's definition), with proline. The polymerase chain synthesis reaction is carried out using the pYAMVSIGh x/rys19 plasmid as a matrix of the mutagenic seed MVSLI ORb5 (sequence with identification Mo37) and the seed M13 Rgiteg KM.

The resulting DNA fragment is cleaved by Vatni and Nipai and subcloned into the pOS19 plasmid, which is previously cleaved by Watni and Nipa!. Plasmid DNA is sequenced and cleaved by VviRI and Aogo1NI, after which the obtained DNA fragment is ligated with the PMVSI ah/rOSC19 plasmid, which is previously cleaved by Vv8iRI and Aogo1NI. The obtained plasmid is named "MVS c5/pOsC19". This plasmid DNA is cleaved by EsokiI to obtain a sequence encoding a humanized I chain. This sequence is introduced into the EsoKI site of the pCO51 plasmid so that the initiating codon 70 of the humanized I chain is located in the direct direction from the EP1 y promoter. The plasmid obtained in this way is named "MVS su/rso51".

Variants "a", "be" and are also obtained by introducing a mutagen according to the ROK method. Variants "a" tet are constructed with a substitution in the variants "a", "p" and "c" of the amino acid in the 91-position, that is, tyrosine (amino acid

Mo87 according to Kabat's definition), isoleucine. To obtain variants "a", "e" and "the polymerase reaction of chain synthesis is carried out using plasmids AMVCI ah/rsCo51 (for variant "a"), AMVCI 5x/rsoB1 (for variant "e") Her MVS c 5/rСО5I ( for variant ") as a matrix, mutagenic primer of the Ministry of Internal Affairs of the Republic of Moldova (sequence with identification Mo38) and seed M-51 (sequence with identification Mo44). The resulting DNA fragment is cleaved by Watni and Nipai and subcloned into pOS19, which is prepared by cleavage of pOSC19 by Watni and Nipai. After sequencing, the plasmid is cleaved by Nipaii and Vipi and the resulting cleaved fragment is ligated with the CX/ryC19 plasmid, which is prepared by cleavage of the Nipaii and Vipi plasmids.

The resulting plasmids are respectively named "MVS ah/pOst", "MVS ex/pOS19" and "nMVSII 7/pOS19". All these plasmids are cleaved by EsoKI to obtain a DNA fragment containing DNA encoding a humanized I chain.

This DNA fragment is introduced into the EsokI site of the pCO51 plasmid so that the initiating codon of the humanized I-chain c is located downstream of the EE protomore! in this plasmid. Plasmids obtained in this way are named Ge) "Me ahrsSoz1", "MVesi eu/pCo51" and "yMVSI E/pCoB1".

Variants "d" and "n" are also obtained by introducing a mutagen using the ROK method. Variants "d" and "p" are constructed with the substitution of amino acids in the Zb-position, i.e., histidine (amino acid Mo36b according to Co. 20 definition of Kabat), with tyrosine in variants "a" and "a". Polymerase chain synthesis reaction is carried out using the mutagenic primer MVSI SOROK (sequence with identification Mo39), primer M13 Rgiteg RM using "- plasmid PMVSIIakh/risC19 as a matrix. The product of the polymerase chain reaction is once again subjected to the polymerase chain reaction using the primer M 13 Rgiteg MA and the plasmid NMVSII ah/pOS19 as a matrix. The resulting DNA fragment is cleaved by Nipai and Vipi and subcloned in the C x5/pOS19 plasmid, which)

Зз5 is prepared by cleavage of the Nipai!y! plasmid. and Vipi. Using this plasmid as a matrix, the polymerase chain reaction is carried out from the primer MVSIIORIZK (sequence with identification Mo40) and

MVSIIM51. The obtained product of RSK is split by AiYia! and Nipaii and introduced into plasmids AMVSIIa x/rist19 and

MVs ah/rOst19, which are prepared by cleavage of both plasmids Aiaii and Nipaii. DNA sequences are determined in the obtained plasmids. Plasmids in which the presence of the correct sequences was confirmed, 70 are named "MVS 45/pOSC19" and "imVeSi pX/pOSC19", respectively. These plasmids cleave EsoK! with obtaining - from the sequence encoding the humanized I -chain. This sequence is introduced into the EsoKiI site of the pCO51 plasmid so that the initiating codon of the humanized I-chain is located downstream of the EE1 promoter. The obtained plasmids are named "NMVSI 9u/rSoz1" and "nMVSI pu/rsoBt", respectively

Variants "b, M, "KU MM, "t", "ia" Her "o" is also obtained by introducing a mutagen according to the ROK method. Polymerase chain synthesis reaction is carried out using the mutagenic primer of MVS cRI45 and (sequence with identification Mo41), primer MKM (5) (sequence with identification Mo43) and plasmid with MVS ah/pOS19 as a matrix. The resulting DNA fragment is cleaved by Arai and Vipi and subcloned into a plasmid

MVS 495/рОС19, which is prepared by splitting the Ara plasmid! and Vipi. In the resulting plasmid

Mothers determine the sequence of bases and select a clone with a mutation corresponding to each of the indicated options. - 70 The plasmid obtained in this way is called "pMVSIIhurOsS19 (x-i, |, K, I, t, p or 0). This plasmid is cleaved by EsoKI to obtain the sequence encoding the humanized I-chain. The resulting sequence is inserted into the EsoKI site pCO51 plasmid so that the initiation codon of the humanized I chain is located downstream of the EETo promoter. The resulting plasmid is named "NMVSII hu/pso51" (x-i, |, K, I, t, p or o). The DNA sequences (including the corresponding amino acid sequences) of variants "|, "I", "t" and "o" are expressed respectively and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and sequences of identification MoMo and 67, 68, 69 and 70. The amino acid sequences of these variants are expressed

ГФ) according to the sequences with the identification MoMo 48, 49, 50 and 51. from Options "p", "ba", "t", "v" and izh are modified forms of options "i", "M, "t", "I" Her " o", in which the amino acid in the 87th position, i.e. tyrosine, is replaced by isoleucine. These options are obtained by using the site in the Aogo1MI restriction of the IVF area to replace the "n" option with the "i", "t", "M" or "o" option in this way. A restriction fragment of Aogb1H (514 base pairs) containing the hypervariable site of SOKZ, part of the IVF region and the entire EC4 region is removed from the expressing plasmid AMVSII hu/rsCo51 (x-i, |, t, I or o). The Aogo1NI restriction fragment (514 base pairs) containing the hypervariable site of SOKZ, part of the ECZ region and the entire EC4 region is ligated to the deleted part of the expressing plasmid, replacing the amino acid in the 91-position, 65 i.e. tyrosine (amino acid Mo87 according to Kabat's definition), with isoleucine . In the obtained plasmids, the DNA sequence is determined and a clone of variants "i", "M, "pt", "I" and "o" is selected, in which the amino acid in

91-positions, i.e. tyrosine (amino acid Mo87 according to Kabat's definition), is replaced by isoleucine.

Variants corresponding to variants "i" "M, "t", "My "o" are respectively named variants "p" "a" "c", those "P", and plasmids for these variants are named "NMVSII xx//irsoB1" ( x-r, ad, 5, g or |). DNA sequences (incl

Corresponding amino acids) of variants "Я" 0", "78" Her b are expressed respectively by sequences with identification MoMo 71, 72, 73 and 74. Amino acid sequences of these variants are expressed respectively by sequences with identification MoMo 52, 53, 54 and 55.

Plasmid AMVSII 4u7/pCO51 is cleaved by Nipai and EsoKII and subcloned into plasmid pOS19, which is prepared by cleavage of plasmid Nipai and EsoKiI. The plasmid obtained in this way is named "MVS 4u/pOS19". 710 Position of substituted amino acids in each variant of humanized | - the chain is shown in the following table 3. oowarant 13643454? | 4917601 a in pi I PNYA POLYA PON OH PO TONYA NO

PI TI PNYA PNYA POLON PON PON PON NOT ev

PIL ZY PIL PON FIELDS FIELDS PON POST NKT in at synti1Ya.

PI PI ZA PNYA PO UA POLYA PON PON KOH sc 2 square o

Dust ZY PI ZA PNIA POLO UANYA PO TONNA PON PON NAH km t v

PI YOU PI FOR PON POLON POLYA PON PON KOH ki

Zo ev - va pi i p S I PO ANYA NOYA PO TONYA NOYA PO o pil PO I POLYA PON OO ZON NOYA OH yu kv 1111 te

The following amino acids are marked with capital letters in the table:

Y - tyrosine; P - proline; K - lysine; M - valine; O - aspartic acid and I - isoleucine. "

The Es. soybean strain containing the plasmid NYMVSINKDNKU/rIST9 and the E. soybean strain containing the NtrbsiI 4u/rOst19 plasmid are named, respectively, "Euspegispia soybean /M109 (AMVSINKDNK/rOst19)" and "Espegispia soybean /M109 (pMVSII 45/risC193) " and Z c are included on the basis of the Budapest Treaty of August 15, 1996 in the collection of the National Institute of Bioscience

From" human technology, agency of industrial science and technology, Japan (1-3, Nidazpi 1-spote, TvyKkyva-vpi,

Irsagadi Cape, Japan ) under the number REKMVR-5629 for Ezspegispia soybean U«M109 (pMVSINKDNnNK/rOst9) and under the number

EECM VR-5630 for Ezspegispia soybean UM109 (MVS 4u/rOs19). -I 15 (5) Transfection in cell CO5-7.

The above-mentioned expressing plasmids are temporarily expressed in CO5-7 cells to determine the antigen-binding and neutralizing activity of the hybrid antibody and the humanized antibody Mo23-57-137-1. For temporary expression

Y-chain of the hybrid antibody, the following combinations of plasmids co-transfect CO5-7 cells by the electroporation method with the help of an electroimpulse device of Sepe Ryizeg (Vio Kai): - 70 NMVSINKDNKYU/rCoO51 and p/tMVve (XX) /peo;x NYMVSINKDNKYU/pSO51 and t/ pMVsiI al/peo;. AMVSINKDNYurSO51 and so t/pMVsI ah/peo; NMVSINKDNK/rCoO51 and ptt MV. (5) /peo; and nmMVSINKDNA/pCo51 and titMVveii. (5) /peo. To

0.vml of a suspension of CO5-7 cells in a phosphate-buffered physiological solution (РВ5(-)) with a concentration of 1x10 cells/ml is added to 1 Ωkg of all plasmid DNA. The resulting solution is exposed to pulses at an electrostatic capacity of 15008 and an amperage of 25 μF. The solution is kept for 10 minutes at room temperature, after which the electroporated cells are suspended in a modified Dulbecco method

F, Igla's medium containing 2956 fetal calf serum with an ultra-low Ids content (SIVSO), and cultured in a 1Osm cup in an incubator with a CO atmosphere. Cells are cultured for 72 hours, after which the culture supernatant is collected and centrifuged to remove plaque debris. The resulting product will be used as a sample for solid-phase enzyme-linked immunosorbent assay (ELISA).

For the temporary expression of the humanized antibody Mo23-57-137-1, the plasmid combination АМВСИКДНКюрСО51 or

NtbvesiY hu/pCO51 (x-a-) is transfected into cells CO5-7 with the help of an electropulse device Sepe Riiize (Vio Kai) similar to the procedure described above for the hybrid antibody. The culture supernatant is used as a sample for solid-phase enzyme-linked immunosorbent assay (ELISA). 65 The hybrid antibody or humanized antibody is purified from the culture supernatant of CO5-7 cells using the AiiSai Rgoivip A MARZIA kit (Vio Cad) according to the instructions attached to it.

(6) Solid-phase enzyme-linked immunosorbent assay (ELISA) () Determination of antibody concentration

A solid-phase enzyme-linked immunosorbent assay (ELISA) tablet designed to determine the concentration of antibodies is prepared as follows. Into all wells of a 9b-well plate for EGI5A (Makhizogr, MOMS) inject 10 μl of surface sensitization buffer (0.1 M solution of Manso with, 0.0296 Mom), supplemented with 1 μg/ml of goat anti-human DO antibody (TASO), and block 200 μl buffers for dilution (5 MM Tris-HCl, mM MoSiI, 0.1 M Masi, 0.059 Tween-20, 0.0295 Mamma, 195 bovine serum albumin (BSA); pH 7.2). In all wells, add the culture supernatant of CO5 cells, in which the hybrid antibody or humanized antibody was expressed, 7/0 Obtained by serial dilution. The contents of the wells are incubated at room temperature for 1 hour and washed with a mixture of phosphate-buffered saline and Tween-20, after which 100 μl of a solution of alkaline phosphatase-conjugated goat anti-human IDO antibody (TASO) is added to each well.

The resulting mixture is incubated at room temperature for 1 hour, washed with a mixture of phosphate-buffered saline and Tween-20, and 1 mg/ml of the substrate solution ("Zidta 104", /5 paranitrophenylphosphoric acid, ZIZMA) is added to each well. The optical density of the resulting solution is measured at a wavelength of 405 nm using a microplate reader (Vio Kai). Purified immunoglobulin IdDSiA, human (binding site) is used as a standard for determining the concentration of antibodies. (i) Determination of antigen-binding capacity

A tablet for solid-phase enzyme-linked immunosorbent assay (ELISA) designed to determine 2o antigen-binding capacity is prepared as follows. In all wells of a 9b-well tablet for EGISA (Makhivogr,

MOM) inject 100 μl of buffer for surface sensitization, supplemented with 1 μg/ml of human RTNGR (1-34), and block 200 μl of buffer for dilution. In all wells add the culture supernatant of CO5-7 cells, in which hybrid antibody or humanized antibody was expressed, or solutions of purified hybrid antibody or purified humanized antibody obtained as a result of serial dilution. The contents of the wells are incubated at room temperature and washed with a mixture of phosphate-buffered saline and Tween-20, after which 100 μl of a solution of alkaline phosphatase-conjugated goat anti-human antibody (TASO) is added to each well. The resulting mixture is incubated at room temperature, washed with a mixture of phosphate-buffered saline and Tween-20, and 1 Tmg/ml substrate ("Zidta 104", paranitrophenylphosphoric acid, ZIZMA) is added to each well. In the obtained solution, the optical density is measured at the length of the lake

From Wave 405nm, using a device for reading microplates (Vio Cad). (7) Confirmation of activity -- () Evaluation of humanized H-chain p

It was established that the antibody containing the humanized H-chain of variant "a" and the chimeric I1-chain are characterized by the same level of RTHgR-binding activity as the chimeric antibody (see Fig.5). This result allows us to predict that in option "a" satisfactory humanization of the M-region of the H-chain will be achieved. Therefore, the humanized H-chain of variant "a" was used as the H-chain of the humanized antibody in the following experiments. (i) Hybrid antibody activity (i-a) EC1,2/ECZ3,4 hybrid antibody

When the I-chain has the structure p/tmBvCs a (5), the antibody does not have antigen-binding activity. However, "

When the I-chain of the hybrid antibody has the structure t/pMVsSiH a) or t/pMVsIH ah, this antibody is characterized by the same level of antigen-binding activity as the chimeric antibody Mo23-57-137-1 (Fig.b). These results suggest that the EKZ and EK4 regions are suitable for a humanized antibody, and the EK1 and EK2 regions have "» one or more amino acid residues that need to be replaced. (ii-5) EKT/EK2 hybrid antibody

When the I-chain of the hybrid antibody has the structure of tpt MV (5), the antibody does not have antigen-binding activity. However, when the I-chain of the hybrid antibody has the structure pttMVei|/5), this antibody is characterized by the same level of antigen-binding activity as the chimeric antibody Mo23-57-137-1 (Fig. 7). These results suggest that the EC1 region is suitable for a humanized antibody, and the EC2 region has (95) one or more amino acid residues that need to be replaced. -l 20 (ii) Activity of the humanized antibody

Determine the antigen-binding activity of a humanized antibody in which all variants are used as the I chain

IR f) from "ta" to "SG. The obtained results show that humanized antibodies having an I-chain according to variants of "M. "M, "t", "a" and "c" of its E are characterized by the same level of RTHgR-binding activity as the chimeric antibody (Fig. 8-11). (8) Creation of a stably produced Chinese hamster ovary cell line. o To create a stable transformant for the humanized antibody, the above-mentioned expressing plasmids are introduced into the CHO cell (ОХВ11). A stable transformant of a humanized antibody is created using the following combinations of plasmids as an expression vector for CHO cells, namely pPMVSINKDNyurSNOiY and pMVSsSI t X/rsoZvi; 60 pMVsSINKDNU/rSNOT and pMVeSiI 4x/rCo511; NMVSINnKDNKrsSNOT and Ministry of Internal Affairs puroSo1. These plasmids are cotransfected into CHO cells using the electroporation method using the Sepe Ryizeg electropulse device (Vio Kai). All expressing vectors are cleaved with the restriction enzyme Ruci to obtain linear DNA. The specified DNA is extracted with phenol and chloroform and precipitated with ethanol. The DNA obtained in this way is subjected to electroporation. Ten micrograms of all plasmid DNAs are added to 0.8 ml of a suspension of CHO cells in RV5(-) with a concentration of 1x10 cells/ml. The resulting mixture is exposed to pulses with an electrostatic capacity of 15008 and an amperage of 2μF. The electroporated cells are kept at room temperature for 10 minutes and suspended in MEM-o medium (SIVSO), supplemented with 1095 fetal calf serum (SIVSO), and cultured on 96-well plates (Raisop) in an incubator with CO." The next day after the start of cultivation, the medium is replaced with selective medium MEM-o, supplemented with 1095 fetal calf serum (518С0О) and 500 mg/ml

SEMETISIM (54185jMafe; SIVSO) without ribonucleoside or deoxyribonucleoside. Cells into which the antibody gene has been introduced are selected from the culture medium. The selective medium is replaced with fresh medium before cultivation and after two weeks of cultivation, after which the cells are examined under a microscope. In the cells characterized by satisfactory growth, the number of antibodies obtained as a result of performing 70 solid-phase enzyme-linked immunosorbent assay (ELISA), which is usually used to measure the concentration of antibodies according to the procedure described above, is determined. Cells producing the largest number of antibodies are selectively collected.

Raising the level of the culture of a stable transformant for the obtained antibodies is carried out in a rotating flask using MEM-o minimum support medium supplemented with 295 fetal calf 75 serum with an ultra-low content of K without ribonucleoside or deoxyribonucleoside. On the 3rd and 4th day of cultivation, the culture supernatant is collected and filtered through a 0.2 µm mesh filter (MiPiroge) to remove cellular debris. Humanized antibodies are then purified from the culture supernatant of CHO cells in a ROKO5 column filled with protein A (RegZeriime Viozuzietv), on SopZer I! C100 (MiPiroge) according to the attached instructions. Purified humanized antibodies are used as samples to determine neutralizing activity and to study pharmacological effectiveness in animal models of hypercalcemia. The concentration and antigen-binding activity of purified humanized antibodies are determined using a similar system for solid-phase enzyme-linked immunosorbent assay (EGIZA).

Example 4

Determination of the neutralizing activity of He

The neutralizing activity of the mouse antibody, chimeric antibody and humanized antibody is determined using the rat myeloma cell line KO517/2.8-5. KO517/2,8-5 cells are cultured in E-12 medium

Heme (SIVSO), supplemented with 1095 fetal calf serum (SIVSO), in an incubator with CO." KOZ17/2,8-5 cells are inoculated into all wells of a 9b-well plate with a concentration of 104 cells/1OOμl/well and cultured for 1 day. The culture medium is replaced with E-12 Hema medium (518C0), supplemented with 4 mM hydrocortisone and 10905 (ee) fetal calf serum. Cells are cultured for three to four days, washed with 26 µl of E-12 medium

Hema (SIVSO) and add 8 μl of E-12 Hema medium, supplemented with 1 mM isobutyl-1-methylxanthine (IVMH, 5ISMA), - 10965 fetal calf serum and 10 mM NEREZ. The resulting mixture is incubated at a temperature of 372C for 30 minutes. yu

The mouse antibody, chimeric antibody and humanized antibody intended for testing neutralizing activity are prediluted stepwise as follows: -

POmkg/ml.

Bbmkg/ml, 1.25mcg/ml, 0.bZmkg/ml and 0.Zimkg/ml. Each solution of the diluted antibody sample is mixed with an equivalent amount of 4ng/ml RTNHR (1-34). Into each well is injected 8 VOmcl of the solution of the obtained mixture. The final concentration of each antibody is equal to the fourth part of the initial concentration of the above-mentioned antibody, therefore the concentration of RTNHR (1-34) is Ing/ml. The culture is maintained for ten minutes at room temperature, after which the culture supernatant is removed and the residue is washed three times with physiological saline and a solution with a phosphate buffer. Cyclic adenosine monophosphate (CsCAMP) is extracted from the obtained product in cells, using 1 μl of a mixture of 0.395 NCI and 9595 ethanol, and evaporated with the help of an injection aspirator to remove NCI ethanol. The residue is dissolved in 120 μl of enzyme-linked immunosorbent assay (EIIA) buffer, added to the kit for CAMP analysis by the EIA method (SAUMAM SNEMISAG 5). The level of CAMP is determined using the kit (SAUMAM SNEMISA!/ 5) in accordance with the instructions attached to it. The obtained results show that humanized antibodies containing different variants of the I-chain are characterized by the same level of antigen-binding activity as a chimeric antibody, while humanized antibodies with the I-chain variants "ad", (95) "g. "v" and Z, in which tyrosine in the 91-position is replaced by isoleucine, have neutralizing activity, which -l 20 most closely corresponds to the similar activity of the chimeric antibody, especially those with the I-chain of the "a" variant, have the strongest neutralizing activity (Fig. 12-14).

IR f) Example 5

Study of the pharmacological effect on an animal model of hypercalcemia (1)

The therapeutic effect of a chimeric humanized antibody against RTNGR, which has the I-chain of "p", "hi" and "a" variants, was studied in an animal model of hypercalcemia (a human tumor transplanted into "naked" mice). o As an animal model of hypercalcemia, "naked" mice were used , which were transplanted with a malignant tumor of the human pancreas RAM-7 mice were purchased from the Central Institute of Experimental Animals. Name) It is known that in "naked" mice transplanted with a malignant tumor of the human pancreas RAM-7, the concentration of calcium in the blood increases as the tumor volume and hypercalcemia develops, 60 which causes a decrease in body weight and a reduction in locomotor activity.In accordance with this example, the therapeutic effect of the chimeric antibody and the humanized antibody of the present invention on hypercalcemia caused by a malignant pancreatic tumor PAM-7 was investigated by measuring the mass body and calcium concentration in the blood of the tested animal.

Malignant tumor of the human pancreas RAM-7 was inoculated into "naked" mice V/s-pi/pi (Mirrop 65 SPapevz Kimeg) ip and mimo. To evaluate the pharmacological action of the antibodies according to the present invention, male "naked" mice VAI V/s-pi-pi (Mirrop Spagiez Kimeg) were purchased at the age of 5 weeks, which were acclimatized for 1 week and used to perform the specified experiment at the age of b-week. Tested mice with symptoms of hypercalcemia were prepared and divided into groups as follows. Passivated malignant tumor of human pancreas

RAM-7 was cut down and a Zmm cube of slices was cut out of it. Each mouse was transplanted subcutaneously with one section of the tumor. Two or three weeks after transplantation, when the cancer tumor in the mice reached a sufficiently large volume, the mice were divided into groups taking into account the average tumor volume, blood calcium concentration and body weight, after which they were used as animal models of hypercalcemia.

The therapeutic effect on hypercalcemia was studied as follows. A single dose of chimeric or humanized antibodies against RTNGgR, having the I-chain of the "t" or "g" variant, was injected into the tail vein of 70 tested mice in the amount of 10 or 30 μg/mouse. A single dose of the humanized antibody having the I-chain of the "d" variant ", was injected into the tail vein of tested mice with symptoms of hypercalcemia, in the amount of 20 or bOmkg/mouse. To evaluate the pharmacological effect of the antibodies in all mice, the concentration of calcium in the blood and body weight was measured on the 1st, 4th, 7th and 11th day after administration. The volume of the tumor was determined by measuring the largest diameter (a mm) and the smallest diameter (6 mm) of the tumor and performing calculations using both obtained 75 values according to the Galant equation (ab 2/21. The concentration of calcium in the blood was determined based on the concentration of ionized calcium in whole blood, which was taken from all mice through the eye socket using a hematocrit tube and analyzed in an automatic 643Ca""/pH analyzer (SIVA-SOBMIMO).

It was established that the introduction of a chimeric antibody and humanized antibodies having the I-chain of variants "t", "g" and "4" causes a rapid improvement in relation to changes in body weight and calcium concentration in the blood and the improvement persists for a long period of time. The obtained results show that the chimeric antibody and humanized antibodies of the present invention are useful for the treatment of hypercalcemia caused by a malignant tumor (see Fig. 15 and 16).

Example 6.

Study of the pharmacological effect on an animal model of hypercalcemia (2). with

The therapeutic effect of a chimeric humanized antibody against RTNGR, which has the I-chain of the "d" variant, was studied on an animal model of hypercalcemia (a human tumor transplanted into "naked" mice).

The therapeutic effect on hypercalcemia was studied as follows. A single dose of a chimeric or humanized antibody against RTNGR, which has the I-chain of variant "d", was injected into the tail vein of tested mice with symptoms of hypercalcemia in the amount of 10 or ZOmkg/mouse. To evaluate the pharmacological effect of antibodies in all (ee) mice, calcium concentration in the blood and body weight were measured on the 1st, 3rd, 7th and 11th days after administration.

The concentration of calcium in the blood was determined on the basis of the concentration of ionized calcium in whole blood, which was taken from all mice through the eye socket using a hematocrit tube and analyzed in an automatic analyzer "9 643Ca""/pH (SIVA-SOVMIMS). yu

It was established that the introduction of a chimeric and humanized antibody, the one with the I-chain of the "ad" variant, causes a rapid improvement in relation to the change in body weight and the concentration of calcium in the blood, and the improvement is maintained for a long period of time. The obtained results show that the chimeric antibody and the humanized antibody of the present invention are useful means for the treatment of hypercalcemia caused by a malignant tumor (see Fig. 17). "

Example 7

Study of the pharmacological effect of an animal model of hypercalcemia (3) t s The therapeutic effect of a chimeric and humanized antibody against RTNGR, which has the I-chain of the "4" variant, was studied in an animal model of hypercalcemia (malignant human lung tumor I C-6, transplanted "naked" mice). » As an animal model of hypercalcemia, "naked" mice were used, which were transplanted with a malignant human lung tumor I C-6 (the mice were purchased from the Central Institute of Experimental Animals). It is known that in "naked" mice transplanted with a malignant human lung tumor IS-6, the concentration of calcium in the blood increases as the volume of the tumor increases and hypercalcemia develops, which causes a decrease in body weight and a reduction in movement activity.

According to this example, the therapeutic effect of a chimeric antibody and a humanized antibody according to this invention on hypercalcemia caused by a malignant human lung tumor I C-6b was investigated by measuring the body weight and calcium concentration in the blood of the tested animals.

Malignant human lung tumor I C-6 was inoculated into "naked" VAI V/s-pi/pi (Mirrop Spagiez Kimeg) mice i.p. with mimo. To evaluate the pharmacological action of the antibodies according to the present invention, male "naked" mice VAI V/s-pi-pi (Mirrop Spagievz Kimeg) were purchased at the age of 5 weeks, which were acclimatized for 1 week and used to perform the specified experiment at the age of b-weeks. 22 tested mice with symptoms of hypercalcemia were prepared and divided into groups as follows.

Gee! A passivated malignant human lung tumor I C-6 was dissected and a Zmm cube of slices was cut from it. Each mouse was transplanted subcutaneously with one section of the tumor. Two or three weeks after transplantation, when the cancer tumor in the mice had reached a sufficiently large volume, the mice were divided into groups taking into account the average tumor volume, blood calcium concentration and body weight, after which they were used as animal models of 60 hypercalcemia .

The therapeutic effect on hypercalcemia was studied as follows. A single dose of a chimeric or humanized antibody against RTNGR, which has the I-chain of the "d" variant, was injected into the tail vein of tested mice with symptoms of hypercalcemia in the amount of 10 or ZOmkg/mouse. To evaluate the pharmacological effect of the antibodies in all mice, the concentration of calcium in the blood and body weight on days 1, 3, 6b, and 10 after administration.65 Blood calcium concentration was determined based on ionized calcium concentration in whole blood taken from all mice through the eye socket using a hematocrit tube and analyzed in an automatic analyzer

643Са""/рН (SIVA-SOVMIMS).

It was established that the introduction of a chimeric antibody and a humanized antibody having the I-chain of the "9" variant causes a rapid improvement in relation to the change in body weight and blood calcium concentration in tested mice with a malignant human lung tumor I C-6, and the improvement persists over a long period of time. The obtained results show that the chimeric antibody and the humanized antibody of the present invention are useful means for the treatment of hypercalcemia caused by a malignant tumor (see Fig. 18).

Example 8

Kinetic analysis of the interaction between PtTgR and an antibody against RTNGR using VIASOKE 70 In this experiment, a kinetic analysis of the antigen-antibody interaction using VIASOKE was performed. RTNHGR (1-34-Suv) was used as an antibody, which was adsorbed on the end of the sensor, especially on the C-end. Purified antibodies with different concentrations were used as an analyte. On the basis of the received sensorgram, calculate the kinetic parameters (the rate constant of binding "Kazv" and the rate constant of "dissociation of kaizv"). Kinetic analysis was performed according to the method described in the article "Kipeis apaiuziz oi toposiopa 75 apirodu-apiiidep ipiegasiopz om/yy a pem/ bBiozepzog brazei apaiuiisa! zvuziet", Kagizzop, MK. (1991),

Ulttipoi. Me (yodz 145, p. 229-240). (1) Immobilization of RTNGR (1-34-C) at the end of the sensor

RTNGR (1-344-C) was adsorbed at the end of the CM5 sensor (Riagtasia). As a test buffer, HBO was used (10 mM NEREB5, pH 7.4, 0.15 M Mass, 34 mM EOTA, 0.00595 surfactant P2O0) with a flow rate of 5 μl/min. The carboxyl groups of carboxymethyldextran at the end of the CM5 sensor were activated by introducing 100 μl of 0.05 M hydroxysuccinimide (MNZUO, 2 M hydroxychloride M-ethyl-M'-(3-dimethylaminopropyl) carbodiimide (EOS) and 100 μl of voMM 2-(2-pyridinyldithio)-ethanamine (ROEAUO ,1M of borate buffer (pH 8.5) and additional introduction of 1Okl

Bmkg/ml RTNGR. (1-34442)/10 mM of sodium-acetate buffer (pH 5.0), as a result of which the C-ends of RTNGR. (1-34) adsorption occurred, especially of Suz-residues. Then 1000 μl of 50mM (I)-cysteine/1M Mass of 1mM Ha sodium formate buffer (pH 4.3) was injected to block excessively activated groups. Following this, 1 μl of 0.1 M glycine-HCI buffer (pH 2.5) and 100 μl of 10 mM HCl were introduced to wash substances with non-covalent binding. i9)

The amount of immunized RTNHR (1-344-C) was 226.4 resonance units (KO) (Fig. 19). (2) Interaction between immunized RTNgR (1-344-C) and purified mouse anti-RTNgR antibody

HB5 was used as a buffer for distillation with a flow rate of 20 μl/min. Antibody-forming hybridomas ee) were injected into the abdominal cavity of VaiBr/s mice. After several weeks, ascites was collected and injected into a column filled with protein A to purify antibodies. The purified antibody Mo23-57-137-1 was named "MVS" and the purified antibody ЗЕ5 was named "ЗЕ5". These antibodies were diluted with HB5 to obtain several solutions with concentrations of 1.25, 2.5, 5, 10 and 20 μg/ml.

When performing this analysis, 4O0μl of antibody solution was injected within 2 minutes to reach the o phase

Bonding, then within 2 minutes HB5 was injected to achieve the dissociation phase. After the dissociation, 1 μl of 10 mM NS was administered! to restore the sensor end. The analysis was carried out according to the binding - dissociation - recovery procedure in the form of one cycle and the introduction of various antibody solutions in order to obtain a sensorgram. (3) Interaction between immobilized RTNHgR (1-34-C) and purified humanized antibody against RTNHgR

HB5 was used as a buffer for distillation with a flow rate of 20 μl/min. The antibody was obtained from Chinese hamster ovary cells and purified in a column filled with protein A. The purified chimeric antibody was named "SMS" and the purified humanized antibodies of variants t and d were named "yMVSt" and "nMVsCa". These antibodies were diluted with HB to obtain several solutions with concentrations of 1.25, 2.5, 5, 10, and 20 μg/ml.

When performing this analysis, 4O0μl of antibody solution was injected for 2 minutes to achieve the binding phase, then HB5 was injected for 2 minutes to achieve the dissociation phase. After the end of the first dissociation, 1 µl of 10 mM NS was administered! to restore the sensor end. The analysis was carried out according to the binding - dissociation - recovery procedure in the form of one cycle and the introduction of various antibody solutions with the aim of obtaining a sensorgram. оз (4) Kinetic analysis of the tsu interaction The file with the data of interest was read and the reaction models were compared by superimposing the reaction regions of interest on each other (Fig. 20-24). Fig. 20-24 shows the data for the concentration of antibodies (Che 1.25, 2.5, 5, 10 and 20 μg/ml. Then the kinetic analysis of the interaction was performed using the analysis program specially designed for the "VIASOKE ViAemuiIchaiop 2/1 sensor" (Rpaptasia), which allows you to calculate the kinetic parameters (the binding rate constant of "kazv" and the "dissociation rate constant of kKaizv") by processing the obtained graphs (tables 4 and 5). evnevkhinmvst|nmvsya ;

In this experiment, an analysis model of type 4 70 was used to determine the binding rate constant.

Example 9

Suppression of phosphorus excretion in a model of hypercalcemia caused by a malignant tumor

Hypercalcemia caused by a malignant tumor (NNM) is a disease caused by RTGNR, it is known that

RTNGR accelerates the resorption of bone tissue and the resorption of calcium in the kidneys and renal tubules, which leads to the development of hypercalcemia. On the other hand, if we consider phosphorus, then RITNGR suppresses the resorption of phosphorus in the kidneys and renal tubules, which leads to the excretion of phosphorus, therefore, hypophosphatemia is often observed in subjects suffering from hypercalcemia. The effect of a humanized antibody against RTNGR on the excretion of phosphorus in the kidneys was studied in rats, which served as an animal model of hypercalcemia caused by a malignant tumor.

As an animal model, "naked" rats were used, which were transplanted with a malignant human lung tumor C-6 (rats were purchased from the Central Institute of Experimental Animals). It is known that in "naked" rats, which are subcutaneously transplanted with a malignant human lung tumor I C-6, the concentration of calcium in the blood increases as the volume of the tumor increases and hypercalcemia develops, which causes a decrease in body weight and a reduction in movement activity. Using this animal model, the influence of the humanized antibody against RTNHR according to the present invention on the excretion of phosphate in the kidneys was investigated by the method of renal clearance based on the fractional excretion of phosphate described below. at

Malignant human lung tumor I C-6 was inoculated into "naked" rats of VAI V/s-pi/pi (Mirrop Kygea) i.p. mimo.

To evaluate the pharmacological action of the antibodies according to the present invention, male "naked" rats EZ44M/)sigpa (Mirrop Kygea) were purchased at the age of 5 weeks, which were acclimatized for 1 week and used to perform the indicated experiment at the age of b-week. (uh)

Tested rats with symptoms of hypercalcemia caused by a malignant tumor were prepared as follows. A passivated malignant human lung tumor 1 C-6 was cut and a Zmm cube of slices was cut from it. One section of the tumor was transplanted subcutaneously to rats. Thirty days after transplantation, when the cancerous tumor in the rats reached a rather large volume (3000 mm U), the rats were divided into groups taking into account the concentration of calcium in the blood and body weight, after which they were used as animal models of hypercalcemia.

Excretion of phosphate by the method of renal clearance was studied as follows. - (1) Renal clearance method

The tested animal with symptoms of hypercalcemia caused by a malignant tumor was anesthetized with pentobarbital (Metbrsha!, Ogvipyrrop RNpagtaseciisa! So., Tsa.), immovably fixed on its back on an incubation mat at a temperature of 372C, and a cannula (polyethylene tube, REBO, Mirrop) was inserted into the urinary bladder Veskiop Viskepzop) to collect urine. This animal was then cannulated (polyethylene tube, REBO, Mirrop VesKiop ts OisKipzop) into the femoral vein and through this cannula, with the help of an infusion pump, the infusion solution "" (0.790 inulin, 595 mannitol, 0.296 pentobarbital and 0.995 sodium chloride) was administered at a rate flow rate of 2 ml/hour. After "achieving physiological balance, i.e., after 50 minutes, urine was collected through the cannula for 5 minutes at 20-minute intervals (starting with period 1 and ending with period 5), resulting in urine samples. In the middle of the urine collection procedure, approximately 0.25 ml of blood was taken from the right jugular vein using a heparinized injection syringe. sl (2) Administration of antibodies

During the clearance test described above, at the time of the start of urine collection in period 2, the animal was intravenously injected with a humanized antibody against RTNGR in the amount of Tmg/ml/kg. -oUu 70 (3) Determination of the concentration of inulin and phosphorus in urine and blood

The volume of urine samples obtained from period 1 to period 5 was measured, and the content of inulin and phosphorus in them was determined. The blood samples obtained above were centrifuged under cooling conditions to obtain a plasma sample, which was used to determine the concentration of inulin and phosphorus. The content of inulin was determined by the anthrone sulfate method | Koe, Y. ei. aiYi., 9. Vioi. Spent 178, 839-845, 1949| and the phosphorus content was determined using an automatic analyzer model 7170 of the Nigasny company using a reagent for the determination of inorganic phosphorus, Atschozeg IR (Oaisnpi Ryge Spetis!v) in accordance with the attached manual (RpuzKe-Zabragopy method). (4) Calculation of inulin clearance, phosphorus clearance, and fractional phosphorus excretion. Calculate inulin clearance (Sip), phosphorus clearance (Cr), and fractional phosphorus excretion (Ber) according to the following equations. 60 Calculation of inulin clearance (Sip)

Sip-Oipm/Rip de Sip means inulin clearance (ml/kg/min); Oip means concentration of inulin in urine (mg/ml); M means the amount of urine per unit time (ml/kg/min) and Rip means the concentration of inulin in the blood (mg/ml).

Calculation of phosphorus clearance (Cr): 65 Cr-Oru/Pp where Cr means clearance of phosphorus (ml/kg/min); Or means phosphorus concentration in urine (mg/ml); M means the amount of urine per unit time (ml/kg/min) and Pp means the concentration of phosphorus in the blood (mg/ml).

Calculation of fractional phosphorus excretion (PEp):

EEr-Sr/Sip where EEr means fractional excretion of phosphorus; Sip stands for inulin clearance and Cr stands for phosphorus clearance. Four animals were used for this study. The results were determined as the mean value - standard error.

The results of fractional excretion of phosphorus and the value of phosphorus concentration in the blood are shown in Fig. 25 and 26. 70 Fig. 25 shows a graph illustrating the fractional excretion of phosphorus (phosphorus clearance/inulin clearance) depending on the clearance periods (1 period-20 minutes). . Humanized anti-RTHNR antibody (mg/kg) was administered (intravenously) at the beginning of period 2.

Figure 26 shows a graph showing the concentration of phosphorus in the plasma depending on the clearance periods (1 period - 20 minutes). Humanized anti-RTHNR antibody (mg/kg) was administered (intravenously) at the beginning of period 2.

These results show that the fractional excretion of phosphorus after the administration of the antibody (ie, from period 2 to period 5) is significantly lower compared to the figure obtained before the introduction of the antibody (ie, period 1). IN OTHER WORDS, it was established that the administration of a neutralizing antibody to a subject suffering from hypophosphatemia due to increased excretion of phosphorus (PEp»0.2) restores the subject's phosphorus resorption to almost a normal level (fractional resorption of phosphate-1-PEp 20.895), while it is observed the tendency to Normalize the concentration of phosphorus in the blood of the subject being examined. These results indicate the effectiveness of antibodies according to the present invention as a means for the treatment of increased excretion of phosphorus and hypophosphatemia caused by the presence of RTNHR.

Since RTNGR is a substrate that causes hypercalcemia caused by a malignant tumor, with a high degree of probability it is possible to predict an increase in phosphorus excretion and a reduction of high-energy organic phosphorus in tissues under the influence of RTNGR. It is believed that many diseases caused by hypophosphatemia, such as hypophosphatemic rickets and hypophosphatemic rickets resistant to vitamin OO, arise as a result of increased excretion of phosphorus in the urine, so the antibodies of the present invention should also be useful for the treatment of these diseases.

Example 10 p

Positive dynamics of various clinical symptoms of hypercalcemia caused by a malignant tumor

It is known that hypercalcemia caused by a malignant tumor occurs under the influence of RTNHRI produced by the tumor, and that RTNHRI accelerates the resorption of bone tissue and the resorption of calcium in the kidneys and renal tubules, which leads to hypercalcemia. In addition, a subject suffering from hypercalcemia has worsening clinical symptoms, namely poor physical condition, loss of consciousness, persistent malaise, hydrodipsia,

Nausea and vomiting (anorexia). The effect of an antibody against RTNGR on such clinical symptoms was studied using the system of transplanting a malignant human tumor into "naked" mice and "naked" rats, which are used as animal models of hypercalcemia.

As an animal model of hypercalcemia, "naked" mice and "naked" rats were used (mice and rats were purchased from the Central Institute of Experimental Animals), which were transplanted with a malignant lung tumor "human GS-6". It is known that in "naked" mice and "naked" rats transplanted with a malignant tumor of the lungs of a human 1-6, the concentration of calcium in the blood increases as the volume of the tumor increases and hypercalcemia develops, which causes a decrease in body temperature and a decrease body weight "" The positive effect of a mouse anti-RTHgR antibody on the general clinical symptoms of hypercalcemia caused by a malignant tumor was investigated using a system of transplantation into "naked" mice of a malignant human lung tumor I C-6. The obtained results are presented in the photos. The effect of the specified antibody on - and improvement in relation to reduced movement activity, body temperature and anorexia was studied using the system of transplanting "naked" mice with a malignant human lung tumor I C-6. others 1. Positive dynamics of pronounced clinical symptoms caused by hypercalcemia

Ge) Malignant human lung tumor I C-6 was inoculated into "naked" VAI V/s-pi/pi (Mirrop Kygea) mice i.p. mimo.

To evaluate the pharmacological effect of the antibodies, male "naked" mice VAI-V/s-pi-pi (Mirrop Kygea) were purchased at the age of 5 weeks, which were acclimatized for 1 week and used to perform the specified experiment at the age of 7 weeks.

Tested mice with symptoms of hypercalcemia were prepared and divided into groups as follows.

A passivated malignant human lung tumor I C-6 was dissected and a Zmm cube of slices was cut from it. Each mouse was transplanted subcutaneously with one section of the tumor. Twenty-seven days after transplantation, when the cancer tumor in the mice had reached a sufficiently large volume, the mice were divided into groups taking into account the average iF) tumor volume, blood calcium concentration and body weight, after which they were used as animal models. models of hypercalcemia.

The volume of the tumor was determined by measuring the largest diameter (a mm) and the smallest diameter (6 mm) of the 60 tumor and performing calculations according to the Galant equation (ab2/21.

The concentration of calcium in the blood was determined on the basis of the concentration of ionized calcium in whole blood, which was taken from all mice through the eye sockets using a hematocrit tube and analyzed in an automatic analyzer 643Ca""/pH (SIVA-SOVMIMO).

The therapeutic effect of this antibody on hypercalcemia was studied as follows. Mouse antibody against RTNgR 65 was injected in the amount of 1000μg/mouse into the tail vein of all tested animals with symptoms of hypercalcemia on the 27th, 30th, 34th, and 37th days after transplantation of a malignant tumor. The control animal was similarly injected with phosphate-buffered saline instead of the antibody. On the 41st day after tumor transplantation, one representative mouse was selected from each group of animals injected with the specified antibody and from the control group and photographed next to a normal mouse.

As a result, a model was obtained from a human I C-6 lung tumor transplanted with a malignant tumor, although the mass of the tumor in mice injected with the antibody (shown in the center in Fig. 27 and 28) corresponded to the weight of control mice (shown on the right in Fig. 27 and 28) , only mice injected with the antibody looked the same as normal mice (shown on the left in Figures 27 and 28). The obtained results allow predicting that the administration of antibodies against RTNHR causes positive dynamics of pronounced clinical symptoms (Figs. 27 and 28). 70 2. Restoration of movement activity, reduced due to hypercalcemia

Malignant human lung tumor I C-6 was inoculated into "naked" VAI V/s-pi/pi (Mirrop Kygea) mice i.p. mimo.

To evaluate the pharmacological action of the antibodies according to the present invention, male "naked" rats EZ44/M si-hyp (Mirrop Kygea) were purchased at the age of 5 weeks, which were acclimatized for 1 week and used to perform the indicated at the b-week age.

Tested animals with symptoms of hypercalcemia were prepared as follows. A passivated malignant lung tumor of a human I/S-b was cut and a Zmm cube of slices was cut from it. Each mouse was transplanted subcutaneously with one section of the tumor. Thirty days after transplantation, when the cancer tumor in the mice had reached a sufficiently large volume, the mice were divided into groups taking into account the average volume of the tumor, the concentration of calcium in the blood and body weight, after which they were used as animal models of hypercalcemia.

The concentration of calcium in the blood was determined on the basis of the concentration of ionized calcium in whole blood, which was taken from all mice through the eye sockets using a hematocrit tube and analyzed in an automatic analyzer 643Ca""/pH (SIVA-SOVMIMO). (1) Method of determining movement activity

Movement activity was determined using an AMIMEH type ZE activity meter (RAKA, Epiesigopisv, Sweden), which was placed in a certain position in a polyethylene cage with the tested animal receiving water and food. This device is designed to measure the mobility of a rat. With the help of this device, the mobility i9) is recorded in the form of the number of movements performed in a certain period of time. Measurements were made for 13 hours (from 7:00 p.m. one day to 8:00 a.m. the next day). The results are given in the form of the number of movements in one hour. ee) (2) Administration of antibodies

The humanized antibody against RTNHR was injected in the amount of 5 mg/O.5 ml/kg into the tail vein of all the above-mentioned rats suffering from hypercalcemia. The control group of rats was injected with a physiological solution in the same way. The measurements were performed alternately in the tested rats that were injected with the antibody and in the control rats.

Rats injected with the antibody were examined on day 0 (i.e., the day before administration), 2nd, 4th, 7th, and 14th days, and control rats were examined on 1st, 3rd, 5th, 8th and 15th day.

The obtained results show that in control rats there was no change or decrease in locomotor activity during the test period, while in rats injected with the antibody, locomotor activity increased on the 4th day of antibody administration and on subsequent days (Fig. 29 ).

Q. Increase in body temperature, reduced due to hypercalcemia - s The test animal was inoculated with a malignant tumor of human lungs 1-6, hypercalcemia caused by the malignant tumor was also induced in them, and prepared for the experiment according to the procedure described in stage 2. (1 ) Method of measuring body temperature

The body temperature of the tested animals was measured using a digital thermometer. For this, the animals were anesthetized with pentobarbital (Metria!Y, Oavipyrrop RpagtaseciisaI So., Tsa.) and a temperature sensor was inserted into the rectum. i-th (2) Administration of the anti-RTNGR antibody Humanized antibody against RTNHR was injected in the amount of Tmg/ml/kg into the tail vein of all tested rats suffering from hypercalcemia. The control rat was similarly injected with physiological solution. In addition, the body temperature was also measured in a normal rat that was not injected with the indicated antibody. o The body temperature of all rats injected with the antibody, a control rat and a normal rat was measured on the 0th day (ie, the day before the injection), the 1st, 2nd and 3rd days after the injection.

As a result of this experiment, no change in body temperature (34.2 - 34.42) was detected in a normal rat during the entire period of the test, while in rats suffering from hypercalcemia caused by a malignant tumor, a decrease in body temperature was observed by about 2 2C in compared to normal rats. Three days after the start of administration of the humanized anti-RTNGR antibody to the tested rats, their body temperature rose to the level characteristic of normal rats. These results allow us to predict that the humanized anti-RTHNR antibody of the present invention is effective for increasing the reduced body temperature in animals suffering from hypercalcemia caused by a malignant tumor (Fig. 30). 4. Improvement of appetite in tested animals, reduced due to hypercalcemia

The test animal was inoculated with a malignant human lung tumor I1!C-6b, hypercalcemia was induced in them and prepared for the experiment according to the procedure described in section 2. Rats were divided into 65 groups taking into account the average concentration of calcium in the blood and body weight, after which they were used in the experiments described below.

(1) Measuring the amount of food consumed

During the test, each rat was placed in a separate metabolic cage, where it received water and food.

The amount of feed eaten by each rat was determined as the amount (g) eaten in 24 hours (from 9:00 a.m. one day to 9:00 a.m. the next day). To do this, the total weight of the feeder with food was measured at 9:00 a.m. one day (that is, together with the weight of the container) and at 9:00 a.m. the next day, after which the difference between them was calculated. (2) Administration of antibody

The humanized antibody against RTNHR was injected in the amount of 5 mg/O.5 ml/kg into the tail vein of all tested rats suffering from hypercalcemia (NNM rats). The control group of rats was similarly injected with physiological solution. In addition, saline was injected into the tail vein of normal rats. The amount of food eaten by rats injected with the antibody, control rats, and normal rats was determined on day 0 (i.e., the day before the introduction of the antibody), day 1 (i.e., between the day of administration and the next day), C3 day (that is, in the period three days after the introduction to the next day) and day 5 (that is, in the period five days after the introduction to the next day).

As a result of this experiment, it was established that before the introduction of the antibody, the amount of food eaten by rats with symptoms of hypercalcemia (5-9 rats) was on average 8.11g, while normal rats gave an average of 12.0bg, which indicates a clear reduction in the amount of feed consumed by the tested rats suffering from hypercalcemia. When the test rats were injected with a humanized antibody against RTNGR, on the day of the antibody injection and on subsequent days, their food intake increased to the level characteristic of normal rats, while there was practically no change in the amount of food consumed in the control rats. These results suggest that the humanized anti-RTHNR antibody of the present invention is effective in ameliorating decreased appetite in animals with symptoms of hypercalcemia due to malignant tumor (Table 6). sch shchi o

Animal Mo rat Introduction "so - i: i: not zv 5 Fviololchnyromin| 559. 882 259 8 m in dniploprotirtnt| 1200/1238 soy 32 in dniploprotyetyat| 335 be 0 2ode 0 21ya " i z s ;"

The above results show that chimeric antibodies and humanized antibodies according to the present invention are useful means of influencing the positive dynamics of various clinical symptoms of hypercalcemia caused by a malignant tumor. 5. Improvement of the blood pH index, reduced as a result of hypercalcemia 1 The tested animals were inoculated with a malignant human lung tumor 1C-6b, induced hypercalcemia caused by the malignant tumor in them, and prepared for the experiment according to the procedure described in stage 2. The rats were divided into groups with taking into account the average concentration of calcium in the blood and body weight. -th (1) Determination of the blood pH index of all tested animals, blood was taken using a heparin-treated syringe for injections according to the method of taking blood samples from the heart, and the obtained blood samples were analyzed in an automatic analyzer 643Са""/РН (SIVA-SOEMIMS) in order to determine the pH indicator. (2) Administration of antibody

A humanized antibody against RTNGR was injected in the amount of 5 mg/O.5 ml/kg into the tail vein of all the tested (F. rats suffering from hypercalcemia (NNM rats) (p-3). Control rats were similarly injected with physiological solution into the tail vein (p- 2).The pH of the blood in the rats injected with the antibody and in the control rats was determined on day 0 (i.e., the day of administration), day 1, and day 7. The results are presented as the average value of the pH values.

The obtained results show that before the introduction of the antibody, the pH value of the blood in the tested rats with symptoms of hypercalcemia was equal to approximately 7.49 (while in normal rats the specified value was equal to 7.40-0.02); this probably indicates that the tested rats had metabolic alkalosis.

When the test rats were injected with the humanized anti-RTNGR antibody of the present invention, they experienced a restoration of pH to nearly that of normal rats seven days after administration of the antibody, whereas control rats showed virtually no change in blood pH . It is known, -d42-

that alkalosis is one of the clinical symptoms of hypercalcemia caused by a malignant tumor, and occurs as a result of inhibition of the excretion of bicarbonate ion (HSO 37) in the kidneys. Since the introduction of a humanized antibody against RTNHR according to the present invention normalizes the pH of the blood in tested animals with symptoms of hypercalcemia, it can be predicted that this antibody is able to restore metabolic alkalosis arising as a result of hypercalcemia caused by a malignant tumor (Fig. 31).

The above results show that chimeric antibodies and humanized antibodies according to the present invention are useful means of influencing the positive dynamics of various clinical symptoms of hypercalcemia caused by a malignant tumor. 169 70 This invention relates to the preparation of a chimeric antibody and a humanized antibody against RTNGR. These antibodies are characterized by low antigenicity relative to humans and are therefore useful for the treatment of hypercalcemia, hypophosphatemia, and similar diseases.

List of sequences (2) Information for the sequence with identification Mo1: (Y) Sequence characteristics: (A) Length: 20 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type molecule: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo1

АААТАОССССТ ТОАССАОСсСА 20 сх и и 2. и но (2) Information for the sequence with identification Mo2: (о) (Й) Sequence characteristics: (А) Length: 38 base pairs (В) Type: nucleic acid со 20 (С) Structure chain: single-stranded (OB) Topology: linear - (ii) Molecule type: other nucleic acid c (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identifying Mo2 iv)

Зо сСТОаТтССОсС ССАССТСТОА AOSTISSАСА ATССАТАСО 38 te (2) Information for the sequence with the identification Moz: (Y) Sequence characteristics: « du (A) Length: 28 base pairs with (B) Type: nucleic acid c (C) Chain structure: single-stranded : z» (OB) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /opis - "synthetic DNA" - 15 (xi) Sequence description: sequence with identification MoZ and СSATSSSOOO SSASTOSATA SASASATOo 25 o 50 (2 ) Information for the sequence with identification Mo4: - (Y) Sequence characteristics: c (A) Length: 29 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid (F. (A) Description: /description - "synthetic DNA" co (s) Sequence description: sequence with identification Mo4

ССАТСССОБОС TСАСКОСААС ОТООКААсСА 29 60 (2) Information for the sequence with identification Mob: (Y) Sequence characteristics: (A) Length: 17 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear

(ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mob and STTTTSSSAS TSASSAS I7 (2) Information for sequence with identification Mob: (Й) Sequence characteristics : 70 (A) Length: 17 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA " (xi) Description of sequence: sequence with identification Mob

SASSAAASAS 0 STATOSAS 17 (2) Information for the sequence with identification Mo7: (Y) Sequence characteristics: (A) Length: 31 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii ) Type of molecule: other nucleic acid ch 29 (A) Description: /description - "synthetic DNA" Ge) (xi) Sequence description: sequence with identification Mo7

ОТСТААСТИ ССАССАТОАА АСТИСОСОСТ С 31 со (2) Information for the sequence with identification Mo8: «h- (Y) Sequence characteristics: (A) Length: 30 base pairs o (B) Type: nucleic acid oyu (C) Chain structure: single-stranded 3o (OB) Topology: linear in (ii) Molecule type: other nucleic acid (A) Description: /opis - "synthetic DNA" (xi) Sequence description: sequence with identification Mov "- so TOTTOBATOS STOSASASAS ASTOASSAbA 30:z" (2 ) Information for the sequence with identification Me9: (Y) Sequence characteristics: (A) Length: 36 base pairs 45 . -1 (B) Type: nucleic acid (C) Chain structure: single-stranded 1 (0) Topology: linear sequence (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA" - (xi) Description sequence: sequence with identifier Me9 co OTSTSAATTS AAOSTISSAS SATOOSSOTTT sbOStTo 36 (2) Information for sequence with identifier Mo10: (Y) Sequence characteristics: (A) Length: 41 base pairs

F) (B) Type: nucleic acid ka (C) Chain structure: single-stranded (OB) Topology: linear in (ii) Type of molecule: other nucleic acid (A) Description: description - "synthetic DNA" (xi) Sequence description : sequence with identification Mo10 ttsСsСпсОосС ССТТОоСТОобА СОСТОАОСАС АССОТОАССА P. 41 65 (2) Information for sequence with identification Mo11: (Y) Sequence characteristics:

(A) Length: 109 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description -- "synthetic DNA" (xi) Sequence description: sequence with identification Mo11

STSTOAATTS AAOSTTASTA STTOOSSSAOS SSALOSSSSAA SSSSASOOTS ASSSTOTTOS sob

СОСССТСТС ТСАСИЛОСТО СААОСССААСА АСОССАСАСТ АСОТОТОИТСТ 109 (2) Information for the sequence with identification Mo12: (Y) Sequence characteristics: (A) Length: 110 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo12

ОOTTTOSTI TSTSSASTOS SOSSTTSASO SOSSSTOSSAT STOSSTTSSA SOSSASTOTTS 50 dv ASASSSTSSSO SSTASAASTS ASTOATSASA SASASTASTO TOOSSTTOTT po s (2) Information for the sequence with identification Mo13: o (Y) Sequence characteristics: (A) Length: 98 base pairs (B) Type: nucleic acid (ee) (C) Chain structure: single-stranded «- (OB) Topology: linear (ii) Molecule type: other nucleic acid co (A) Description: /opis - "synthetic DNA" and (xi) Sequence description: sequence with identification Mo13 and -

OSBAOTOSASA SSASSALASS STSSALASAS DOSAASAASA AOTASOISOSOS SASSAOSTAS 60

STOAOSSTOA СОСССОСАССА СТОСААСТОС САСАСААО 98 « ! . g. še | - (2) Information for the sequence with the identification Mo14: c (Y) Sequence characteristics: :c" (A) Length: 106 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded 15 (OB) Topology: linear (ii) Molecule type: other nucleic acid 1 (A) Description: /description - "synthetic DNA" c (xi) Sequence description: sequence with identification Mo14 ayu TITTOAATTS TTASTATOAA SATISTOTAO OSOSSASTOT STISTOSASASo STOSTOSSTT va so SATOSSTOAS STOSSASSTO TAOSTTSTOT SOOBASTTSSA STOSTS 106 (2 ) Information for the sequence with identification Mo15: (Y) Sequence characteristics: (A) Length: 43 base pairs (F) (B) Type: nucleic acid co (C) Chain structure: single-stranded (OB) Topology: linear in (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo15

STSTODATTS DAOSTTASTA STTOOSSSAOS SSADSOSSAA ССС ж 65 (2) Information for the sequence with identification Mo16: (Y) Characteristics of the sequence:

(A) Length: 20 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA" ( xi) Sequence description: sequence with identification Mo16 to TOTTSAATTS TTASTATSAA 20 (2) Information for sequence with identification Mo17: (І) Sequence characteristics: (A) Length: 39 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo17

СААААСТАС ОСООССАОСА ОСТАССТСАСb ССТОАСОСС 39 (2) Information for the sequence with identification Mo18: (Y) Sequence characteristics: c (A) Length: 39 base pairs o (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid co (A) Description: /description - "synthetic DNA" - (xi) Sequence description: sequence with identification Mo18

STAOSTOSTO bSSOSOTAST TOTTOTTOST STOTTTOSA 39 o (2) Information for the sequence with identification Mo19: in (Y) Sequence characteristics: - (A) Length: 46 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded « (ОБ) Topology: linear (ii) Type of molecule: other nucleic acid ts (A) Description: /opis - "synthetic DNA" h» (xi) Sequence description sequence with identification Mo19 " ОСТСТОААТТС ААОСТТАСТСТАСТСОАА СТОТООСТОС АССАТС 46 (2) Sequence information with identification Mo20: -i (Y) Sequence characteristics: (A) Length: 34 base pairs and (B) Type: nucleic acid

Ge) (C) Chain structure: single-stranded (OB) Topology: linear - (ii) Molecule type: other nucleic acid co (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo20

TOTTSAATTS TTASTAAAS TSTOSSSTOT TOAA 34 (2) Information for the sequence with identification Mo21:

HF) (Y) Sequence characteristics: (A) Length: 35 base pairs and (B) Type: nucleic acid (C) Chain structure: single-stranded 60 (p) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo21 v5 СТСТААОСТТ ССАССАТОСОС СТОСАСТОСТ СТО . 35

(2) Information for the sequence with identification Mo22: (Y) Sequence characteristics: (A) Length: 48 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /opis- "synthetic DNA" 70 (xi) Sequence description: sequence with identification Mo22

TOTTOAATTS АSATSTAAST ASTTASTAS САСАСТОАСсС ТТООаТСССС 45 (2) Information for the sequence with identification Mo23: (Y) Sequence characteristics: 75 (A) Length: 128 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description sequence with identification Mo23

STSTAASSTT SSASSATOOSS STTTOBOSTO ASSTOSOTTT TOSTOOTTOS TSTTTTAAbA 60

OSTOTSSAST OTSAbOTOSA OSTOSTOSAS TSTOPOObAO OSOTOSTOSA OSSTOSOSASO 120 Ha

TteSSstbAS 128 He) (2) Information for the sequence with identification Mo24: (Y) Sequence characteristics: (A) Length: 125 bp (B) Type: nucleic acid (C) Chain structure: single-stranded «- (OB) Topology: linear c (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (ii) Sequence description: sequence with identification Mo24

ASSATTASTA SOTOSOTOSTAS TTASASSTAS TATSSASASA STOTOAAOSO OSOATTSASS 60

ATSTOSASAb ASAATTSSAA SAASASOSTO TATSTOSAAA TODASAOSST BASAOSTOAO 0 120 «

САСАС 25 - с (2) Information for the sequence with identification Мо25: (Y) Characteristics of the sequence: ;» (A) Length: 132 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded -I (OB) Topology: linear (ii) Type of molecule: other nucleic acid o (A) Description: /description - "synthetic DNA" 2) (xi) Sequence description: sequence with identification Mo25 - STASSASSAE TASTAATOST TOSSASSSSAS TSSASSOSSST TOSSTOSAOS STOSSSSSASS syu so SAASASATOS SATAOSTAST SAASOSTSAAT SSASASOSTO SASASSASAS TSTSAOOSAS 120 rya СstosSAsSOosT oo 132 (2) Information for the sequence with identification Mo26:

F, (Y) Sequence characteristics: ko (A) Length: 110 base pairs (B) Type: nucleic acid vo (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo26 b5

TOTTOBATSS STOAOSASAS OSTOASSAOO STTSSSSTOOS SSSASTAAOS AAASTAASTS o

АТАСТАСТСТ СТСТСОСАСА ОТААТАСАСА ОССОТаТтОСтТ САОСТСТСАС on (2) Information for the sequence with identification Mo27: (Y) Sequence characteristics: (A) Length: 30 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo27 to STSTAAAOSTT SSASSATOSS bttTosOosto 30 (2) Information for sequence with identification Mo28: (І) Characteristics sequence: (A) Length: 30 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA " c (xi) Sequence description: sequence with identification Mo28 Ge)

ТОТТОСАТСС СТСАбСАбАС ОТСАСАБО 30 (2) Information for the sequence with identification Mo29: Ge) (Y) Sequence characteristics: (A) Length: 133 base pairs - (B) Type: nucleic acid co (C) Chain structure: single-stranded (OB) Topology : linear o (ii) Molecule type: other nucleic acid che (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo29

АСАААОСТTS САССАТОССОССТОБАСТОСТСТСТТСТИСТТСТТТОТСТТ САТТОСТСАО 60 « s tho OTTSTTTTSTS ССАОСТТОТОо СТОАСТСААТТ СОСССТСТООС СсToТоССстТОС stoOobАОСсСстТ 120 not "from СОСТСААОСТ САС 33 (2) Information for the sequence with identification number Mo30: (Y) Sequence characteristics: -i (18) Base pairs Length: B) Type: nucleic acid o. (C) Chain structure: single-stranded (95) (OB) Topology: linear shu 20 (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA" co (hi ) Sequence description: sequence with identification Mo3ZO0

АССААСАТОО ААСССАСАСС АСAbSTOATO СSATISSTOA TSOSTISTSA СОСТССАОСТ 60

STOSSOSTOA OSOSTASTSTS ASSATSTOSA OSSTSSASTS TSAbSATSAb OSTOASTA 118 o (2) Information for the sequence with identification Mo31: ю (Й) Sequence characteristics: (A) Length: 128 base pairs 60 (B) Type: nucleic acid (C) Chain structure: single-stranded (OB ) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" b5 (xi) Sequence description: sequence with identification Mo31

STOSTOOSTTS SATSTTOST AASTITSATS AASTASSbAS OBSSSTISTIS TOOSTOSTOS OS

TOATOSSATT SAATOSTOTA SOTASTOTOS TOASTASTSA ASOTOSASSST SAOSTTSASS 120.

SASOSTOS 128 (2) Information for the sequence with identification Mo32: (Y) Sequence characteristics: 70 (A) Length: 114 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ии) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo32

STTObATSSO SOSTSASSTA OBASOSTSAO TTTOOTOSST SSOSSOAASA SSSTSASAAA bo

TISTTOSTTA ATTOTATATSAS ССАСАССАСА СТАТАСОТSA ОССТСАТССТ САбСА 114 720 (2) Information for the sequence with identification Mo33: (Y) Sequence characteristics: (A) Length: 17 base pairs (B) Type: nucleic acid o s (C) Chain structure: single-stranded (OB ) Topology: linear (o) (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identifying Mo33 co

ASAAAOSTTS SASSATO I7 - (2) Information for the sequence with identification Moz4: Che (Y) Sequence characteristics: ю (A) Length: 19 base pairs (B) Type: nucleic acid - (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid « (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo34 in - СТТОбАТССО ООСТОАССТ 19 p (2) Information for sequence with identification Mo35: ( Y) Sequence characteristics: -I (A) Length: 75 base pairs (B) Type: nucleic acid o (C) Chain structure: single-stranded c (OB) Topology: linear (Y) Type of molecule: other nucleic acid - (A) Description: /description - "synthetic DNA" with (xi) Sequence description: sequence with identification Mo35

STTObATSSO SOSTSASSTA SSASOSTSAS TITOSTOSTST SSOSSOAASA SOTASASADA 60

TOTTSSTTA ATTOT 75

Ф) co (2) Information for the sequence with identification Mo36: (Y) Sequence characteristics: 60 (A) Length: 43 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii ) Molecule type: other nucleic acid 65 (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo3b

ALAOSATOSST TAASATOSSAT SAASTASSOA SOOPOSTIST Sto 43 (2) Information for the sequence with identification Mo37: (Y) Sequence characteristics: (A) Length: 46 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identifier Mo37 5 АСАААОСТТА ССОСТАССТСАСТОСА ОССТОСАбСС TbAbСА 46 (2) Information for sequence with identifier Mo38: (І ) Sequence characteristics: (A) Length: 111 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: /description - " synthetic DNA" with (xi) Sequence description: sequence with identification Mo38 o

STTObATSSO SOSTOASSTA OSASOOTSAO TITOOTSSST SSOSSOAASA SOTASASAAA 6o

TISTTSTTTA ATTOTATATSAS SSASASASSASA SATATASTSA OSSTTSATOSST C NI c (2) Information for the sequence with identification Mo39: - (Y) Sequence characteristics: (A) Length: 42 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded

Zo (0) Topology: linear i- (ii) Molecule type: other nucleic acid (A) Description: /opis - "synthetic DNA" (xi) Sequence description: sequence with identification Mo39 «

СстИСТСТСОИС ТОСТОСТОСАТ АССАТИСААТ СОСТСТАСОСТА СТ 42 c) c (2) Information for the sequence with the identification Mo40: c (Y) Sequence characteristics: (A) Length: 26 base pairs (B) Type: nucleic acid -I (C) Chain structure: single-stranded (OB) Topology: linear 1 (i) Molecule type: other nucleic acid co (A) Description: /opis - "synthetic DNA" and yu (xi) Sequence description: sequence with identification Mo40 co СссАдсобсСссСт tststoOosto stasStTo 26 (2) Information for the sequence with identification Mo41: (Y) Sequence characteristics: oo (A) Length: 35 base pairs o (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid 60 (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo41

SAPpAAsSOaSS STAKSTASUT SATOKAMUSTI AASSA 38 bo (2) Information for sequence with identification Mo42: -BO0-

(Y) Sequence characteristics: (A) Length: 35 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (Y) Molecule type: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo42 to SASSAATTSA STATSSATTS TOSAASSTTTS AbASb 35 (2) Information for sequence with identification Mo43: (Y) Sequence characteristics: (A) Length: 18 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo43

SOSTTObAOS TSSTSAbA 18 (2) Information for the sequence with identification Mo44: c (Y) Sequence characteristics: Ge) (A) Length: 20 base pairs (B) Type: nucleic acid (C) Chain structure: single-stranded (OB) Topology: linear so (ii) Type of molecule: other nucleic acid "- (A) Description: /description - "synthetic DNA" (xi) Sequence description: sequence with identification Mo44 and

SASASOTOOTT SAAASTITIT 20 Is)

Зо (2) Information for the sequence with identification Mo45: c. (Y) Characteristics of the sequence: (A) Length: 118 amino acids (B) Hyp: amino acid " (OB) Topology: linear (i) Type of molecule: protein C c (xi) Description of the sequence: sequence with identification Mo45;" -And 1 (95) - 50

IR e)

F) ime) 60 b5 bip Se Mai Sen Tpg o bip bZeg beg Zeg Aia bek Re bet Gen biu 1 5 10 15 b "Aa 5eg A1a gub ep Tig Sub TP Tez bek bZeg bip Niye beg TSHg v Tuye Tish Pe bi Tgr Tug bip b1p b)p Rgo Ge uv Rgo Rgo Guv 20 Tuk Ua! Meb Avr Te Mu5 bip Azr b1iu bek Ni bek Tig 01U ABr 59 bo s shchi b1iu Pe Rgo Ar Agr Re beg biu beg bet beg S1u Aia Ar Aga o s 30 "- . so 655 to 75 yu 35 h-

Tug Sen bek Pe 5eg Ap I1e bip Rgo bIv dr bi Aza Mei Tug «

Y 80 bo 90 - s z» Pe Su 01u Ua! (Tu Abr Te Te uye 010 01p Re Uai1i Tug Uai 45 95 SHO 105 -And her Re b1iu biu biu Tig Guz Ma! Tg Hai Ge b1u bip Rgo - 50 119 115

IR e) (2) Information for the sequence with the identification Mo46: (Y) Sequence characteristics: (A) Length: 118 amino acids iF) (B) Type: amino acid ko (OB) Topology: linear (y) Type of molecule: protein vo ( хи) Description of the sequence: the sequence with identification Mo46 b5 and bi Ua! b1p Bez Ma1i 010 Zeg biu b1u Abo be Mai Gu Rgo 01u 1 Z 10 15 y biu 5eg Geo Gu5 Ge 5eg Su5 AIa Ai1a beg 01u Rpe TIPg Riye beg 20 25 30 bFeg Tut biu Mesye beg Tgo Ge Agya b1y Tit Rgo Ar Tu5 Age Gen 7 35 40 45 bij TgroMai Aia Tig o 112 bet beg b1u b1u beg Tug Ti Tut Tug sch o 50 55 bo so zo Rgo Avor beg Ma! Tsuz b1iu Agv Rpe TIig Ge beg Age Azr Asp A1a - so sh-- iv) with 55 70 5 k

Mus Ap Tng ben Tug bec biy Mes beg 5eg Ge Gu Zeg biy Ar « shi s » Vo 85 90 o TVg ATa Met Rye Tut Sub AЯa Aga b1p TNK TygoMei TNg o Tug Re 1 g 95 100 105 - 50 so Aia Tut Tgr b1iu b1p biu TBgoSeo Ua) Trade oUa) 5eg dia 110 115

Ф) о (2) Information for the sequence with identification Mo47: (І) Sequence characteristics: bo (А) Length: 116 amino acids (В) Type: amino acid (OB) Topology: linear (І) Type of molecule: protein (хи) Description sequence: sequence with identification Mo47 b5 bip mesh Mai Ge Tibg o bii beg Rgo beg AIa beh AIa Zeg Ge 01u 1 t 10 15 70 A1a beg Ma! Du5 Sei Tg Suye Tpg Se beg beg beep Ni beg Tag 23 30 7 Tut Tk This would Ter Niv beep bil bil Rgo bish Tuz b1u Rgo Agye 35 40 45 20

Here Petz Met Gu5 Sen Guz beep Or biu beg Ni Zeg Tg bu dor 4) 55 bo sch o b1u Pe Rgo der Age Rpe behg (01u bZeg beg bek Siu Aia 010 Age zo b5 shk 75 y

Tug Hech Tig I1e beg Zeg Tvy bip beg byu Avr Si Aa Avr Tug y

IS) h- 8o0 85 90 « shi s Tut Suz (Tu Ma! b1u Ar Ts Pe Mu b1y biv Re Uai Tug Uai z 95 100 15 -I sl Re biu b1u b1u Ti Gu Mei Tit Ma! Ge bih (95) ayu 110 115 42) (2) information for the sequence with identification Mo48: (Y) Characteristics of the sequence: (A) Length: 118 amino acids (B) Type: amino acid (B) Topology: linear o (y) Type of molecule: Imo protein) ( хи) Description of the sequence: sequence with identification Мо48 60 б5 -Б4-

beep beach Ma! Pet Tu beep beg Rto bBei A1ia beg A1a bZeg Hep si1u 1 5 10 15 to Aia beg Mai Sub Ge Tag o Sub5 Tk Ge beg bek beep Ni bek TTG 7 Tut TVg Pe Sis Tgr Tukh b1p beep beep Rgo bIsh Tuz S1u Rgo yuv 20

Tuk Mech Mebs Azr Ge) Suz bij Ayer biu beg Ni bet TNK (01u Ar zv 50 55 bo o biu Pe Rgo Abr Age Re beg Si beg bek 5eg F1u Aa Ti Agr zo 65 70 75 y

Tug SCsesh Tg o TIe beg 5Zek Mei bilo bet bin A5r bin Aa Abr Tug her » Vo Vo 90 y.

Tuk Sub b1u Ma1i Sbiu A5r TK Tie Tu5 bio beep Rpe Ma! Tut Uai « 40 shi - 95 100 105 z 18 Rre b1u Siu biIu Tig obu Be TgoUa) Teo 01u bil Rtgo -I s 119 115 (95) - 50 i42) (2) Information for the sequence with the identification Mo49: (Y) Characteristics sequence: (A) Length: 118 amino acids (B) Type: amino acid (B) Topology: linear o (u) Type of molecule: ko protein (hi) Sequence description: sequence with identification Mo49 60 b5

Sip Tecp Uai yeech Tv bip bet Rgo beh Aa bet Aia Zeg Te 01U y | 1 5 10 15 dia beg Wow! Ace Gets Tpg Suv Tig Hey beg beg beep No beg Tig

ИЙ Tukg Tpg Pe b1im Tgr Tukh bip bil bib Rgo bish іu5 b1u Rgo Tuz opt yannia: .

Tut Mai Meb Azr Gei Guz bip Abr b1iu bek Ni beg Tig (in Azo

BO sch 25 o b1u Pe Rgo Avr Age Re Beh biu beg beg Beh 01u Aia b1y Age 65 70 75 so 30 | "-

Tug (em Tag Pe beg beg Ivo bip beg bish Azr b1m Aia Avr Tug o

IS) 35 80 85 90 th-

Here Suz 01u Ma! S1u Ar Ty Pe Gu biy bil Re Uai Tuk Uvi « - с 70 95 100 105 и» Rve biu b1u b1u Tyh uv Ge Tig Uzi Mem b1u Sip Rgo 45 - - 110 115 1 o (2) Information for the sequence with identification Mo50: -oUu 70 (Y) Sequence characteristics: (A) Length: 118 amino acids c (B) Type: amino acid (B) Topology: linear (y) Type of molecule: protein 22 (xi) Sequence description: sequence with identification Mo50

F) ime) 60 b5 -58c-

beep Se Ma! Se TI beep beg Rgo beg A1a 5eg Aia beg Gets S1u 1 5 10 15 b ATa bek Mai Guz Kets Tg Suz TK Gets bet beg 01p Ni Zeg Tg 29 30 5 Tut Tig Pe 01 Ter Tut beep beep b1p Rgo b1y Guv b1u Rgo Age 35 40 45 20 sho Sh

Tug Te Mes Ar Te uz bil Avo b1u 5eg Niye beg Tpt (u der 5O 55 60 o biu Te Rgo Avr Agya Rpe beg b1iu 5eg b5eg Zeg S1u Aa 010 Ago s b5 to 75 - so

Tug Mei TygoOe bek obek Gets 012 beg bij A5r 01 A1a Ar Tug yu 7 vo What 90 y

Tug Suz S1u Ua! b1u Ayer Tit Pe tsu Sich bil Re Ma Tug Ua! h shi s 95 MO 105 z y Re 01u b1u B1u Tpg Guz Ge TvgoUa! Geo b1iu b1ya Rgo -I sl 110 115 (95) -oUu 70 (2) Information for the sequence with identification Mo51: (Y) Sequence characteristics: so (A) Length: 118 amino acids (B) Hyp: amino acid (O) Topology: linear оо (и) Hyp of the molecule: protein о (хи) Sequence description: sequence with identification Мо51 име) 60 b5 bip iets Uai Tem TBg bip beg Rgo bek Aia beg A1a Zeg yvi 0b1u 1 Z 10 15 to A1a beg Ua! Sue Ge Tg Suz Tg Sam 5eg beg beep Ni5 Beg Tpg 20 25 30

Tut Tk Te bish Ter Tug b1p bip bij Rgo bis Buz b1u Rgo AgeE 35 40 45

This is Ma! Me Ar iey Guz bip der biu beg Ni beg Tg Siu Ar 50 55 60 sch o

Siu Te Rgo Aho Agb Rpe bZeg yiu bek 5eg bBeg biu Aia bij Age s zn | . rent - for 70 75 so

IS)

Tuk Sets Tag Le beg bek Ge bil bek beach Ar biy Aa Abr Tug m- vo 85 Zo « -7 Tug Su b1u Ua! b1u Ar Tng Pe uv Sim biya Rve Uai Tug Uai 7 ;" 95 100 105

V. Rpe b1iu b1u b1u Tai Sub Seo Teg Ma! Beo biu bid Rgo 1 s 1 115 - 50 "so (2) Information for the sequence with identification Mo52: (Y) Sequence characteristics: (A) Length: 118 amino acids 550 (bugology: linear, (F) (y) Type of molecule: white

GI (xi) Description of sequence: sequence with identification Mo52 60 b5

(ip oben Ua! Se Te o bip bet Rgo beg Aa beg Aia bet Gen (iu

I 5 10 15 7 AIa beg Hai Gu5 Tez Teg Su TygoTets beg beg bil Niz beg TPg 20 23 Zo

Tug TP Pe bij Tgr Tut Tl (o bip Rgo 010 Su5 01u Rgo Gu yu 35 | 40 45

Tug be Mebs d5r o bech Su 01p Abr b1iu bek Ni5 bek Tig F1u Azr s 50 56 bo o

Siu Pe Rgo Ar Age Re beg biu beg beg bek 01u ATa 01 Age shshe nnya. - t i shoyu che 65 7o 75 y

IS) shchi Tut This Tyg Ge bek beg tse bip beg bits Ar bi Aa Azr Tug y, 80 55 that « shi s Pe Sub b1iu Ua! biu Ar Tpg Pe Guv bi bip Rpe Ua! Tug Uai z v 95 0 105 -I sl Rpe p1u 1u (Tu Tyg Suhe Tvg Ua! Teo b1u (poro (95) pub 110 115 s I (2) Information for the sequence with identification Mo53:

HF) (Y) Characteristics of the sequence:

A) Length: 118 amino acids di (c) Type: amino acid (OB) Topology: linear 60 (i) Molecule type: protein (hi) Sequence description: sequence with identification Mo53 b5 bil without Uai Sen TBg o bip beg Rtgo beg AIa beg AtTa beg Getch 01U and 5 10 15

A1a back Ma! Guz es Tpg Su5 TNg o GSech beg ip Ni Zeg Tg 20: 25 30 75 Tut TP Pe bio Tur Tut beep beep beep Rgo b1y Sub S1u Rgo Ag 35 40 45 7 Tuk Sei Met Azr bez Gu beep Ar biu beg Ni5 beg TI S1u Avr 50 93 bo sch o (Mu Pe Rgo dr Age Rpe beg SIu beg bek beg Shu Aa bin Age s 65 70 75 )y so

Tug Ge Tag T1e beg bZeg Ge bip bek beach Ar SI Aa Avr Tug v chvo 85 90 « liu Pe Suz b1u Ha! b1u Ar Tg Pe Guye b1y beep Rne Ua! Here Wow! from the village

І» 95 100 105 what Rpe Siu biu biu Tig Tuz Gey Tg Uai Te b1u bip Rgo 1 s 1yuЮ 115 - 50 s (2) Information for the sequence with the identification Mo54: (Y) Characteristics of the sequence:

A) Length: 118 amino acids o (c) Tit amino acid name) (O) Topology: linear (ii) Type of molecule: protein bo (hi) Sequence description: sequence with identification Mo54 b5 bip Se Ma! Sei Tyg o b1lobeg Rgo bek AZa beg A1a beh Gey s1u 1 5 10 15 b A1a Chek Ma! muz Se Tlg Sub Tgobeiy beg o beg 01p Ni beg Tig 5 Tut Tsh Pe 010 Tgr Tut bij bil bij Rgo 010 buye b1u Ro Gu 45 40 4 7 Tug Ua! Met Ar Getch Guye bip Ayer S1u beg Ni beg Tig S1U Azr 50 55 bo sch 25 o

Siu She Rgo Ar Ago Re beg biu beg bek bek siu A1a bicp AgE . s 30 "- 65 70 15 How

Tug (em Tpt I1e beg bek Geu bip Zeg bij Azr SI Aa Abr Tug y 80 85 90 «

Pe Suz 01u Ma! S1u A5r Tvg Pe Guz bish beep Re Ua! Tug Whoa! From the village

I» 95 100 105 shchi Rre biu b1u b1u Tpg Guz Cez Tvk Khai Se b1u bip Rgo 1 i 110 ц5 - 50 s (2) Information for the sequence with identification Mo55: (i) Characteristics of the sequence:

HF) (A) Length: 118 amino acids (B) Type: amino acid di (OB) Topology: linear (i) Type of molecule: protein 60 (xi) Sequence description: sequence with identification Mo55 b5

; beep Gen Ma! ep Tng bil obeg Rko Zeg A)a 5eg A1a beg Gei S1u 1 o Yu 15 to Aia beg Ha! Gu ep TK o Suz Tho be bZeg o beg 01p Niz bet Tig 20 25 30

Tuk Tig I1e bib Ter Tug bip bib bij Rgo bish (u5 bu Rgo Age

From 40 45

Here Ua1! Mei bro Hep Guz b1p Azo 01u beg Niye beg Tk b1u Ar z | 50 55 60 o b1u I1e Rgo Avzr Age Re beg biu beg beg beg s01u Aa (Ti Ate s «-

IS) zv Tut Te Tig Pe beg 5eg Gey bij beg bij Avr Sich dia Ar Tug te vo 85 90 « shi s Pe Sub b1u Ma! bIu A5r Tigo Pe uz 010 bip Re Uai Tug Uvi z 95 100 105 -I sl Rpe bIu biu b1u Tpg uz ep Tit Ua! tep b1u bip Reo (95) a yu 110 115 s (2) Information for the sequence with identification Mo56: i) Sequence characteristics: (c) Length: 118 amino acids (F) (B) Type: amino acid

Ge (OB) Topology: linear (u) Molecule type: protein vo (hi) Sequence description: sequence with identification Mo56: b5 beep Uai beep Hey Uai 01 bek biu 017 b1u Mai Ma) beep Rgo b1u

And at 10 15 and Agy beg Ge Age Geo bZeg Suz A1a A1a bek biu Rbe TNg Rpe 5eg 20 20 30 beg Tug biu Mei beg Tgr Ma! Age ()p Aa Rgo 1u Tuz schu Ge" yu 35 40 45,

So Ter Mai ATa TBg o Y12e beg o beg biu biu bZeg Tut TVgoTug TuUG cm 7 50 55 60 o

Rgo Aet beg Ma1 1u5 biu Agro Re Ti G1e beg Agro Ar Abp beg so so 65 70 5 oyu ch- uz A5p TK Sem Tug be bip Met Avp beh Ge Aga ATa 01 Or 80 h

Y 85 90 - s g Tik Aa Ua! Tut Tug Su5 Aa Age bip TB Tk Met Tg Tug Re 95 100 105 -I t A1a Tut Ttr biu biu biu TU Se Ua! Tu Wai Seg beg (95) 7 Pb 115 s ch 231 i i iz ications Mo57: o nn v vin i pic ko (A) Length: 411 base pairs (B) Type: nucleic acid vo (C) Chain structure: double-stranded ( OB) Topology: linear (i) Molecule type: cDNA - me NK (xi) Sequence description: sequence with identification Mo57 b5

ATO AAS TS bob Sto AOS OTTO ATI TS ST OS STO ATT TTA AKA 045

Me Azp Re S1u Petz beg Gen Pe Re Petz AZa Getch Te Geny uv -5 -yu -5

SOT OT SA TOT bai Oto SAA Sto OTO AS TST bo bbA BAS OTTA 090

Sbiu ZhMai beep Su5 fight Ma! b1ip Gen Uai 01 beg 01u Shu Ar Ge

And 5 10

STO AdO SST bbA 000 TOS STO AAA STO TOS tot oSA oS TST (SA 0135

Wow! tsu Rgo Siu b1u 5eg Pech Mu5 Se bZeg Su Aia ATa Zeg (Pu s 29 15 20 25 o

TS AST TS AST AOS TAT bOS ATO TST TO AT SOS SAb AST SSA 180 so

Zo Rve Tig RNe beg Zeg Tug Siu Mei beg Tgo Pe Agro b1p Tig Rgo - so yu 5 САС AD AOS STO САС TOB STS OSA ASS ATT AST AST OST OST ASTO 225 t

Avr Guz ygv Sets bij Tgr Uai Aia Tk Pe beg beg 01u (Lu beg " 40 - s 45 50 55 ;"

TAS ASS TAS TAT SSA BAS AST bot AAb bob SbA TT ASS ATS TO 0270 - Tukh TiK Tukh Tut Rgo Avr eh Mai Yiu5 b1u Agya Re Tyt I1e beg 5. s - 50 so (F)

Po) bo 65 bo bo 10

ASA SAS AAT OSS AAb AAS AOS STA TAS STO SAA ATO AOS AST STO 0315 o Ag Abr Ahp Aza Sue Azy o TBg o Ge Tug Hey b1ip Mei beg beg I eu 75 80 85

IY AAS TST pub SAS ASA OSS ATS TT TAS TT bSA ASA SAS OAST AST o 0/360

Sh back bi Asr TK ATa Meb Re Tut Sub ATa As bip Te Te 90 95 100

ATO AST TAS TT OST TAS tob boss SAA bob AST STO ST AST STO 0-05 s

Mest Tpg Tug Rpe yIa Tut Tgr S1iu bii b1iu Tk Te: Uai Tne Uvi (8) 105 o 15 s and TST OSA 411 - so bek Aa o m. (2) Information for the sequence with the identification Mo58: (Y) Characteristics of the sequence: (A) Length: 411 base pairs (B) Type: nucleic acid « (C) Chain structure: double-stranded - s (OB) Topology: linear ц (i) Type of molecule: cDNA - me NK "» (xi) Sequence description: sequence with identification Mo58 t ATE bbo TT bob STO ABS Tos STI TS STO STT OST STTOTTA ASA 45 1 o Meb biu Re biu Te beg Tgr Udi Rie Gen Ua1 Aia Pec ep Atge - 50 «so -15 -10 -y dv ot OTO SAB TOT SAB STO SAB STO OTO bAb TST Ob (A b0S To 090 o Sbiu Ua! beep Suz beep Ua! beep ieey Mai bi bek b1u Su b1u Ua! 60 b5

1 5 10th hundred САС сСТ bob Or TOS Sto AbA STO TOS ToT oSA OSS TST OSA 0135

Mai b1p Rgo b1u Aga beg Ge AgeE Gets bet Su A1a Aza beg Su 7 15 20 25

TS ACC TS AST ABS TAT boss ATO TST TOb ST SOS SAS ST SA 180 t Rbe TENg Re Zeg beg Tuk b1u Met beg Tgr Ua! Age b1p Aza Rgo 30 35 40

BOS Ado bob STO bas TO To bSA AS ATT AT AT AT SOT OT AT 0225 biu buh b1u Gen bi Ttr Ma! Aza Tg T1e beg bek b1u biu beg sch 2 | at 45 50 55

TAS ACC TAS TAT Sba BAS AST OTO AAb oh SA TS ACC ATS TOS 0270 so

Tuk Tizh Tut Tut Rgo Avr beg Ma! Tu5 b1u Ag Rye Ti Pe beg so

Y yu zv 8О 65 70 shcha

АСА бАСОААТ ТОС АС DАС АСО STO TAT STO SAA ATO AAS AS STO 0315 " du Age Ar Asp bek Gu Abp o TpgoTGeu Tut Me bil Mee Ap beg Te z s khz" 15 VO 85 sh 45 dod OST ba SAS AS OST OTO TAT TAS TOT oS AbA SAS AST ASTO 0360 i-y Agy Aia biz Avr TVg Aa Ua! Tug Tug Su Aa Age bip TNg Teg g pz OO 95 4,9) so ATO AST TAS TT OST TAS To 00S SAi bbA ASS STO TO AS ST 0405 z Mes Tvg Tug Rne Ia Tug Tgr biu biu b1u Tlg cem Uvi Tng o Uai

F) Oh! 105 110 115 sh TOS TA 411 bek beg bo (2) Information for sequence with identification Mo59:

(Y) Sequence characteristics: (A) Length: 11 amino acids (B) Type: amino acid (0) Topology: linear (ii) Molecule type: peptide (xi) Sequence description: sequence with identification Mo59

Guz Aa bek OS!p Avr Ma! Avzp Tit Aa Ma! Aa (2) Information for sequence with identification Mobo: (Y) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (OB) Topology: linear (ii) Molecule type: peptide (xi) Sequence description: sequence with identification Mobo

Zeg Aa beg Ap Age Tut TP s 5 o (2) Information for the sequence with identification Mob1: so (Y) Sequence characteristics: -- (A) Length: 9 amino acids (B) Type: amino acid o (OB) Topology: linear y (ii) Type of molecule: peptide (xi) Sequence description: sequence with identification Mob1 t

Sip o bSip No Tut o bego Tig o Rgo Rbe Tit « || 5 and s;" (2) Information for the sequence with identification Mob2: (Y) Sequence characteristics: -I (A) Length: 5 amino acids (B) Type: amino acid o (OB) Topology: linear c (ii) Molecule type: peptide (xi) Description sequence: sequence with identification Mob2 -

IR e)

Rgo Tut Tr Me biv oh ! 5 (2) Information for the sequence with identification MobZ3Z: (Y) Sequence characteristics: 60 (A) Length: 16 amino acids (B) Type: amino acid (OB) Topology: linear (ii) Molecule type: peptide (xi) Sequence description: sequence with identification Mob3 b5 beg Pe Rie Ou Ar Su Avzr big Ata TukgobegoSip o iuz Re Tuz Ou y 1 5 10 (2) Information for sequence with identification Mob4: (Y) Sequence characteristics: (A) Length: 11 amino acids (B) Type: amino acid to (OB) Topology: linear (ii) Molecule type: peptide (hi) Sequence description: sequence with identification Mob4

SLu ep Atgo Ago SLu (u Lug Tut RrBe Avr o Tug (2) Information for the sequence with identification Mobb5: (Y) Sequence characteristics: (A) Length: 411 base pairs (B) Type: nucleic acid c (C) Chain structure : double-stranded (OB) Topology: linear o (i) Molecule type: kKDNA - me NK (xi) Sequence description: sequence with identification Mob5 с «- со

IS) m. "shi s"; - I 1 (95) - 50 p

F) name) 60 b5 and ATO SSS tob ST SST STO TT TS TT TT st ST SAT tob TSA 45

Mes ATa Tr TpgoRgo Gen Rpe Rpe Rbe Rpe Mai yiv) Niv Suz Zeg 10 -15 -10 -5

SST TS OTTS TOS SAA ST Sto ST AST SA TSA TST TSA SSS TS sh S1u bek Rre beg b1p Gey Ua! This is TBg o beep back back bBeg A1a beg 20

TTS tob sto bbA OSS TSA bSA ALLA STO ASo TOS ASS To AT AT 0135 r Rne bek ieu biu Aza bek A1a Guz Sem TbgoSuz Th Geo Zeg 5eg sch o so

SAb SAS AST ASV TAS ASS ATT SALO TOb TAT SAS SAA SAS OSA ST O180 her beep Ni bek Tpg Tuk Te Pe bij Ter Tut (12 bija bij Rgo And you and | M

Zo 5 30 « yu AAS OST OST AAS TAT ST ATO SAT STT AAS SA ObAT bSbA AOS Sas o 0225 sho s with Muz Rgo Rgo Guz Tug Ua! Met Ayer Tei Gu bip Azo biu beg Niye -I s s - 50 so (F.

Because b5

45 50 nn 2 AOS ASA bot PAT bob ATI OST ObAT SOS TS ST OSA TS AS TT 270

Bek Tpt (in Azr bIu T12e Rto Ayer Agro Re be S1u beg beg beg 7 bo 55 7o

Obt OST bAT SOS TAS STT AbS ATT TOS AAS ATS SA SSA baAA SAT OO 315 biu Aa Abr At Tut b bek 12 bek Azp Te sel Rgo by Avr 7 vo 85

SAD SA ATO TAS ATS TT bot Oto OT AT ASA ATT AAb SAA SAA 360 (io Aia Mei Tut T1ve Su5 b1u Ma! (Mu Ar Tk Pe Kub bu bip sch o 90 : 95 100 s zo TI OTO TAT OT TS bos ob 000 ACC A TS AST OTO STA OT 405 -

Wow! Here Wow! Rne b1u S1u biu Tpg Guz Ua! Tvg Ma! Gay S1 and her with 5 105 and 115 m

САб ССС 01 "shi s bip Rgo" (2) Information for sequence with identification Mobb: (Y) Sequence characteristics: (A) Length: 405 base pairs -I (B) Type: nucleic acid (C) Chain structure: double-stranded o (OB) Topology: linear 2) ( i) Type of molecule: cDNA - me NK a yu (xi) Sequence description: sequence with identification Mobb

IR e)

F) name) 60 b5

ATO bSS tob AST OST STO TS TS TS TT OOTT STT SAT TOS TSA 45 y Mek dia To Tg o Rgo Pech Rbe Re Re Re Ua! Geo Ni Sub 5eg -13 -140 -5 bot TS TS tSb SAb STI Oto Sto AST SAA TO SSS TS SS ST 90 b1u beg Rne Zeg beep Geo Ma! Gay Tk bib beg Rgo bet A1a Zeg 18 1 Z 10

SSS ToS st boA OSS TO oto Ai Sto ASS TOS ASS TT AST AT 0135 yu A1ia beg Iei Sb1u A1a bek Haiii Tuye vi TpgoSue Tbgoieo bek beg

ИЗ 20 25

SAS SAS AST ASO TAS ASS ATT SAA TO SAT SAB SAS SAS SSA bAb 180 sch 7 bij Nie beg Trg Tug Tag Pe bio Tgr Niz beep beep beep Rto bi o so 7 AAS SOS OST Sat TAS TTS ATS ALA STT AAS SAA SAT OSA AbS SAS 225 -

Mus b1u Rko Agya Tut Ge Met MSu5 Men bu bip Avr 01u beg Niv. she is 35 45 50 55 -

ABS ASA OST SAT bob AT SST AT SS TS TSA bos TOS AS TS 0270 yu Zeg Tg biu Der biu 12 Rgo Ar Age Re beg biu beg beg beg ih i 60 55 79 in bob bST BA SOS TAS STS ASS ATS TOS ABS STO SAS TST BAS SAT 0315 ih Siu Aa Siv Ag Tug Bez Tr Pe beg bek Gen bel bek bi Abr her 75 8o 85 so 7 SAS ST BAS TAT TAS TOT O0T OTO 007 SAT ASA ATT AAb bla SAA 360 bin ATa d5roTue Tut Suz biu Ua! biu Azr Tg o Pe iu5 (y bil d 55 to 95 ik to TT Sto TAS Sto TS bos sbA bob AS AAA STO ASS OT STA OST 405 60 Rne Ua Tut Ua! Rpe b1u (Tu biu Trg Guv Geo Track in Te) S1u 105 | 115 (2) Information for the sequence with identification Mob7: Vol

(Y) Sequence characteristics: (A) Length: 411 base pairs (B) Type: nucleic acid (C) Chain structure: double-stranded (OB) Topology: linear (i) Type of molecule: cDNA - me NK (xi) Sequence description: sequence with identification Mob7

ATO BOS tob AST OST ST To TS TS Tit OT ST SAT TOS TSA 45

Met AIa Ter Te Rtgo Se Rre Re Re Rne Ua! Se Ni Suz beg -45 -10 -5 7 01 TST TS TOS SAS STI oto STO AST SAA tob SOS TT OSS So s1u beg Rne beg bij Gen Ma! Se) Tbg o b01b beg Rgo beg Dia beg sch

Ii 5 10 o oS TOS Hundred both OSS TO TS AL Hundred ASS ToS ASS TT AT AST 0135 so

ATa bek Gesh b1iu A1a bek Ma! Su Se Tyg Sub Tg o Ge beg beg so iv) zv 15 20. 25 schi

SAC SAC AST ASO TAS ASS JSC SAA TOb TAT SA SAb SM; ССА САС 180 « 7 white Mi5 bet Tk Tut Tvg Pe b1ish Ter Tug bip b1p white Rgo b ne z che For 40

V. Allo boss SST AAS TAS CO JSC SAT OST ALLO SALA OAT BOA AOS SAC 0205 1

Mn y Mu biu Rgo Sub Tuk Sen Mee bro Ge Guz bil Azr b1iu beg Ni so 45 5O0 oo 5 OS ASA bot bAT obo AT OST pAT SOS TS TsA oS TS AS TT 0270

F) yuyu Zeh TiK biu Azr u Pe Rgo Abr Agya Re bek biu bet bek Zeg 60 b5

60 ba to bob ost bab SS TAS ST ASS ATS TOS AOS STO SA TST bAb bAT 0315

S1u Aza bish Age Tut Ge TagoYi1Ye 5eg beg Gbesh beep back bio Avr 7 75 80 85

SAS OST SAS TAT TAS TOT 00 bo 01 bAT ASA ATT AAb OSAA SAA -/360

Fiv Aza Azo Tug Tut Suye O1u Uai biu Or TVh Pe (in b1y bii 90 9 109

TT Sto TAS Oto TS 000 ObA bob ASS AAA ST ASS STO STA SOS oS 405 y Rne Ua! This is Ma! Rpe b1u b1u b1u Tnt Guz Temy TngoUai Ge Su o 105 110 115 zo SA SSS 41 o "- bip Rko o

IC) (2) Information for the sequence with identification Mob8: them (Y) Sequence characteristics: (A) Length: 411 base pairs (B) Type: nucleic acid (C) Chain structure: double-stranded « 20 (OB) Topology: linear - c (i) Type of molecule: cDNA - me NK c (xi) Sequence description: sequence with identification Mob8 c

ATO SS TO AST OST STO TS TS TS TS TT OT STI SAT TOS TSA (45

V. Mee Aa Tgr TngoRgo Ty Rbe Re Rpe GRye Ua! Gay Niz Suz beg 1

ГК) 0-18 -І0 -5 - 50 Sat TSt TS TOS Sa ST bto STO AST SAA tob SOS IST oOS TST090

S1iu bek Re beg bip cen Mai Gez Tig bil bBeg Ro beg Aa beg o 1 5 10 ime) boss TO Sto boA bob tob sto AAb STO AS TOS ASS TO AST AT 0135 60

Aia beg iyey biu Aia beg Uai uh be TNg Sub Tag Pen bek o Zeg b5

15 20 25

SAb SAS AST AS TAS ASS ATT SAA TOb TAT SAS SAS SAS OSA SAS 180 bip Niv beg Tishzh Tut Tik Te Siy Ter Tug Sil Sip bij Rgo C1y 3 35 shk

AS spos OST AAb TAS bTo ATO SAT STI AAb SAA SAT OSA AOS SAS 0229

Guz biu Rgo Mu Tut Ua! Meb Azr Gen uz biy Ar O1u beh Ni 45 50 5o

AOS ASA bot AT obo ATT SST bAT SOS TS TSA OO TO AYU TS 270 zv Zeg Ty biu Aer biu Tie Rgo Der Ak Rpe behg (yiu Zeg Zeh Zeh Zeg sch (8) bo 65 70 zo sob ost bab SOS TAS STO ASS AT TO AOS ST SAS TT BAS SAT 315 so «- biu dia bii Age Tug o Gech Tg o 1T1e be beg Geu bip beg bi) Avr s 85 o 75 o shcha

SAS OST SAS TAT TAS TOT ObT OT OT SAT ASA ATT AAb oAA SAH 360 « yu Siv Aza Ahr Tug Tug Sub b1u Ua! biu Ar Ti Pe Guz (To b1p - s t with 90 95 10 15 TT To TAS sto TS 0bS bbA bob ASS AALA STO ASS OTO STA bos o0S405 -I 1 g) Rne Uli Tut Ma! Rne biu bi S1u TNg obu Med TNgoUa)! bets biu - 50 so 105 i1a PU

SA SS those

GF! bIap Rtgo name) 60 (2) Information for the sequence with identification Mob9: (Y) Sequence characteristics: (A) Length: 411 base pairs (B) Type: nucleic acid (C) Chain structure: double-stranded 65 (OB) Topology: linear (i) Type of molecule: cDNA - me NC

(xi) Sequence description: sequence with identifying MobO9

ATO SSS tob AST SST STO o TC TIS TT OSIT STT SAT TC TA 45

Met A1ia Ter Trg Rko Gen Rpe Rpe Rpe Rpe Ua! levi No Suz 5eg 70 -15 -10 -5 obt TstT To ToS SAS STI Sto Sto AST SAA tob SOS TS OSS TS 090 iz Fu bek Re beh bip Ge Uai Tem Tig o b1ip beg Ro beg Aa beg 1 no 10 7 person tos hundred obA boss TOb STS AAb STO ASS TOS ASS TO AST LOT OO135

ATa bag Gen biu Aza beg Ma! Su5 Geo Tbg Su TNgoi en beg Zeg sch

I5 20 25 o

SAS SAS DST ASO TAS ASS ATT LA TOb TAT SAS Sai SAb SSA bAb at 0180 so

Sbip No beg Tag o Tug TbgoTiv sii Tgo Tug bSbip beep beep Rgo fight her yu

ZY 5 A cha

Aa OSS SST A TAS STO ATO PAT ST AAb SAA bAT OA AOS SAS 0225 « s 70 Mu bi Ro Ake TUg Mei Me Ar be Guz bil Ar biu bog Mi ne g» 45 5 vv 10700 ABS ASA OST SAT 00S ATT SST SAT OS TS TSA 0bS TOS ABS TT 279 her Chek Tk bIu Abr b1u Tse Rgo A5r Age Re beg biu beg beg beg - 50 I; 65 70 so

SOS OST bab SOS TAS STO AS ATS TS AS STO SA TST bAb ObAT 0315 o b1iu A1a bij Ake Tut Tez TVh T1e Zeg o beg Gech bip beg bi Avr 9) 75 o 85 bo

SAS OST BAS TAT TAS TO 001 OTO OT SAT ASA AT AS BAKA SAA 360 bis Aia d5r Tug Tut Suh b1u Ua1! Biu Ar Tg Pe Guye Bish 01p 65 -1й5-

90 95 10 y TIT TS TAS STO TTS SOS obA bbS ASS AAA STO ASS ST STA 00 0405

Re Wai Tuh Ma! Rre biu b1u biu TC o Gu Tay TV Uai Gen 019 7 105 10 15

САС бий Рго (2) Information for the sequence with identification Mo70: (Y) Sequence characteristics: (A) Length: 411 base pairs (B) Type: nucleic acid (C) Chain structure: double-stranded (OB) Topology: linear (i) Type of molecule: cDNA - me NC cm (xi) Sequence description: sequence with identification Mo70 (8)

ATO OSS TO AST SST STO TS TS TS TT OT ST SAT TOS TSA 045 so

Met dia Tkr TigoRgo Ge Re Rne Rie Rpe Ha! Ge Niz Sub beg t so -15 -40 -a o m. sot TST TS TO Sas STI oto STO AST SALOTOB SSS TST OSS TT 90 biu bek Re beg beep ep Ua! Ge Tk fight Zeg o Rgo beg A1a beg ih and - and 5 10 and? in bOS OS sto ObA BOS TOb TS AAS ST ASS TOS AOS TT AST AST 135 -I sl dia bek Ge Siu Aia bek Chai yub (ep Tpt o Suye Tu Ge beg 5eg (95) z yu 15 20 25 so SA SAS AST ASO TAS ASS ATI BAHA TO» TAT SAb SAb SAb SSA bAb 0180 biv Ni beg TP Tut TP Pe bib Tor Tuk biv bip bip Rgo b1y

F) name) 60 b5

From 35 to 40

AAb (S SST AYU TAS STO ATO SAT ST AAS SAA SAT SSA AS SAS 0225 uz Siu Rgo Agsh Tut Ua! Mebs Asr Tez Buv 01vp Azr b1u bek Nik 45 50 Zo

ABS ASA OT SAT bob ATT OST SAT SOS TS TSA OS TOS AOS ST 0270

Seg Tyg biu Abr 1u Te Rgo Abr Age Rpe beg biu Beh beh beg bo bo 70 z bo OST bab SOS TAS STO AS ATS TS AOS STO SAb TST bab bAT 0315 zv biu Aza Si) Age Tuk Gey Te Pe bek bek Ge bil Zek fight Ar at 75 Vo | 85 zo САС OST BAS TAT TAS TOT 00 Oto OT SAT ASA ATT AAS BALA SAA 300 that «- biz A1ia der Tug Tuk Sub biu Mai b1u Ar Te Ce Gu 1 (18 Shk

IS) 90 95 100 m tt sta TAS sto TS oS oba bob SS AllA STO ASS ot STA oS 0 lob « shi s Rye Uai Tus Ua! Rbe PIh bih b1u Tk gu Be Tvg Uvi Gbech S1u

And" 5 More; и15 и САС ССС 1 1 о п1в Рго - 50 (2) Information for the sequence with identification Mo71: со (Y) Sequence characteristics: (A) Length: 411 base pairs (B) Type: nucleic acid (C) Chain structure: double-stranded

HF) (OB) Topology: linear kyu (i) Molecule type: cDNA - me NK (xi) Sequence description: sequence with identification Mo71 60 b5

ATO boss tb AST SST ST To TT TS TI TT ST SAT TOS TSA 045

Met Aza Ter Ts Rko ep Rrbe Rbe Rbe Re Ua! This is Ni Su bZeg -15 - -5 o bot tSt TS TOS SA STI OTO ST AST SAA TOB OSS tot OSS ST 90 b1u bet Rne beg beep Hey Ma! bep Tye bep beg Rro bek Aa beg 1 5 10 with SOS TOS Sto OSA SOS TOb STS AAS STO ASS TOS ASS OTTO AST AST OS 135

ATa beg Sei b1u A1a beg Ma! Гу5 Геч Тг Su Тг Hey beg beg

From 20 25

SAS SAS AST Or TAS ASS ATT SALA TOb TAT SAS SAb SAb SSA bAbo 0180 biv Nie Heg Tys Tut Ts Pe bio Ter o Tuk bip bio bip Rgo bin s o

Kn) 5 I) from AAS boss SST AL TAS STO ATO SAT STT AL SAA BAT ObA AOS SAS 0295 (ee)

Shh piu Reo Gb Tut Se Meb Azr Their uz bio Ahr o (1u Zeg Pi5 her 45 5 55 oyu

DOS ASA OT bAT OO ATT OST bAT Ob TT TSA bOS TOS AOS TT 270 th-

Zst TVg obiu dr biu Pe Rko zr Ago Re beg S1u betg beg beg « du BO 55 70 Z - sob ost bab SOS TAS ST ASS ATS TOS AbS STO SAC TST bAb SAT 315 ? biu Aia bish Age Tug Gay TVg o Pe beg bek Ge) bip beg b1y Avr shchi t5 vo 85

Mn bAb OST SAS TAT ATS TT ObT OTO ObT SAT ASA ATO AAS OAA SAA 360 iy 50 Siv AiTa Avr Tuk Pe Suye b1u Mai biu Ayer Tih Pe Tsu5 bi bid so 90 95 100 5 TTT oto TAS oto TS OS ObA 000 ASS AAA STO ASS OTO STA boss 405

F! Re Wow! Tuk Uvi Rpe C01u b1u biu Tig Guye Te Tu Ha! Geo y from 5 to 15 bo CA SSS 11 bip Rgo 65 (2) Information for the sequence with identification Mo72: (Y) Characteristics of the sequence:

(A) Length: 411 base pairs (B) Type: nucleic acid (C) Chain structure: double-stranded (OB) Topology: linear (i) Type of molecule: cDNA - me NK (xi) Sequence description: sequence with identification Mo72 o ATO OSS TOb AST OST STO TT TS TS TTT OTTO STT SAT TOS TA C 45

Meb Aia Tur Tvg o Rko Bez Rbe Rre Rie Rie Ua! Gen Ni Su Zeg 5 -15 -10 -5 sot TStT TS ToS sSlb ST bob Sto AST o SlA TOb SSS tST ss ts 90 20 Siu bek Re 5eg bij Sei Udi Te Tk bip bek Rgo Cheg d1ia Set 1 o 10 s 7 SOS TOS Sto boA boss TO oto LAB ST ASS TS ASS TTO AST ASTO 135 o

Aia Zek Iep b1u A1ia bek Ma! Gu Gen Tk o Sue Tk Ge beg beg so 30 "- s with SAb SAS AST ASS TAS AOS ATT SA TOS TAT SAS SAS SAb OSA SAb 180 y bip Ni beg Tyt Tut TPg o Ce bio Ter Tut bil bip bip Rgo b « 30 From 40 With p

From AS boss OST AS TAS STO ATO SAT ST AAS SAA SAT OA AS SAS 0725 sh 45 Buv b1u Rgo Ake Tug iey Met Abr Tem Guz 610 Avr biu beg Nib y 45 50 55 (95) - 0 AOSASA SOT SAT 006 ATT OST SAT SOS TT TSA boss TOS AS TS 270 s beg Tishzh b1u Ayer biu Pe Rgo Azr Age Riv bZeg Siu beg beg 5eg o vo 65 70 y bob OST bab sbS TAS Sto ASS ATS TOS AOS STO SAS TST bab SAT 0315 bo

Siu Aia Si» Age Tuk Ge Ty Pe bek bek Ge b)ip beg 010 Azr b5

GE 80 85 и САС OST САС TAT ATS TOT OST oto b0T OAT ASA ATT AAb OSAA САА 360

Si dia Ayer Tut Te Suz b01u Ua! biu Ar Tig Pe Gu5 bi b1p len 95 100

TTT Hundred TAS Hundred TC bbS bbA bOb ASS AAA STO ASS bTS STA 00S 0405

Re udi Tut ma! be biu b1u b1u Tbgouzh5 Hey So Udi oo O1U » 105 at 15

SA SOS 11 p

Sbip Rtgo (o) (2) Information for the sequence with identification Mo73: (Y) Sequence characteristics: (A) Length: 411 base pairs o (B) Type: nucleic acid (C) Chain structure: double-stranded - (OB) Topology: linear so (i) Type of molecule: kKDNA - me NK (hi) Sequence description: sequence with identification Mo73 that m. « shi s z -I 1 (95) - 50

IR e)

F) name) 60 b5

ATO oSs To AST SST Sto TS TIS PS TT TT STI SAT OS TSA 045 2 Me Aa Ter Tig o Rgo Sem Re Riye Riye Rpe Ua! you Niv Sub beg -15 -0 -5 70 I about TST TS ToS SAS ST OTO STO AST o SAA TO SSS tTST OSS TS 90

SIu bBeg Rpe bek bij Hey Whoa! besh TBg o bil beg Ro beg AiTa b5eg 1 Z 10

OSS TS Sto bbA OSS TO TS AB STO ASS TS ASS TT AST AT 0135

A1a beg Ge S1u Ai1a bak Ua! uh Sech TVgoSuye TBg o Hech back bZeg sch z 15 20 25 (o)

САС САС AST ASO TAS АСС АТТ САА TO TAT САС САС САС ССА САС 180 со 30 beep Niz Zeg Ti Tuh Tik Te bish Tho Tug beep biv bil Rko beat her

IS i- « - s ;» -I c d) - 50 so

F)

PF) because 65

30 35. 40

ALO OSS SST ALLO TAS Oto ATO bAT STT ADO SAD PAT OSA AOS SAC 0225

Ku piu Rgo Muz Tut Ma! Mei Ar Ge (in b1p Avr S1u Zeg Ni 45 50 99

ABS OASA OT AT bob AT SST AT SOS TS TSA ObS TO AOS TS 0270 bat Tag obiu Ar b1u (1e Ro Avo Age Re beg S1u beg beth beg bo 65 70 bo OST bab SOS TAS STO ASS ATS TS AS STO SAb tot SAS SAT 0315 b1u A1a bij Age Tut Gey Tpg o Я1e beg beg Mem bip bei biiy Avr sch (8) nya 80 85 z САС OST BAS TAT ATS TT 001 ST 001 bAT АСА АТАС САА САА / 360 со «-

SI ATa Ar o Tut Te Sub Su Ma! S1u Ar Tg Te Yiu5 01 b1p so zv 90 95 100 y.

TIT Oto TAS oto TS boss boA bob ASS DAd STO AS STO STA boss o 405 « - 47 Rye Ua! Tuh Whoa! Rpe b1u b1u biu Tng Tsuv Ge Tih Ua) Gen bu ch 105 110 115 7 SA SOS I 1 bip Pto (95) - 50 (2) Information for the sequence with identification Mo74: so (Y) Sequence characteristics: (A) Length: 411 base pairs (B) Type: nucleic acid (C) Chain structure: double-stranded

HF) (OB) Topology: linear kyu (i) Molecule type: cDNA - me NK (xi) Sequence description: sequence with identification Mo74 60 b5

ATO ССС That AST OST STO TT TC To TT ot ST SAT ТС ТСА 45 ? Mes Aza Tgr TNi Rgo iey Re Rre Re Ree Ua) IGey Niye Suye beg -15 -10 -5 70 ost tSt TT tToS Sat ST oto Sto AST SAA TO SOS TST bos ST 090

S1u bek Riye bek beep Ge Uai Mep Tph 01p beg Rgo Zeg Aa Zeg 1 9 10

SOS tes sto bbA boss tob TS AS ST AOS TOS ACC TT AT AST 0135 dia bZeg Be b1u Aa beh Ua! Sub em Tpg Sub Tk Geo beg beg 7 15 20 25

SAS SAS AST AS TAS ASS ATT SAA TOb TAT SAb SAb SAS SSA SAS at 0180 in white Niv bek Trg Tut Peak Pe Ti Ter Tut b1p beep beep Rgo Siyu at 30 35 40

AAb SOS OST Or TAS STO ATO bAT STI AAb SAA SAT bbA AbSOSAS 0225 so sh Tuz biu Rgo Age Tut Ua! Met Avr (ep Tuz Sb1p Avr S1u Seg Niv t 45 50 5o her 35 AOS ASA O0T SAT Ob AT SST SAT SOS TS TSA OS TOS AOS TS 0270 -

Zeg Tpg b1u Ar 01u I1e Rho Ar Agai Rre bZeg (1u beg back beg 60 55 70 and 40 in) with bob ost SAS Sat TAS STO AS ATS TOS ABS STO SAS TST bab SAT 0315 t S1U A1a bi Auya Tug Geo TigoT1e Beh bet Gen bii bek bi Agr shchi 15 NN o i-y SAS ST SAS TAT ATS TOT 0bT To bot bAT ASA ATT AAS SAA SAA 360 ii 50 (Tv Aa Avr Tug Pe Sub (Lu Hai S1u Ar Te Pe Gu bi p so 90 95 100 -5 TTT bo TAS Oto TC bob ObA obo ACC AAA STO AS STO STA SOS 0405

F) kyu Re Hua! Here Wow! Rye S1u biu b1u Tit Guz Teo Tr o Uai Tv SOIU 105 110 | 115

SAC SOS 411

I SiP Ro with bo (2) Information for the sequence with identification Mo75:

(Y) Sequence characteristics:

(A) Length: 34 amino acids

(B) Type: amino acid

(OB) Topology: linear (ii) Molecule type: peptide (xi) Sequence description: sequence with identification Mo75

Claims (49)

The formula of the invention 1. A chimeric I-chain of an antibody against human parathyroid hormone-related protein, which includes the C-region of the I-chain of a human antibody and the M-region of the I-chain of a mouse monoclonal antibody against a human parathyroid hormone-related protein, where the M-region of I The !-chain includes the amino acid sequence shown in ZEO IYUO MO: 45. 2. A chimeric I-chain according to claim 1, where the C-region is a Cx-chain. 3. A chimeric H-chain of an antibody against human parathyroid hormone-related protein, comprising the C-region of the human H-chain antibody and the M-region of the H-chain of a mouse monoclonal antibody against human parathyroid hormone-related protein, where the M-region of H - chain includes the amino acid sequence shown in ZEO IU MO: 46. 4. Chimeric H-chain according to claim 3, where the C-region is a SG1-chain. 5. A chimeric monoclonal antibody against a protein related to human parathyroid hormone, which includes a chimeric I-chain according to claim 1 or claim 2 and a chimeric H-chain according to claim 3 or claim 4. b. A polypeptide including the M-region of the I-chain of a humanized antibody against a protein related to human parathyroid hormone, where framework regions 1-3 of the indicated M-region | -chain originates from the M-region of the Y-chain of the human antibody NZIOZ868, and framework region 4 of the specified M-region I -chain originates from the M-region o) Y-chain of the human antibody 525755, and regions 1-3 of the specified M-region I- chains that determine complementarity include the amino acid sequences shown in BZEO IU MO: 59-61. 7. A polypeptide including the M-region of the I-chain of a humanized antibody against a protein related to human parathyroid hormone, where the framework regions 1-3 of the indicated M-region of the I-chain are essentially identical to the framework regions 1-3 of the human antibody NZSHIOZ868, and the framework region 4 of the indicated M-region of the I-chain is essentially (87 identical to the framework region 4 of the human antibody 525755, and the regions 1-3 of the indicated M-region of the I-chain, which co determine complementarity, include the amino acid sequences shown in BEO IU MO : 59-61. 8. Polypeptide according to claim 7, in which in the framework region the 36th and 49th amino acids according to Kabat's definition are tyrosine and aspartic acid, respectively. Cha 9. Polypeptide according to claim 8, which includes the amino acid sequence shown in any of ZEO IU MO: 48-51. 10. Polypeptide according to claim 7, in which in the framework region the 45th and 87th amino acids according to Kabat's definition are lysine and isoleucine, respectively. 11. Polypeptide according to claim 10, which includes the amino acid sequence shown in any of ZEO IU MO: 52-55. " 12. A polypeptide comprising the M-region of the H-chain of a humanized antibody against a protein related to human parathyroid hormone, where framework regions 1-4 of the indicated M-region of the H-chain are derived from the M-region of the H-chain of a human antibody subgroup I, and regions 1-3 of the specified M-region of the H-chain, which determine "with" complementarity, include the amino acid sequences shown in BZEO IIU MO: 62-64. 13. A polypeptide comprising the M-region of the H-chain of a humanized antibody against a human parathyroid hormone-related protein, where the framework regions 1-4 of the indicated M-region of the H-chain are essentially identical to the -I framework regions 1-4 of the human antibody of the human subgroup I, and regions 1-3 of the specified M-region of the H-chain, which determine complementarity, include the amino acid sequences shown in KEO IU MO: 62-64. at 14. Polypeptide according to claim 12 or claim 13, where the human antibody is human antibody 531679. oo 15. Polypeptide according to claim 12, which includes the amino acid sequence shown in zZEO IJU MO: 56. 16. I-chain of a humanized antibody against a protein related to human parathyroid hormone, which - includes a polypeptide comprising the C-region of the I-chain of a human antibody, and a polypeptide according to any of claims 6-11. with 17. The I-chain of the humanized antibody according to claim 16, where the C-region is a C and X-chain, the specified framework regions 1-3 are essentially identical to the framework regions 1-3 of the human antibody NZIOZ3868, respectively, and the specified framework region 4 is essentially identical framework region 4 of human antibody 525755, and regions 1-3, which determine complementarity, include the amino acid sequences shown in ZEO IU MO: 59-61, respectively. 18. The H-chain of a humanized antibody against a human parathyroid hormone-related protein that F) includes a polypeptide comprising the C-region of the H-chain of a human antibody and a polypeptide according to any of claims 12-15. co 19. The H-chain of the humanized antibody according to claim 18, where the C-region is a C 51-chain, the indicated framework regions 1-4 are derived from the framework regions 1-4 of human H5ONI, respectively, and regions 1-3, which determine the complementarity , include the amino acid sequences shown in BEO IJOO MO: 62-64, respectively. 20. A humanized antibody against a protein related to human parathyroid hormone, which includes the I-chain of the humanized antibody according to claim 16 or claim 17 and the H-chain of the humanized antibody according to claim 18 or claim 19. 21. DNA including the base sequence encoding the M-region | -chain of a mouse monoclonal antibody against human parathyroid hormone-related protein, wherein the M-region of the I-chain contains the amino acid 65 sequence shown in ZEO IO MO: 45. 22. DNA including the base sequence encoding the M-region | -chain of a mouse monoclonal antibody against human parathyroid hormone-related protein, where the sequence of bases encoding the M-region of the Y-chain is shown in ZEO and MO: 65. 23. DNA comprising the base sequence encoding the M-region of the H-chain of a mouse monoclonal antibody against human parathyroid hormone-related protein, wherein the M-region of the H-chain contains the amino acid sequence shown in ZEO IU MO: 46. 24. DNA comprising the base sequence encoding the M-region of the H-chain of a mouse monoclonal antibody against human parathyroid hormone-related protein, where the base sequence encoding the M-region of the H-chain is shown in 5EO IU MO: 57. 70 25. DNA encoding a chimeric I chain according to claim 1 or claim 2. 26. DNA according to claim 25, where the DNA encoding the chimeric I-chain contains the base sequence shown in the ZEO IU MO: 65. 27. DNA encoding a chimeric H-chain according to item C or claim 4. 28. DNA according to claim 27, where the DNA encoding the chimeric H-chain contains the base sequence shown in ZEO IU MO: 75.9. 29. DNA comprising a base sequence encoding a polypeptide according to any one of claims 6-11. 30. DNA according to claim 29, comprising the sequence of bases shown in any of ZEO IU MO: 66-74. 31. DNA comprising a base sequence encoding a polypeptide according to any one of claims 12-15. 32. DNA according to claim 31, which includes the sequence of bases shown in ZEO IU MO: 58. 33. DNA encoding the I chain of the humanized antibody according to claim 16 or claim 17. 34. A DNA I-chain of a humanized antibody against a human parathyroid hormone-related protein comprising a base sequence encoding the amino acid sequence shown in any one of SEQ ID NO: 48-55. 35. The DNA according to claim 34, wherein the DNA of the I-chain of the humanized antibody contains the sequence of bases shown in any of ZEO IU MO: 66-74. with 36. DNA encoding the H-chain of the humanized antibody according to claim 18 or claim 19. 37. DNA of the H-chain of a humanized antibody against a protein related to human parathyroid hormone, i) comprising a sequence of bases that encodes the amino acid sequence shown in zZEO IU MO: 56. 38. DNA according to claim 37, where the DNA of the H-chain of the humanized antibody contains the sequence of bases shown in ZEO IU MO: 58. 39. Recombinant vector including DNA according to any of claims 21-38. 40. A transformant transformed by a recombinant vector according to claim 39. -- 41. The method of obtaining a chimeric antibody against a protein related to the human parathyroid hormone, which includes: cultivation of a transformant transformed with an expression vector containing DNA according to any of the PPs. 21, 22, 25 and 26 and an expression vector containing DNA according to any of claims 23, 24, 27 and 28; and collecting a chimeric antibody against a human parathyroid hormone-related protein from the obtained culture. 42. The method of obtaining a humanized antibody against a protein related to the human parathyroid hormone, which includes: "cultivation of a transformant transformed by an expression vector containing DNA according to any of pt") with claims 29, 30, and 33-35 and an expression vector , which contains DNA according to any of claims 31, 32 and 36-38; and collecting a humanized antibody against a human parathyroid hormone-related protein from the obtained cultures 43. A pharmaceutical composition including a humanized antibody according to claim 20 as an active ingredient. 44. A means for suppressing hypercalcemia, including the humanized antibody of claim 20 as an active ingredient. -AND 45. An agent for suppressing hypercalcemia associated with a malignant tumor, comprising the humanized antibody of claim 20 as an active ingredient. 1 46. Means for suppressing hypercalcemia according to claim 45, where the malignant tumor is at least one selected from the group consisting of pancreatic cancer, lung cancer, pharyngeal cancer, larynx cancer, tongue cancer, gum cancer, esophageal cancer, stomach cancer , bile duct cancer, breast cancer, kidney cancer, bladder cancer, - uterine cancer, prostate cancer and malignant lymphoma. with 47. A remedy for hypophosphatemia, comprising the humanized antibody of claim 20 as an active ingredient. 48. The agent according to claim 47, wherein hypophosphatemia is hypophosphatemic rickets. 5B 49. The agent according to claim 47, wherein the hypophosphatemia is hypophosphatemic, resistant to vitamin 0, rickets. F) name) 60 b5