UA112168C2 - ANTIBODY THAT SPECIFICALLY Binds TO THE HUMAN Glucagon Receptor - Google Patents

Abstract

The invention relates to an antibody that specifically binds to the human glucagon receptor (hGCGR), the method of producing the antibody, and the pharmaceutical composition containing it. The invention also relates to a method of reducing glucose or ketone levels in the blood or treating a disease that is associated or partially characterized by high levels of glucose or ketone in the blood, and the use of a dedicated antibody for the preparation of a medicament for the treatment of diseases associated with hGCGR, including the treatment of diabetes, diabetic ketoacidosis and long-term complications caused by diabetes, or other metabolic disorders that are partially characterized by increased levels of glucose or ketones in the blood.

Description

(54) AN ANTIBODY THAT SPECIFICALLY BINDS TO THE HUMAN GLUCAGON RECEPTOR (57) Abstract:

The invention relates to an antibody that specifically binds to the human glucagon receptor (passa), a method of producing the antibody, and a pharmaceutical composition containing it. The invention also relates to a method of reducing the levels of glucose or ketones in the blood or the treatment of a disease associated or partially characterized by high levels of glucose or ketones in the blood, and the use of an isolated antibody for the preparation of a medicinal product for the treatment of diseases associated with NaSOV, including the treatment of diabetes, diabetic ketoacidosis and long-term complications caused by diabetes, or other metabolic disorders, which are partially characterized by an increased level of glucose or ketones in the blood.

The field of technology to which the invention relates

This invention relates to human antibodies and antigen-binding fragments of human antibodies that specifically bind to the glucagon receptor, and therapeutic methods of using these antibodies.

Information on prior art

Glucagon is a hormone of 29 amino acids produced by the alpha cells of the pancreatic islets. Glucagon maintains normal glucose levels in animals, including humans, by balancing the action of insulin. It is the imbalance of glucagon and insulin that can play an important role in some diseases, such as diabetes and diabetic ketoacidosis. In particular, studies have shown that elevated basal glucagon levels and insufficient inhibition of postprandial glucagon secretion contribute to diabetic disease in humans (MiiPeg yei a!., M Epu U Mesa 283: 109-115 (1970)).

It is believed that the effect of glucagon on increasing the level of glucose in the blood is partially mediated by the activation of some cellular pathways after the binding of glucagon (SS) to its receptor (which is denoted by OWL). ZSOC is a member of the secretin subfamily (family

B) receptors associated with (c-proteins, and is expressed mainly in the liver. Binding of glucagon to its receptor triggers a cascade of signal transduction reactions along the pathway mediated by c-proteins and which activates intracellular cyclic AMP and leads to increase in the output of glucose in the synthesis of de pomo (gluconeogenesis) and the breakdown of glycogen (glycogenolysis) (Umakeiat ei ai., Maiige, (1986) 323: 68-71; Opzvop ei ai., Reriidevs, (1989), 10: 1171-1177; and Ritsdpeg apa Raip, Viospet. 9. (1991)277: 371-378).

The rat glucagon receptor was isolated and purified for the first time by Eijpek et al. Then the sequence of the rat receptor was used to identify and clone the sequence of the human glucagon receptor, which contains 477 amino acids (I OK, 5. ei a. (1994) Sepe 140: 203-209; MasmMeii, 0... ei ai. (1994)

Viospet. apa Viorpuz Nez. Sott). US Patent No. 5,776,725 discloses an isolated nucleotide sequence encoding a human or rat glucagon receptor.

Targeting glucagon production or function with a glucagon receptor antagonist such as an antibody to (CON) may be one way to regulate or reduce

From the level of glucose in the blood, and because of this, it can be useful for the treatment of diseases such as diabetes or diabetic ketoacidosis. In addition, lowering glucose levels may prevent or reduce some of the long-term complications associated with elevated glucose levels in patients with diabetes.

The first studies showed that polyclonal antibodies to the rat glucagon receptor were able to block the binding of glucagon (pvop, S... (1996) РМАБбБ 93(1): 310-315).

Monoclonal antibodies to the human glucagon receptor were described by Viddu ei ai!. (Editor,

In... her a. (1995) 9. VioI. Spent 270(13): 7474; Viadu, ch.. her a). (1996) Nogt Meiai Res. 28(5): 215-9). The antibody described by Widau, et al., competed with glucagon for the receptor binding site and recognized both human and rat glucagon receptors, but not the mouse receptor.

M weeks of her aI. discovered a monoclonal antibody produced in a mouse against the human glucagon receptor and carried out a detailed study of the protein structure of the monoclonal antibody to the receptor (MU/gidni, I.M. (2000) Asia StuziaPodgarnisa besiyop 0. 56(5): 573-580). Other antibodies to the glucagon receptor are described in US patents 5,770,445 and 7,947,809; European patent application EP2O74149A2; European patent ЕРО65820081; published patent applications

US 2009/0041784; 2009/0252727; and 2011/0223160; and the published International Application

MO2008/036341.

The essence of the invention

In a first aspect, the invention provides fully human monoclonal antibodies (tAbBv) and antigen-binding fragments thereof, which bind to the human glucagon receptor (HgR) and inhibit or block its activity, for example, block the binding of glucagon to its receptor, thereby blocking the increase in blood glucose levels. Antibodies or antigen-binding fragments thereof can be used to lower blood glucose levels in a subject suffering from a disease characterized by elevated blood glucose levels, such as diabetes. Antibodies can also be used to treat a wide range of diseases and disorders, in which it is necessary to block the interaction of glucagon with the glucagon receptor, that is, they show a therapeutic effect. Finally, the antibodies can be used to prevent long-term complications caused by elevated blood glucose levels in diabetic patients, or to reduce the intensity of at least one symptom caused by elevated blood glucose levels in diabetic patients (516).

Antibodies of the invention can be full-length (for example, an antibody of si or Ids4) or can contain only an antigen-binding region (for example, a fragment of Rab, E(ab")2 or 5cEm), and can be modified in order to affect the functionality, for example, exclude residual effector functions (Nedau et al., 2000, 9. Ittipoi. 164: 1925-1933).

According to one embodiment, the invention provides an isolated human antibody, or an antigen-binding fragment thereof, which specifically binds the human glucagon receptor (peso), and the antibody binds to the ectodomain and/or to the extracellular (EC) loop of the human SSOV, where the ectodomain is the M-terminal domain of the CSO receptor, and the EC loop is one or more extracellular loops of ECI, EC2, and ECZ.

In one embodiment, the invention provides an antibody, or a fragment thereof, that binds to an M-terminal domain containing amino acid residues starting from amino acid residue number 27 to approximately amino acid residue 144 of the sequence

ZEO IO MO: 153, or binds to the EC loop of the PAS receptor, where EC loop means one or more extracellular loops of ECI, EC2, and ECZ, and ECI1 includes amino acid residues starting from about amino acid residue 194 to about amino acid of the remainder of the 226 sequences of ES IYU MO: 153; EC2 includes amino acid residues starting from about amino acid residue 285 to about amino acid residue 305 of the 5EO sequences

IO MO: 153; The ESZ includes amino acid residues starting from approximately amino acid residue 369 to approximately amino acid residue 384 of the sequence FEO IJU MO: 153.

According to one variant of the implementation of the invention, a human antibody, or an antigen-binding fragment of a human antibody that binds NSS, contains a heavy chain variable region (HCHV) having an amino acid sequence selected from the group consisting of BEO

IO MO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 90 95, at least 9595, at least 9895 or at least 99 95. According to certain variants of the implementation of the invention, the antibody or antigen-binding fragment of the antibody that binds NOSIN contains NSUV having an amino acid sequence selected from the group consisting of 5EO IU

MO: 34, 70, 86, 110 and 126, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 9095, at least 9595, at least 9895 or

From at least 99 95.

According to one embodiment of the invention, a human antibody, or an antigen-binding fragment of a human antibody that binds PSO, contains a light chain variable region (LRV) having an amino acid sequence selected from the group consisting of 5EO

IO MO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 90 95, at least 9595, at least 9895 or at least 99 95. According to certain embodiments of the invention, an antibody or an antigen-binding fragment of an antibody that binds PUMP contains

A CME having an amino acid sequence selected from the group consisting of 5EO IUO MO: 42, 78, 88, 118 and 128, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 9095, at least 9595, at least 9895 or at least 99 95.

According to certain variants of the implementation of the invention, the human antibody, or its fragment, which binds PASSIV, contains a pair of amino acid sequences NSUV/SUV, selected from the group consisting of 5BEO IU MO: 2/10, 18/26, 34 /42, 50/58, 66/68, 70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/138 and 146/148. According to certain variants of the implementation of the invention, a pair of amino acid sequences NSUV//SUK is selected from the group consisting of 5EO IYUO MO: 34/42, 70/78, 86/88, 110/118 and 126/128.

According to a related variant, the invention includes an antibody, or an antigen-binding fragment of an antibody, which (which) specifically binds NEASSIV, and the antibody, or its fragment, has COM domains of heavy and light chains contained in pairs of heavy and light chain sequences , selected from the group consisting of 5EO AND MO: 2/10, 18/26, 34/42, 50/58, 66/68, 70/78, 86/88, 90/98, 106/108, 110/ 118, 126/128, 130/138 and 146/148.

Methods and techniques for identification of SOV in amino acid sequences of NSMNA and I! SAUs are well known in the art and can be used to identify SAUs in specific amino acid sequences of NSOMA or SUA disclosed in this application. Typical rules that can be used to determine the boundaries of the SOC include, for example, the Kabaya definition, the Spoipia definition, and the AVM definition. In general features of the definition

Cabrai is based on variable sequences, Spoipia's definition is based on the localization of regions of structural loops, and ABM definition is a compromise between KaBbai's and Spoipia's approaches. 60 See, for example, Kabai, "Zedoepsev oi Rgoieip5 oi Ittipoiodisa! Ipiegevi", Maiiopai! Ip5iishev oyi

Neash, WeiPezaa, Ma. (1991); A1-I agikapi eyi ai., U. Moi. Cheers! 273:927-948 (1997); and Mapip eyi ai., Rgos.

May Asad 5 BOTH 86: 9268-9272 (1989). Public databases are also open for identification of antibody sequence IDs.

In certain embodiments, the present invention provides an isolated human antibody, or an antigen-binding fragment thereof, that specifically binds to PASA, wherein the antibody contains an NSMA comprising three regions of heavy chain SOB (HSOBI, HOOH2, and

NOSOVZ) in the NSUR sequence selected from the group consisting of BEO IU MO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146; and ISMV containing three light chain SOVA regions (I SOV1, SOV1 and I SOV3) in I SOV sequences selected from the group consisting of 5EO IU

MO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148.

According to one embodiment, the present invention provides an isolated human antibody, or an antigen-binding fragment of a human antibody, which binds a PNSOV containing

NOSOVZ domain having an amino acid sequence selected from the group consisting of 5EO IU

MO: 8, 24, 40, 56, 76, 96, 116, and 136, or a sequence essentially similar to them, and the degree of identity of these sequences is at least 90 95, at least 95 95, at least 98 95, or at least 99 95; and And! SOVZ domain having an amino acid sequence selected from the group consisting of BEO IJU MO: 16, 32, 48, 64, 84, 104, 124 and 144, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 90 95, at least 95 95, at least 98 95 or at least 99 95.

According to one embodiment, the invention provides an antibody, or its fragment, which (which) additionally contains an NSAIDs domain having an amino acid sequence selected from the group consisting of 5EO, IUO, MO: 4, 20, 36, 52, 72, 92, 112 and 132, or a substantially similar sequence, and the degree of identity of these sequences is at least 90 95, at least 9595, at least 9895 or at least 9995; NSON2 domain having an amino acid sequence selected from the group consisting of 5EO IUO MO: 6, 22, 38, 54, 74, 94, 114 and 134, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 9095 , at least 9595, at least 9895 or at least 99 95; ISOV1 domain having an amino acid sequence selected from the group consisting of 5EO IO MO: 12, 28, 44, 60, 80, 100, 120 and 140, or a sequence substantially similar thereto,

Moreover, the degree of identity of these sequences is at least 90 95, at least 9595, at least 98595 or at least 9995; and ІСОВ82 a domain having an amino acid sequence selected from the group consisting of 5EO IХО MO: 14, 30, 46, 62, 82, 102, 122 and 142, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 90 95, at least 95 95, at least 98 95 or at least 99 95.

According to one embodiment of the invention, an antibody, or an antigen-binding fragment of an antibody, contains: (a) an HCVZ domain having an amino acid sequence selected from the group consisting of

ZEO IO MO: 8, 24, 40, 56, 76, 96, 116 11136; and (6) And! SOVZ domain having an amino acid sequence selected from the group consisting of

ZEO IYUO MO: 16, 32, 48, 64, 84, 104, 124 and 144.

According to one embodiment of the invention, the antibody, or antigen-binding fragment of the antibody, further comprises: (c) an NSAIDs domain having an amino acid sequence selected from the group consisting of

ZEO IO MO: 4, 20, 36, 52, 72, 92, 1121 and 132; (gu HCOV2 domain having an amino acid sequence selected from the group consisting of

ZEO IO MO: 6, 22, 38, 54, 74, 94, 114 and 134; (e) an EDUCOUS domain having an amino acid sequence selected from the group consisting of

ZEO IO MO: 12, 28, 44, 60, 80, 100, 1201140; (e) GSOV2 domain having an amino acid sequence selected from the group consisting of

ZEO IYUO MO: 14, 30, 46, 62, 82, 102, 122 and 142.

According to one variant of the implementation of the invention, the antibody, or its antigen-binding fragment, contains an NSUMA that includes the NSONI domain, which has an amino acid sequence selected from one of the sequences of 5EO IUO MO: 4, 20, 36, 52, 72, 92, 112 and 132; HCOV2 domain having an amino acid sequence selected from one of the sequences of 5EO IU MO: 6, 22, 38, 54, 74, 94, 114 and 134; NSOVZ domain having an amino acid sequence selected from one of the sequences 5EO IO MO: 8, 24, 40, 56, 76, 96, 116 and 136; and | SUMA, including the I SOVI domain, having an amino acid sequence selected from one of the sequences of 5EO IU MO: 12, 28, 44, 60, 80, 100, 120 and 140; AND! SOV2 domain having an amino acid sequence selected from one of the sequences of 5EO IO MO: 14, 30, 46, 62, 82, 102, 122 and 142; and an I1COV8Z domain having an amino acid sequence selected from one of the sequences of 5EO IJOO MO: 16, 32, 48, 64, 84, 104, 124 and 144.

According to certain variants of the implementation of the invention, a human antibody, or an antigen-binding fragment of a human antibody, which (which) specifically binds to human CSO, contains a pair of amino acid sequences selected from the group consisting of 5EO IU MO: 8/16, 24 /32, 40/48, 56/64, 76/84, 96/104, 116/124 and 136/144. Non-limiting examples of antibodies to

SSOVs having these NSOVZ/LSOVZ pairs are antibodies labeled NANIZ345M, H4ANI1617M,

NANI765M, NaANI13218 and NA4NITZ21R, NANI1327V and N4NI327R, NANI13288 and N4NI1Z28R, NAN1IZ3Z1V and

NANIZZ1TR, NANI3398B and NANI1ZZ9R, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains the NSOVI, NOSOV, and NSOVZ domains, having the amino acid sequences of FEO IU MO: 4, 6, and 8, respectively, and SOVA, I CON2, and I SOVZ3 domains, having amino acid sequences 5EO IU MO: 12, 14 and 16, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains domains NSOVI, NOSOV and NSOVZ, having the amino acid sequences FEO IJUO MO: 20, 22 and 24, respectively, and domains I! SOVI1, I SON and II SOV3, having amino acid sequences 5EO IU MO: 28, 30 and 32, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains domains NSOVI, NOSOV and NSOVZ, having amino acid sequences FEO IU MO: 36, 38 and 40, respectively, and domains I! SOBI1, I SON and II SOB3, having amino acid sequences 5EO IYUO MO: 44, 46 and 48, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains domains NSOVI, NOSOV and NSOVZ, having amino acid sequences FEO IU MO: 52, 54 and 56, respectively, and domains I! SOVI1, I SON and II SOV3, having amino acid sequences 5EO IU MO: 60, 62 and 64, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains domains NSOVI, NOSOV and NSOVZ, having the amino acid sequences FEO IJUO MO: 72, 74 and 76, respectively, and domains I! SOBI1, I SON and Ii SOB3, having amino acid sequences 5EO IYUO MO: 80, 82 and 84, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains domains NSOVI, NOSOV and NSOVZ, having the amino acid sequences FEO IJUO MO: 92, 94 and 96, respectively, and domains I! SOVI1, I SON and II SOV3, having amino acid sequences 5EO IU MO: 100, 102 and 104, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains the domains of NSOVI, NOSOV and NSOVZ, having the amino acid sequences of 5EO 10 MO: 112, 114 and 116, respectively, and the domains of Y1SOVI, ISON2 and I SOVZ , having the amino acid sequences 5EO IO MO: 120, 122 and 124, respectively.

According to one embodiment of the invention, the human antibody, or its antigen-binding fragment, contains the domains of NSOVI, NOSOV, and NSOVZ, having the amino acid sequences of FEO 10 MO: 132, 134, and 136, respectively, and the domains of Y!SOVI, YSON2, and I SOVZ3 , having amino acid sequences 5ХЕО ИО МО: 140, 142 and 144, respectively.

According to one variant of the implementation of the invention, the antibody against pPOSSOV, or its antigen-binding fragment, contains the sequence of NSOVI, which has the formula X'-X2-X3-X1-ho-X6-X7-XV (5EO IU MO: 202), where X ' means Su, X? means РнНе, ХЗ means ТНг, Х" means

Rne or 5eg, X" means 5eg, X9 means beg or Avp, X" means Tug or Rne, and X8 means Agvr,

Yi em or Spu; sequence НСОН2, having the formula Х'-Х2-Х3-Х7-Х5-Х5--Х" - ХВ (ZEO Ir

MO: 203), where X! means Ie, X? means 5eg, Sp, A5r or Tr, ХЗ means 5eg, Sim, ТНg or РНе, X" means Avr or Aa, X? means Siu or Sim, X9 means Ago, Pe or absent, X" means Azr or Sim and ХУ? means I uz or Tig; the sequence NSOVZ, having the formula X'-X2-X3-X1-X5 -

Хе - а - ХВ - Хо - Хо - ХХ" - хг - ХЗ - Х - Хо - Х - ХХ - ХВ - ХХ - Хго - а (ZEO IO MO: 204), where X' means Aa or Tng, X? stands for Huz or Agu, XZ stands for Sim, X" stands for Mei, Rgo, Spu or Avr, X? means Mai, 5eg, Guz, Agu or absent, ХУ means Tug, Ni5, Azp or absent, X" means Tug, ХУ? means Azvr or Si, X? means Iee, X'"? means Gei, X" means Tpg, X'"? means

SIu, ХЗ means Tug, Avr or Niv, X"" means No, Avr, Tug or absent, X'" means Azp, Tug,

Niz or Absent, X'E means Tug, X'" means Tug or No, X'8 means Siu or Aa, X"? means

Mei, Ho means Azr and X"! means Mai or Ie; the sequence | SOV1, which has the formula X'- X2 - хз АХ - X» -X6 (5EO IYUO MO: 205), where X! means Sip, X? means Siu or Aa, ХЗ means Pe, X" means Ap or Aha, X? means Azp and X9 means Tug or Avr; sequence | СОН2, which has the formula Х'-Х2 - ХЗ (5EO IU MO: 206), where X means Tng or Aa, X? means Aa or Tng, and xz 60 means 5eg or PnNe; and the sequence Y/SOVZ having the formula X'-X2-X3-X1-X5-X585-X-

hv - ХР" (5EO I MO: 207), where X' means aSip or I ey, X? means Sip, ХЗ means Tug, Niz or Avr,

X" means Avp or Tug, X? means Tng or 5eg, X9 means Tug, Azp or Ni, X" means Rgo, X8 means I ey, Riye, Agu or absent and X? means TnHg.

According to one embodiment of the invention, the antibody, or its antigen-binding fragment, binds human, monkey, mouse and rat SSOV.

According to one embodiment of the invention, the antibody, or its antigen-binding fragment, binds human, monkey and mouse CSC, but does not bind rat CSC.

According to one embodiment of the invention, the antibody, or its antigen-binding fragment, binds human, monkey and rat CSC, but does not bind mouse CSC.

According to one embodiment of the invention, the antibody, or its antigen-binding fragment, binds human and monkey CSC, but does not bind rat or mouse CSC.

According to one embodiment of the invention, the antibody, or its antigen-binding fragment, binds human CSC, but does not bind monkey, mouse or rat CSC.

According to one embodiment, the invention provides a fully human monoclonal antibody, or an antigen-binding fragment thereof, which neutralizes the activity of PASSIV, and the antibody, or fragment thereof, has one or more of the following features: (i) contains a region

HCMV having an amino acid sequence selected from the group consisting of 5EO IU MO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146; (ii) contains a region of ISMA having an amino acid sequence selected from the group consisting of 5EO IJOO MO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148; (ii) contains an NSONZ domain having an amino acid sequence selected from the group consisting of BEO IUO MO: 8, 24, 40, 56, 76, 96, 116 and 136, or a substantially similar sequence, and the degree of sequence identity is at least 9095, at least 9595, at least 9895 or at least 9995; and domain

And SOVZ having an amino acid sequence selected from the group consisting of BEO IU MO: 16, 32, 48, 64, 84, 104, 124 and 144, or a sequence substantially similar to it, and the degree of sequence identity is at least 9095, at least 9595, at least 9895 or at least 99 95; (m) contains an NSAID domain having an amino acid sequence selected from the group consisting of 5EO IO MO: 4, 20, 36, 52, 72, 92, 112 and 132, or a substantially similar sequence, and the degree of sequence identity is at least 90 95,

Co) at least 9595, at least 9895 or at least 9995; the NSONB2 domain, having an amino acid sequence selected from the group consisting of 5EO IUO MO: 6, 22, 38, 54, 74, 94, 114 and 134, or a sequence essentially similar to it, and the degree of sequence identity is at least 90 95 , at least 9595, at least 9895 or at least 99 95; domain I SOVI1 having an amino acid sequence selected from the group consisting of 5EO IU

MO: 12, 28, 44, 60, 80, 100, 120 and 140, or essentially a similar sequence, and the degree of sequence identity is at least 90 95, at least 95 95, at least 98 95 or at least 99 95; and an HER SOV2 domain having an amino acid sequence selected from the group consisting of 5EO IO MO: 14, 30, 46, 62, 82, 102, 122 and 142, or a sequence substantially similar to it, and the degree of sequence identity is at least 90 95, at least 95 95, at least 98 95 or at least 99 95; (m) binds any one or more SOVA: human, monkey, mouse or rat SSO; (mi) may or may not block SS activity in at least one non-human species; (y) demonstrates

KO in the interval from about 10-8 to about 10-12; (mi) lowers blood glucose levels by at least about 25 95 - 75 905 in a mammal having elevated blood glucose levels; (mi) may or may not lower triglyceride levels to levels observed in a healthy mammal; or (mii) does not have any deleterious effect on the level of OA, NOA or total cholesterol in the blood of the mammal.

According to another related variant, the invention provides an antibody, or its antigen-binding fragment, which (which) competes for specific binding to pOSOMK with an antibody, or its antigen-binding fragment, containing SOE domains. of heavy and light chains present in pairs of sequences of heavy and light chains selected from the group consisting of BEO IUO MO: 2/10, 18/26, 34/42, 50/58, 66/68, 70/78, 86 /88, 90/98, 106/108, 110/118, 126/128, 130/138 and 146/148.

According to another related embodiment, the invention provides an antibody, or an antigen-binding fragment thereof, that binds the same epitope on the TOE that is recognized by an antibody containing pairs of heavy and light chain sequences selected from the group consisting of BEO IYUO MO: 2/10, 18/26, 34/42, 50/58, 66/68, 70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/ 138 and 146/148.

According to one embodiment, the invention provides an antibody to POSOK having one of 60 or more of the following properties:

a) capable of lowering blood glucose levels by approximately 25 95 - 75 95 for a period of at least 7 days when administered in a dose range from about 1 mg/kg to about 30 mg/kg; b) capable of reducing body weight by at least 10 95 when administered to a mammal requiring such treatment; c) is able to reduce the level of ketones in the blood, approximately, by 25 95 - 75 95 when administered in the range of doses from about 1 mg/kg to about 30 mg/kg; or d) capable of lowering blood glucose levels by approximately 20 95 - 4095 without causing appreciable increases in blood lipids or cholesterol when administered with an antibody specific for subtilisin-kexin type 9 (RSB5BK)-9 proprotein convertase, and maintaining reduced blood glucose levels for at least 7 days after administration.

In the second aspect, the invention provides nucleic acid molecules encoding anti-pass antibodies or their fragments. Recombinant expression vectors carrying the nucleic acids of the invention and host cells into which such vectors are introduced are also encompassed by this invention, as are methods of producing antibodies by culturing host cells under conditions conducive to antibody production and isolation of produced antibodies.

According to one embodiment, the invention provides an antibody, or a fragment thereof, containing the NSMEA region, which is encoded by a nucleotide sequence selected from the group consisting of 5EO, IUO, MO: 1, 17, 33, 49, 65, 69, 85, 89, 105, 109, 125, 129 and 145, or an essentially identical sequence homologous to these sequences by at least 90 95, at least 9595, at least 9895 or at least 99 95. According to one variant of the invention, the NSMA region is encoded by a nucleotide sequence selected from the group consisting of BEO IYUO MO: 33, 69, 85, 109 and 125.

According to one variant of the implementation of the invention, the antibody, or its fragment, additionally contains the SUMA region encoded by a nucleotide sequence selected from the group consisting of 5EO, IUO, MO: 9, 25, 41, 57, 67, 77, 87, 97, 107 . of the group, which consists of 5 EO IUO MO: 41, 77, 87, 117 and 127.

According to one embodiment, the invention also provides an antibody, or an antigen-binding fragment of an antibody, which (which) contains a domain of NSOVZ, encoded by a nucleotide sequence selected from the group consisting of 5EO, IUO, MO: 7, 23, 39, 55, 75 , 95, 115 and 135, or essentially similar sequence, with the degree of identity of these sequences at least 90 95, at least 95 95, at least 9895 or at least 99 95; and domain

IISOVZ encoded by a nucleotide sequence selected from the group consisting of 5EO 10

MO: 15, 31, 47, 63, 83, 103, 123 and 143, or a substantially similar sequence, with a degree of identity of these sequences of at least 90 95, at least 95 95, at least 98 95, or at least 99 95.

According to one embodiment, the invention provides an antibody, or its fragment, which (which) additionally contains the NCOMH1 domain encoded by a nucleotide sequence selected from the group consisting of 5EO IO MO: 3, 19, 35, 51, 71, 91, 111 and 131, or a substantially similar sequence, with a degree of identity of these sequences of at least 90 95, at least 95 9565, at least 98 95 or at least 99 95; the HCOV2 domain, which is encoded by a nucleotide sequence selected from the group consisting of FEO IUO MO: 5, 21, 37, 53, 73, 93, 113 and 133, or a substantially similar sequence, with a degree of identity of these sequences of at least 9095, at least 9595, at least 9895 or at least 99 95; domain

II SON encoded by a nucleotide sequence selected from the group consisting of 5EO 10

MO: 11, 27, 43, 59, 79, 99, 119 and 139, or essentially similar sequence, with the degree of identity of these sequences at least 90 95, at least 95 95, at least 98 95 or at least 99 95; and the ІСОВ2 domain encoded by a nucleotide sequence selected from the group consisting of 5EO, ІХО, MO: 13, 29, 45, 61, 81, 101, 121, and 141, or a substantially similar sequence, with a degree of identity of these sequences of at least 90 96, at least 95 95, at least 98 95 or at least 99 95.

According to the third aspect, the invention includes a human antibody to pPOSOV, or an antigen-binding fragment of an antibody, which contains the region of HOSMV, which is encoded by the nucleotide sequences of the segments obtained from the Mn, On and dn sequences of the germ line, and the Y region of SUEN, which is encoded nucleotide sequences of segments obtained from Mk and

Uk germline sequences, in the combinations shown in Table 2.

The invention covers PUMP antibodies having a modified glycosylation pattern. In some areas of application, modification with the removal of unnecessary glycosylation sites, or, for example, with the removal of a fucose fragment to increase the function of antibody-dependent cellular cytotoxicity (AOSS) can be useful (see ZPieiYia eyi ai. (2002) UVO 277: 26733) .

For other applications, galactosylation can be modified to modify complement-dependent cytotoxicity (CTO).

In a fourth aspect, the invention includes a pharmaceutical composition containing a recombinant human antibody, or a fragment thereof, which specifically binds PASI, and a pharmaceutically acceptable carrier or solvent.

According to one embodiment, the invention includes a composition that is a combination of an antibody or an antigen-binding fragment of an antibody according to the invention and a second therapeutic agent. The second therapeutic agent can be any agent that binds preferentially to the antibody or its fragment according to the invention.

According to one embodiment of the invention, the second therapeutic agent may be an agent capable of lowering blood glucose levels or reducing the intensity of at least one symptom in a patient suffering from a disease or condition characterized by elevated blood glucose levels, such as diabetes.

According to certain embodiments of the invention, the second therapeutic agent may be an agent that helps to neutralize or attenuate any possible side effect(s) associated with the antibody or with the antigen-binding fragment of the antibody according to the invention, in case of side effect(s). For example, in the event that any or the antibody to pOSOK increases lipid or cholesterol levels, it may be appropriate to introduce a second agent that effectively lowers lipid or cholesterol levels.

The second therapeutic agent can be a low molecular weight drug, a protein/polypeptide, an antibody, a nucleotide molecule such as an antisense molecule or 5igRnK.

The second therapeutic agent can be synthetic or natural.

According to one embodiment of the invention, the second therapeutic agent can be a glucagon antagonist, or an antagonist of the second glucagon receptor, such as another antibody to the glucagon receptor, which differs from the antibodies of the present invention. It should also be taken into account that antibodies and pharmaceutically acceptable compositions of the present invention can be used in combination therapy, i.e. antibodies and pharmaceutically acceptable compositions

Zo can be administered simultaneously with, before or after the administration of one or more other necessary drugs or medical procedures. When implementing a specific combination of treatment methods (drugs or medical procedures) for use in a combined scheme, the compatibility of given drugs and/or medical procedures with achieving the required therapeutic effect is taken into account. It should also be taken into account that the drugs used can provide the desired effect in relation to the same disorder (for example, an antibody can be administered simultaneously with another agent used to treat the same disorder), or they can provide different effects ( for example, managing any side effects). In this context, additional therapeutic agents that are commonly used to treat or prevent a particular disease or condition are appropriate (adequate) for the disease or condition to be treated.

According to one variant of the implementation of the invention, the anti-PpOSOC antibodies of the invention can be used in combination with one or more of the following drugs currently available for the treatment of type 2 diabetes. These drugs include biguanide (metformin), sulfonylureas (such as glyburide, glipizide) , agonists of gamma receptors activated by peroxisome proliferators (PPAK) (pioglitazone, rosiglitazone); and alpha-glucosidase inhibitors (acarbose, voglibose). Additional medications include injectables such as exenatide (EHEMATIOEF (glucagon-like peptide 1)) and Symlin (ZUMI IM (pramlintide)).

According to certain variants of implementation of the invention, the composition may include a second agent selected from the group consisting of secretory stimulants that do not belong to the class of sulfonylureas, insulin, insulin analogs, exendin-4 polypeptide, beta-3 adrenoreceptor agonists, PPAK agonists, dipeptidyl- MI peptidases, statins and statin-containing combinations, cholesterol and/or bile acid absorption inhibitors, II-cholesterol antagonists, cholesterol ester carrier protein antagonists, endothelin receptor antagonists, growth hormone antagonists, insulin sensitizers, amylin mimetics or agonists, cannabinoid receptor antagonists, agonists glucagon-like peptide-y, melanocortins, melanin-concentrating hormone receptor agonists, ZMKII5, mimetics of fibroblast growth factor 21 (BOE21) (see, for example, US patents I520110002845 and 0520080261236), fibroblast growth factor receptor type 1c agonist (ЕСЕ1c) (see , because, for example, US patent Sh052011 0150901), an inhibitor of excess formation of glycosylation end products, such as, but not limited to, aminoguanidine, and protein tyrosinase inhibitors.

According to certain embodiments of the invention, the composition may contain a second agent that helps lower lipid and cholesterol levels and may include an agent such as a 3-hydroxy-3-methylglutaryl coenzyme A (HMO-CoA) reductase inhibitor (eg, a statin such as atorvastatin (Lipitor, GIRITOKF), fluvastatin (Lescol, I E5BSOIF), lovastatin (Mevacor,

MEMASOKF)), pitavastatin (IMAGO), pravastatin (Pravahol, RKAMASNOI F)), rosuvastatin (Krestor, SKEZTOKF)) and simvastatin (Zokor, 2OSOKF)) etc.

According to certain embodiments of the invention, it may be appropriate to administer the antibody of the invention in combination with any one or more of the agents listed below: (1) niacin, which increases lipoprotein catabolism; (2) fibrates or amphipathic carboxylic acids, which reduce low-density lipoprotein (0), improve high-density lipoprotein (HDL) and To levels, and reduce nonfatal heart attacks; and (3) activators of the HC transcription factor that plays a role in cholesterol elimination, such as 22-hydroxycholesterol, or combination drugs such as

MUTONIM) (vitorin, ezetimibe plus simvastatin); statin plus bile acid sequestrants (onoexchange resin) (eg, colestyramine, colestipol, colesevelam), combination drug niacin plus statin (eg, niacin plus lovastatin); or lipid-lowering agents such as ethyl esters of omega-33 fatty acids (eg, omacor). In addition, the second therapeutic agent can be one or more glucagon inhibitors or

SSOCs, as well as inhibitors of molecules of other substances, such as angiopoietin-like protein C (AMORTI 3), angiopoietin-like protein 4 (AMORTI 4), angiopoietin-like protein 5 (AMORTI 5), angiopoietin-like protein 6 (AMORTI 6) , which are involved in lipid metabolism, in particular, in the homeostasis of cholesterol and/or triglycerides. Inhibitors of these molecules include low molecular weight substances or antibodies that specifically bind to these molecules and block their activity.

According to certain variants of the implementation of the invention, it may be appropriate to administer antibodies to

CSO according to the invention in combination with a nucleic acid that inhibits the activity of PROZKO, such as an antisense molecule, a double-stranded RNA molecule or a 5iRNA molecule. Examples of nucleic acids that inhibit ROKU activity are described in US patents 052011/0065644,

From 052011/0039914, 052008/0015162 and 052007/0173473.

According to certain variants of the implementation of the invention, it may be appropriate to introduce antibodies to

POSOK according to the invention in combination with an antibody that specifically binds to PROBKU (human proprotein convertase subtilisin/kexin) and inhibits its activity, thereby inhibiting its activity, while such an antibody reduces lipid or cholesterol levels. Examples of antibodies to

SNAPS are described in US patent O52010/0166768. An isolated antibody that specifically binds to human RSOBKO, or an antigen-binding fragment thereof, can be administered in a dose range of about 0.01 mg/kg to about 30 mg/kg. It can be administered as a single dose or in multiple doses. The PRS5KOU antibody can be administered simultaneously with the MAMM antibody, or it can be administered before or after the administration of the CPR antibody.

According to one variant of the implementation of the invention, the second therapeutic antibody for use in combination with the antibody of the invention is an isolated antibody that specifically binds to human RSBKO, or an antigen-binding fragment thereof, where the antibody to PRSB5KO contains three heavy chain SOC domains (HSOKI1 , NSOM2 and NSOKZ) in one of any NSUV sequences selected from the group consisting of ZEO IU MO: 173 and 177; and three SOC domains of the light chain (-COM1, ICOM2 and |COM) in one of any I! SMC of sequences selected from the group consisting of BEO IU MO: 175 and 185.

According to one embodiment of the invention, the isolated antibody that specifically binds to human RSBCOU, or its antigen-binding fragment, contains a heavy chain variable region (HCHV) selected from the group consisting of BEO IU MO: 173 and 177.

According to one variant of the implementation of the invention, the isolated antibody that specifically binds to the human RSBKOU, or its antigen-binding fragment, contains a variable region of the light chain (SUK), selected from the group consisting of BEO IU MO: 175 and 185.

According to one variant of implementation of the invention, the isolated antibody that specifically binds to human ROBZKO, or its antigen-binding fragment, contains NSUK having an amino acid sequence selected from the group consisting of 5EO IU MO: 1731177, and SM, which has an amino acid sequence selected from the group consisting of BEO IU MO: 175 and 185.

According to one embodiment of the invention, an isolated antibody that specifically binds to human RSBKOU, or its antigen-binding fragment, contains a heavy chain variable region (HCHV) and a light chain variable region (LCH), and pairs of 60 HCH/HCH sequences selected from the group consisting of 5BEO IU MO: 173/175; and ZEO IU

MO: 177/185.

According to one embodiment of the invention, the selected antibody, which specifically binds to the human ROZKO, or its antigen-binding fragment, contains: the NSOKI domain, which has an amino acid sequence selected from the group consisting of 5EO IU MO: 161 and 179; a domain of NSOK2 having an amino acid sequence selected from the group consisting of ZEO IU

MO: 163 and 181; the NSOKZ domain having an amino acid sequence selected from the group consisting of ZEO IO MO: 165 and 183; the GSOKI domain having an amino acid sequence selected from the group consisting of ZEO IU MO: 167 and 187; domain I! SOK2 having an amino acid sequence selected from the group consisting of 5EO IU MO: 169 and 189, and the Y SOCZ domain having an amino acid sequence selected from the group consisting of 5EO IU MO: 171 and 191.

According to certain variants of the implementation of the invention, antibodies to PROBKU for use in combination with antibodies to SSOK according to the invention are encoded by nucleic acid molecules according to this description. For example, according to one embodiment, the invention provides an antibody to

PROBKU, or its fragment, which (which) contains the NSMK region encoded by a nucleotide sequence selected from the group consisting of 5BO IUO MO: 172 and 176, or a sequence essentially identical to them, at least 90,956, at least 95,956 , at least 98 95 or at least 99 95 homologous to them, and the G/SMC region encoded by a nucleotide sequence selected from the group consisting of 5EBO IUO MO: 174 and 184, or a sequence essentially identical to them, at least 90 956 , at least 95 956, at least 98 95 or at least 99 95 homologous to them.

According to one embodiment, the invention provides an antibody against PROBCU for use in combination with antibodies against CPR. according to the invention, and the antibody to PROZKO, or its fragment, contains the NSOKI domain, which is encoded by a nucleotide sequence selected from the group consisting of 5EBEO IJOO MO: 160 and 178, or a sequence essentially similar to them, and the degree of identity of these sequences is at least 90 95, at least 95 9565, at least 98 95 or at least 99 95; the NSOK2 domain encoded by a nucleotide sequence selected from the group consisting of ZEO IU MO: 162 and 180, or a substantially similar sequence, and the degree of identity of these sequences is at least 9095, at least 9595, at least 9895 or at least 9995; domain

NSOKZ encoded by a nucleotide sequence selected from the group consisting of ZEO

IOJUO MO: 164 and 182, or a sequence essentially similar to them, and the degree of identity of these sequences is at least 9095, at least 95595, at least 9895 or at least 9995; domain YSOMK1I encoded by a nucleotide sequence selected from the group consisting of BEO IU MO: 166 and 186, or a sequence essentially similar to them, and the degree of identity of these sequences is at least 9095, at least 95 95, at least 9895 or at least 9995; the YCOM2 domain encoded by a nucleotide sequence selected from the group consisting of 5EO IU MO: 168 and 188, or a sequence essentially similar to them, and the degree of identity of these sequences is at least 90 965, at least 95 95, at least 98 95 or at least 99 95; and the domain of the YISOKZ encoded by a nucleotide sequence selected from the group consisting of ZEO IU MO: 170 and 190, or a sequence substantially similar to them, and the degree of identity of these sequences is at least 90 95, at least 95 95, at least 98 95 or at least 99 95.

If multiple drugs are co-administered, dosages may be adjusted as is accepted in the art.

In the fifth aspect, the invention includes methods of inhibiting the activity of NOSOC using an antibody to pOSOC, or an antigen-binding region of an antibody according to the invention, and therapeutic methods include the introduction of a therapeutically effective amount of a pharmaceutical composition containing an antibody or its antigen-binding fragment. Antibodies of the invention can be used to treat any condition or disorder that is ameliorated by

BO is alleviated, inhibited, or prevented by eliminating, inhibiting, or reducing PpOSO activity. It is contemplated that the antibodies of the invention may be used alone or as adjunctive therapy in combination with other agents or methods known to be standard therapy in the treatment of patients suffering from diseases or conditions characterized, in part, by elevated blood glucose or ketone levels, such as, but not limited to, diabetes. Such standard therapy may include infusion therapy or the administration of any other pharmaceutical agents that lower blood glucose, ketones, or lipids or reduce body weight.

Antibodies to pOSOM according to the invention can block the interaction between glucagon and its receptor, thereby inhibiting the effect of glucagon, which increases the level of glucose. The use of glucagon receptor antagonists, such as the antibodies of this description, can be an effective way to achieve normal glucose levels, thereby reducing the intensity or preventing one or more symptoms or long-term complications due to, for example, diabetes. The use of glucagon receptor antagonists, such as the antibodies of this description, may also be an effective way to achieve normal blood glucose levels in non-diabetic patients who have hyperglycemia as a result of conditions or disorders not related to diabetes, such as perioperative hyperglycemia (hyperglycemia, observed in patients immediately before surgery or after surgery). According to certain variants of implementation of the invention, methods of reducing the levels of glucose or ketones in the blood in diabetic ketoacidosis with the use of antibodies according to the invention are proposed. According to certain variants of the invention, methods of treating patients with the aim of reducing body weight, or preventing weight gain, or maintaining normal body weight with the use of antibodies according to the invention are also proposed.

Antibodies of the present invention can also be used to reduce the intensity of pathological conditions, for example, such as impaired glucose tolerance, obesity, or to treat diabetic conditions, or to prevent and reduce the severity of any one or more long-term complications associated with diabetes. such as nephropathy, neuropathy, retinopathy, cataract, stroke, atherosclerosis, delayed wound healing, and other diabetes-related complications known to those skilled in the art.

Other pathological conditions or disorders amenable to treatment by the therapeutic methods of the invention include diabetic ketoacidosis, hyperglycemia (including perioperative hyperglycemia, intensive care unit hyperglycemia, and hyperosmolar hyperglycemia syndrome), hyperinsulinemia, metabolic syndrome, insulin resistance syndrome, impaired fasting glycemia or glycemia associated with hypercholesterolemia, hypertriglyceridemia, hyperlipidemia, and dyslipidemia in general. According to certain embodiments, the invention provides an isolated antibody, or an antigen-binding fragment thereof, specific for CKD, as described herein, for use in reducing blood glucose or ketone levels or in treating a patient suffering from a disease or condition caused by or characterized, in part, by high levels of glucose or ketones in the blood, and this condition or disease is selected from diabetes, impaired glucose tolerance,

From obesity, nephropathy, neuropathy, retinopathy, cataract, stroke, atherosclerosis, delayed wound healing, diabetic ketoacidosis, hyperglycemia, hyperosmolar hyperglycemia syndrome, perioperative hyperglycemia, hyperglycemia in a patient in the intensive care unit, hyperinsulinemia, metabolic syndrome, insulin resistance syndrome and impaired fasting glycemia. According to certain embodiments, the use of an isolated antibody or antigen-binding fragment of the invention is contemplated for the purpose of preparing a medicament for lowering blood glucose or ketone levels or for treating a patient suffering from a disease or condition caused or characterized, in part, by high levels blood glucose or ketones, or to reduce the intensity of at least one symptom of such a disease or condition, wherein said condition or disease is selected from the above diseases or conditions.

Antibodies may also be used to treat patients with inoperable glucagonoma (pancreatic endocrine tumor with or without necrotizing erythema migrans and hyperglycemia).

Other embodiments of the invention will become apparent from the following detailed description.

Brief description of the drawings

In Fig. 1 shows the change in the percentage of glucose levels in the blood of C57VI 6 mice after administration

H4iNI1I327P (antibodies against OSOK) and/or NINZ16P (antibodies against RSOB5KO), individually or in combination. Control (X solid line); H4NI1T327P in a dose of 3 mg/kg (Ш solid line);

H4ANI327P in a dose of 10 mg/kg (A solid line); NINZ16R at a dose of 10 mg/kg (solid line); NANI1327R in a dose of 3 mg/kg - NINZI16R in a dose of 10 mg/kg (dotted line); NANI1327R at a dose of 10 mg/kg - NINZIbR at a dose of 10 mg/kg (O dashed lines).

In Fig. 2 shows the levels of GOI-C in the plasma of C57VI6 mice after administration of H4NI1327R and/or

NINZI16R, individually or in combination. Control (X solid line); НА4НИ1327Р at a dose of 3 mg/kg (Ш solid line); H4NI1327P in a dose of 10 mg/kg (A solid line); NINZI16bR at a dose of 10 mg/kg (t solid line); H4NI1327P in a dose of 3 mg/kg - NINZI16P in a dose of 10 mg/kg (dotted line); H4NI1327R at a dose of 10 mg/kg - NINZI6R at a dose of 10 mg/kg (O dashed lines).

In Fig. 3 shows NOI-C levels in the plasma of C57VI 6 mice after administration of H4NI1327R and/or

NINZI16R, individually or in combination. Control (X solid line); H4NI1327P in a dose of 3 mg/kg (Ш solid line); H4NI1327P in a dose of 10 mg/kg (A solid line); NINZ16R at a dose of 10 mg/kg (solid line); H4NI1327P in a dose of 3 mg/kg - NINZI16P in a dose of 10 mg/kg (dotted line); H4NI1327R at a dose of 10 mg/kg - NINZI6R at a dose of 10 mg/kg (O dashed lines).

In Fig. 4 shows the levels of total cholesterol in the plasma of C57VI 6 mice after administration

H4ANI327R and/or NINZI16R, individually or in combination. Control (X solid line);

NANI327P in a dose of 3 mg/kg (Ш solid line); НА4НИ1327Р at a dose of 10 mg/kg (A solid line);

NINZI16R at a dose of 10 mg/kg (t solid line); H4NI1327P in a dose of 3 mg/kg - NINZI16P in a dose of 10 mg/kg (dotted line); H4ANI1327R at a dose of 10 mg/kg and NINZI1I6R at a dose of 10 mg/kg (O dashed lines).

In Fig. 5 shows the levels of glycerides in the plasma of C57VI 6 mice after administration of H4NI1327P and/or

NINZI16R, individually or in combination. Control (X solid line); НА4НИ1327Р at a dose of 3 mg/kg (Ш solid line); H4NI1327P in a dose of 10 mg/kg (A solid line); NINZI16bR at a dose of 10 mg/kg (t solid line); H4NI1327P in a dose of 3 mg/kg - NINZI16P in a dose of 10 mg/kg (dotted line); N4AN1327R at a dose of 10 mg/kg - NINZI6R at a dose of 10 mg/kg (O dashed lines).

In Fig. 6 shows hepatic triglyceride levels in C57VI 6 mice after administration of NANI1327R or NINZI16R, individually or in combination. Control (X); H4NI327P in a dose of 3 mg/kg (AI);

NANI327R at a dose of 10 mg/kg (4); NINZ1I6R in a dose of 10 mg/kg (t); NANI1327R in a dose of 3 mg/kg - NINZI16R in a dose of 10 mg/kg (3); H4NI1327R at a dose of 10 mg/kg - NINZI16R at a dose of 10 mg/kg (0).

Detailed description of the invention

Before describing the methods of the present invention, it should be noted that the present invention is not limited to the specific experimental methods and conditions described, as such methods and conditions may vary. It should also be noted that the terminology used in this description is only for the purpose of describing a specific variant of the invention and is not intended to be limiting, since the scope of this invention is limited only by the attached Formula of the invention.

Unless otherwise indicated, all technical and scientific terms used in this specification have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention relates. Although any methods and materials similar or equivalent to those presented herein are used in the practical application or testing of the present invention, the preferred methods and materials are described herein.

Definition "Glucagon receptor", "glucagon receptor", also called "Glucagon" in this application, refers to the family of C-protein-coupled class 2 receptors and consists of a long amino-

From the terminal extracellular domain (see 5EO IUO MO: 158 for DNA encoding the M-terminal extracellular domain and 5EO IUO MO: 159 for the amino acid sequence of the M-terminal extracellular domain), seven transmembrane segments and the intracellular C-terminal domain ( a!., Zsiepse 259: 1614-1616 (1993), 5edge ei ai., Ttepaz Epaostipoi. Meyarb 4:309- 314 (1993)). Glucagon receptors are expressed, in particular, on the surface of hepatocytes, where they bind to glucagon, and transmit the resulting signal into the cell. Accordingly, the term "glucagon receptor" also refers to one or more receptors that specifically interact with glucagon, resulting in a signal. DNA sequences encoding glucagon receptors of rat and human origin were isolated and described in the prior art (European patent ЕРО658200В81). Mouse and monkey (cynomolgus monkeys) homologues were isolated and sequenced (Vigseiip, ei aI., zepe 164 (1995) 305-310; MeMaiU ei a!., Reridez5 25 (2004) 1171-1176). The terms "glucagon receptor", "glucagon receptor" and "ZSOoK" are used interchangeably. The expression "cSOe", "peso" or its fragments, according to this description, refers to human (MAMMA protein or its fragments, unless it is specifically stipulated that this protein is of non-human origin, for example, "mouse CSO", "rat

SSOC" or "monkey MAMMAL." Additionally, "SSOC" or "peso" herein refers to a human SSOC encoded by the nucleotide sequence shown in ZEO IUO MO: 157, which has the amino acid sequence ZEO IU MO: 153 , or its biologically active fragment.

There are a number of sequences related to the ZSOK gene and have the following accession numbers in Seprapk: MR О000151.1 (human), MR 742089.1 (rat), ЧР. 001111894.1 (rhesus macaque) and MR 032127.2 (mouse). Other sequences disclosed herein include human MAMMAL (5EBO IUO MO: 153), mouse SSOC (5EBO IUO MO: 154), cynomolgus monkey (ZEO IUO MO: 155), rat SSO (5EO IUO MO: 156). According to certain variants of the implementation of the invention, fusion (hybrid, chimeric) proteins in this invention may include ZEO IU

MO: 149 (pOSOB-pPEs fragment, residues 27-144 of protein MR 000151.1, fused with ES fragment of human Ids) ZEO IU MO: 150 (fragment PpOSOK-PpEs, residues 27-144 of protein MR 0001511, fused with ES fragment of human Ids) .

MyaSOaVv-pEs containing the M-terminal Mi sequence of the cynomolgus monkey is identical to residues 27-144 of the SSOC of the rhesus macaque, Masasa tiiatsa, having the accession number XP. 001111894.1, and merged with a fragment of the human to).

The term "human proprotein convertase subtilisin/kexin type 9", or "PROBCU 9", as used herein, refers to the PROBCU encoded by the nucleotide sequence shown in ZEO

IUO MO: 192, and has the amino acid sequence 5EO IUO MO:193, or its biologically active fragment.

The term "antibody" in this description is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (I) chains linked together by disulfide bonds (i.e., "complete antibody molecules"). , as well as to their multimers (for example, DM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain variable region ("NSMK" or "Me") and a heavy chain constant region (containing the Sni, Snag, and Sn3 domains). Each light chain consists of a light chain variable region C SMK or "Me" and constant region of the light chain (Su. The Mn and M regions can be further divided into hypervariable regions called complement-determining regions (domains), interspersed with more conservative regions called framework regions (EK). Each region of Un and Mi consists of three

SOC and four ECs, located from the amino end to the carboxy end in the following order: EC,

SOCI, EC2, SOC2, ECZ, SOCZ, EC4. According to certain variants of the invention of the EC, the sections of the antibody to ZSOK (or its antigen-binding fragment) can be identical to the sequences of the human germline or can be modified naturally or artificially. The amino acid consensus sequence can be determined based on the analysis of adjacent (located "side by side") two or more SOC.

It is also possible to replace one or more SOEs. remnants or exclusion of one or more areas (one or more sections, domains) of the CPR. Antibodies are described in the scientific literature, in which one or more regions of the SOC can be replaced by a bond (these regions can be dispensed with).

Radiop ei ai. (1995 EABEV 4). 9: 133-139) analyzed the areas of contact between antibodies and their antigens on the basis of published crystal structures and concluded that only one-fifth to one-third of SOC residues actually contact the antigen. Radiap also identified many antibodies in which one or more regions of the SOC did not contain antigen-contacting amino acids (see also Ma)|dose ei aI. 2002 9 My Vioi! 320: 415-428).

Residues of ESR, not in contact with the antigen, can be identified based on more

From early studies (e.g., HbO-NE5 residues in SOCN?O are often not needed), with the SOM of Cabrai sites that lie outside of the SPOIPia SOC sites, by molecular modeling and/or empirically. If SOC or its residue(s) are eliminated, they are usually replaced by an amino acid that occupies the corresponding position in the sequence of another human antibody or their consensus sequence. The position for substitution in the CRP and amino acids for substitution can also be selected experimentally. Empirical substitutions can be conservative and non-conservative substitutions.

The fully human anti-MAP antibodies of this description may contain one or more amino acid substitutions, insertions and/or deletions in the framework or SOC regions of the variable domains of the heavy and light chain compared to the corresponding germline sequences. Such mutations can be easily detected by comparing the amino acid sequences of this description with germline sequences, for example, from a publicly available antibody sequence database. This invention includes antibodies, antigen-binding fragments thereof, which are derived from any amino acid sequence disclosed in this application, where one or more amino acids in one or more framework and/or SOC regions have been mutated to the corresponding residue(s) ( residues) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid replacement of the corresponding residue(s) (such sequence changes in this application collectively called "germline mutations"). One of ordinary skill in the art, based on the sequences of the variable regions of the heavy and light chain disclosed in this application, will be able to easily obtain a variety of antibodies and antigen-binding fragments that contain one or more individual mutations or combinations thereof in the germline. According to certain variants of the implementation of the invention, all residues in the framework and/or SOC regions in the Mn and/or Mi domains of type5 are mutated "back" to the residues of the initial sequence of the germ line from which this antibody was obtained. According to other variants of the implementation of the invention, only some residues are mutated "back" to the residues of the initial sequence of the germ line, for example, only mutations of the residues are found among the first 8 amino acids of EC! or among the last 8 amino acids of EKA, or only residue mutations were detected among SOMK1, SOK2, or SOKZ.

According to other embodiments of the invention, one or more residues in the framework and/or SOC regions are mutated to the corresponding residue(s) of another germline sequence 60 (ie, a germline sequence different from the germline sequence from which it was originally derived In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework and/or SOC regions, for example, where some individual residues are mutated to the corresponding residue of a particular germline sequence, while some other residues, that differ from residues in the original germline sequence are conserved or mutated to a corresponding residue in another germline sequence.The resulting antibodies and antigen-binding fragments that contain one or more germline mutations can be readily screened for one or more desired properties, such as increased binding specificity, increased binding affinity zation, improved or enhanced antagonistic or agonistic biological properties (depending on the situation), reduced immunogenicity, and so on. Antibodies and antigen-binding fragments obtained by this general method are covered by the present invention.

This invention also includes antibodies to PSO containing any variants of the NSMK, SUK or COP amino acid sequences disclosed in this application, and having one or more conservative substitutions. For example, this invention includes antibodies to POSOK having the amino acid sequences of the NSMK, GSMK and/or SOR regions, and containing, for example, 10 or less, 8 or less, b or less, 4 or less and so additionally conservative amino acid substitutions in comparison with any of the amino acid sequences of NSURM, I SUK and or SOR disclosed in this description.

The term "human antibody" as used herein is intended to include antibodies having variable and constant regions of human germline immunoglobulin sequences.

Human TAs of the invention may include amino acid residues not encoded by nucleotide sequences for human germline immunoglobulin sequences (e.g., mutations introduced using random or site-specific mutagenesis of ip migo or by somatic mutation of ip mimo), e.g., in SOC :z and, in particular, in

catfish However, it is not intended that the term "human antibody" as used herein includes tAb5 in which SOC sequences derived from the male germline of another species (eg, mouse) have been grafted to human EC sequences. Antibodies to human CSCs of the invention may be designated as "anti-cSCs" or "anti-C(B).

The term "specifically binds" or a similar term means that the antibody, or its

The antigen-binding fragment forms a complex with the antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 105 M or less (for example, a lower value of Ko indicates stronger binding). Methods for determining the fact of specific binding of two molecules are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds pPOSOK may show cross-reactivity to other antigens, such as molecules of ZSOK from another species. Moreover, in this context, multispecific antibodies that bind to PpOSOK and one or more other antigens, or bispecific antibodies that bind to two different regions of pPOSO, however, are considered as antibodies that "specifically bind" pOSOK.

The term "high-affinity" antibody refers to those tAbrB5, the binding affinity of which with

POSOR, expressed as Ko, is at least 1079 M; mostly, 1079 M; more acceptable, 1071 M, even more acceptable, 1072 M, as determined by the method of surface plasmon resonance, for example, VIASOKE "M, liquid-phase EG IA.

By the term "slow (low) dissociation rate", "Koi" or "ka" is understood that the antibody dissociates from the complex with POSOK with a rate constant of 1 x 103 sec" or less, preferably 1 x 102 sec! or less, according to the definition by the method of surface plasmon resonance, for example, VIASOKE "M,

The terms "antigen-binding region" of an antibody, "antigen-binding fragment" of an antibody and the like in this context include any natural, enzyme-derived (enzymatic), synthetic or genetically engineered polypeptide or glycoprotein that specifically binds antigen with the formation of a complex. The terms "antigen-binding region" of an antibody, "antigen-binding fragment" of an antibody, and the like in this context refer to one or more fragments of an antibody that retain the ability to specifically bind to antibodies.

According to specific embodiments of the invention, the antibody or antibody fragments of the invention can be associated with a therapeutic agent ("immunoconjugate"), such as a second antagonist of the MAPS, or a biguanide (metformin), a sulfonylurea (such as glyburide, glipizide), an agonist A gamma PPAR (such as pioglitazone or rosiglitazone), an alpha glucosidase inhibitor (such as acarbose or voglibose), EHEMATIOEF (exenatide, glucagon-like peptide 1), ZUMI IMF bo (Symlin (pramlintide)), a chemotherapy agent, a radioisotope, or any other therapeutic an agent used to treat a disease or condition caused, in part, by unwanted glucagon activity.

The term "isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies (AB5) having distinct antigenic specificities (eg, an isolated antibody that specifically binds POSOK, or a fragment thereof, substantially does not contain Abv, which specifically bind antigens other than PNOSOC).

It is assumed that the term "blocking antibody" or "neutralizing antibody" (or "antibody that neutralizes SOC activity") refers to an antibody, the binding of which to pOSOOK leads to the inhibition of at least one biological activity of ZSOK. For example, an antibody according to the invention can contribute to the prevention of an increase in blood glucose levels due to an increase in glucagon levels. Alternatively, the antibody of the invention may exhibit the ability to block the production of CAMP in response to glucagon. This inhibition of the biological activity of the ZSOC can be assessed by quantifying one or more parameters of the biological activity of the ZSOC by one or more of some standard ip migo or ip mimo assay methods known in the art (Examples section, see below).

The term "surface plasmon resonance" in this context refers to an optical phenomenon that allows real-time analysis of biomolecular interactions by detecting changes in protein concentration in the biosensor matrix, for example, using a system

VIASOKE "m (Rpagtasia Viozepzog AV, Orrzaia, Zuedep apa Rizsagamzhau, M..).

It is assumed that the term "Koi" in this context refers to a specific antigen-antibody interaction.

The term "epitope" refers to an antigenic determinant that interacts with a specific binding site in a variable region of an antibody molecule known as a paratope. One antigen can have more than one epitope. Thus, different antibodies can bind to different areas on the antigen and can have different biological effects. The term "epitope" also refers to a site on an antigen to which B and/or T cells respond. It also refers to the area of the antigen that is bound by the antibody. Epitopes can be defined as structural or functional.

Functional epitopes are usually a set of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e. consist of non-linear amino acids. According to certain embodiments of the invention, epitopes may include determinants that are chemically active groups on the surface of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, according to certain embodiments of the invention, may have specific three-dimensional structure characteristics, and/or specific charge characteristics.

The term "substantially (virtually) identical" or "substantially (virtually) identical" with respect to a nucleic acid or fragment thereof indicates that in optimal alignment, with appropriate nucleotide insertions or deletions, to another nucleic acid (or complementary to chain) nucleotide sequence identity is observed for at least approximately 90 95, and more preferably at least approximately 95 95, 96 95, 97 95, 9895 or 9995 nucleotide bases, as determined using any known sequence identity algorithm, such as EA5TA, VI A5T or SAR discussed below. A nucleotide molecule that is substantially identical to a reference nucleotide molecule may in some cases encode a polypeptide that has the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleotide molecule.

As applied to polypeptides, the term "substantially (substantially) similarity" or "substantially (substantially) similar" means that the two peptide sequences when optimally aligned, such as alignment by the programs CAP or BE5ZTRIT, using default hepa weights have a degree of sequence identity of at least 90 95, more preferably a degree of sequence identity of at least 9595, 9895 or 99 95.

Preferably, the positions of residues that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitution" represents a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (LC group) with similar chemical properties (eg, charge or hydrophobicity). As a rule, a conservative amino acid substitution essentially does not change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to adjust for the conservative nature of the substitution. Methods of implementing such regulation are well known to specialists in this field of technology. for See, for example, Reagzop (1994) Meipodz Moi. Vioi 24: 307-331. Examples of groups of amino acids having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl-containing side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; b) acidic side chains; aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of amino acids for conservative substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the РАМ250 matrix of the log-likelihood function, which is revealed in the article by Soppei et al. (1992) bsiepse 256: 1443-45. A "moderately conservative" replacement is any change that has a non-negative value in the RAM250 matrix of the log-likelihood function.

Sequence similarity of polypeptides is usually quantified using a sequence analysis program. The protein analysis program allows matching pairs of similar sequences using similarity scores assigned to various substitutions, deletions, and other modifications involving conservative amino acid substitutions. For example, the CO program package contains programs such as CAP and WEB5TRIT that can be used with default parameters to determine sequence homology or sequence identity of closely related polypeptides, such as homologous polypeptides from organisms of different species or a wild-type protein and its mutein. See, for example, SO Megviop 6.1. Polypeptide sequences can also be compared using EA5TA with default or recommended parameters; program SSO Megvsiop 6.1. EAZTA (for example, EAZTA? and

EABZTAZ) provides for the alignment and identity (in percentage) of the sequences of the regions of greatest overlap between the requested and sought sequences (Reagzop (2000) 5irga).

Another preferred algorithm for comparing the sequence of the invention with a database that includes a large number of sequences of different organisms is the computer program VIA5T, especially VI A5TR or TVI A5TM, which use default parameters. See, for example, Aivspii eyii ai. (1990) 9. Mine. Vioi 215: 403 410 th (1997) Mysivys Asiah Nev. 25: 3389 402.

According to specific embodiments of the invention, the antibody or antibody fragment for use in the method according to the invention can be monospecific, bispecific or

With multispecific. Multispecific antibodies may be specific to different epitopes on the target polypeptide or may contain antigen-binding domains specific to epitopes of more than one target polypeptide. A typical format of a bispecific antibody that can be used in the context of the present invention includes the use of the first immunoglobulin (4) SnZ3 domain and the second immunoglobulin Id Sn3 subtype, where the first and second Id Sn3 domains differ from each other by at least one amino acid, and where the difference is at least one amino acid weakens the binding of a bispecific antibody to Protein A compared to a bispecific antibody that does not have such a difference between amino acids. According to one variant of the implementation of the invention, the first Id Sn3 domain binds Protein A, and the second Id Sn3 domain contains a mutation that weakens or abolishes binding to Protein A, such as a NOK modification (according to IMOT exon numbering; NiZ5K EO numbering) . The second Сn3 may additionally contain the modification М9бЕ (IMOT numbering; У436Е according to EU numbering). Other modifications that may be in the second SNZ include: ЮО16Е, И18М, М445, К52М, М57М and мМ821И (IMOST numbering; ОЗ356Е, И 358М, М3845, К392М, М397М and М422И BSHO numbering) in the case of (901 tAbr5; М445,

K52M and M82I (IMOT; M3845, KZ392M, and M4221I; E) in the case of Ids2 tAbr5;i 15, M445, K52M, M57M,

Eb69K, E790 and M82I (IMOT; OZ355E, M3845, K392M, M397M, K409K, E4190, and M4221 according to EM) in the case of 39054 tAbB5. Variants of the bispecific antibody format described above are considered within the scope of the present invention.

The term "therapeutically effective amount" means the amount that produces the desired effect for which it is administered. The exact amount depends on the purpose of treatment and is established by a specialist in this field of technology using known methods (see, for example, PIoua (1999) Tpe Agi, zsiepse apa TesppoYodu oi Rpagtaseshisa! Sotroypaipo).

The term "normal glucose levels" refers to average human plasma glucose levels of less than about 80 mg/dL for fasting glucose, less than about 110-120 mg/dL for postprandial glucose levels. Plasma glucose levels can be determined according to

Eyuep ei ai., (Meyaroiizet 2000; 49(5): 684-688) or calculate by transferring from the concentration of glucose in whole blood according to O'Ogalio ei ai., (Siip. Snet. I ab. Mei. 2006; 44( 12): 1486-1490). The terms "cholesterol normalization" or "normal cholesterol levels" refer to a person's total cholesterol level of less than approximately 200 mg/dL, with the range of about 200-240 mg/dL considered the upper limit. Taking into account the normal value of 60 for total cholesterol, the average value of I 0 in a person, approximately, from 100 to approximately 129 mg/dL is considered normal, and for NOI, the value of 45 mg/dL is considered normal.

The normal level of triglycerides in humans is less than 150 mg/dL. Normal ratio of total cholesterol/NOI. is less than 4.5, and the normal ratio

GOY/NOI. less than 3. These values can be determined according to standard laboratory practice (see also Egiedezhmaiy, UMT, Siip. Spet (1972), 18: 499-502; Spep, U. ei ai. I irid5 Neain riv. (2010); 9: 52; Keeemii, Ua, et al., Sixtsayop (2007), 115: 1363-1370; and WaiigaKiagi, E. et al., Siip.

Viospet. (2000)33: 549-555). According to certain variants of the implementation of the invention, antibodies to aCaB can be used to reduce blood glucose levels to normal values. According to certain variants of the implementation of the invention, antibodies to 5SOK can be used to increase the levels of NOI -C. According to certain variants of the implementation of the invention, antibodies to CSO B can be used to reduce the level of triglycerides

General description

Because glucagon produces its physiological effects by signaling through the glucagon receptor, the glucagon receptor may be a potential target in the treatment of diabetes and other glucagon-related metabolic disorders. The use of glucagon receptor antagonists, such as the antibodies of the present invention, can be an effective way to achieve normal glucose levels, thereby reducing the intensity or preventing long-term complications due to diabetes. Antibodies of the present invention may also be used to ameliorate conditions associated with, for example, impaired glucose tolerance, to treat obesity, to prevent weight gain, to treat metabolic syndrome, to treat diabetic conditions, including diabetic ketoacidosis, or to prevent and /or reducing the risk of developing one or more complications due to diabetes, such as nephropathy, neuropathy, retinopathy, cataracts, stroke, atherosclerosis, delayed wound healing and other complications associated with diabetes known to those skilled in the art.

Antibodies to PPOSOC described in this application may also be used to treat other conditions, including hyperglycemia, hyperosmolar hyperglycemia syndrome (iopeg, (.0.,

Ategisap Ratiiu RNuzisiap, (2005), 71(9): 1723-1730; Oiareye5 Zresipt, Shotriete, A.E., (2002)15(1): 28-36; Mydepi, V.MU/., Etegdepsu Meadisipe Siipisvo oi Moi Ategiisa, (2005), 23: 629- 648)), perioperative hyperglycemia (Rgizsp, A. ei aYi. Oiareyez Sage, (2010), 33(8): 1783 -1788;

Napagaki, K. ei aI. Uuopa )U Savigoepiegoi, (2009), 15(33): 4122-4125; Ztiieu, O.O. her a. 5th Thursday

Meadis! Choshgpai, (2006), 99(6): 580-589; Negtapidezv, u. eyi aI., Tpe Meipepapovz .). Oi Med. 67(6): 226-229; Mayega, I.I... ei aI., Syctepi Oripiop ip Stiiisa! Sage, (2011), 17: 370-375), hyperglycemia in patients of the intensive care unit ((zipvi, «). ,

M-V., Styisa! sage, (2010), 14: 231), hyperinsulinemia and the syndrome of insulin resistance and glucagonoma (pancreatic endocrine tumor, accompanied or not accompanied by necrotic migratory erythema and hyperglycemia (see, for example, Vodep, A. Eiai., M Epai U Mea (1986); 314: 1686-1689).

According to certain embodiments of the invention, antibodies according to the invention were obtained from mice immunized with a primary immunogen, followed by immunization with a secondary immunogen.

The immunogen can be a cell line expressing the HR protein or its biologically active fragment, or DNA encoding the SOVA protein or its active fragment, or (SOVA protein or its active fragment. For example, according to certain variants of the implementation of the invention, the primary immunogen can be a cell line obtained by standard methods known in the art to overexpress a full-length pSOCA (e.g., the murine MOI87 cell line). Alternatively, DNA-mediated immunization can be performed using DNA encoding a full-length pSOCA (e.g., constructs of using a protein with MP accession number 000151.1), or DNA encoding its biologically active fragment, for example, DNA encoding the M-terminal domain (COM (see

(eg, 5EO IO MO:158, which encodes 5EO IO MO:159), or a soluble M-terminal protein, including a protein with the sequence 5EO IO MO:159, or amino acids encompassing residues 27-144 in 5EO IO MO:153 The secondary immunogen can be a C2OSSOV protein, or its biologically active fragment, or a fusion protein, such as NESOeV-ttH (VESM547, 5EO IUO MO: 151) or naSav-nes (VESMZ315, 5EO IO MO: 150; VESM316, 5EO IU MO: 149).

According to certain embodiments of the present invention, the M-terminal domain having the amino acid sequence shown in BEO IJOO MO: 159 (without the signal sequence), or any one or more ectodomains of the ectodomain, e.g., any one or more extracellular regions (or their fragments) can be used to obtain antibodies that bind (SON) and inhibit its function, for example, its ability to bind glucagon, which would result in a decrease in blood glucose levels.

The full-length amino acid sequence of human OWL is shown as 5EO IO MO: 153.

The signal peptide covers amino acid residues 1-26 of the sequences 5EO and MO: 153; M- terminal domain covers residues 27-144 of 5EO IU MO: 153; extracellular region 1 (ECI1) covers amino acid residues 194-226 5EO IO MO: 153; extracellular region 2 (EC2) covers amino acid residues 285-305 5EO IO MO: 153; and extracellular region C (ESZ) covers amino acid residues 369-384 of 5EO IO MO: 153.

According to certain embodiments of the invention, antibodies that specifically bind to ccCaB can be obtained using fragments of the above-mentioned extracellular regions or peptides that are outside the named regions, extending them by approximately 5 to 20 amino acid residues from any M or From the end or from both ends of the regions according to this description. According to certain embodiments of the invention, any combination of the above-mentioned regions or their fragments can be used to produce antibodies specific for MAMMALS. According to certain embodiments of the invention, any one or more of the above-described regions or their fragments can be used to obtain monospecific, bispecific, or multispecific antibodies.

Antigen-binding fragments of antibodies

Unless specifically stated otherwise, the term "antibody" in this context is understood to encompass antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (ie, "complete antibody molecules"), as well as antigen-binding fragments thereof.

The terms "antigen-binding region" of an antibody or "antigen-binding fragment" of an antibody and the like in this context include any natural, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex . The terms "antigen-binding region" of an antibody or "antigen-binding fragment" and the like in this context refer to one or more fragments of an antibody that retain the ability to specifically bind to pOSOK.

The antibody fragment may include a Rar fragment, an E(ab) fragment, an Em fragment, an Ad fragment, a fragment containing SOC, or a designated SOC region. Antigen-binding fragments of an antibody can be obtained, for example, from whole antibody molecules by appropriate standard methods, such as proteolytic cleavage, or by recombinant

From DNA (genetic engineering methods), including manipulation of DNA and expression of DNA encoding the variable and (optionally) constant domains of an antibody. Such DNAs are known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and processed by chemical or molecular biological methods, for example, to inform one or more variable and/or constant domains of the appropriate configuration, or introduce codons, create cysteine residues, modify, add or remove amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Rab fragments; (iii) fragments of KE(ab) 2; (iii) fragments of Ha; (im) fragments of Em; (m) single-chain Em (csEm) molecules; (m) AB fragments; and (my) minimal recognition elements consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., a dedicated complement-determining region (CDR), such as the SOMKZ peptide), or an inhibited (spatially complicated) EK3-SONZ-EK4 peptide . Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, SOC-grafted antibodies, di-antibodies, tri-antibodies, tetra-bodies, mini-antibodies, nano-antibodies (e.g., monovalent nano-antibodies, bi-valent nano-antibodies, and so on) , small modular immunopharmaceutical proteins (ZMIR5) and shark antibody (novel antigen receptor) variable domains (DMAK) are also encompassed by the term "antigen-binding fragment" of this specification.

Antigen-binding fragment of an antibody, as a rule, contains at least one variable domain. The variable domain can be of any size and amino acid composition and usually contains at least one region of SOC that is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having the Mn domain associated with the Mi domain, the Mn and M domains can be arranged relative to each other in any appropriate order. For example, the variable region can be dimeric and contain Mn-Un, Mn-Mi or Mi-Mi dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric Mn or M domain.

According to certain embodiments of the invention, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting typical configurations of variable and constant domains that can be found in an antigen-binding fragment of an antibody 60 include: (i) Mn-Sni; (iii) Mn-Sne; (iii) Mn-SnZ; (him) Mn-Sni-Snge; (m) Mn-Sni-Sna-Snz; (mi) Mn-Sna-

SnZ; (my) Mn-Si; (miii) Mi-Sn1; (their) Mi-Snge; (x) Mi-SnZ; (hi) Mi-Sn1-Sne; (khii) MI-Sn1i-Sne-SnZ; (hiii) Mi-

Sne-SnZ; and (chem) Mi-Si. In any configuration of the variable and constant domains, including any of the typical configurations listed above, the variable and constant domains may be either directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region can consist of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, which as a result provides a flexible or semi-flexible connection between adjacent variable and/or constant domains in one polypeptide molecule. In addition, the antigen-binding fragment of the antibody of the present invention may contain a homodimer or heterodimer (or other multimer) of any configuration of the variable and constant domains listed above, noncovalently linked to each other and/or to one or more monomeric Mn or Mi domain (for example, disulfide bond(s)).

As in the case of complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody usually contains at least two different variable domains, and each variable domain is able to specifically bind to a separate antigen or to different epitopes on the same antigen. Any format of a multispecific antibody, including typical formats of a bispecific antibody of the present invention, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using standard methods known in the art.

Obtaining human antibodies

Methods of obtaining monoclonal human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to human SOM.

When using MEGOSIMMYOME "M" technology (see, for example, US patent 5 6,596,541,

Kedepegop RPagtasecsiyaye, MEGOSIMMYOMEF) or any other method of obtaining monoclonal antibodies, high-affinity chimeric antibodies to ZSOK, having a human variable region and a mouse constant region, were initially isolated. MEGOSIMMYOME TM technology includes the production of a transgenic mouse whose genome contains variable regions of heavy and light

From the chain, functionally related to the endogenous loci of the mouse constant region, so that in response to antigenic stimulation, an antibody containing the human variable region and the mouse constant region is produced in the mouse. The DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and functionally linked to the DNA encoding the human constant regions of the heavy and light chains. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a MEGOSYMMIOME M mouse is immunized with the desired antigen and lymphoid cells (such as B cells) are taken from mice that express antibodies. Lymphatic cells can be fused with a myeloma cell line to obtain immortalized hybridoma cell lines, and such hybridoma cell lines are screened to identify hybridoma cell lines that produce antibodies specific for the target antigen. DNA encoding the variable regions of the heavy chain and the light chain can be isolated and linked to isotypic, which are similar, constant regions of the heavy chain and the light chain. Such an antibody protein can be produced in a cell such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or variable domains of light and heavy chains can be isolated directly from antigen-specific lymphocytes.

First, high-affinity chimeric antibodies containing a human variable region and a mouse constant region are isolated. As shown below, in the experimental part, antibodies are characterized and selected according to the required characteristics, including affinity, selectivity, epitope, and so on. Mouse constant regions are replaced with the desired human constant region to obtain a fully human antibody of the invention, for example, wild-type or modified Ids1 or Ids4. While the selected constant region may vary according to the specific application, the variable region is responsible for characteristics such as high-affinity binding to the antigen and specificity to the target.

Typically, the antibodies of the present invention have a very high affinity, typically

The KO, which is determined by binding to an antigen immobilized either on a solid or in a liquid phase (solution), is approximately from 10-72 to approximately 109 M. Mouse constant regions were replaced with the required human constant regions, yielding fully human antibodies of the invention. While the selected constant region may vary according to the specific application, the variable region is responsible for such characteristics as high-affinity binding to the antigen and specificity to the target.

Bioequivalents

Antibodies to 5SOK and fragments of antibodies according to the present invention include proteins having amino acid sequences that differ from the sequences of the described antibodies, but retain the ability to bind human CSO. Such variants of antibodies and antibody fragments contain one or more additions, one or more deletions or substitutions of amino acids compared to the original sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the described antibodies. Similarly, DNA sequences of the present invention encoding anti-PSA antibodies include sequences that contain one or more additions, one or more deletions or substitutions of nucleotides compared to the test sequence, but which encode an anti-PSA antibody or antibody fragment that is substantially bioequivalent an antibody to COM or an antibody fragment according to the invention.

Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutically equivalent or pharmaceutically alternative, the rate and extent of absorption of which differ slightly when administered at the same molar dose under similar experimental conditions or as a single dose, or in in the form of a multiple dose. Some antibodies are considered equivalent or pharmaceutically alternative if the degree of absorption but not the rate of absorption is equivalent, and yet they may be considered bioequivalent because such difference in rate of absorption is intentional and reflected in the label, not essential to achieve effective addictive concentrations. for example, with continuous administration, and is considered medically insignificant for the particular medicinal product being studied.

According to one embodiment, two antigen-binding proteins are bioequivalent if there is no clinically significant difference in their safety, purity and effectiveness (activity).

According to one embodiment, two antigen-binding proteins are bioequivalent if the patient can switch, one or more times, the reference product to the biological product without an expected increase in the risk of harmful side effects, including a clinically significant change in immunogenicity or reduced efficacy compared to continuous therapy without such a change (switching).

According to one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or common mechanisms on the condition or conditions, to the extent that these mechanisms have been studied.

Bioequivalence can be demonstrated by IP mimo and IP migo methods. Methods for quantifying bioequivalence include, for example, (a) an IP mimo test in humans or other mammals, in which the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid is determined over time; (b) ip migo test, which is correlated with ip mimo data on bioavailability and which allows reasonable prediction of these data; (c) ip mimo test in humans or other mammals, in which the corresponding acute (short-term, sharp) pharmacological effect of the antibody (or its target) is determined as a function of time; and (d) a rigorously controlled clinical trial in which the safety, efficacy, or bioavailability or bioequivalence of the antibody is established.

Bioequivalent variants of antibodies to SSOC according to the invention can be constructed, for example, by introducing various substitutions of residues or sequences or by making deletions of terminal or internal residues or sequences that are not required from the point of view of biological activity. For example, cysteine residues, which are not essential for biological activity, can be removed or replaced with other amino acids in order to prevent the formation during renaturation of optional or incorrect intramolecular disulfide bridges. In another context, bioequivalent antibodies can include variants of antibodies to MAMMALS containing amino acid substitutions that modify the glycosylation characteristics of the antibodies, for example, mutations that eliminate or delete glycosylation.

Biological characteristics of antibodies

As a rule, the antibodies of the present invention can act by binding to at least one of the extracellular regions of pOSOMK. According to certain embodiments, the antibodies of the present invention can bind to an epitope located in at least one M-terminal region, or to an epitope located in at least one of the extracellular (EC) loops of the passav.

According to certain embodiments, the antibodies of the present invention can act by blocking or inhibiting SOSOC activity by binding to the extracellular M-terminal region, the amino acid sequence of which is given in ZEO IU MO: 159, and which is encoded by the nucleotide sequence 60 given in 5EO IU MO: 158.

In certain embodiments, the antibodies of the present invention may act by blocking or inhibiting COP activity by binding to at least one of the EC loops or segments of loops in the entire receptor. According to one embodiment of the invention, the antibodies of the invention can bind to an epitope located in EC1, approximately between amino acid residue 194 and amino acid residue 226 of the sequences 5EO IO MO: 153.

Or, additionally, antibodies according to the invention can bind to an epitope located in

ES2, located approximately between amino acid residue 285 and amino acid residue 302 of the sequence 5EO IJOO MO: 153. Alternatively, or in addition, the antibodies of the invention can bind to an epitope located in ESZ, located approximately between amino acid residue 369 and amino acid residue 384 of the sequence 5EO IYUO MO: 153.

According to certain embodiments, the antibodies of the present invention may be bispecific antibodies. Bispecific antibodies according to the invention can bind one epitope in EC1 and can also bind one epitope in the NOSOC region other than EC1. According to certain embodiments, the bispecific antibodies of the invention can bind a single epitope in ECI and can also bind a single epitope in EC2 or ECZ, or in the M-terminal region, or any other region in EC1, EC2, ECZ POSOK receptor, or any combination thereof. According to certain embodiments, the bispecific antibodies of the invention can bind to two different sites in the same extracellular region.

More specifically, the anti-COM antibodies of the invention may exhibit one or more of the following properties: (1) the ability to bind to human MAMMA or a fragment thereof and to non-human MAMMA (e.g., mouse, monkey, rat, rabbit, dog, pig, and so on) or its fragment; (2) the ability to connect with the human

a CSC or a fragment thereof, but not with a CSC other than human (for example, mouse, monkey, rat, rabbit, dog, pig, etc.), or a fragment thereof; (3) the ability to bind to a human SSC or fragment thereof and to a non-human primate (eg, monkey) SSC or fragment thereof, but not to a mouse, rat, rabbit, dog, or porcine SSC or fragment of SSC; (4) the ability to bind to a human SOR or a fragment thereof and to SOR. non-human primate (e.g., simian) or a fragment thereof, and with a murine ZSOC or a fragment thereof, but not with a rat

From SSO; (5) the ability to bind to human COM or a fragment thereof and to non-human primate (eg, monkey) MAMMALS or a fragment thereof, and to rat COM or a fragment thereof, but not to mouse SON; b) blocks binding of glucagon to ZSOK; 7) blocks glucagon-induced production of CAMP (CAMP); 8) demonstrates the ability to reduce glucose levels in the blood of people suffering from diabetes and in animal models of diabetes; 9) may or may not reduce triglyceride levels to levels in healthy mammals; or 10) does not have a harmful effect on the level of lipids in the plasma.

Certain antibodies to (against) OSOB of the present invention are capable of inhibiting or attenuating OSOR activity in ip migo or ip mimo assays. The ability of the antibodies of the invention to bind to OSOB and to inhibit glucagon binding to OSOC can be quantified by any standard method known to those skilled in the art, including binding assays, reporter gene assays, such as assays using luciferase reporter.

Non-limiting examples of these assays to quantify SSOC activity are illustrated in Examples 4 and 5 of this application. In Example 4, binding affinities and kinetic constants of human antibodies to pOSOK were determined by the method of surface plasmon resonance and measurements were carried out on a T100 device. In Example 5, a bioassay was performed on HEK293 cell lines expressing full-length human, monkey, and mouse SSOC together with a luciferase reporter to detect activation by co5, and the subsequent increase in CAMP levels and transcriptional activation. Examples 6, 7, 8, 9 and 10 show the effects of antibodies on lowering blood glucose levels, blood ketone levels and weight loss in various animal models.

The present invention also includes anti-MAP antibodies and antigen-binding fragments thereof that bind to at least one biologically active fragment of any of the following proteins or peptides: cZEO IU MO: 153 (full-length PSOC), residues 27-144 of the zEO IU sequence MO: 153 (M-terminal domain of pOSOK); residues 194-226 of the sequence

ZEBEO AND MO: 153; residues 285-305 of sequences 5EBEO IU MO: 153; residues 369-384 of the sequence of FEO IU MO: 153. Any of the ZSOK peptides according to this description, or its fragments, can be used to obtain antibodies to SOM.

Peptides can be modified, including the addition or replacement of some residues for the purpose of introducing a label or for the purpose of conjugation with carrier molecules such as CIN (snail lymph hemocyanin 60). For example, you can add cysteine either at the M-terminus or at the C-terminus of the peptide, or you can add a linker for conjugation, for example, with KIN for immunization. Antibodies specific to

CCCs may not contain additional labels or agents, or they may contain a label or agent at the M-terminus or C-terminus. According to one embodiment of the invention, the label or agent is biotin. In a binding assay, the position of the label (if any) can determine the orientation of the peptide relative to the surface to which the peptide binds. For example, if a surface is coated with avidin, a peptide containing M-terminal biotin will be oriented so that the C-terminal region of the peptide is located away from (distal to) the surface.

According to one embodiment, the invention provides a fully human monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds PSOC and neutralizes the activity of PPOSOC, and the antibody or fragment thereof has one or more of the following characteristics: (i) contains NSUC , having an amino acid sequence selected from the group consisting of ZEO IJUO MO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146; (iii) contains GSMEK having an amino acid sequence selected from the group consisting of 5EO IU

MO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148; (iii) contains any one or more SOKI1 heavy chain sequences selected from the group consisting of 4, 20, 36, 52, 72, 92, 112 and 132; any one or more SOMK2 heavy chain sequences selected from the group consisting of 6, 22, 38, 54, 74, 94, 114, and 134; and any one or more sequences

A heavy chain SOKZ selected from the group consisting of 8, 24, 40, 56, 76, 96, 116 and 136; and any one or more SOMB1 light chain sequences selected from the group consisting of 12, 28, 44, 60, 80, 100, 120, and 140; any one or more SOC2 light chain sequences selected from the group consisting of 14, 30, 46, 62, 82, 102, 122 and 142; any one or more sequences of SOCZ light chain selected from the group consisting of 16, 32, 48, 64, 84, 104, 124 and 144; and their combinations; (m) exhibits binding specificity to any element from the following: the M-terminal region of the ZSOK containing amino acid residues 27-144 of the sequence 5EO IO MO: 153, or to one or more extracellular loops of the ZSOK, including, e.g. , EC1, EC2 or ECZ, where ECI1 contains amino acid residues in the approximate interval from residue 194 to residue 226 of the sequences

ZEO IUO MO: 153, and wherein EC2 contains amino acid residues in the approximate range from residue 285 to residue 305 "E 5EO IU MO: 153; and wherein ECZ contains amino acid residues in the approximate range

From the interval from residue 369 to residue 384 of the sequence "EO IU MO: 153; (m) binds one or more receptors selected from human, monkey, mouse or rat MAMMALS; (mi) blocks the binding of glucagon to the nipples; mi) blocks glucagon-induced production of CAMP (cAMP); mii) has shown the ability to reduce blood glucose levels or blood ketone levels in people with diabetes and in animal models of diabetes; mii) may or may not reduce triglyceride levels to levels observed in healthy mammals; or ih) does not have a harmful effect on plasma lipid levels.

Epitope mapping and related methods

To screen for antibodies that bind to a specific epitope, a cross-blocking assay can be used, such as the assay described in Apiibodie5, Nagiom/ apa I ape (Soy Zrgipd Nagrog Rgezz, Soy Zrgipd Nagr., MU). Other methods include alanine-scanning mutagenesis, peptide blotting (Keippe, 2004, Meiod5 Mo! Bioi 248:443-463) or peptide digestion. In addition, such methods as epitope exclusion, epitope extraction and chemical modification of antigens can be used (Toteg, 2000, Rgoivip 5siepse 9: 487-496).

The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed either from adjacent amino acids, or not from adjacent amino acids, which converge during protein folding to form a tertiary structure. Epitopes formed from adjacent amino acids are usually preserved when exposed to denaturing solvents, whereas epitopes formed by folding into a tertiary structure are usually lost when treated with denaturing solvents. Usually, the epitope includes at least 3, and more often, 5 or 8-10 amino acids in a unique spatial conformation.

Model-based (antibody) profiling (MAP), also known as antigen structure-based antibody profiling (SAB), is a method that allows the classification (categorization) of large numbers of monoclonal antibodies (tAb5) specific to the same antigen , based on the similarity of the binding profile of each antibody to antigen surfaces modified by chemical or enzymatic methods (see patent

USA Z 2004/0101920). Each category may represent a unique epitope that is either markedly different from an epitope represented by another group, or that partially overlaps with an epitope represented by another group. This method allows for rapid filtering of genetically identical antibodies so that characterization is focused on genetically different antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma cell clones that produce tAbB5 with desired characteristics. MAP can also be used for the distribution of antibodies to SCC by groups of antibodies that bind different epitopes.

According to certain variants of the implementation of the invention, an antibody to COM. or the antigen-binding fragment of the antibody binds an epitope in at least one of the extracellular regions of MAMMALS, where the extracellular region is the M-terminal domain or one of the EC loops, including EC1, EC2, or

EHS as previously described.

According to one embodiment of the invention, the antibody binds an epitope in the M-terminal region

ССО, or its fragment containing an amino acid sequence in the approximate interval that includes amino acid residues 27-144 of the sequence BEO IU MO: 153. According to one embodiment of the invention, the antibody binds the EC1 epitope, or its fragment containing an amino acid sequence in in the approximate interval that includes amino acid residues 194-226 of the sequences 5EO IU MO: 153. According to one embodiment of the invention, the antibody binds an epitope in EC2 of the CSC, or its fragment, containing an amino acid sequence in the approximate interval that includes amino acid residues 285- 305 of the ZEO IJU MO: 153 sequence. According to one embodiment of the invention, the antibody binds an epitope in the ECZ, or its fragment containing an amino acid sequence in the approximate interval that includes amino acid residues 369-384 of the ZEO IJU MO: 153 sequence.

According to certain variants of the implementation of the invention, an antibody or antibody fragment binds an epitope that includes more than one of the listed epitopes of ZSOK in the M-terminal domain or in

EC1, EC2, or ECZ, and/or in two or three other extracellular regions (eg, epitopes in

In the M-terminal region, EC1, EC2 and ECZ loops, or in EC1, EC2 and ECZ loops, or in the M-terminal region, EC2 and ECZ loops, or in the M-terminal region, EC1 and ECZ loops.

According to certain variants of the implementation of the invention, the antibody is a bispecific antibody that binds one epitope in one extracellular region of the SOSC, and the second epitope in another extracellular region of the OSOR, including the M-terminal domain, or EC1, EC2, or ECZ.

According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in the M-terminal region of NOSOC, and the second epitope in EC1 of POSOC. According to one variant of the implementation of the invention, the antibody is a bispecific antibody that binds one epitope in the M-terminal region of PSOR, and the second epitope in EC2 pOSOR. According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in the M-terminal region

POSOB, and the second epitope in ESZ pOSOK. According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in EC1 pOSOK, and the second epitope in EC2 pOSOK. According to one embodiment of the invention, the antibody is a bispecific antibody, which binds one epitope in ES1 POSOK, and the second epitope in ESZ pOSOK. According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in EC2 pOSOB, and the second epitope in ESZ pPOSOK.

According to one variant of the implementation of the invention, the antibody is a bispecific antibody that binds one epitope in the M-terminal domain of pOSOK, and this one epitope is located in the approximate interval from residue 27 to residue 144 of the sequence c"EO IU MO: 153, and the second epitope in EC1 of pOSOK, and this second epitope is located in the approximate interval from residue number 194 to residue number 226 of the sequences 5EO IJU MO: 153. According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in the M-terminal domain of pOSOK , and this one epitope is in the above-mentioned interval, and the second epitope is in EC2 pOSO, and this second epitope is in the approximate interval from residue number 285 to residue number 305 of the sequences 5EO IU MO: 153. According to one variant of the implementation of the invention, the antibody is a bispecific antibody , which binds one epitope in the M-terminal domain of PpOSOK, and this one epitope is in the above-mentioned interval, and the second epitope in EC2 pOSOM, and this second epitope is located in the approximate interval from residue number 369 to residue number 384 of the sequence ZEO IU MO: 153.

According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in EC1T POSOK, approximately from residue 194 to approximately residue 226 of the sequences ZEO IU MO: 153 and a second epitope in EC2 COR, approximately from residue 285 , approximately to residue 305 of the 5EBO IU MO sequences: 153. According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in EC1 pOSOB, approximately, from residue 194, approximately, to residue 226 of the ZEO IU MO sequences: 153 and the second epitope in the ESZ of MAMMALS, approximately, from residue 369, approximately, to residue 384 of the sequence ZEO IU MO: 153. According to one embodiment of the invention, the antibody is a bispecific antibody that binds one epitope in EC2 pOSOKK, approximately, from residue 285, 60 approximately, to residue 305 of sequences 5EO IU MO: 153 and the second epitope in ESZ3 SOSOK,

approximately, from residue 369, approximately, to residue 384 of the sequence 5EO IU MO:153.

The present invention includes antibodies to SSOC that bind to the same epitope as any of the specific illustrative antibodies herein (eg, NAN1345M, NANI1617M,

NANIT765M, NANI13218 and H4NI1321R, NANI1327V and H4NI1327R, NANI13288 and NANI1328R, NANI13318 and

NANIZZ31R, NA4NIZ339V and NaA4NIZ339R). The present invention also includes antibodies to SSC that compete for binding to the COM or to a fragment of SSC with any of the specific illustrative antibodies of this description.

One can easily determine whether an antibody actually binds to the same epitope as a reference anti-SCC antibody, or competes for binding with a reference anti-SCC antibody, using standard methods known in the art. For example, in order to determine whether the test antibody binds to the same epitope as the reference antibody to the MAT according to the invention, the reference antibody is left to bind to the protein or peptide of the COP under saturating conditions. Then the ability of the studied antibody to bind to the molecule is determined

SSO If the test antibody is able to bind to the SMA after saturation of binding with the reference antibody to the SMA, it can be concluded that the test antibody binds to a different epitope than the reference antibody to the SMA. On the other hand, if the test antibody is unable to bind to the CO molecule after saturation of binding with the reference antibody to CSO, then the test antibody can bind to the same epitope with which the reference antibody to CSO according to the invention binds.

In order to determine whether the antibody really competes for binding with the reference antibody to OSOBK, the above-described binding method is carried out in two directions: in the first case, the reference antibody is left to bind to the SSOK molecule under saturation conditions, followed by an assessment of the binding of the studied antibody with the SOC molecule. In the second case, the tested antibody is left to bind to the molecule of the SSOK under conditions of saturation with the subsequent assessment of the binding of the reference antibody to the molecule of the SSOK. If in both cases (in two directions) only the first (saturating) antibody is able to bind to the MAMP molecule, then it is concluded that the tested antibody and the reference antibody compete for binding to COM. One of ordinary skill in the art will appreciate that an antibody that competes for binding with a reference antibody may not necessarily bind

Zo with the same (identical) epitope as the reference antibody, but can spatially block the binding of the reference antibody by binding an overlapping epitope or an adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each of them competitively inhibits (blocks) binding to the other antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding to another by at least 50 95, but preferably by 75 95, 9095, or even 9995 as determined by competitive binding assays (see, for example, Uypodpapz ei aI., Sapseg Res. 1990 50: 1495-1502).

Or, two antibodies have the same epitope if virtually (essentially) all amino acid mutations in the antigen that weaken or eliminate binding of one antibody weaken or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that weaken or eliminate the binding of one antibody weaken or eliminate the binding of the other.

Additional standard experiments (eg, peptide mutation analysis and binding analysis) can then be performed to confirm whether the absence of binding of the test antibody is indeed due to the fact that it actually binds to the same epitope to which it binds. the reference antibody binds, or spatial blocking (or another phenomenon) is responsible for the lack of binding in the experiment. Such experiments can be carried out by the methods of EGISA (ELISA), KIA (RIA), surface plasmon resonance, flow cytometry, or any other method of quantitative or qualitative analysis of antibody binding known in the art.

Species selectivity and species cross-reactivity

According to certain variants of implementation of the invention, antibodies to COM are associated with human SSOK, but not with another species' SSOK. Or, according to some variants, antibodies to

The CSCs of the invention bind to the human CSC and to the SOM of one or more non-human species. For example, anti-SOC antibodies of the invention may bind to human SOC and may or may not bind, respectively, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, or mouse SOC. dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus macaque or chimpanzee.

Immunoconjugates 60 The invention encompasses a monoclonal antibody to human SCC conjugated with a therapeutic agent ("immunoconjugate"), such as an agent capable of lowering blood glucose levels, or a radioisotope, or a chemotherapeutic agent. When choosing the type of therapeutic agent that can be conjugated with an antibody to COM, take into account the condition to be treated and the intended therapeutic effect. For example, for the treatment of diabetes or any other condition requiring the lowering of blood glucose levels and/or the maintenance of normal blood glucose levels, an agent such as a biguanide (eg, metformin), a sulfonylurea (eg, glyburide, glipizide), an agonist Gamma PRAK (for example, pioglitazone, rosiglitazone); an alpha glucosidase inhibitor (eg, akrbose, voglibose), an inhibitor of excess formation of glycosylation end products (eg, aminoguanidine), or a second SOC inhibitor can be conjugated to an antibody to MAMM. Alternatively, if the desired therapeutic effect is the treatment of complications or symptoms associated with diabetes, or any other condition caused by high or uncontrolled glucose levels, it may be preferable to conjugate an agent suitable for treating the complications or symptoms of the disease. Examples of agents suitable for the preparation of immunoconjugates are known in the art, see, for example,

International patent application UMO 05/103081.

Multispecific antibodies

Antibodies of the present invention can be monospecific, bispecific or multispecific. Multispecific antibodies may be specific to different epitopes of one target polypeptide or may contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tiy ei ai., 1991, 9. Ittipoi. 147: 60-69;

Kiieg evy a!., 2004, Ttepaz VioyesNpoi. 22: 238-244. Antibodies to SOV according to the present invention can be associated with or can be co-expressed with another functional molecule, for example, with another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (eg, by chemical linkage, fusion by genetic engineering, non-covalent association, or otherwise) to one or more molecular particles, such as another antibody or antibody fragment, to form a bispecific or multispecific antibodies with a second binding specificity. For example, this invention includes bispecific antibodies in which one arm of the immunoglobulin is specific for human cSOV or a fragment thereof, and the other arm of the immunoglobulin is specific for a second

From a therapeutic target or conjugated to a therapeutic agent. According to certain variants of the invention, one arm of the immunoglobulin is specific to the epitope on the M-terminal domain of NASOIV, or its fragment, and the other arm of the immunoglobulin is specific to the epitope on one of the EC loops of PSO, or its fragment. According to certain variants of the implementation of the invention, one arm of the immunoglobulin is specific to one EC loop, or its fragment, and the second arm is specific to the second EC loop, or its fragment. According to certain variants of the implementation of the invention, one arm of the immunoglobulin is specific to one epitope on one EC loop of the band, and the other arm is specific to the second epitope on the same EC loop of NASIV.

A typical bispecific antibody format that can be used in the context of the present invention includes the use of the first Sn3 domain of immunoglobulin (19) and the second Sn3 domain

Id, where the first and second SnZ3 domains of Id differ from each other by at least one amino acid, and at the same time, the difference by at least one amino acid weakens the binding of the bispecific antibody to Protein A compared to a bispecific antibody that does not have this difference. According to one embodiment, the first Id SnZ3 domain binds the Protein

A, and the second IdSn3 domain contains a mutation that weakens or cancels the binding of Protein A, such as a modification of NOBA (according to exon numbering in BMI; Na4Z5A according to EU numbering).

The second SnZ3 domain may additionally contain the U9bE modification (according to IMOST; U436E according to ESH). Other modifications that may be present in the second domain of Cn3 include: O16E, I18M, M445, K52M, M57M, and

M821I (according to IMOT; YuZ356E, I 358M, M3845, KZ392M, M397M and M422I according to EE) in the case of Ids1 antibodies; M445,

K52M and M821I (IMOT; M3845, KZ392M and M4221I according to EE) in the case of Ids2 antibodies; and 015, M445, K52M, M57M,

Eb69K, E790O and M821I (according to IMOST; 0355, M3845, K392M, M397M, K409K, E4190 and M422I according to EU) in the case

Id9c4 antibodies. Variants of the format of bispecific antibodies, described above, are considered within the scope of the present invention.

Medicinal forms and their introduction

The invention provides pharmaceutical compositions containing antibodies to SOC or their antigen-binding fragments according to this invention. The pharmaceutical compositions of the invention are administered with appropriate carriers, excipients, and other agents included in the composition to provide better tolerability, better delivery, tolerability, and the like.

A huge number of suitable dosage forms can be found in the pharmacological reference book known to all chemists-pharmacists: Ketipdiopye RPpaptaseciisa! 5siepsez, Mask Rybiyepipad 60 Sotrapu, Eaziyuop, RA. These dosage forms include, for example, powders, pastes, ointments, jellies,

wax, oils, lipids, lipid (cationic or anionic) - containing vesicles (such as LIPOFECTIN M),

DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbovac emulsions (polyethylene glycols of different molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. See also Romei! her a. "Sotrepaisht oi eksiriepiv og ragepivga! Toptiaiopv"

ROA (1998) ) Ripagt 5si TesNnpoi! 52: 238-311.

The dose of the antibody may vary depending on the age and size of the patient to whom it is administered, the disease, condition, route of administration, etc. When an antibody of the present invention is used to lower blood glucose levels due to COM activity in various conditions or diseases, such as diabetes, in an adult patient, it is usually appropriate to administer the antibody of the present invention intravenously in a single dose of about 0.01 to about 30 mg/kg body weight, more preferably from about 0.02 to about 7, from about 0.03 to about 5 or from about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, an antibody or antigen-binding fragment thereof of the invention may be administered in an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, up to about 100 mg or up to about 50 mg. According to certain variants of the implementation of the invention, the initial dose may be followed by the introduction of a second or several subsequent doses of the antibody or its antigen-binding fragment in an amount that may be approximately equal to or lower than the initial dose, and subsequent doses are administered at intervals of at least 1 days to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least b weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known that can be used to introduce the pharmaceutical composition according to the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, UMy et al., 1987, 9 262: 4429-4432). Methods of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural (and oral) routes. The composition may be administered by any conventional route, such as by infusion or bolus injection, by absorption through the epithelial or skin-mucosal lining (eg through the oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents.The administration can be systemic or local.

The pharmaceutical composition can also be delivered in vesicles, in particular in liposomes (see, for example, IGapdeg (1990) 5siepse 249: 1527-1533).

In some cases, the pharmaceutical composition can be delivered in the form of a controlled release system. According to one embodiment, a pump can be used. According to another embodiment, polymeric materials can be used. According to another variant of the implementation of the invention, the system with controlled release can be placed near the target of the composition, while only a part of the systemic dose is required.

Preparations for injections may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These drugs for injection can be prepared by generally known methods. For example, preparations for injections can be prepared by dissolving, suspending or emulsifying the antibody or its salt, described above, in a sterile aqueous medium or oil medium, which is usually used for injections. As an aqueous injection medium, for example, physiological saline solution, isotonic solution containing glucose and other additives, and so on, which can be used in combination with a suitable solubilizing agent, such as an alcohol (for example, ethanol), a polyol ( for example, propylene glycol, polyethylene glycol), nonionic surface-active substance I, for example, polysorbate 80, HCO-50 (hydrogenated castor oil/polyoxyethylene adduct (50 mol.))|, and so on. As an oil medium, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent, such as benzyl benzoate, benzyl alcohol, etc., are used. Preparations for injections obtained at the same time are preferably placed in a suitable ampoule.

The pharmaceutical composition of this invention can be administered subcutaneously or intravenously using a standard needle and syringe. In addition, a syringe pen is used for subcutaneous delivery of the pharmaceutical composition according to this invention. Such a pen can be reusable or disposable. A reusable syringe pen usually uses a replaceable cartridge that contains a pharmaceutical composition. After all the pharmaceutical composition in the cartridge is used and the cartridge becomes empty, the empty cartridge can easily be discarded and replaced with a new one containing the pharmaceutical composition. After that, the pen can be used again. There is no replaceable cartridge in the disposable syringe pen. More specifically, a disposable pen comes with a reservoir pre-filled with a pharmaceutical composition within the device. After the pharmaceutical composition in the reservoir is used, the entire device is thrown away.

Numerous reusable syringe pens and autoinjectors (syringe tubes) are used for subcutaneous delivery of the pharmaceutical composition according to this invention. Examples include, but are of course not limited to, AOTOREM M (Ou/"ep Myptioga, Ips., Uosaziosk, OK), a syringe pen

RIBETKOMIS "m (bizeigopis Meais! Zuyetv5, Vegodaogi, Zm/y2epapa), syringe pen NOMASOS MIH 1715/2574, syringe pen NOMAG OS tm, syringe pen NOMASIM 70/30 "m (E|yi Shu apa So., Ipaiaparoi 5,

IM), MOMOREM M|, Poi SHI (Momo MogaizK, Soreppadep, Oeptagk), MOMOREM UOMIOK M (Momo

MogaiiK, Soreppadep, YuOeptagk), PTO syringe pen (Vesiop bBisKipzop, EgapKiip Iakev5, MU),

ORTIREM"M, ORTIREM RKO"M, ORTIREM 5TAKIGET"M and ORTISGIK mm (drinking amepiivs, Egapkishn,

Segptapa), and these are just some of them. Examples of disposable pens used for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are of course not limited to, ZOGOBTAK"M (Zapoii-Amepiii), RGEHREM"M (Momo Mogai5K) and

KUMIKREM "M (Eii and Shu), auto-injector ZOKESI ISKTM (Atdep, Tpoizapa Oak5, SA), REM'ETTM (Nazeitviseg, biiNdap, Septapu), ERIREM (Oeu, GR.) and syringe-pen NOMIKATM (AbBroi I arz,

Arroi Ragk IM), and these are just some of them.

It is advisable to prepare the above-described pharmaceutical compositions for oral or parenteral use in dosage form in the form of a single dose, depending on the dose of the active ingredients. Such dosage forms in the form of a single dose include, for example, tablets, pills, capsules, ampoules for injections, suppositories, etc. The amount of the above-mentioned antibody is usually from about 5 to about 500 mg per dosage form in the form of a single dose; in particular, it is preferable that the injection form contains from about 5 to about 100 mg, and for other dosage forms from about 10 to about 250 mg of the above-mentioned antibody.

Therapeutic use of antibodies

By interacting with the glucagon receptor, the antibodies of this invention can

Zo is used to reduce blood glucose levels, as well as for the treatment of a wide range of conditions and disorders in which blockade of the interaction of glucagon with its receptor is beneficial. These disorders and conditions may be selected from any glucagon-mediated metabolic disorder that involves glucagon receptor signaling that ultimately leads to the pathophysiology of the disorder or to the body's homeostatic response to the disorder. Therefore, the antibodies may find use, for example, in the prevention, treatment or mitigation, alleviation of diseases or conditions, or associated symptoms or complications, of the endocrine system, the central nervous system, the peripheral nervous system, the cardiovascular system, the bronchopulmonary system and gastrointestinal tract by reducing or eliminating one or more unwanted side effects caused by modern drugs. Metabolic disorders associated with glucagon include, but are not limited to, type 1 and type 2 diabetes, diabetic ketoacidosis, hyperglycemia, hyperosmolar hyperglycemia syndrome, perioperative hyperglycemia, intensive care unit hyperglycemia, hyperinsulinemia, postprandial hyperglycemia, impaired fasting glycemia (IS), metabolic syndrome, hyper/hypokalemia, low II/NOG ratio, eating disorders, weight gain, obesity as a result of diabetes, childhood diabetes, gestational diabetes (pregnancy diabetes), late complications of diabetes, micro-/macroalbuminuria, nephropathy , retinopathy, neuropathy, diabetic foot syndrome, wound healing, impaired glucose tolerance (IST), insulin resistance syndrome, X syndrome, glucagonomas, gastrointestinal disorders, obesity, obesity-induced diabetes, and so on. In addition, this invention provides a method of treating conditions caused by excess glucagon in a mammal; a method of inhibiting the glucagon receptor in a mammal; a method of inhibiting the cellular response mediated by the glucagon receptor in a mammal; or a method of reducing the level of glucose (glycemia) in a mammal, which consists in administering to the mammal in need of such treatment, an amount of an antibody to ESA, or a fragment thereof, sufficient to inhibit the glucagon receptor.

Antibodies of the present invention effectively lower both fasting and postprandial glucose levels.

In certain embodiments, the antibodies of the present invention are useful in the preparation of a pharmaceutical composition for the treatment of type 2 diabetes. In yet another embodiment, the antibodies of the present invention are useful in the preparation of a pharmaceutical composition for delaying or preventing disease progression from impaired glucose tolerance to type 6 diabetes 2. According to another embodiment, the antibodies of the present invention are useful for the preparation of a pharmaceutical composition for delaying or preventing disease progression from non-insulin-dependent diabetes to insulin-dependent diabetes. According to another embodiment, the antibodies of the present invention are useful for the preparation of a pharmaceutical composition for the treatment of type 1 diabetes. Such treatment is usually accompanied by insulin therapy.

Combined medicines

Combined medicinal products may include an antibody to pOSO according to the invention and any additional therapeutic agent, which preferably can be used with an antibody according to the invention or with a biologically active fragment of an antibody according to the invention.

For example, the second therapeutic agent can be used to further reduce glucose levels or to reduce the intensity of at least one symptom in a patient suffering from a disease or condition characterized by high blood glucose levels, such as diabetes. Such a second agent can be selected, for example, from a glucagon antagonist or another SSOC antagonist (eg, an antibody to glucagon or to

SSRIs or low molecular weight glucagon inhibitor or SSRIs), or it may include other therapeutic agents useful in the treatment of diabetes or other diseases or conditions associated with elevated blood glucose levels or impaired glucose metabolism, or resulting from elevated glucose levels in the blood or impaired glucose metabolism. These agents include biguanides, which decrease hepatic glucose production and increase insulin sensitivity (eg, metformin), or sulfonylureas, which stimulate insulin production (eg, glyburide, glipizide). Other drugs aimed at maintaining glucose homeostasis include PRAC gamma agonists that act as insulin sensitizers (eg, pioglitazone, rosiglitazone); alpha glycosidase inhibitors, which slow down the absorption of starch (carbohydrates) and the production of glucose (for example, acarbose, voglibose). Other medications include injectable medications such as exenatide F (glucagon-like peptide 1) and simlinF (pramlintide).

According to certain variants of implementation of the invention, the composition may include a second agent selected from the group consisting of non-sulfonylurea secretion stimulators, insulin, insulin analogs, exendin-4 polypeptides, beta-3 adrenoceptor agonists, PPAK agonists, dipeptidyl inhibitors IM peptidases, statins and statin-containing combinations, cholesterol absorption inhibitors, IO-cholesterol antagonists, cholesterol ester carrier protein antagonists, endothelin receptor antagonists, growth hormone antagonists, insulin sensitizers, amylin mimetics or agonists, cannabinoid receptor antagonists, glucagon-like peptide agonists -1, melanocortins, melanin-concentrating hormone receptor agonists, ZMPRKI5, and protein tyrosine phosphatase inhibitors.

According to other certain embodiments of the invention, the combination therapy may include the administration of a second agent that counteracts any potential side effect(s) due to the administration of the antibody of the invention, if such side effect(s) -th) effect(s) occur(s). For example, if any of the anti-MAP antibodies raise lipid or cholesterol levels, it may be appropriate to administer a second lipid- or cholesterol-lowering agent, such as an NMO-CoA reductase inhibitor (eg, a statin such as atorvastatin (Lipitor, GIRITOCF ), fluvastatin (Lescol, GEBSOY F), lovastatin (Mevakor, MEMASOKF)), pitavastatin (IMAGOF)), pravastatin (Pravahol, RKAMASNOIF), rosuvastatin (Krestor, SKEBZTOKF) and simvastatin (Zokor, 27OSoOKF)) etc. Alternatively, the antibodies of the invention can be administered in combination with an agent such as

MUTOVIMF) (Vytorin), which is a combination of a statin and another agent, such as the ezetimibe/simvastatin combination.

According to certain variants of the invention, it may be appropriate to administer the antibody of the invention in combination with any one or more of the agents listed below: (1) with niacin, which increases lipoprotein catabolism; (2) with fabrates or amphipathic carboxylic acids, which lower low-density lipoprotein (0), increase high-density lipoprotein (HDL) and triglyceride (triglyceride) levels, and reduce nonfatal heart attacks; and (3) activators of transcription factor I XE, which plays a role in cholesterol elimination, such as 22-hydroxycholesterol, or a statin plus bile acid sequestrants (onoexchange resin) (eg, colestyramine, colestipol, colesevelam), a combination drug niacin plus a statin (eg , niacin plus lovastatin); or with other lipid-lowering agents such as ethyl esters of omega-

C-fatty acids (for example, omakor).

In addition, the second therapeutic agent can be one or more other inhibitors of glucagon or OSOB, as well as inhibitors of molecules of other substances, such as angiopoietin-

C-like protein (AMORTI 3), angiopoietin-like protein 4 (AMORTI 4), angiopoietin-like protein 5 (AMORTI 5), angiopoietin-like protein 6 (AMORTI 6), which are involved in lipid metabolism, in particular in cholesterol homeostasis and / or triglycerides. Inhibitors of these molecules include low molecular weight substances or antibodies that specifically bind to these molecules and block their activity.

According to certain embodiments of the invention, it may be appropriate to administer the antibodies of the invention in combination with an antibody that lowers lipid or cholesterol levels, such as, but not limited to, an antibody to ROSBKOU (proprotein convertase subtilisin/kexin type 9), for example, as described in US patent I52010/0166768. Other antibodies to RSOB5KOU are described in US patents O052010/0040611, O52010/0041102, I52010/0040610, O52010/0113575,

ОБгО09/0232795, ИБ2009/0246192, О52010/0233177, И52009/0142352, . 052009/0326202,

О52О10/0068199, О52011/0033465, О52011/0027287, О52010/0150937, 052010/0136028 and in

International patent application UMO2009/055783.

According to certain variants of the implementation of the invention, it may be appropriate to introduce antibodies to

C2SOV according to the invention in combination with a nucleic acid that inhibits the activity of PROBKU (proprotein convertase subtilisin/kexin type 9), such as an antisense molecule, a double-stranded RNA or a 5iRNA molecule. Examples of nucleic acids that inhibit activity

RSBZKO, described in US patents I52011/0065644, 052011/0039914, 052008/0015162 and

O52007/0173473.

Additional therapeutically active component(s) may be administered prior to, concurrently with, or after administration of the antibody to MAMMALS of the present invention. For the purposes of this disclosure, such administration regimens are contemplated as administration of an anti-SCC antibody "in combination with" a second therapeutically active component.

The use of antibodies in diagnostics

Antibodies to SOSOK according to the present invention can also be used for detection (detection) and/or quantification of SOM in a sample, for example, for diagnostic purposes.

For example, an anti-SOC antibody or fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, reduced expression, or lack of expression, etc.) of SOS. Examples of diagnostic methods

SSOCs may include, for example, contacting a sample obtained from a patient with an anti-SOSC antibody of the invention, wherein the anti-SOSC antibody is labeled with a detectable label or reporter group or is used as an immobilized ligand for selective isolation

SSOC from samples obtained from the patient. Alternatively, an unlabeled antibody to 5SOC can be used for diagnosis in combination with a secondary antibody that is itself labeled with a detectable label. The detected label or reporter group can be a radioisotope such as UZN, 7yC, 52p, 555 or 725! a fluorescent or chemiluminescent molecule, such as a fluorescein, isothiocyanate or rhodamine molecule; or an enzyme such as alkaline phosphatase,

B- galactosidase, horseradish peroxidase or luciferase. Specific examples of assays that can be used to detect or quantify MAPS in a sample include solid-phase enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (KIA, RIA), and fluorescence-activated cell sorting (EAS5).

Samples that can be used in the diagnosis of ESSOC of the present invention include any tissue or fluid sample that contains detectable amounts of ESSEC protein, or a fragment thereof, that can be obtained from a patient in a normal or pathological condition. Generally, PSA levels in a particular sample obtained from a healthy patient (eg, a patient not affected by a disease or condition associated with abnormal PSA levels or activity) are quantified in order to first establish a baseline or standard level. NIPPLE. This baseline level of OSK can then be compared to the levels of the SOM. in specimens obtained from individuals suspected of having a disease or condition associated with OSA, or with symptoms associated with such a disease or condition.

Examples

The following examples are offered for the purpose of providing those of ordinary skill in the art with a full disclosure and description of the preparation and use of the methods and compositions of the invention, and are not intended to limit the scope of what applicants consider to be their invention. Attempts were made to confirm the accuracy of the quantities used (for example, quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1. Preparation of human antibodies to human CSF. To obtain antibodies to CSF, you can use an immunogen, which is any

which of the objects listed below. For example, according to certain variants of implementation of the invention, cells expressing

TOE. In addition, according to certain variants of the implementation of the invention, as an immunogen for obtaining antibodies according to the invention, DNA encoding pOSOK was used. In addition, according to certain variants of the invention, peptides containing amino acid sequences from the M-terminal domain were used as an immunogen for the preparation of antibodies to human MAMP

POSOK Further, according to certain variants of the invention, peptides containing amino acid sequences from any regions of the extracellular loops EC1, EC2 or EC3 of the PpOSO receptor were used as an immunogen for the preparation of antibodies to human MASOC. Cells, DNA, or peptides used as immunogens as indicated above were administered directly, with an adjuvant to stimulate an immune response, to mice MEGOSIMMYOMEF", containing DNA encoding the variable regions of heavy and kappa light immunoglobulin chains. The antibody immune response was monitored using a sCOCK-specific immunoassay. When the required immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells in order to preserve their survival and form hybridoma cell lines. Hybridoma cell lines were subjected to screening and selection in order to identify cell lines that produce SOSSOV-specific antibodies. Using this method and the various immunogens described above, several chimeric antibodies to CSO (ie, antibodies containing human variable domains and mouse constant domains) were obtained; some typical antibodies produced in this way are labeled as

Н4нНИТ345М, Н4НИ1617М and Н4НИ1765М.

Antibodies to OSOR. was also isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in the US patent application |I.5. 2007/0280945A1.. Several fully human antibodies to CO (ie, antibodies having human variable domains and human constant domains) were obtained by this method; typical antibodies obtained in this way are labeled as follows: H4NI13218, H4NI1321R, H4NI1327B, H4NI1327R, nNan1iz288, NANI1Z328R, NANIT3318, NANITZ331R, N4NIT3398 and H4NITZ339R.

The biological properties of typical antibodies to (SOCk), obtained according to the methods given in this Example, are described in detail in the Examples presented below.

Example 2. Amino acid sequences of the variable regions of the heavy and light chains

In Table 1, pairs of sequences of the variable regions of the heavy and light chain of selected antibodies to MASOC and designations of the corresponding antibodies are presented. If the antibodies have the same numerical designation, but different end indices M, B or P, this means that the antibodies have heavy and light chains with identical SOC sequences, but with sequence variants outside the SOC sequences (ie, in the framework regions). Thus, options

M, B and P specific antibodies have identical SOC sequences in the variable regions of the heavy and light chain, but differ from each other in their framework regions.

Table 1 nnnnnnnnnnIshnnnnIIInIInnIIIIII here we string | 2 | 4 | 6 | 8 | 0 | 12 | 714 | 76 Nanizaves. | 50 | 52 | 54 | 56 | 58 | 60 | 62 | 64 nanizatR | 66 | 52 | 54 | 56 | 68 | 60 | 62 | 64 nanizauV | 70 | 72 | 74 | 76 | 78 | 80 | 82 | 84 nanizauR | 86 | 72 | 74 | 76 | 88 | in | 82 | 84 nanizavv | 890 | 92 | 94 | 96 | 98 | tl00 | 702 | 104 nanizavR | 7106 | 92 | 94 | 96 | 708 | tl00 | 702 | 104

Example 3. Analysis of the use of variable region genes

To analyze the structure of the obtained antibodies, the nucleic acids encoding the variable regions of the antibodies were cloned and sequenced. Based on the nucleotide sequence and the predicted amino acid sequence of antibodies, gene usage was identified for each heavy chain variable region (HCHV) and light chain variable region (LRV). IN

Table 2 presents gene usage for selected antibodies of the invention.

Table 2

NSUV isSUV

NSUVL SVV

NANI617M 18/26 M1-24 M2-28

NANIZ45M 2/10 M1-24 M2-28

NANI765M 34/42 m3-48 106-696 0 1M2-28

NANIZ21R 66/68 U3-30 M1-16 nNantZa7R 86/88 M1-17

NANIZ28R 106/108 3-13 M1-17

NANIZZ1R 126/128 U3-33 M1-17.. 191794

NaNTZZoR 146/148 M3-13

Example 4. Binding of an antibody to a soluble (SSOK, determined by the method of surface plasmon resonance

Affinity and kinetic constants of binding of human monoclonal antibodies to pOSOK with the ectodomain of human and monkey soluble recombinant pOSOK (POSOmK m MISSOK, respectively) were determined by the method of surface plasmon resonance both at 252C and at 37"C. Measurements were carried out on a T100 device. Antibodies was captured on the surface of the chip with an anti-human kappa chain antibody covalently linked to the capture surface, and soluble bileus of ZCO were applied to the surface either in a monovalent format (pOSOK expressed with a C-terminal tus-tus-hexahistidine tail) or in a bivalent format (pOSOK m MIOSOK, expressed with an M-terminal Es fusion).

Designation of the amino acid sequences of the reagents used in this example are presented in Table 3.

Table C

Construction of 5EO and MO: anti-SSOK positive control (See map, Nai ev! ai. pIChSA(81О8Р 150 kDa, dimer UGO2008/036341 tesSav-M-end PES 160 Kk paSavV-tEs 80654.42 Yes, dimer paSavV-tti 18,965 Yes, monomer

Soluble ZSOK was fed into the flow cell as separate injections at various concentrations ranging from 3.1 nM to 50 nM, and the kinetic rate constants of association (Ka) and dissociation (Ka) were determined by fitting to the data for a 1:1 binding model using programs for building curves 5sgyrreg m2.0a. The equilibrium constants of dissociation and the half-life of dissociation were calculated from the rate constants: Ko-Ka / Ka; H/2 - (Ip2/Ka).

Table 4a:

Binding results at 25 "C, obtained using the Viasoge system

Tested antigen T1/2 (min) paSOaV-ttip 1.06E--06 | 3.74E-03 | 3.54E-09

NANIZ2gtR paSSOV-NES 1.20E--06 | 2.85E-04 | 2.38E-10 ti aSOaVv-pes 1.76E-406 | 4.18E-05 | 2.38B-11 pasSOoV-tti 9.52E--05 | 3.52E-04 | 3.69E-10

NaNIZ327R pasov-pes 1.214406 | 4.10E-05 | 3.38B-11 ti aSOaVv-pes 1.60E--06 | 1.44B-05 | 9.02B-12 passSOoV-tti 1.03E-4-06 | 2.12E-0O3 | 2.06E-09

NaANIZ28R pasov-pes 1.13E--06 | 2.26E-04 | 2.01B-10 ts SOVv-пес 1.60E--06 | 8.46B-05 | 5.296B-11

Zo

Continuation of table 4a

From nowhere

NantZeR

NantzaBm

Nantv17Mm nat7v5m

Corresponding to peSov-tty | 667E405 | 168E-04 | 252ELOY 69 isotype antibody comparison

Table 46:

Binding results at 37 "C, obtained using the Viasoge system

NaNIZatR paSov-tty | 748E406|2.00E-03 | 135e09| 6 (

NanIza?R

NaNTZagVR paSovgs 0 | 7155E506|1.92E-04 | 124E10| 60 (

NANIZZ1R

NantZZoR paSOv-tty | 928E405|1.97E-03 | 212e-09| 6 Ts (nantiZa5M

Nantucket 17m

NanI765M

Corresponding to paSOv-tty | 873E-05|146E-03 | 168g09| 8 ( isotype antibody comparison

As shown in Tables 4a and 4p, in the case of the antibodies given as an example, high-affinity binding to soluble proteins of both human and monkey SSOK was observed. A significant increase in binding affinity (from 5-fold to 15-fold) was observed when passing bivalent PPOSO compared with monovalent PpOSOK. Antibodies consistently bound to the monkey version of the MIZSOK with a higher affinity (from 3-fold to 10-fold) compared to pOSOK.

Example 5. bioassay for the purpose of quantitative determination of the effect of antibodies to CSO. on the activation of mammals

MAMMAL is a receptor associated with C protein, and its ligand, glucagon (50), stimulates the activity of adenylyl cyclase (adenylate cyclase) through Я5 and the turnover of phosphoionositol through Ca (Uapu apa 7Napa, (2003), At U РНузиои Epadostipoi Meiyar 284: Еб71 -E678). A bioassay was developed to detect Ca5-mediated activation, subsequent elevation of CAMP levels, and transcriptional activation. Cell lines NEK293 were obtained for stable expression of full-size: human (CO (accession number in zepVapk MR 000151.1; 5EO IU MO: 153), monkey (Masasa

Tazsisshiagiii) of the ZSOK (5EO IUO MO: 155), and of the mouse SOSOK (MR 032127.2; 5ZEO IU MO: 154) in combination with a luciferase reporter assay. Stable cell lines were isolated and maintained in 10 95 EV5, OMEM, MEAA, Rep/z5iger and 500 mg/ml 418. For rat MAMMALS, the NEK293 cell line expressing the ISKE (4X)-luciferase-IVE5-SERI reporter gene was transiently transfected using full-sized rat SOM (MR 742089.1; 5EO IU

MO: 156) using Gyroitesiatipe2000 (Lipofectamine) (Ipmigodep).

For bioassays, 293/5SOK cells were seeded in 96-well assay plates at a density of 20,000 cells/well in low-serum medium, 0.196 EU5 and ORTIMEM, and incubated at 37 °C and 5 95 °C overnight. The next day, serial 1:3 dilutions of CCO were added to the cells starting from 100 nM to 0.002 nM, without including any control to test the dose effect.For inhibition, serial dilutions of 1:3 antibodies were prepared and added to the cells starting from 200 to 0.003 ( for PpOSOK cells) or from 100 nM to 0.002 nM (for cells with monkey, mouse and rat MAMMARS), not including any control using antibodies with a constant concentration of 100 pM CO. Luciferase activity was tested after 5.5 hours of incubation at 37 "C and 5 95 SO".

ES50 values for stimulation of each "reporter" cell line with 100 pM

CSOs are shown in Table 5a. The results of the determination of IC50 values for antibodies blocking stimulation of cells with 100 pM Co are shown in Table 50, including the results of two control experiments, Control(no) tAb1 (positive control expressed as pIdsa4 isotype; see, for example, UMO2008 International Patent Application /036341 relative to the antibody designated as "A-9" having NSC with the sequence 5EO IU MO: 275, NSOKTI with the sequence

ZEO IU MO: 102, NSOKZ with sequence 5EO IU MO: 128 NSOKZ with sequence 5EO IU MO: 169 ii GSMK with sequence 5EO IU MO: 229, YSOKI1 with sequence 5EO IYUO MO: 14, YSOK2 with sequence ZEO IYU MO: 50 and ISOKZ with the sequence ZEO IU MO: 74) and Control(-not) tAb2 (isotype-related negative control).

With respect to inhibition of human sCO by anti-SOC antibodies, activation of sCO by CO was shown to stimulate luciferase activity with an EC5O of 113 pM, and all antibodies except Control tAba2 (an isotype-related negative control) blocked CSO activation at 100 pM and decreased luciferase activity.

With regard to inhibition of monkey MASC by anti-SCO antibodies, activation of SC by CO was shown to stimulate luciferase activity with an EC50O of 36 pM, and all antibodies except Control tAb2 (an isotype-related negative control) blocked CO activation at 100 pM and decreased luciferase activity. In the case of NANI1765M, partial CO inhibition was observed at the highest concentrations of the tested antibody, 100 nM.

With regard to inhibition of murine SSC by anti-SSC antibodies, activation of SSC by Co was shown to stimulate luciferase activity with an EC50 of 83 pM, and all antibodies except H4NI345M, H4ANI617M, H4iNI765M, and Control tAbB2 (an isotopically related negative control) blocked C at a concentration of 100 pM it reduced luciferase activity.

With respect to inhibition of rat COM by anti-SCO antibodies, activation of CSO by CO was shown to stimulate luciferase activity with an EC50 of 252 pM, and all antibodies except H4NI1765M and control tAb (an isotype-related negative control) blocked CSO at 100 pM and reduced luciferase activity.

Table 5a

Table 5B5 11111111... | laid | toSov | toSov | sasosov nanizzIR 77777777 17711066 | 8292 | 7.62 | 387 shshch

Control tAbB1 positive control 00009012 161 positive control

Thus, eight fully human anti-PSOC antibodies were tested and demonstrated to block the activation of human PSOC by CO at a concentration of 100 pM in a reporter cell line, with an EC50 value of 113 pM upon stimulation with CO alone. In the case of a reporter cell line transfected with murine MAMMALS, seven out of eight antibodies tested completely inhibited activation by 100 pM Co. Antibody H4H1765M did not completely inhibit SOR at the highest concentration of the tested antibody, 100 nM. Five of eight antibodies completely inhibited activation by 100 pM CO in a reporter cell line transfected with murine MAMM, and seven of eight antibodies completely inhibited activation by 100 PM CO in reporter cells transfected with rat cZSOC.

Example 6. Study of the effect of antibodies to OSOK on or/06 mice

Selected anti-NAS antibodies, all of which cross-react with murine SCC, were tested for their ability to lower blood glucose levels in oB/0ob mice, a mouse model of type 2 diabetes.

oB/o0r mice were grouped into ten groups of five or six animals. Each group was injected subcutaneously with each antibody at a dose of 1 or 10 mg/kg. The control group was injected with an isotype control antibody that does not bind to any known mouse protein. Two days or seven days after administration of the antibody dose of 1 or 10 mg/kg, respectively, a few drops of blood were collected from the tail vein of the mice. Specifically, in the group that received the H4H1327R antibody at a dose of 10 mg/kg, blood was collected from the tail vein more often, on 2, 4, 7, 9, 11, 14, 16, 18, and 21 days after the dose. Blood glucose levels in samples taken from the tail vein were determined with a glucometer (system) ASSO-SNEKF Sotrasi Riiz (Kospe). The percent reduction in blood glucose levels compared to mean blood glucose levels in the control group was calculated for each animal at each time point. The average decrease in glucose levels in percent was calculated for each group of animals. Table b shows the average blood glucose levels of the control group. Results expressed as (mean x BEM) percent reduction in glucose levels are presented in Tables 7a and 765.

Table 6

Coo)

Table 7a

Dose Time (days)

Table 7

Name of antitiles 77777771 Chasidnd///////711111СсСС 2 14 1 7 | 9 | 11 | 14 | 716 | 18 | 2

In the tested mice that received antibodies to NOSOC (shown in Tables 7a and 75), a significant decrease in blood glucose levels was observed compared to mice that were injected with control antibodies.

Example 7. Study of the effect of antibodies to ZOSOK on transgenic mice expressing the human protein ZOSOK

The effect of antibodies to pOSOK on the level of glucose in the blood and the level of lipids in the plasma was determined in transgenic mice expressing the human protein zSOKk ("mice humanized by CO").

ZSOK humanized mice were obtained by replacing the mouse ZSOK gene with a human gene

SSO (5EO IU MO: 157; encoding full-length protein, accession number in zZepVapk MR 000151.1;

ZEO IU MO: 153) in C57VI 6/129 (E1H4) embryonic stem cells. After the transmission of the germ line was proven, heterozygous mice (Sov cht) were crossed, obtaining homozygous mice (bo dgttit) based on C57VI 6. Homozygous humanized according to OSOV. mice were divided into ten groups of three or four animals per group. Each group was administered each antibody in a dose of 3 mg/kg as a subcutaneous injection. Control group I was injected with a control antibody of an isotype that does not bind to any known mouse proteins. In the Control Group II, pIDs4 antibody to pOSOK was administered (in the form of injections), which, as it was proven, lowers glucose levels in the blood of humanized ZOSOK mice. Blood was taken from the mice three days after the introduction of the antibody, and blood glucose levels were determined with a glucometer (system) ASSO-SNEKFO Sotrasi Rii (Kospe). The decrease in blood glucose levels in percentages compared to the average blood glucose levels in the Control I group was calculated for each animal. The average decrease in glucose levels in percent was calculated for each group of animals. Table va shows the average blood glucose levels of the control group. The results, expressed as (mean 5EM) reduction in glucose levels in percent, are presented in Table 86.

In addition, in the Control I, Control II and H4ANI1T765M groups, blood was taken from mice before and after 3 and 8 days after administration of the antibody dose, and plasma lipid levels were determined on an automatic biochemical analyzer AOMIAF 1650 SPpetivig Zubiet (bietep5). Means were calculated

From the value for each determination of the levels of low-density lipoprotein cholesterol (I 01-05), high-density lipoprotein cholesterol (NOI-C), total cholesterol (TOTAI-C), triglycerides (TO), non-esterified fatty acids (MEA) for each of the three groups

The results expressed as (mean and 5EM) plasma lipid concentrations are shown in

Tables of all

Mice receiving most of the tested antibodies to NOSOC (shown in Table 85) had a significant decrease in blood glucose levels compared to 3 mice receiving a control antibody. Mice receiving some of the tested antibodies to pOSOK (shown in Table 8c) showed significant reductions in triglyceride levels compared to mice receiving a control antibody. In particular, a reduction in triglyceride levels was observed with two anti-SSOC antibodies, one called H4NI1765M (results shown below in Table 8c) and the other called H4NI1327P (results not shown).

Table v

Table 8B

Name of antibody and blood (90)

Table 8p

Control I 8 | 98522 | 4b:32 | 9954 | 120319 / 08850.05

HER control 8 | l44k21 | 5743 | 11457 | 743415 | 0735004

NanI765m 8 | 92-16 | 46:36 | 9258 | 75:37 | 0705005

Example 8: The effect of combined therapy with an antibody to ZSOBK and an antibody specific to ROZKOU (proprotein-convertase subtilisin-kexin type 9) on the level of blood glucose, plasma lipids and liver triglycerides in mice

Reagents

The following antibodies were used to study the effect of combination therapy with an anti-ESOK antibody and an antibody specific for ROBCO on blood glucose, plasma lipids, and hepatic triglyceride levels in C57VI/5 mice: Anti-PI AK antibody labeled KESMA496, which is a pIdC4 control isotype; antibody to COM (pPIds4), labeled NANI1327R; and an antibody to RSZKOU (subt), labeled NINZI16R. Amino acid sequence identifiers NSMK, GSMK,

Neo Сом5 are presented below, in Table 9.

Table 9 11111111 BEOYUMOMVENVYG/:/ССС/:/ССС7777797

VESM AV NanizauR | 86 | 72 | 74 | 76 | 88 | 80 | 82 | 84

Methodology of the experiment

The combined effect of НА4НИ1327Р, an antibody to NOSOK, and NINZI6R, an antibody to PROBKU, on the level of blood glucose, plasma lipids and liver triglycerides (TO) was determined in C57VI/6 mice.

Na4NI327R cross-reacts with murine SSOK, and NINZI16R cross-reacts with murine

WORK C57VI/6 mice were divided into six groups of six animals in each group. Each group was given an antibody or a combination of two antibodies as a subcutaneous injection once a week. The first group was injected with a control antibody of the pIds4 isotype, which does not bind to any known mouse protein, at a dose of 10 mg/kg. Animals of the second and third groups received H4NI1327P in a dose of 3 mg/kg and 10 mg/kg, respectively. The animals of the fourth group were injected with 10 mg/kg NINZ16R. Animals of the fifth group received a combination of 3 mg/kg H4NI1327P and 10 mg/kg NINZIb6R, and animals of the sixth group received a combination of 10 mg/kg H4NI1327P and 10 mg/kg NINZI6R. 11 and 19 days after the initial dose, mice were bled to determine blood glucose and plasma lipid levels. On the 19th day, the liver was removed to determine the content of hepatic TO. Blood glucose levels were determined using the ASSO-SNEKF Sotrasi Riiz (Cospe) system.

The percent reduction in glucose level compared to the average blood glucose level in the isotype control group was calculated for each animal. Then, for each experimental group, the percentage decrease in blood glucose level and the associated error were calculated by averaging the values for individual animals in each group. The results, expressed as (mean x SEM) percent reduction in blood glucose, are shown in Table 10a and Figure 1.

Plasma lipid levels were determined on a biochemical analyzer AOMIDF 1650 Spetivig 5uziet (Zietep5). The content of hepatic TO was determined by colorimetric analysis (Teso Oiadpobviysv5) after extraction of TO from the tissue with chloroform/methanol. Mean values were calculated for each measurement of plasma levels of low-density lipoprotein cholesterol (I 01 -C), high-density lipoprotein cholesterol (HOH-C), total cholesterol (TOTAIH-C), TO and liver TO for each group. Results expressed as (mean x EM) plasma and liver lipid concentrations are shown in Table 1065 and Figure 2 (levels 0 -C in plasma); Figure C (NOG-C levels in plasma); Figure 4 (levels of total-C in plasma); Figure 5 (TO levels in plasma); and Figure 6

Zo (content of liver TO).

Summary of results and conclusions:

Table 10ba. NaNI327R Nantz27R

From mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg

Table 10 r liiyu | whose | cable T cable Done Liver TO (tissue clearance) (mg/dL) (mg/dL) (mg/dL) | (mg/dL)

CONTROL

TSINZIVR then mg

Continuation of table 100

MA: Not determined

Brief conclusions from the results given in the Tables:

In mice that received only H4NI1327P, a marked decrease in blood glucose levels and an increase in TOI, NOI, and total cholesterol levels were observed compared to mice that received control tAb. Mice receiving only NINZ16R had a significant decrease in cholesterol levels and no changes in blood glucose levels. Mice receiving a combination of HA4ANI1327P and NINZI6P antibodies had a marked decrease in blood glucose levels and no changes in cholesterol levels compared to mice receiving control tAB. The content of hepatic TO did not change in any of the experimental groups in comparison with the isotype control group.

Example 9. Study of the effect of an antibody to ZOSOK on a mouse model of alimentary obesity

The effects of H4NIT327R, an anti-PSOC antibody that cross-reacts with murine PSOC, on blood glucose levels and body weight were studied in a mouse model of dietary obesity (IS), type 2 diabetes (120). The pIO model was obtained by keeping mice on a diet with a high (60 90 kcal) fat content (NEO) from 5-6 weeks of age. Approximately 6 weeks after being on this diet, the mice developed metabolic abnormalities, including insulin resistance, impaired glucose tolerance, and obesity. This model induces in mice a physiological state closer to T20 in humans than that induced in two other widely used 120 models, the or/ob and ar/ar mice, because the latter two models result from mutations in leptin or leptin receptor genes, respectively, which are rarely the cause of T20 in humans.

In this study, 11-week-old male C57VI/bB mice, which had been on NEO since 5 weeks of age, were obtained from the company Tasopis Tagtv, Ips., and kept on this diet for another 8 weeks. Mice were divided into 4 groups of 10 animals per group at the age of 19 weeks. The animals of each group received weekly (on days 0, 7, 14 and 21) the H4NI1327P antibody as a subcutaneous injection at a dose of 3, 10 or 30 mg/kg, or 30 mg/kg of the control antibody pPidsa4 isotype, which does not bind with no known mouse protein. Blood glucose levels and body weight were determined periodically, and after 6 days

After the administration of the last dose of the antibody (on day 27), the mice in each group were killed. During the next 6 weeks, blood glucose levels and body weight were monitored in the remaining 4 mice/group.

The decrease in blood glucose levels (in percent) compared to the average blood glucose level of animals in the isotype control group was calculated for each animal. Then, for each experimental group, the percentage change in blood glucose level and the associated error were calculated by averaging the values for individual animals in each group. The results, expressed as (average of 5 WEM) percent blood glucose reduction, are shown in Table 11. The percent change in body weight from baseline (day 0 body weight) was calculated for each animal. Then, for each experimental group, the percentage decrease in body weight and the associated error were calculated, averaging the values for individual animals in each group. The results, expressed as (average of ZEM) body weight change in percent, are shown in Table 12.

Summary of results and conclusions:

Antibody NANI1327R, at all tested doses, reduced blood glucose levels and body weight of OIO mice compared to animals in isotype control groups. The greatest relative reduction in blood glucose levels (48.5 95) was observed in the group receiving the highest dose (30 mg/kg) on day 10, and the greatest relative reduction in body weight (12.8 95) was observed in the group receiving the highest dose, on the 28th day. In animals from the groups that received the lowest dose (3 mg/kg), the average value of the relative decrease in blood glucose levels and the average value of the decrease in body weight, which were at least 70 95 of the values observed in the groups of animals that received the highest dose, were achieved on day 28 (a week after the last dose). The observed effects of lowering blood glucose levels and reducing body weight 21 days after the last administration (ie, 28, 47 and 68 days) persisted longer in the NANI1T327R high dose groups compared to the lower dose groups.

Table 11,768 | ...rurrs43a79 | 77771118 | fragile

Table 12 30 mg/kg Z mg/kg 10 mg/kg 30 mg/kg 768 | --( lodge 17 97507 | 10684 | vol

Example 10. Study of the effect of an antibody to ZSOK on a mouse model of streptozotocin (512)-induced diabetic ketoacidosis

The effect of Н4НИ1327Р, an antibody against pOSOK, which cross-reacts with mouse SOSK, on the level of ketones in the plasma and glucose in the blood, was determined in the mouse model of streptozotocin (512)-induced diabetic ketoacidosis (DKA). 512 is a chemical toxic to mammalian pancreatic beta-cells that, due to its toxicity, destroys this type of cell when administered to rodents. A single injection of a high dose (200 mg/kg) in 5-2 C57VI/6 mice for 3 days leads to the development of severe hyperglycerinemia and ketonuria, conditions observed in OCA in humans. Nine-week-old male C57VI/6 mice obtained from the company Tasopis Tagt5, Ips., were divided into 2 groups of 10 animals each, and animals in each group were injected intraperitoneally with either 512 (in citrate buffer) at a dose of 200 mg/kg, or vehicle (also in citrate buffer). Three days later, severe hyperglycerinemia (blood glucose levels of 2400 mg/dL) and ketonuria were confirmed in all 517-treated animals. The next morning, 517-treated mice were divided into 2 groups of 5 animals, and animals in each group were administered intravenous injection of either NANI1T327R or pIDS4 isotype control at a dose of 10 mg/kg. Mice receiving citrate buffer were also divided into 2 groups of 5 animals each, and each group was injected intravenously with either H4NI1327P or pIds4 isotype control at a dose of 10 mg/kg. Blood for obtaining plasma from mice was taken 18 hours before the introduction of the antibody dose (2.5 days after the introduction of 517) and 28 hours after the introduction of the antibody under isoflurane anesthesia. Ketone levels in plasma were determined by the beta-hydroxybutyrate assay (Viomysiop), and glucose levels in plasma were determined on the AOMIAF 1650 Spetibig Zulet analyzer (Zietep5). Average values of beta-hydroxybutyrate and glucose levels were calculated for each of the four groups. Results expressed as (mean x 5EM) plasma concentrations of beta-hydroxybutyrate and glucose are shown in Table 13.

A brief summary of the results and conclusions:

A decrease (on average by 67 95 in comparison with plasma levels 18 hours before antibody administration) in the plasma concentration of beta-hydroxybutyrate (ketone) was observed in mice with 512-induced ketoacidosis 28 hours after administration of HAN1I327R, this indicates that the H4ANI1327R antibody effectively reduces plasma ketone levels in a mouse model

OKA For 5T12-treated mice receiving isotype control antibody, serum samples collected 28 hours after antibody administration showed an average increase in blood glucose levels of 14 95 compared with samples collected 18 hours before antibody administration, whereas in the case of mice dosed with NANI1327R in the 517-treated group, the change in serum samples collected at these two time points averaged less than 1 95 difference between the two.

Table 13 035 EL ashe nya" antibodies (hour) control Nan1tzag7R control nNan1tzg7R

Betavia

Example 11. Preparation of bispecific antibodies

For the practical application of the methods according to the invention, various bispecific antibodies were obtained. For example, CEEC-specific antibodies have been produced in a bispecific antibody format (a "bispecific element") in which variable regions binding to different epitopes in the ectodomain region and/or EC loops of the PAD are linked together to confer specificity to two loops to one binding molecule. Appropriately designed bispecific antibodies can increase the overall effectiveness of the blockade of the glucagon receptor both by increasing the specificity for CSO and by increasing the binding affinity. Variable regions with specificity for individual ectodomain epitopes (e.g. segments of the M-terminal domain or EC1, EC2, or EC3 papillary loops), or able to bind to different regions within the same ectodomain segment or loop, were connected pairwise based on the structural framework, which allows each variable region to simultaneously bind to separate epitopes or to different regions within the same ectodomain or EC loop. In one example, to obtain bispecificity, variable regions of the heavy chain (Mn) from the linker region ("links" "binding molecules") with one specificity for the ectodomain or loop were joined (recombined) with variable regions of the light chain (Mi) from a group of binding sites ("connection") with a second specificity to the ectodomain or EC loop to determine non-cognate Mi chain partners, which can be combined in pairs with the initial Mn, without violating the primary specificity of this Mn. Thus, one Mi segment (for example, Mi1) can be combined with two different Mn domains (for example, Mni and Mn2) to obtain a bispecific element consisting of two binding "arms" (Mn1i-Mi1 and Mn2-mi1 ).

The use of a single Mi segment reduces the complexity (multiplicity) of the system and thereby simplifies and increases the efficiency of the cloning, expression and purification processes used to obtain a bispecific element (antibody) (see, for example, US patents

О5ЗМ13/022759 and О052010/0331527).

Alternatively, antibodies that bind both SSOC and a second target, such as, but not limited to, human proprotein-convertase subtilisin-kexin type 9 (PROBCU), can be prepared in a bispecific antibody format by methods described herein or by other methods , known

With specialists in this field of technology. Variable regions of antibodies that bind to different regions of OSOB, with an exposed extracellular region, bind together with variable regions that bind to the corresponding site, for example, on РОБ5КОУ, reporting specificity for two antigens (dual antigenic specificity) in one binding molecule.

Appropriately designed bispecificities (bispecific molecules) of this nature perform a dual function. For example, in the case of a bispecific antibody that binds both CSO and RBCOU, it is possible to lower blood glucose due to binding of one arm of the bispecific antibody to ROSCO, while lowering plasma lipid levels due to binding of the second arm of the antibody to RBCOU . Variable regions with specificity for individual epitopes of the ZSOK ectodomain were linked to a variable region with specificity for ROB5KU and combined in pairs on a structural framework that allows each variable region to bind to individual antigens.

Bispecific bonds were tested for binding and functional blocking of target antigens, for example, ZSOK and/or ROBKOU, in various assays described above for antibodies.

For example, standard methods of quantitative determination of soluble protein binding, such as Viasoge, EG IZA, gel chromatography, multi-angle laser light scattering, scanning calorimetry and other methods were used to evaluate the bispecific-RS5KOU interaction. The binding of bispecific antibodies to cells expressing SOC was determined using a fluorescently labeled secondary antibody that recognizes either target antigen or both target antigens associated (or associated) with the cell. Cross-reactivity with respect to different ectodomains or loops of ZSOK within different species variants and between different species variants was determined by EHIZA binding assay, in which synthetic peptides representing different ectodomains or loops were applied to the wells of microtiter plates, and the binding of bispecific molecules were determined using a secondary detection antibody. Loop peptide binding experiments can also be performed using the surface plasmon resonance method, where the binding interaction is determined in real time by passing a current of the peptide or bispecific molecule along the surface of the sensor on which the bispecific molecule or peptide is captured, respectively. Functional IP of blockade of the -SOC receptor by a bispecific molecule was determined by any of the bioassays described above or by determining IP by blood glucose levels in appropriate animal models, such as those described in this application. The functional IP of RSOB5KO blocking by a bispecific molecule was determined by any bioassay, such as the bioassay described in International Patent Application UMO2010/077854 or in US Pat.

О52010/0166768, or by determining ip mimo plasma lipid levels in appropriate animal models, such as those described in this application.

LIST OF SEQUENCES

"1105 REGENERON PHARMACEUTICALS, INC. "1205 Human antibodies to the glucagon receptor "1305 1400A-WHAT "1405 To be assigned "1415 Submitted together with this document "150" 61/416,409 "1515 2010-11-23 "150" 61/481,958 "1515 2011-05-03 " 1505 61/551,032 "1515 2011-10-25 "1605 207 "1705 BavEe5EO for version 4.0 Mipdomz "2105 1 "2115 375 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic

З5 «4005 1 saddeEssade sddsasades gddddssdas deEdaadaads sEddddssis adgdaaddes 60

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AND

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Sip bBeg Gets Gets Niv Avp Avzp biu Tut Avzp Tug 1 5 10 "2105 45 "2115 9 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 45

From ЕСсСОЯЫЯАЕССЯ З "2105 46 "2115 З "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 46

Те с1и1у б5ег 1 "2105 47 "2115 27 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 47 ayesdsaadses sasaaasss dEddasd 27 "2105 48 "2115 9 "2125 PZT "213" Artificial sequence

"223" Synthetic "4005 48

Mee bip Aia Ge sip TPt Rkgo Tkr TPg 1 5 "2105 49 "2115 384 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 49 chadadeEdsads sddeddades eddddddads dEddESsSSsSads sedddaddes sseEdadasts 60

ЕссЕдеєдсад ссЕСтДдаєс сассеЕєЕсаді адеЕсаєдаса єдсассддді ссдссаддсе 120 ссаддсаадд дасеЕддадед дагаддсадее асаєссаєстуд асддасдеєда гааасастатє 180 чдсадассссд сдаадддссу аєссассаєс сссададаса ассссаадаа сасдсетває 240 сеЕдсаааєда асадсстдад адседаддас асддсЕЧЕЄс асстасеєдедс дааададчаєа 300

ChdECHEaeesasu atassevdas eddeEvayesae aastastasu dsayeddasdi srddddssaa 360 chddadassasdd EsassudEsts sssa 384 "2105 50 "2115 128 "2125 PZT "213" Artificial sequence

Zo "2205 "223" Synthetic "4005 50 si Mai sbip Gets Uai Sits back siu si1u siu Ua1i Ua1 sbip Rgo s1u Agd 1 5 19 15

Zzek Gay Agd Getsp bek Suv Aia Aia bek s1u Rpe TPti Ribe bek bek Tuk 20 25 30

Avr Mee Niz Ter uUai Agd bip Aza Rgo sbi1u Guz SsS1u iets sb1ts Tgr Uai1i 35 40 45

A1ia uaii Iie Bek bBet Avr s1u Agd Avr Guz Tut Tut Uai Avr bBet Uai 50 zo bo

Tuz s1u Akd RpPe TPi Iie bek Agud Avr Avp o bet Guv Avp TPt Gets Tuk 65 70 75 80 tep sip Mesb Avzp o b5et Hey Agud Aia siy Avr TPt Aia Uai Tut Tuk Suv 85 30 35

A1ia puv si Meyes uai Tukhk Tug Avr I1e eat TPh SsS1iu Tut Niv Azp Tug 1600 195 110

Tuk siu Mee Avr Uai Tgr b1u beep b1iu TPt TPt Ua1! TPti Uai back back 115 120 125 "2105 51 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic

"4005 51 chchayeesassya ssadsadeEsa edas 24 "2105 52 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 52 s1iu Rpe TPI RPe bBet 5bet Tut Avr 1 5 "2105 53 "2115 24 "2125 DNA 213" Artificial sequence "2205 "223" Synthetic "4005 53 asayesaestd ayeddasdyeda gaaa 24 "2105 54 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 54

I1e Bek 5Bet Avr s1u Agd Avr Guz 1 5 "2105 55 "2115 63 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 55 dsdaaadacha seddeedtaesta sdataestevd aseEddeEtaes asaasstasta soddeaeddas 60 hEs2C15 63 "112 65 "212 "2 PZT "213" Artificial sequence "2205 "223" Synthetic

"4005 56

A1ia puv si Meyes uai Tukhk Tug Avr I1e eat TPh SsS1iu Tut Niv Azp Tug 1 5 19 15

Тук с1у Меєс Авр Уаї 20 «2105 57 «2115 324 «2125 ДНК «213» Штучна послідовність «2205 «223» Синтетична «4005 57 часаєсудєда сдасссадес Ессаєсстса сЕдЕСтЕДдсає ссдтаддада сададісасс 60 ассасЕсдеЕс дддсдадесса дддсаєсаас аасаєрьвад сстддеЕссса дсадааасса 120 чдададааадссс стаадесссІ дасссасасе дсаєссадсс єдсааадсдд ддЕСсСссаєса 180 aadesssadsud dsadsddayes edddasadaye sssasestssa ssayesadsad ssudsadsst 240 chaachaeeesstd saasetayesta sedssaasad bagaarases asssESSesEsas EEESOdsdda 300 chdddadassaaad sYeddadayesaa asda 324 "2105 58 "2115 108 "2125 PZT "213" "2205 Artificial sequence

From "223" Synthetic "4005 58

Avr Iie Maii Mes TPtI sip bet Rko bBeti bBeit Hey begt Aia bBet Ua1i 01Uu 1 5 19 15

Avr Akud Ua1i TPg Iie TPti Suv Agud Aza beg bip sS1u Iie Avp Avp Tug 20 25 30 tep Aia Tkr Rpe sSip bip puv Rgo biu Guv Aia Rgo Guz Bek Getsp Te 35 40 45

Niz TrI ATia bBetg bek yeets sbip bek biu UMa1i Rgo bBet Iiuv Rre beh 01U 50 zo bo bek biu bBek bi1u TPh Avr Rpe TPti TIetsy TPI Iiee bBet Bek Gay sip Rgo 65 70 75 80 biCh Avr Rpe Aia TPtI Tut Tut Suv sbip bip Tut Avp TPtI Tut Rgo TGets 85 90 35

TPg Rpe siu siu biu TPi puz Ua1i! siy I1e puz Agd 1600 195 "2105 59 "2115 18 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 59 sadddsayeta asaayetaye 18

"2105 60 "2115 6 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 60

Sip b1u Iie Avp Avp Tug 1 5 "2105 61 "2115 9 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 61 asedsayess Z "2105 62 "2115 Z "2125 PZT "213" Artificial sequence "2205 " 223" Synthetic "4005 62

TVPg Aia bek 1 "2105 63 "2115 27 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 63 saasadsata asassstass ssesase 27 "2105 64 "2115 9 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 64 beep beep Here Avp o TPt Here Rkgo Gets TPg 1 5

"2105 65 "2115 381 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 65 saddeEdsads sddeddades eddddddads dEddESsSSsads sedddaddes sseEdadasts 60

ЕссЕдеєдсад ссЕСтДдаєс сассеЕєЕсаді адеЕсаєдаса єдсассддді ссдссаддсе 120 ссаддсаадд доассддадед ддеддсадее агаєсаєсьд аєддасдєда сааавгастає 180 чдсадассссд сдаадддссу аєссассаєс ссададаса ассссаадаа сасдсеЕттає 240 сЕдсаааєда асадсстдад адссдаддас асдасеЕдеЕєт астасеєдедс дааадчачаєуд 300

ЧдЕЧЕаєсасу атассєвдас єддеЕваєсає аастастасу дсаєддасді срддддссаа 360 чддадассасдд сассдЕсстс с 381 «2105 66 «2115 127 «2125 ПЗТ «213» Штучна послідовність «2205 «223» Синтетична «4005 66 сбіп Маі сбіп Гец Уаі сіц бехт сі1у сі1у с1у Уаї Уаії біп Рго с1у Акгд 1 5 10 15

Zo zet Gay Agd Pec bek Suv Aia Aia bBet sSiu Rpe TPtI RpPe bBetg bek Tug 20 25 30

Avr Mee Niz Ter uUai Agd bip Aza Rgo sbi1u Guz SsS1u iets sb1ts Tgr Uai1i

A1ia uaii Iie Bek bBet Avr s1u Agd Avr Guz Tut Tut Uai Avr bBet Uai

Z5 50 zo bo

Tuz s1u Akd RpPe TPi Iie bek Agud Avr Avp o bet Guv Avp TPt Gets Tuk 65 70 75 80 tep sip Mee Azp bet Gets Agd Aia bi Avr TPtI Aia Uai Tut Tut Suv 85 30 35 40 A1a puv sats Mes Ua1i! Tug Tug Avr I1e te TPg s1u Tug Niv Avp Tug 1600 105 110

Tuk siu Mee Awr Ua! Ткгр сС1у біп бі1у ТПкх ТПт Уа1ї ТПптІ Уаї 5ег 115 120 125 45 «2105 67 «2115 321 «2125 ДНК «213» Штучна послідовність «2205 «223» Синтетична «4005 67 часаєссада сдасссадеЕс Ессаєсстса сЕдЕСтЕДдсає ссдтаддада сададісасс 60 ассасеєвдЕс доадсдадесса дддсаєстаас аасстасєсвтад сстддеЕсЕса дсадааасса 120 чдададааадссс стаадесссІ дасссасасе дсаєссадсс єдсааадсдд ддЕСсСссаєса 180 аадесЕсадсуд дсадсддаєс єдоддасадає єЕСсасесеса ссаєссадсад сстдсадссе 240 чаачаєєсстд саасетаєста седссаасад багаарасес асссЕСсЕсас ЕЕЕСОдсдда 300 чдадассаадд сддадаєсаа а 321 «2105 68 «2115 107 «2125 ПЗТ «213» Штучна послідовність «2205 «223» Синтетична «4005 68

Avr Iie sbip Meyese TPtI sip bet Rko bBeti bBeit Hey begt Aia bBet Ua1i 01Uu 1 5 19 15

Avr Atud UMaii TPt Iie TPt Suv Agkd Aia bek bip biu Iie Avp Avp Tug tep Aia Tkr Rpe sSip bip puv Rgo biu Guv Aia Rgo Guz Bek Getsp Te

Niz TrI ATia bBetg bek yeets sbip bek biu UMa1i Rgo bBet Iiyuv Rre beh 01U zo bo 20 zek biu Bek biu TPh Avr Rpe TPt iets TPi Iiee bBeg bBet Hey beep Rgo 65 70 75 80 biCh Avr Rpe Aia TPtI Tut Tut Suv sbip beep Tut Avp TPtI Tut Rgo TGets 85 30 35

TPE Rpe siu si1u s1u TPzhi Kpuz Uai bi Iie Guz 25 1600 195 "2105 69 "2115 384 "2125 DNA 30 "213" Artificial sequence "2205 "223" Synthetic

З5 "4005 695 saddedsads sdeedsades eddddddads EEddESsSSsSads seddddoddES, sseEDadasis 60

EssEdeyedsad ssEsSsuddayes sasseEEsade aastayeeeda edaassdddi ssssaddse 120 ssadddaadd dodssdddaded dseddssaas agasaddaad asddaaaeesda daaasaste 180 cheEddasestd sdaadddssu ayessassayes ssadadasa asdssaadaa stisastdnae 240 40 sgEdsaaaeda asaddasseddasseddasseddasseddasseddassedda0

Essesevdas Eddeevaydas Gastaeasu DSAEDDASDY SRDDDDSSAA 360 HDDADASDASDDSDSDSDSDSDSDSDSDSDASEADASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDASDA SRDDDDSDSAA 360 SIASUSSDASDDSDSDSDSDSDSDASDASUA sS1i1u 1U 1 5 19 15 zet Hey Agd Petz bek Suv Aia Aia bBet sSiu Rpe TPtI Rpe bBetg Avp Tug 20 25 30 tep Mee Avzp Tktr Uai Agd bip Aza Rgo siu yuv o1Uu Ge si Ter Gets 35 40 45

Aza Avp chI1e sip sats Avr s1iu I1e siy puz Tut Tut Uaiyi Avr bBet Uai 50 55 bo

Tuz s1u Akd RpPe TPi Iie bek Agud Avr Avp Aia Guz Avp bek Gay Tuk 65 70 75 80 tep sip Mesb Avzp o b5et Gay Agd Aia siy Avr TPt Aia Uai Tut Tuk Suv 85 Zo 95

A1i1a Agud si Rgo bBet Niv Tut Avr I1e eat TPh SsS1iu Tut Avr Tug Tug 100 105 110

Tuk si1u Mee Avr Uai Tgr sb1u bip b1iu TPg TPtI Ua1i! TPI Uai back back 115 120 125 "2105 71 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 71 chchayeesassya etadsaasa ES 24 "2105 72 "2115 8 "2125 PZT "213"220 5 Artificial sequence " "223" Synthetic

Zo «4005 72 siu Rpe TPI RPe bBetg Avp Tut Gets 1 5

C5 "2105 73 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 73 asasaddaad ayeddaaeda daaa 24 "2105 74 "2115 8 "2125 PZtT "213" Artificial sequence "2205 "223" Synthetic "4005 74

Iie bip siy Avr si1u Iie si Guv 1 5

"2105 75 "2115 63 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 75 dsdadadads sseEsssayeta schastasyesstud astddeetayed astastaesta sddsayeddases 60 hEsS 63 "2105 76 "2115 21 "2125" "2225" PZtu "223" » Synthetic «4005 76

A1i1a Agud si Rgo bBet Niv Tut Avr I1e eat TPh SsS1iu Tut Avr Tug Tug 1 5 19 15

Tuk si1u Mee Avr Uai 20 "2105 77 "2115 324 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic

З5 «4005 77 часаєссаде сдасссадес єссаєсстсс сЕдЕСЕдсає ссдтаддада сададісасс 60 ассасеЕсдсс дддсаадсса дддсаєсада ааєдарєвсад дссддсаєса дсадааасса 120 чддадааадссс стаадсдссі дассвтаєдсе дсаєссадсс єдсааадсдд ддЕСсСссаєса 180 аддеЕЕсадсуд дсадсддаєс єдддасадаа ЕСсасесеса садуісадсад ссеЕдсадсст 240 40 чаачасеЕєсд саасетсаєта седессвасад басаасадса асссарссас ЕЕсСсСоддссст 300 чдадассаадд сЄддадаєсаа асда 324 «2105 78 «2115 108 45 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic 50 "4005 78

Avr Iie sbip Gets TPt sip bet Rko bBeti bBeit Hey begt Aia bBet Ua1i 01Uu 1 5 19 15

Avr Atud Uaii TPt Iie TPt Suv Agkd Aia bek bip b1iu Iie Agd Avp Avr 55 20 25 30 tep s1u Tkr Tut bip bip uv Rgo bi1u Guv Aia Rgo Guz Akd Tec Te

Tut Aia A1ia bet bBet Gay beep bek biu Ua1 Rgo bBet At9d Rpe Beh schu

50 55 bo bek biu bek biu TPkh bi Rpe Iie iey TPtI Uai1i bBet Bek Gay sip Rgo 65 70 75 80 bis Avr Rpe Aia TPI Tut Tut Suv Gets sip Tukhk Avp bBegt Avp o Rgo RIe 85 90 35

TPE Rpe siu Rko si1u TPh Kguz Ua1i bitsh Iie Guv Agd 100 105 "2105 79 "2115 18 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 79 sadddsayeta daaaedvaye 18 "2105 80 "2115 6 "2 "125 PZT 213" Artificial sequence "2205 "223" Synthetic "4005 80 bip b1u Iie Agd Avp Avr 1 5 "2105 81 "2115 9 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 81 dsEdsayess Z "2105 82 " 2115 Z "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 82

Aza Aia 5eg 1 "2105 83 "2115 27 "2125 DNA "213" Artificial sequence

"223" Synthetic "4005 83 stasadsata agadsaasss ayessase 27 "2105 84 "2115 9 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 84 tep sip Tut Avp bBet Avp Rgo RoPe TiK 1 5 "2105 85 "2115 381 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 85 chadadeEdsads sdaddyddades eddddddadds EEddESsSSsSads seddddoddES, sseEDadasys 60

Zo Essedsdsad sseEssddavre sasseEjetade aasstayevieda edaassdddaye ssdssaddse 120 ssadddaadd dodssddaded dseddssaas agasaddaad asddaaaeesda daaasastaye 180 cheEddasestd sdaadddssu ayessassayes ssadadasa asdssaadaa sysasednae 240 seEdsaadeda asadsstrdad asssdaddassssssssss30

Essesevdas Eddeevaydas Gastaeasu DSADDASDI SRDDDDSSAA 360 HDDASDASDDSDESDESTESS C 381 "2105 86" 227 "2125" 2205 "2205" 2205 "2205 1 5 19 15

Zzek Gay Agd Getsp bek Suv Aia Aia bek s1u Rpe TPtI Rppe bek Avp Tukg 20 25 30

Te Mes Avp o Tgr Uai Agud sip Aza Rkgo sSiu Ppuv sb1u Gets sich Tgr Gets 35 40 45

Aza Avp chI1e sip sats Avr s1iu I1e siy puz Tut Tut Uaiyi Avr bBet Uai 50 zo bo

Tuz s1u Akd RpPe TPi Iie bek Agud Avr Avp Aia Guz Avp bek Gay Tuk 65 70 75 80 tep sip Mee Azp bet Gets Agd Aia bi Avr TPtI Aia Uai Tut Tuk Suv 85 30 35

A1i1a Agud si Rgo bBet Niv Tut Avr I1e eat TPh SsS1iu Tut Avr Tug Tug

1600 195 110

Tuk siu Mee Awr Ua! Ткгр с1у біп бі1у ТПхкх ТПт Уа1ї ТПтІ Уаї 5ег 115 120 125 «2105 87 «2115 321 «2125 ДНК «213» Штучна послідовність «2205 «223» Синтетична «4005 87 часаєссада сдасссадес Ессаєсстсс сЕдЕСЕДдсає седтаддада сададісасс 60 ассасеєвдсс доадсаадсса доддсаєстада аасдчаєстад дседдваєса дсадааасса 120 чддадааадссс стаадсдссі дассвтаєдсе дсаєссадсс єдсааадсдд ддЕСсСссаєса 180 аддеЕЕсадсуд дсадсддаєс єдддасадаа ЕСсасесеса садуісадсад ссеЕдсадсст 240 чаачдасеЕстд саасеєтаєста сеЕдеЕСсСвтасад гагаавадса асссаєєссас ЕЕЕСДДдДСссСсТ 300 чддадассааад сЄддасаєсаа а 321 «2105 88 «2115 107 «2125 ПЗТ «213» Штучна послідовність «2205 «223» Синтетична «4005 88

Zo Avr Ii1e sbip Mayes TPg bip betg Rkgo bek bBeg Hey bek Aia Beh Uai U 1 5 10 15

Avr Atud Uaii TPt Iie TPt Suv Agkd Aia bek bip b1iu Iie Agd Avp Avr 20 25 30 tep s1u Tkr Tut bip bip uv Rgo bi1u Guv Aia Rgo Guz Akd Tec Te

Z5 35 40 45

Tut Aia A1ia bet bBet Gay beep bek biu Ua1 Rgo bBet At9d Rpe Beh schu 50 zo bo bek biu bek biu TPkh bi Rpe Iie iey TPtI Uai1i bBet Bek Gay sip Rgo 65 70 75 80 bi Avr Rpe Aia TpTI Tut Tut Suv Gay sip Tut Avp bek Avp o Rgo RIPe 85 30 35

TPE Rpe siu Rko s1u TPI Kguz Uaii Avr Iie Guz 1600 195 "2105 89 "2115 381 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 89 chaddeEdsads sddedsades eddddddadss EEDddsasads sedddddddES ssEEDadasis 60

Есседсасад ссЕСТддеЕЕ сассеєсадеі аассасдаса гдсассддде ссдссаааст 120 асаддааагад чЕСсддадед дасстсадсе аєрдасасьд седдеЕдасас агастаєсса 180 чдасЕСсСсдєда адддссдаєс сассдЕСтІсс ададааааєсуд ссаадаасіс стеЕЕсатєсеє 240 саааєдааса дссідададс сддддасасуд дсеЕдЕЧдтаєсє ассдедсаад ддаддддаад 300 таєсасдата ЄЕЕсддассд Єдасвассас гассасддра єддасдеєсьд дддссааддуа 360 ассасддеіса ссдуістІсстс а 381 «2105 90 «2115 127 «2125 ПЗТ «213 » Artificial sequence «2205 «223» Synthetic «4005 90 si Mai sbip Gets Maii sip beht siu s1u Ai1a yetsy Uai bip Rgo sS1iu 01U 1 5 10 15 zet Hey Agkd Pec bek Suv TPt Aia bBet siu Rpe TPtI Rpe bBetg Avp Tug

Avr Mee Niz Ter Uai Agd bip TPrt Trh s1u yuv SsS1u yets s1c Tgr Ii1e zehk Aia Iie Avr TPh Aza sb1u Avr TPI Tut Tut Rgo siu bek Uai1i Guz 20 50 zobo s1u Akud Rpe TPtI Uaiyi bBet Akd s1its Avp Aip Aip Guz Avp bet R Tut Tiets 65 70 75 80 bip Mesie Avp o bBet Gets Agud Aia s1u Avr TPi Aia Uai Tut Tut Suv A1a 85 Zo 95 25 Atkud bi sS1u Ppuv Tuk Tug Avr I1ie TiTecp TPtg SsS1u Avr Tuhg Niv Tuhg Tug 100 105 110 Avr U siau Mesieu Ttr siu sip siu TPti TPti Ua1 TPg Uai bek bekg 115 120 125

From "2105 91 "2115 24 "2125 DNA "213" Artificial sequence

З5 "2205 "223" Synthetic "4005 91 дЯдЕСЕСсСася ssadsaasta sdas 24 40 "2105 92 "2115 8 "2125 PZT "213" Artificial sequence 45 "2205 "223" Synthetic "4005 92 с1у Рпе Тпх РпПе бБег Авп Тут Авр 1 5 2105 93 "2115 21 "2125 DNA "213" Artificial sequence "2205

"223" Synthetic "4005 93 asedayeasd srddsdasasa 21 "2105 94 "2115 7 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 94

I1ie Avr TPg Aza C1u Avr TIPg 1 5 "2105 95 "2115 63 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 95 dsaadddadd ddaadtaesta sdataestevd aseddedasi assasstasta soddeaddas 60 hEsS 63

From "2105 96 "2115 21 "2125 PZT "213" Artificial sequence

Z5 "2205 "223" Synthetic "4005 96

Aza Agd si s1u Guz Tut Tut Avr I1e eat TPh SsS1u Avr Tuk Niv Tug 1 5 19 15

Тук сі1у Меє Авр Уаї 20 «2105 97 «2115 324 «2125 ДНК «213» Штучна послідовність «2205 «223» Синтетична «4005 97 дссаєссдудда сЄдасссадес Ессаєсстсс сЕдЕСЕДдсає седтаддада сададісасс 60 ассасЕсдеЕс дддсаадсса дддсаєсада ааєдаєєвсад дссддсаєса дсадааасса 120 чдадааадссс ссаадсдасе даєстаєдсе асаєссадеє єдсааадедд ддЕсссаєса 180 addeEEsadsud dsadsddayes edddasadaa sEsasesesa saayessadsad sseEdsadsst 240 chaachaeeesstd saaseetayesta seEdeESsvtasad sayegaatadesi asssdsesas EEESOdsdda 300 chddadassaadd Yeddaaayesaa asda 324

"2105 98 "2115 108 "2125 PZtT "213" Artificial sequence "2205 "223" Synthetic "4005 98

A1ia Iie Aktu Mes TPtI sip bet Rko bBeti bBeit Gay begt Aia bBet Ua1i 01Uu 1 5 10 15

Avr Atud Uaii TPt Iie TPt Suv Agkd Aia bek bip s1u Iie Agd Avp Avr 20 25 30

Tecp s1u Ter Tut bip sbip Guv Rgo biu puv Aia Rgo Guz Agd Iets Iie 35 40 45

Tut Aia TPt bBet bBet Gay beep bek biu Ua1 Rko bBet Akd Rpe bBeh SHU 50 55 bo bek biu bek bi1u TPkh bi Rpe TPt TIetsy TPI Iiee bBet Bek Gay sip Rgo 65 70 75 80

Sish Avr Rpbe ATia Tit Tut Tuk Suv Gets Sip Niz Azp bet Tut Rgo Iets 85 Zo 95

TPE Rpe siu s1i1u s1u TPih Kguz Uai1i bish Iie Guv Agd 100 105 "2105 99 "2115 18 "2125 DNA "213" Artificial sequence

Zo "2205 "223" Synthetic "4005 99 saddaddsayeta daaaesdvaye 18 "2105 100 "2115 6 "2125 PZtT "213" Artificial sequence "2205 "223" Synthetic "4005 100 bip b1u Iie Agd Avp Avr 1 5 "2105 101 9 "2115 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 101 dsvEasess Z

"2105 102 "2115 Z "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 102

A1ia TpPkK 5eg 1 "2105 103 "2115 27 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 103 stasadsata agadesstass dsesase 27 "2105 104 "2115 9 "2125 PZT "213" Artificial sequence

From "2205 "223" Synthetic "4005 104

Petz sb1ip Niv Avp beg Tuk Rgo Iets Tig

C5 1 5 "2105 105 "2115 378 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 105 chadadeEdsads sdddyddades eddddddadss EEDddsasads sedddddddES sseEDadasys 60

ЕссЕдвасад ссЕСЕДДЕЄС сасстсаде аассасдаса гдсасвдддЕ ссдссаааст 120 асаддааагад чЕСсддадед дассеЕсадсе аєєдавтастд ссддеЕдасас агастаєсса 180 чдасЕСсСсдєда адддссдаєс сассдЕСтІсс ададааааєсуд ссаадаасіс стеЕЕсатєсеє 240 саааєсдааса дссідададс соддадасасу дстдеЕЧтаєст асесдедсаад даваддддаад 300 таєсасдата ЄЕЕсддассд Єдасвассас гассасддра єддасдеєсьд дддссааддуа 360 ассасддеіса ссдеісстсс 378 «2105 106 «2115 126 «2125 ПЗТ «213» Artificial sequence

"223" Synthetic "4005 106 si Mai sbip Gets Uai Sits back sbiu si1u Ai1a Hey Uai1i sip Rgo si1u schu 1 5 19 15

Zzek Gay Agd Gay bek Suv TPt Aia bek s1u Rpe TPtI RPpe bek Avp Tuk 20 25 30

Avr Mee Niz Tkr Ua1i Agd bip Tph TiBg sb1iu puv SsS1u yets s1p Tgr Ii1e 35 40 45 zehk Aia Iie Avr Tph Aza sb1u Avr TPI Tut Tut Rgo siu bek Uai1i Guz 50 zo bo s1u Akud Rpe TPtI Uaii bek Agd Gup siy Avp Aza bet РПе Tut TIец 65 70 75 80 beep Mee Avp beg Hey Agu Aza sb1u Avr TPtI Aia Uai1i! Tug Tuk Suv Ayia 85 30 35

Atd bi s1u Puv Tut Tut Avr I1iee yetssh TPt C1u Avr Tut Niv Tug Tug 1600 195 110 siu Mesie Avr Uaii Ttr siu sip siu TPti TPti Ua1 TiPg Uai bBekg 115 120 125 "2105 107 "2115 321 "2125 DNA sequence "2 "2205 "223" Synthetic

Зо «4005 107 часаєссада сдасссадес Ессаєсстсс сЕдЕСЕДдсає седтаддада сададісасс 60 ассасЕсдеЕс дддсаадсса дддсаєсада ааєдаєєвсад дссддсаєса дсадааасса 120 чдададааадссс стаадсдасі дассвтаєсдсе асаєссадес єдсааадсдд ддЕсссаєса 180 аддеЕЕсадсуд дсадсддаєс єдддасадаа сЕсасесеса сааєссадсад ссеЕдсадсст 240 чаачаєєеєсдд саасесаєта ссдессвасад саєсаасадеє асссдсссас ЕсЕСЯОДсдда 300 чддадассаадс сддадаєсаа а 321 «2105 108 «2115 107 « 2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 108

Avr Iie sbip Meyese TPtI sip bet Rko bBeti bBeit Hey begt Aia bBet Ua1i 01Uu 1 5 19 15

Avr Atud Uaii TPt Iie TPt Suv Agkd Aia bek bip b1iu Iie Agd Avp Avr 20 25 30 tep s1u Tkr Tut bip bip uv Rgo bi1u Guv Aia Rgo Guz Akd Tec Te 35 40 45

Tut Aia TPt bBet bBet Hey bip bek biu Ua1 Rgo bBet At9d Rpe Beh schu 50 zo bo zek biu bBet biu TPh si Rpe TPt iets TPi Iie bBegk bBetg Hey bip Rgo 65 70 75 80

Sish Avr Rpbe ATia Tit Tut Tuk Suv Giets Sip Niz Avp bet Tut Rgo Iets 85 30 35

TPE Rpe siu siu s1u TPzhi Kpuvz ge bi Iie Guz 1600 195 "2105 109 "2115 372 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 109 saddedsads sddedsades edddddadds dEddESsSsads sedddaddes sseddasts 60

ЕссЕдЕдсад сдЕСЕДДДЕЄ сассеЕЕсаде аасеЕсвддса єдсассддді ссдссаддсе 120 ссаддсаадд дасеЕддадед дагадсадеє асасдаєєсу асдааасєтда гааасастатє 180 дсадассссд сЄдаадддссуд аєссассаєс ссададаса ассссаадаа сасдседнає 240 ссдсаааєсда асадсстрдад адссдаадас асддсеЕдеЕЧді асстасеєдедс дсдадаачас 300 тасчдаваєєє сдаседдеєста свтаєвтасдсе аєддасдЕСсСї ддддссаадд дассасддіс 360 ассдЕСстсся са 372 «2105 110 «2115 124 «2125 ПЗТ «213» Artificial sequence "2205 "223" Synthetic "4005 110

Zo sbip Mai sbip Gets Uaii Sip back siu si1u siu Ua1i Ua1 sbip Rgo s1u Agd 1 5 19 15

Zzek Gay Agd Getsp bek Suv Aia Aia bek s1u Rpe TPti Rpe bek Avp Rie 20 25 30 sSiu Mesye Niz Ter Uai Agd SsSip Aza Rgo sS1u Guz sS1u htc bis Tgr Uai

Z5 35 40 45

A1ia uaii Iie Tgr Rpe Avr sit I1e Avr Guz Tut Tut Aia Avr bBet Uai 50 zo bo

Ace s1u Akd RpPe TPi Iie bek Agud Avr Avp o bet Guv Avp TPt Gets Tuk 65 70 75 80

Rgo Sip Mee Avp bek Gets Agd Aia Sbits Avr TPI Aia Ua1i Tuk Tuk Suv 85 30 35

Aza Atud bi Avr Tut Avr I1e Getsp TPi Sbiu Tut Tut Tut Aia Mee Avr 1600 195 110 uai TEr s1u sip siu TPti TPt Ua1! TPtiI Uaii bBektk bekg 115 120 "2105 111 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 111 chadEEsassya tadeases vdos 24 "2105 112

"2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 112 s1iu Rpe TPI RpPe bBeg Avp Rpe s1U 1 5 "2105 113 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 113 asayesdChdaeestd ayedaaaeeda eaagaa 24 "2105 114 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 114

I1e Ter Rpe Avr sitz I1e Avr Guz 1 5 "2105 115 "2115 51 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 115 chdsdsdachaach asssasdchasaye eeEeChaseEddeE sasvtayesasu stasddasdE s with "2105 "1512 127 "21 213" Artificial sequence "2205 "223" Synthetic "4005 116

Aza Akud bi Avr Tut Avr chI1ie Gecp TPg siu Tukhk Tukhk Tug Aia Mee Avr 1 5 10 15 uai

«2105 117 «2115 321 «2125 ДНК «213» Штучна послідовність «2205 «223» Синтетична «4005 117 часаєссада сдасссадес Ессаєсстсс сЕдЕСтдсає сеЕдстаддада сададссасс 60 аєссасеЕсдсс дддсаадсса дддсаєсада ааєгчаєттад дссддваєса дсадааасса 120 чададааадссс стаадсдссі аасстаєсдсе дсаєсссдЕс гдсааадсдд ддеЕсссаєсу 180 аддеЕЕсадсуд дсадсддаєс гдоддасадаа сссассістса сааєссадсад сстдсадссе 240 chaachaeeeeesud daasesayesta sedESvasad sasaarades asssassasee sddssaaddua 300 assaaddedd adayessaaasch a 321 "2105 118 "2115 107 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 118

Avr Iie sbip Meyese TPtI sip bet Rko bBeti bBeit Hey begt Aia bBet Ua1i 01Uu 1 5 19 15

Avr Atud Uaii TPt Iie TPt Suv Agkd Aia bek bip b1iu Iie Agd Avp Avr

Zo 20 25 30 tep s1u Tkr Tut beep beep uv Rgo bi1u Guv Aia Rgo Guz Akd Tec Te

Tut Aia A1ia bBet Agkd Hey bip bek biu Ua1 Rgo bBet At9d Rpe Beh schu zo bo 35 zek biu bBet biu TPh si Rpe TPt iets TPi Iie bBegk bBetg Hey bip Rgo 65 70 75 80

Sish Avr Rpe sS1u TPt Tut Tuk Suv Gets Sip Niz Avp bet Niz Rgo TEG 85 30 35

Rpe s1u bip b1u TPh puv Ua1 sbish Iie Guv Agd 40 1600 195 "2105 119 "2115 18 "2125 DNA 45 "213" Artificial sequence "2205 "223" Synthetic 50 "4005 119 sadddsaeta daaaedvae 18 "2105 5520 6 "211 PZT "213" Artificial sequence "2205

"223" Synthetic "4005 120

Sbip b1u Iie Agd Avp Avr 1 5 "2105 121 "2115 9 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 121 dsEdsayess Z "2105 122 "2115 Z "2125 PZT "213" Artificial sequence "2205 " 223" Synthetic "4005 122

Aza Aia 5eg 1 "2105 123 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 123 srasadsata agsadssasss sass 24 "2105 124 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223 Synthetic "4005 124 tep sip No Avp betg Niv Rgo TIC 1 5 "2105 125 "2115 369 "2125 DNA "213" Artificial sequence "2205

"223" Synthetic "4005 125 saddeEdsads sdddyddades eddddadadds dEddESsSSsSads sedddaddes sstdadasyis 60

Есссдсдсад сдЕСтдддадеЕЕ сассеЕєтаде аасееЕсддса гЕдсассддде ссдссаддсе 120 ссаддсаадд доассєддадед ддєддсадеЕє авгаєдаєєсу асддаааєєсда сааасастає 180 дсадассссд сЄдаадддссуд аєссассаєс ссададаса ассссаадаа сасдстднає 240 ссдсаааєсда асадсстрдад адссдаадас асддсеЕдеде аєсасвдедс дсдадаачас 300 тасчдаваєєє сдаседдеєста свтаєвтасдсе аєддасдЕСсСї ддддссаадд дассасддіс 360 ассаєсесс 369 «2105 126 «2115 123 «2125 ПЗТ «213» Штучна sequence "2205 "223" Synthetic "4005 126 sbip Mai sbip Gets Uai sitz becht si1u si1u s1u Uai Uaiyi beep Rgo s1u Akgd 1 5 19 15

Zzek Gay Agd Getsp bek Suv Aia Aia bek s1u Rpe TPti Rpe bek Avp Rie 20 25 30 sSi1iu Mee Niz Tkgr Uai1 Agkd sS1ip Aia Rgo s1u Puv sS1u iets si Tkr Uai1

A1ia uaii Iie Tgr Rpe Avr sit I1e Avr Guz Tut Tut Aia Avr bBet Uai zo bo

Ace s1u Akd RpPe TPi Iie bek Agud Avr Avp o bet Guv Avp TPt Gets Tuk

From 65 70 75 80

Rkto beep Mees Avp back Gay Agud Aia bij Avr TPI Aia Uaii Tuk Tuk Suv 85 90 35

Aza Atud bi Avr Tut Avr I1e Gecp Tpi Sbiu Tut Tut Tut Aia Mee Avr 1600 195 110 35 Uua1ii Ter b1u sip sSi1u TPti TPt Ua1i TPg Uai 5eg 115 120 «2105 127 «2115 318 40 «2125 DNA «252» 213 «252» Artificial sequence «223» Синтетична 45 «4005 127 часаєссада сдасссадес Ессаєсстсс сЕдЕСЕДдсає седтаддада сададісасс 60 ассасеЕсдсс дддсаадсса дддсаєсада ааєдарєвсад дссддсаєса дсадааасса 120 чддадааадссс стаадсдссі аассвтаєсдсе дсаєсссдс гдсааадсдд ддеЕсссаєсу 180 50 аддеєссадсуд дсадсддаєс єдддасадаа Ессасссьса сааєсадсад ссеЕдсадсст 240 чаачаєєсстд даасетаєста сеЕдеЕсвасад саєгаавгадес ассссассьсє сддссааддуа 300 ассааддеєдд адаєссааа 318 «2105 128 "2115 106 "2125 PZT "213" Artificial sequence

"223" Synthetic "4005 128

Avr Ii1e sbip Mayes TPg bip betg Rkgo bek bBeg Hey bek Aia Beh Uai U 1 5 19 15

Avr Atud UMaii TPt Iie TPt Suv Akud Aia bek sbip s1u Iie Guv Avp Avr 20 25 30 tep s1u Tkr Tut bip bip uv Rgo bi1u Guv Aia Rgo Guz Akd Tec Te 35 40 45

Tut Aia A1ia bet Agkd Gay bip bek biu Uai Rko bBet Akd Rpe bBeh SHU 50 zo bo bek biu bek bi1u TPkh bi Rpe TPt TIetsy TPI Iie bBet Bek Gay sip Rgo 65 70 75 80 si Avr Rpe s1u TPti Tugk Tuk Suv Tets S1ip Niv Avp bek Niv Rgo TPg 85 30 35

Rpe s1u bip b1u TPkh puvz Uai bij I1ie Guz 1600 195 "2105 129 "2115 381 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 129 chaddeEdsads sdddyeddades ddddddadds ayeddsasads sedddddddES sseEDadasts 60

Зо Есссдсдсад ссЕСТтддаре сассеЕссаді аассасдаса гдсассдддеЕ ссдссаадсе 120 асаддааагад чЕСсддадед ддЕсЕСааді аєєдавастід ссддддасас стастаєсса 180 часеЕссдєда адддссдсЕс бассаєстісс ададааааєд ссааавасіс сстднаєсеєс 240 саааєдааста дсссдададс сддддасасуд дсеЕдЕЧдтаєсс ассднасаад ддадссссда 300 ааєсасдааа ЄЕЕсдассдд єсасвассас гассасддра єддасаєстд дддссааддуа 360 ассасддєса ссдустсстс а 381 «2105 130 «2115 127 «2125 ПЗТ « 213" Artificial sequence "2205 "223" Synthetic "4005 130 si Mai sb1ip Gets Maii sit becht siu si1u s1u Mee Uai bip Rgo s1u 1U 1 5 19 15

Zzek Gay Agd Getsp bek Suv Aia Aia bek s1u RPpe TPti bek bek Avp Tukg 20 25 30

Avr Mee Niz Tgr Uai1 Agd sSip Aia TPkg si1u puv s1u iets si Tkr Uai 35 40 45

Zek Bek I1ie Avr Tph Aia sb1u Avr TPtg Tut Tut Rgo Avr bek Uai1i Guz 50 zo bo s1u Akud Rpe Iie Iie 5Begt Akd sits Avp Aia Guz Avp bet ieets Tut TGec 65 70 75 80 bip Mesye Avp o bBet Gets Agud Aia s1u Avr Tpi Aia UMai Tut Tut Suv TPg 85 30 35

Ahtud bitz Rgo Agd Avp Tut Si I1e iyets TPkg s1i1u Niv Tut Niz Tukg Niv

100 105 110 siu Mesye Avr Iie Ttr siu sip siu TPti TPk Ua1 TPg Uai bek 5eg 115 120 125 "2105 131 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 131 chsadas2 ssadas2 ssadas25 " 132 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 132 s1u Rpe TPt bek bBeg Avp Tut Avr 1 5 "2105 133 "2115 21 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 133 ayesedavjeastd seddddasas 21 "2105 134 "2115 7 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 134

I1ie Avr TPtI Aza c1u Avr TPg 1 5 "2105 135 "2115 63 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic

"4005 135 asaadddads sssdaaaesta sdaaayesevd asrddesasoi assassassa sddeaeddas 60 ayess 63 "2105 136 "2115 21 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 136

TE Agd Si Rgo Agd Avzp Tut S1its I1ie Getsh TPkg SsS1iu Niz Tuk Niv Tug 1 5 19 15

Niz s1u Mee Avr Ii1e "2105 137 20 "2115 324 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 137 chasayessada sdasssades dssayesstss sEdESEDdsaye sedtaddada sadadisass 60 assaseEsdss dddsaadssa ddaessaedsada dssaedsadassadassadassadassadassadassadass20

Zo chadaaasss staaaseisst dahastatase dsaeyesadee gasadadedu ddEsSssayesa 180 addeEEsadsud dsadsaaayes eddsasadas sssasestsa ssayessadsad sstdsadsst 240 chaachaeeesstd sdaseetayesta seEdeESvdsad dahsasassta asssiEsddas dEeSddssaa 300 chdadassaadsad2 sEda

C5 "2105 138 "2115 108 "2125 PZT "213" Artificial sequence 40 "2205 "223" Synthetic "4005 138

Avr Iie sbip Meyese TPtI sip bet Rko bBeti bBeit Hey begt Aia bBet Ua1i 01Uu 45 1 5 19 15

Avr Atud Uaii TPt Iie TPt Suv Agkd Aia bek beep Aia Iie Agd Avp Avr 20 25 30 tep s1i1u Tkr Tut beep beep uv Rgo biu Guv Aia Rgo Guz Getsp Gets I1e

Tut TPg Aia RpPe bBet Te sip beg Sbiu Uai Rgo Beh Agd Rpe bBeh SHU 50 zo bo

Zzek Gpuzv bek bi1u TPh Avr Rpe TPti TIetsy TPI Iie bBet Bek Gay sip Rgo 65 70 75 80 bis Avr Rpe Aia TPtI Tut Tut Suv Gets bip Avr Tut TPti Avp Rgo Agd 85 90 35

TPE Rpe siu sip s1u Tpyh Kguz Uai1i bish Iie Guv Agd 1600 195

"2105 139 "2115 18 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 139 saddssayeta daaaedvaye 18 "2105 140 "2115 6 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 140 bip Aia Iie Ag9d Avp Avr 1 5 "2105 141 "2115 9 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 141 assdsayess Z "2105 142 "2115 Z "2125 PZT "213" Artificial sequence "2205 "223 » Synthetic «4005 142

TVPg Aia RiPre 1 "2105 143 "2115 27 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 143 seEdsaddayee asassaayess eSddasd 27

"2105 144 "2115 9 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 144 tep sip Avr Tut TPII Avp Rgo Agd TOK 1 5 "2105 145 "2115 378 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 145 chaddeEdsads sddeddades dddddddads ayeddsasads sedddddddES sseEDadasts 60

ЕссЕдеєдсад ссЕСвддаєс сассеЕссаді аассасдаса єдсассдддЕ ссдссаадсе 120 асаддаааад деЕсСсддадед ддЕСЕСсааді астдасастд ссддддасас састаєсса 180 часеЕссдєда адддссдсЕс бассаєстісс ададааааєсуд ссааааасіс ссеЕдсатєсе 240 саааєдааста дсссдададс сддддасасуд дсеЕдЕЧдтаєсс ассднасаад ддадссссда 300 ааєсасдааа СЕЕсдассдд єсасврассас гассасддра єддасаєстд дддссаадда 360 ассасддеіса ссдеісстсс 378

From "2105 146 "2115 126 "2125 PZT "213" Artificial sequence

Z5 "2205 "223" Synthetic "4005 146 si Mai sbip Gets Uai Sic bek siu si1u siu Mee Uai1i sip Rgo s1u schu 1 5 19 15

Zzek Gay Agd Getsp bek Suv Aia Aia bek s1u RPpe TPti Bek bek Avp Tukg 20 25 30

Avr Mee Niz Ter uUai Agd bip Aza Tph s1u Guz SsS1u iets s1ts Tgr Uai1i 35 40 45

Zek Bek I1ie Avr Tph Aia sb1u Avr TPI Tut Tut Rgo Avr bek Uai1i Guz zo bo s1u Akud Rpe Iie Iie 5Begt Akd sits Avp Aia Guz Avp bet yeets Tut TGec 65 7o 75 80 50 bip Mee Avp o beg Hey Agu Aza sb1u Avr TPtI Aia Ua1i! Tug Tuk Suv TPg 85 30 35

Ahtud bets Rgo Agd Avp Tut Si I1e iets TPkg s1i1u Niv Tut Niz Tukg Niv 1600 195 110 siu Mesye Avr Iie Tgtr siu sip siu TPti TPti Ua1 TiPg Uai bBekg 115 120 125 "2105 147 "2115 321

«2125 ДНК «213» Штучна послідовність «2205 «223» Синтетична «4005 147 дссаєссада сдасссадес дссаєсстсс сЕдЕСЕДдсає седтаддада сададісасс 60 ассасеЕсдсс дддсаадсса ддссаєсада ааєдарєвсад дссддсаєса дсадааасса 120 чадааадссс стааасеісст даєстатасе дсаєєсадеє гасададеду ддЕсСссаєса 180 аддеЕЕсадсуд дсадсаааєс єддсасадас ЕСасесеса ссаєссадсад сстдсадссе 240 чаачаєєсстд сдасеєтаєста сеЕдеЕСвдсад даєсасасста асссіЕсддас dEeSddssaa 300 chddadassaadd sddaaaesaa a 321 "2105 148 "2115 107 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 148

A1ia Iie sbip Meyese TPt sip bet Rko bBeti bBeit Hey begt Aia bBet Ua1i 01Uu 1 5 19 15

Avr Atud Uaii TPt Iie TPt Suv Agkd Aia bek beep Aia Iie Agd Avp Avr 20 25 30 tep s1i1u Tkr Tut beep beep uv Rgo biu Guv Aia Rgo Guz Getsp Gets I1e

Zo Tut TPg Aia RpPe bBet Te sip beg Sbiu Uai Rgo Beh Agd Rpe bBeh SHU zo bo

Zzek Gpuzv bek bi1u TPh Avr Rpe TPti TIetsy TPI Iie bBet Bek Gay sip Rgo 65 70 75 80 bis Avr Rpe Aia TPtI Tut Tut Suv Gets bip Avr Tut TPti Avp Rgo Agd

Z5 85 30 95

TPE Rpe siu sip s1u TPzhi Kpuz Uai bi Iie Guz 1600 195 "2105 149 40 "2115 345 "2125 PZT "213" Artificial sequence "2205 45 "223" Synthetic "4005 149 sSip Ma1i Mes Avr Rpe Gets Rpe sitz Gpuz Tgr Guz Tut b1u Avr sip 1 5 19 15 50 Suv Niv Niv Avp iets bet Getsp tep Rgo Rgo Rgo TPkg Si Iets Ua1! Suv 20 25 30

Avp Atud TPt Rpe Avr Gpuv Tut bet Suv Tgr Rgo Avr TPtI Rgo Aia Avp 35 40 45

TE TrI ATa Avp I1e bek Suv Rgo Tgr Tut yets Rgo Tgr Niv Niv Guz 50 zo bo wai b1ip Niz Akud Robe Ua1i Ribe ace Agd Suv s1u Rgo Avr s1u bip Tgr 65 70 75 80 wai Aktu s1u Rgo Akd siu sip Rgo Tgr Agd Avr Aza bet beep Suv sip

85 of 95

Mee Avr so1u sic sic I1i1e si Uai sip uz bic Uai Aia puv Mee Tug 100 105 110 bek bek Rre Sip Uai Mes Avr Gpuv TPt Niz TPt Suz Rgo Rgo Suz Rgo 115 120 125

Aza Rgto sbits Gets Gets siu siu Rko bek Uai1i Rbe Gets Rpe Rkgo Rkgo Guz 130 135 140

Rko Guz Avr TPh Ge Mees Iie bBeg Agud TPtI Rgo si Ua1i TPkh Suz Uai 145 150 155 160

UuUai Ma1 Avr Ua1 bek Niv s1ts Avr Rgo Ssitzsch Ua1! Guz Ribe Avp Tgr Tug 165 170 175 uai Avr s1u Ua1! sats Uai Niv Avp Aia yuv Trt Kguv Rgo Agd si b1ts 180 185 190

Sip Tut Avp bek TPt Tut Agd Uai Ua1i bek Ua1i! iets TiPgt Uai Iets Nivs 195 200 205 bip Avr Ttr Gay Avp o siu Gpuv sats Tut puv Suv guv Uai bBet Avp Guz 210 215 220

A1ia tey Rgo Aia Rgo Ii1e sits Gpuv Tpi I1i1e bBet guv Aia Guv siu sip 225 230 235 240

Rgo Akd bi Rgo sbip Ma! Tuk TPt Pets Rkgo Rgo bBet Agud Avr bij Gets 245 250 255

TPE guv Avp o bip Uaii bek Ggetsp TPkhkh Suv Gay Uai puzv s1u Robe Tuk Rgo 260 265 270 zeht Avr Iie Aia Ua1 sbish Tgtr bij bBeg Avp s1iu sip Rkgo b1y Avp Avp 275 280 285

Tuk Gguv TPzhI TPtI Rkgo Rgo Uai Ggetsp Avr bBeght Avr siu bBet RPe RiPe Iets 290 295 300

Tuk bBet puv Gecp TPi Ua1i Avr puv bek Agd Tgtr bip sip s1u Avp Uai1i 305 310 315 320

Zo Rpe bek Suv bek Uaii Mee Niv si Aia iets Niv Avp Niv Tug TPk s1ip 325 330 335

Kpuzv bBet Gets bet Gets bek Rgo b1UuUu Guz 340 345

З5 "2105 150 "2115 348 "2125 PZT "213" Artificial sequence 40 "2205 "223" Synthetic "4005 150 cSip Ma1i Mes Avr Rpe Gets Rpe sitz Gpuz Tgr Guz gay Tut b1u Avr sip 45 1 5 19 15

Suzv Niv Niv Avp iets bek Gay Gets Rgo Rgo Rgo TPt Si Iyets Uai Suvz 20 25 30

Avp Atud TPt Rpe Avr Gpuv Tut bet Suv Tgr Rgo Avr TPgI Rgo Aia Avp

TI Trg AtTa Avp oI1e betg Suv Rgo Tgr Tut Gets Rgo Tgr Niv Nivz Guv 50 55 bo wai b1ip Niz Akud Robe Ua1i Ribe ace Agd Suv s1u Rgo Avr s1u Sip Tgr 65 70 75 80 wai Aktu s1u Rgo Akd siu sip Rgo Tgr Agd Avr Aza bet beep Suv sip 85 30 95

Mee Avr so1u sic sic I1i1e si Uai sip uz bic Uaii Aia puv Mee Tug 100 105 110 bek bek Rre sip Uai Mesye s1iu Rgo b1u Avr Guz TPI Niv TPg Suz Rgo

115 120 125

Rgo Suv Rgo Aza Rgo bitsch Gay Gay s1iu s1u Rgo Bek Uai1i Rpe Gets RiGe 130 135 140

Rko Rgo Gpuv Rgo guv Avr TPth Gay Mes Iie 5bBet Agd TPh Rkgo si Uai 145 150 155 160

TE Suv Ua1i uai Uai Avzr Uai be Niv bit Avr Rgo bit Uai1i! Kpuv RBGe 165 170 175

Avp oTtgr Tut Ua1! Avr cS1u Ua1! Soc Uai Niv Aup Aia Guz Trt Guv Rgo 180 185 190

Akd bi bi sip Tugk Avp obet TP Tut Akd Uai1i Uai1! bBegk Uaii Getch TEG 195 200 205 uai Gets Niv bip Avr Tgr Te Avp sS1u Puv si Tut Guz Suv Kpuv Uai1i 210 215 220 zek Avp guv Aia gay Rgo Aza Rgo Iie bij Ppuv TPI I1ie bek Guz Aia 225 230 235 tuzsipgosi u 240 Agkd bi Rgo bip Ua1! Tuk TPti Gets Rgo Rgo 5Beh Agd 245 250 255

Avr bish Getsp TPtg Guv Avp o sip Uai bBet Gay TPt Suv getc Uai puv 01U 260 265 270

Rpe Tut Rgo bBecht Avr I1ie Aia Uaii Sits Tgr bij bBeg Avp s1u Sbip Rgo 275 280 285 biCh Avp o Avp o Tut Guv TPt TPt Rko Rkgo Uai gay Avr bBet Avr s1u 5Beg 290 295 300

Rpe RiPe Gay Tuk bek Ppuv Gay TPI Uaii Avr Guz bek Akd Tgr Ssip sip 305 310 315 320 sSi1u Avp Ua1 Rie betg Suz bek Uai Mee Niv sb1c Aia Gay Niz Avp Nivs 325 330 335

Tuk TPt sip puv bek Ggetsp bek Getsp bek Rgo so1U Guv 340 345

From "2105 151 "2115 146 "2125 PZT "213" Artificial sequence

Z5 "2205 "223" Synthetic "4005 151

Sip Mai Mes Avr Rpe iets Rpe siy Gpuv Ter Guz Gets Tug o1u Avr Ssip 1 5 10 15

Suzv Niv Niv Avp iets bek Gay Gets Rgo Rgo Rgo TPt Sits Gets Uai Suvs 20 25 30

Avp Atud TPt Rpe Avr Gpuv Tut bet Suv Tgr Rgo Avr TPgI Rgo Aia Avp 35 40 45

TE TrI ATa Avp I1e bek Suv Rgo Tgr Tut Gets Rgo Tgr Niv Niv Guz bo uai b1ip Niz Akud Robe Ua1i Ribe ace Agd Suv s1u Rgo Avr s1u Sip Tgr 65 70 75 80 50 uai Akud b1u Rgo Akd biu biu Rgo Tgr Agd Avr Aia beg Sip Suv Sip 85 Zo 95

Mee Avr so1u sic sic I1i1e si Uai sip uz bis Uaii Aia puv Mee Tug 100 105 110

Zek bek Rpe bip Ua1i Mee si sbip Gpuv Hey Iie bek bi bii Avr Iey 55 115 120 125 sSiu b1u si sSip was iets I1ie beg Sbits Si Avr Iets Niv Niz Niv Nivs 130 135 140

Niv Niv

"2105 152 "2115 351 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 152 s1u Aia Rgo sbip Ua! Mee Avr Rpe Getsp Rpe bitz guv Tgr puv Iets Tug 1 5 10 15 s1u Avr Sip Suv Niv Ni Avzp Hey bBet KGec Gets Rgo Rgo Rgo TPk s1tsy tep Uaii Suv Avp Akd TBPh Rbe Avr puz Tut bBet Suv Tgr Rgo Avr TPkK

Rgo Ai1a Avp TPh TPt Aza Avp o I1ie begt Suv Rgo Tgr Tut Iets Rgo Tgr bo 20 Niv Niv puv Ua1i sip Niv Akd Rpe Ua1! Rpe Puv Agd Suv S1u Rgo Avr 65 70 75 80 siu bip Tktr Mai Agd siu Rgo Akd si1u bip Rgo Tgr Agd Avr Aia 5eg 85 Zo 95 sbip Suv bip Mes Avr siu sitc si Ge bi Uaii bip uv bis Uai A1a 25 1600 105 110 Belly Mee Tugk bBetg behg Ribe sip Ua1i Mee si1u Rgo SsS1u Avr Guv TiPg Niz 115 120 125

TPk Suv Rko Rko Suv Rgo Aza Rgo bicp iey Gets s1u s1i1u Rko bek Uai1i 130 135 140

Zo Rpe Gay RpPe Rgo Rgo Puv Rgo Guv Avr TPti Gay Mee I1i1e Beh Agd TIPg 145 150 155 160

Rgo Si Uai TrI Suv Uai Uai Uai Avr Uai beg Niz sb1ts Avr Rgo s1ts 165 170 175 uai puv Rpe Avzvp Tgr Tut Uai! Avr cS1u Uai! Soc Uai Niv Aup Aia Guz

Z5 180 185 190

TPk Gpuv Rgo Akd bi bi bip Tuk Avp obet TPtI Tut Agtd Uai1i Uai1i bek 195 200 205 uai Gets Trphi Uai Getsshsz Niz Sip Avr Tgr Gets Avp sS1u Puv S1its Tuk Guz 210 215 220 40 Suv puv Uaii bBet Avp Guz Ai Aza iey Rgo Iie si Guz TPk Iie 225 230 235 240

Zzek Guz Aia guv b1u beep Rgo Agud bij Rko sip Ua1i Tuk Tik Gets Rgo 245 250 255

Rko Bek Agd Avr bi Gay TPg Guv Avp o sip Ua1i bek Getsp TPg Suv Gay 45 260 265 270 mai puv s1u RPe Tut Rko bBet Avr I1e Aia Uaii si Tgr sitz bBet Avp 275 280 285 siu bip Rgo bis Avp Avp Tut puv TPti TBPpi Rgo Rgo Uaii yets Avr 5eg 290 295 300 50 Avr siu Bek RPpe Rpe yets Tut Bek Gpuv Gets TPh Ua1i Avr Gguv bBetg Agd 305 310 315 320

Tegr s1p sbip sb1u Azp Ua1 Ribe behg Suv bBet Ua1 Mee Niv SsS1ip AiTa Iets 325 330 335

Niz Avp Niv Tut TPtI Sip Puv beg Gay bBetg KGets beg Rgo s1u Guz 55 340 345 350 «2105 153 «2115 477

"2125 PZT "213" Noshto zariepz "4005 153

Me Rgo Rgo Suv beep Rgo beep Agkud Rgo Gay Gay yeets Gay Gets Gets Gay 1 5 10 15 tep Aia Suv sip Rggo beep Uai Rgo bek Aia Sbip Uai Me Avr RoPe Geeeets 20 25 30

Rpe si Puv Tgr puv Hey Tut S1u Avr sip Suv Niz Niv Azp Gets 5bekg 35 40 45 tep Gets Rko Rko Rkgo TPti bi Ggetsp Ua1i Suv Avp Agud TpPt Rpe Avr Guz 50 55 bo

Tuk bBet Suv Ter Rgo Avr TPhi Rgo Aza Avp TPtI TPI Aia Avp I1e bek 65 70 75 80

Suv Rgo Ter Tut Pec Rgo Ter Niv Niv puv Ma1i SsSip Niz Agd Rie Uai 85 Zo 95

Rpe Guz Agd Suv b1iu Rgo Avr b1u bip Tgtr Uai Akd s1u Rgo Agd Sh1Uu 100 105 110 bip Rgo Tgtr Agud Avr Aia bBet sip Suv sip Mee Azvr sS1u bii bits Ii1e 115 120 125 si Maii beep puv sits Mai Aia puv Mee Tut bBe Rpe beep Waii! It has 130,135,140

Tuk TPt Uai si1iu Tut bek Ggetsp bek Ge b1u Aia Getsp Getsp Gay Aia Gets 145 150 155 160

A1a I1e tiets s1u cS1u Tiets bek Guv Tets Niv Suv TPI Agud Avp AtTa I1e 165 170 175

Niz ATia Avzp iets Rpe Aia bBetg Rire Ua! Ietsch Gpuv Aia bBet bBet Uai1i Iets 180 185 190 uai Iie Avr siu Gets Gets Akud TPtI Agud Tut bet bip uv Iie S1Uu Avr

From 195 200 205

Avr Gay back MUMai! bBet TPtI Tkr Ggets Beh Avr biu Aia uUai Aia 01Uu Suv 210 215 220

Atud uaii Aia Aia Uai Rpe Mee sSip Tukhk s1u Iie Uai Aia Avp Tut Suv 225 230 235 240

TER Gets iets Uai si sSi1iu Pets Tut Pets Niv Avp Tets Iiets S1u Iets A1a 245 250 255

TPE Gets Rko si Akd bek Ribe Ribe bek Hey Tut yets s1u Iie s1Uu Tgr 260 265 270 s1u Aia Rgto Mes Gets Rpe Uai! Uai Rkgo Tgr Aia Uai Uai puv Suv GIets 275 280 285

Rpe b1p Avp Ua1i bip Suv Tgr TPg bBeg Avp Avr Avp Mee siu RoPe Tgr 290 295 Zo

TER Ii1e hets Akud RiPe Rgo Ua1i Rpe Gay Aza Iie iets I1ie Aup Rope RiGe 305 310 315 320

Iie Rpe Uaii Akud I1i1e Uai sip Gets Hey Uai Aia Puv ey Agd Aia Agd 325 330 335

Sip Mesye Niz Niv TI Avr Tuk Puv Rrbe Agd iets Aia Guz bet TrE Iets 340 345 350

TE Gets I1ie Rgo iets iets sSiu Ua1 Niv bis Ua1i Uai Rbe Aia Rie Uai 355 360 365

TE Avr Si Niv ATia SsSip si1u TPi tep Agd bek Aia Guz iets Roie RBGe 370 375 380

Avr Gay RPpe Gets bet bet Rpe sip si1u Gay Ggets Uaii Aia Uai Iech Tug 385 390 395 400

Suv Rpe yeets Avp o puv Si Ma! SsSip bek sSits tey Agd Agd Agd Tgr Niv 405 410 415

Atud Ttr Agd Gets siu puv Uaii Ggetsp Tgr bi bis Agud Avp oTvbPt bet Avp 420 425 430

Niz Agud Aia bet bBeg bek Rgo SsS1u Niv siu Rgo Rgo bBet Kpuv bis TIets 435 440 445 bip Rpe b1u Atd siu siu siu siu bBet bip Avr begt bet Aia bis TPg Rkgo 450 455 460 tep Aia siu si1u Gecp Rgo Agd bek Sigo Ai РПе 465 470 475 "2105 154 "2115 485 "2125 PZT "2135" My5 shivschitsv "4005 154

Mesye Aia tep TPg sip ep Niv Suv Rgo Nivz Gay Yyets Iets IGets Iets Iets 1 5 19 15 wai Gay bek Suv Gets Rkgo si Aza Rgo bek Aza beep Ua! Has Avr RPe

Pecs Rpe si Gpuv Tgr puv Hey Tut beg Avr bip Suvz Niv Niv Avp Iets 20 zet Ggetsy Gets Rgo Rgo Rgo TPt Sbits Gets Mai Suv Avp Agud TPt Rope Avr bo

Ace Tut bBet Suv Tgr Rgo Avr TPh Rgo Rgo Avp TPI TPti Aza AvBp Pe 65 70 75 80 bek Suv Rgo Tgr Tuk Pec Rgo Tgr Tut Niz Ppuv Ua1ii s1ip Niv Agd Iets 25 85 30 95 uai Rpe uv Agd Suv siu Rgo Avr siu bip Ttr Uai Agd si1u Rgo Agd 100 105 110 siu bip Rgo Tgtr Agd Avp Aia bet sip Suzv bip geish Avr Avr sbits s1ts 115 120 125

Zo Iie bis Ua1ii sip uv siu Mai Aia puv Mee Tuk bek bBeg sip sip Uai 130 135 140

Mee Tut TPt UMai siu Tut bBet Gecp bet Ge biu Aia iets Iiets Iets A1a 145 150 155 160

Pecs Uai Iie yeetssh Giets SsS1u Gietssh Agd Guz Gets Niv Suv TrI Agd Avp Tug

Z5 165 170 175

IT1e Niz SsS1u Avp Giets Ribe Aia bet Rie Uai yets puv Aia s1u betg Uai 180 185 190 tep Uaiii I1ie Avr Tkgr Hey gay guv TPtI Akud Tut bet bip Guz I1e SHHU 195 200 205 40 Avr Avr Gets bBeg Uaii bBet Gev Mai1 biii Tgr Avr sb1u Ayia Messia Ayia 0Щ1U 210 215 220

Suzv Akd Uai Aia Tpi Uai Iie Mesye sip Tut s1u I1i1e I1e Aza Avp Tuk 225 230 235 240

Suv Ter Gets Gets Uaii si siu Ua1 Tuk petsy Tut bBet Getsp Getsp bek Gets 45 245 250 255

A1a TPI RpPe bBeg sats Akd bek RpPe RpPe beg Ietsy Tut Gets s1u Iie 01Uu 260 265 270

TER s1u Aia Rko Gets Hey Rbe Uai Iie Rgo Tgtr Uaii Uai Uai puv Suv 275 280 285 50 tep Rpe siy Avp Uaii sip Suv Tgr Tpi 5ek Avp Avr Avp Mee s1Uu RPe 290 295 300

Ter Tgr I1e hets Agud Iie Rgo Ua1 Rpe iey Aia Gets Hey Ii1e Avp Rie 305 310 315 320

Rpe Iie Rpbe Uai Niv I1ie I1ie Nivz Gets Hz Uai Aia Guz Gay Agd A1a 55 325 330 335

Niz sip Mee Niv Tut Aia Avr Tut Kpuv Ribe Agd tey Aia Agd betg TEPg 340 345 350

Pecs TPti Gets I1ie Rgo Gets iets sS1u Uai Niz si Uai Uai Rbe A1ia RBGe

355,360,365

Rpe Avr Gay Ribe Gay bet bet Rpe sip siu Gay Getsp Ua1 A1ia Uai Gay 385 390 395 400

Tuk Suv Rpe Gets Avp Guz bi Uai1i bip Aia sbits Gets Mee Agd Agd Tgr 405 410 415

Atud bip Ter sip sats s1u puv Aia ep bip bi bits Agd yeets Aia 5eg 420 425 430 bek Niv si1u bek Niv Mee Asia Rgo Aza sS1u Rgo Suv Niv Ss1u Avr Rgo 435 440 445

Suv sich puv Gets bSip Gay Mees bek Aza siu bet bBet Beh s1u TPkE schu 450 455 460

Suzv Maii Rko bBet Mer bi TP bek iets Aia bet bBet Getsy Rgo Agd Iets 465 470 475 480

Aza Avr bBeg Rgo TPg 485 "2105 155 "2115 491 "2125 PZT "213" Masasa Havsistiaghiv "4005 155

Mees Aia Rgo Suv bip Rgo Agud Agud Rgo Gay Gay yets Gay Gets Gets Gay 1 5 10 15 tep Aia Suv sip Rgo beep Aza Rgo bBet Aia sip Uai Me Avr Rie Iets 20 25 30

Rpe si Puv Tgr puv Gay Tut S1u Avr sip Suv Niz Niv Azp Gets 5bekg

Zo 35 40 45 tep Gets Rko Rko Rkgo TPiI bij gay Uaii Suv Avp Akd TPt Robe Avr Guz 50 55 bo

Tuk bBet Suv Ter Rgo Avr TPhi Rgo Aza Avp TPtI TPI Aia Avp I1e bek 65 70 75 80

Suv Rgo Ter Tut Pec Rgo Tgr Niv Niv puv Ua1i SsSip Niz Akd RiPe Uai1i 85 Zo 95

Rpe Guz Agd Suv b1iu Rgo Avr b1u bip Tgtr Uai Akd s1u Rgo Agd Sh1Uu 100 105 110 bip Rgo Tgtr Agd Avr Aia bek sip Suv bip Mesie Avr sS1i1u bis sich Gets 115 120 125 si Maii bip puv sic Mai Aia puv Mee Tut bBe Rpe beep Waii! It has 130,135,140

Tuk TPt Uai si1u Tuk bBet Gay bBet iets biu Aia Getsp Getsp Gay Aia Gets 145 150 155 160

A1a I1e tiets s1u cS1u I1e back KGuv Tiets Niv Suv TPI Agud AvBp AtTa I1e 165 170 175

Niz ATia Avzp iets Rpe Uai1i bek Rie Uai Iiets Puv Aia Beh bBet Uai1i Iets 180 185 190 uai Iie Avr siu Gets Gets Akud TPtI Agud Tut bet bip uv Iie S1Uu Avr 195 200 205

Avr Hey bBet UMai1i bBet Iie Tkr Gets Beh Avr biu Aia uUai Aia 01Uu Suv 210 215 220

Atud uaii Aia Aia Uai Rpe Mee sSip Tukhk biu Uaii Uaii Aia Avp Tut Suv 225 230 235 240

TER Gets yets Uai si sS1iu Pets Tut Pets Niv Avp Tets Iyets S1u Iets A1a 245 250 255

TPE Gets Rko si Akd bek Ribe Ribe bek Gay Tut yets s1u Iie s1Uu Tgr 260 265 270 s1u Aia Rgto Mes Ge Ribe Ii1e I1e Rkgo Tgr Uai Uai Uai Agd Suv Iets 275 280 285

Rpe b1p Avp Iie bip Suv Tgr TPg bBeg Avp Avr Avp Mee siu RoPe Tgr 290 295 300

Ter I1ie gay Aku Rpe Rgo Uai Rpe hets Aza Iie Gets I1e Avp Rpe Rpe 305 310 315 320

IT1e Rbe I1ie Agud I1ie Uai Niz Iiets iets Uai Aia guvz Giets Agd A1i1a Agd 325 330 335

Sish Mee Niz Niv TI Avr Tuk Puv Rrbe Agd iets Aia Guz bet TrPE Iets 340 345 350

TE Gets I1ie Rgo iets iets sSiu Ua1 Niv bis Ua1i Uai Rbe Aia Rie Uai 355 360 365

TE Avr Si Niv ATia sSip si1u TPti tep Agd Rbe Aia Guz iets Roie RBGe 370 375 380

Avr tep Rpe Hey bet bBet Rpe sbip siu Hey hey Ua1i! Aia Uai Ge Tug 385 390 395 400

Suv Rpe Gets Avp Guz Si Uai Sip back bitz Tiets Agd Agud Niv Ter Niz 405 410 415

Atud Ttr Agd Gets siu puv Uai Ge sbip bi bi Agd biu TpPBg bet Avp 420 425 430

Niz puzv ATia Rgo bBeg Aia Rgo SsS1u bip siu Hz Rkgo s1u Ppuv Guz Iets 435 440 445 beep bek b1u Agtd Avr siu siu bBet beep Avr begt bBegt Aia bis Iie Rkgo 450 455 460 tep Aia siu si1iu Pec Ag Siu Rgo Agd RpPe bBet TPkhth Gets 465 470 475 480 tep si1u Rko sip Hep bi1u Ggep Avr bek S1u TPg 485 490

From "2105 156 "2115 485 "2125 PZT "213" Vaetiz poguedisiv

Z5 "4005 156

Mee wai tep TBg sip Gets Niv Suv Rgo Tut Iiets yets Giets Ieets Iets Uai 1 5 10 15 wai Gay bek Suv Gets Rko puv Aia Rgo Beh Asia beep Ua! Mee Avr РПе 20 25 30 40 Tietssh Rpe sSi1ts Gguv Ter Guv Gets Tut Bek Avr s1p Suv Niv Niv Avp Iets

Zzek Ggetsp Gay Rgo Rgo Rgo TPh bi Tetsy Maiii Suv Avp Agd TPh Rope Avr bo

Ace Tut bBet Suv Tgr Rgo Avr TPh Rgo Rgo Avp TPtI TPtI Aza Ap Pe 45 65 70 75 80 bek Suv Rgo Tgr Tuk Pec Rgo Tgr Tut Niz Ppuv Ua1ii s1ip Niv Agd Iets 85 90 95 uai Rpe uv Agd Suv siu Rkgo Avr siu bip Tgr.

Iie sbits Uai sip puv s1u Uai Aia guz Meis Tut bet bet Tut beep Uai 130 135 140

Mee Tut TPt UMai siu Tut bBet Gecp bet Ge biu Aia iets Iiets Iets A1a 55 145 150 155 160

Pecs Uai Iie Gietssh Giets SsS1u Giets Agd Guv Gets Niv Suv TPI Agd Azp Tug 165 170 175

IT1e Niz SsS1u Avp Giets Ribe Aia bet Rie Uai yets puv Aia s1u betg Uai

180,185,190

Awr Awr Hey bBet Uai! bBet Uaii Tkr Ggets bek Avr bi1u Aia Uai Aia 01Uu 210 215 220

Suzv Akd Uai Aia Tpi Uai Iie Mee sip Tukh s1u Iie I1ie Aia Avp Tuk 225 230 235 240

Suzv Ter Gets Gets Uai bi siu Ua1 Tuk Gay Tut bBet Getsp Ge bek Te 245 250 255

ТПтИ ТПг Rпе бек биц Гуз Бек Рппе РпПе бет Гпецы Тут Гец Сув И1е У 260 265 270

TER siu bBet Rko Gets Gay Ribe Uai Iie Rgo Tgtr Uaii Uai Uai puv Suv 275 280 285 tep Rpe si Avzp Uai bip Suv Tgr TPg bBeg Avp Avr Avp Mee s1u RiPe 290 295 300

Ter Tgr I1e hets Agud Iie Rgo Ua1i Gay ey Aia Iie Gay Ii1e Avp Rie 305 310 315 320

Rpe Iie Rpbe Uai Agd I1ie I1ie Niv iets iieets Uai Aia Guz Iets Agd A1a 325 330 335

Niv sSip Mee Niv Tuk Aia Avr Tut Puv Rpe Agd iets A1i1a Agd bBet TEG 340 345 350

Pecs TPti Gets I1ie Rgo yets yets S1i1u Uai Niv si Uai Uai Rbe A1ia RBGe 355 Z6o0 365 uai TE Avr bi Niz Aia sbip siu TPhyh Gets Agd bBetg TPtI Guz Gets RBGe 370 375 380

Rpe Avr Gay Ribe RiPe bet back Rpe sip siu Gay Getsp Ua1 A1ia Uai Gay 385 390 395 400

Tuk Suv Rpe Gets Avp Guz bi Uai1i sbip Aia sbits yGecp Gay Agd Agd Tgr 405 410 415

Zo Ag Agd Tgtr sip Si si1u Puv Aza yeetsp sip bij bi Agd Mees Aia 5eg 420 425 430 bek Niv bsiu bBecht Niv Mesie Aia Rgo Aia s1u TPt Suz Niv S1u Avr Rgo 435 440 445

Suv sich puv Gets bSip Hey Mees bek Aia siu bet bBet Beh s1u TPkE schu 450 455 460

Suv sats Rko bBet A1ia Guz TPh bek iets Aia bet bBet Getsy Rgo Agd Iets 465 470 475 480

Аза Авр бБег Рго ТПг 485 «2105 157 «2115 142434 «2125 ДНК «213» Ношто зарієпз «4005 157 аєдссссссЕ дссадссаса дсдасссстд сЕдсЕдЕєдс єЕдсЕдсЕдсЕ додсстдссад 60 ссасаддесс ссЕССдсЕСса ддєдаєддас ЄЕсСсСЕдЕеЕЄвЧд адаадеддаа дсеСТтасддде 120 чассадесдес ассасаассії дадссвдсвд сссссессса сддадсєддЕ деЕдсаасада 180 ассеЕссдаса адтаєсссвд ссддссддас асссссдсса асассасуддс саасаєстсс 240 gdssssgddE assedssssd dsassasaaa dEdsaasass dseEhssYaedEEEE saadadayds 300 chddadsssdasud deEsadedoddE dsdyeddasss sddddddsads seEsddsdeeda Yedssissad 360 gdssadayedd ayeddsdadda chastsdaddes sadaaddadd eddssaadaye dgasadsads 420

Esssaddyeda sdeasasadi dddsvasads sEdEsssvdd dddesssEdseE ssESYADSSEE9 480 dssayessvdu daddssisad saadsedsas gdsasssdsa asdssayessa sdsdaayestd 540

TGEsEdsSdesSsSE ssdEdsvdaa adssadstss dEdStddESYA EsDavddudse dSESaddasess 600 sdstasadss adaavaesdu sdasdasss adeEdssadsa sstddsssad Yedayeddadsu 660

ЧдЕддсСЕдДЯсСЕ дссдєдЕД9ДЯдсС сдсодЕДЕЕС авдсаатаєд дсаєсдеєддс саасстастдс 720 гддсеЕдссдда сддадддессоі деассвдсас аасседстдуа дсстддссас сстуссссдад 780 адчадсеЕєссеЕ ссадссеЕста сстдддсаєєс ддсєЕддддеєд сссссаєдсе дЕЕСЧЯдєЄСЯєЄсС 840 сссгдддсад єддЕсаадід ЕСЕдДЕЕСсСдад аасдіссаді дссддассад сааєдасаас 900 асдддсеЕсєсеЕ ддеЕддаєссі дсуддЕЕСсСсСсс дЕСЕСССвду ссаєсстдає саасеЕсьсс 960 ассеЕЕсдЕСС дсаєсдрєсса дсеЕдсЕсдрд дссаадсвдс дддсасддса даєдсассас 1020 асадассаса адЕсссддсс ддссаадесс асдседассс Есаєуссстсе дсеЕдддсдеЕсС 1080 сасчдаадеєдд ССЕСсдссес sdyedasuddas dadsasdsss adddsasssya dsdseEssdss 1140 aadsEseEsst ssdassEss ssesadsess EESSadddsess tdsEddEdds dESSESTas 1200

БдсеЕЕСсСсСсса асааддадді дсадеЕсддад сЕдсддсддс дЕсддсассд стддсдсестд 1260 чдасааадедс гаєдддадда дсддаасасс адсаассаса дддссісаєс Ессдсссддес 1320 сасддсссіс ссадсаадда дседсадеЕЕ дддаддоддєд деддсадсса ддаєссаєст 1380 дсддадассс ссЕСддседа гЕддссЕСССя адастддсвд ададсссстс сьда 1434 «2105 158 «2115 354 «2125 ДНК «213» Ношто зарієпз «4005 158 саддечаєдуд асеЕссстЯдЕє єдадаачдсдуа аадсестасуд деЕдассадсд Есассасаас 60 сеЕдадсстдс єдсссссесс сасдддадсьд дЕдсдсааса даассессда саадсаєьсс 120 гдсеЕддссдда асасссссудс саатассасуд дссаасаєссі сседсссстд дтасстдсст 180 гддсассаса аадсдсааса ссдсЕЕсСдвд ЕЕСаададає дсдддсесссда сддЕСсадедд 240 чЧдЕдсдЕддас сссдддддса дссеЕєддсдЕ давдсстіссс адсдссадає ддаєддсдад 300 чачаєсдадд сСссадаадда додсддссаад аєдсасадса дсеЕсссададе датд 354 «2105 159 «2115 118 «2125 ПЗТ

From "213" Noshto zariepz "4005 159 sSip Ma1i Mes Avr Rpe Gets Rpe sitz Gpuz Tgr Guz gay Tut b1u Avr sip 1 5 19 15

Suv Niv Niv Avp iets bet KGecp tep Rgo Rgo Rgo TPk Sits Gets Ua1i Suv 20 25 30

Avp Atud TPt Rpe Avr Gpuv Tut bet Suv Tgr Rgo Avr TPgI Rgo Aia Avp 35 40 45

TE TrI ATa Avp I1e bek Suv Rgo Tgr Tut Gets Rgo Tgr Niv Niv Guz 50 zo bo wai b1ip Niz Akud Robe Ua1i Ribe ace Agd Suv s1u Rgo Avr s1u Sip Tgr 65 70 75 80 wai Aktu s1u Rgo Akd siu sip Rkgo Tgr Agud Avr Aia bBet sip Suv sip 85 30 35

Mayes Avr o1u sic Sic I1i1e si Mai sip puv bij Ma1ii Aia Gpuv Mee Tug 1600 195 110 zetk Bek Rpe bip Ua1i Mayes 115 «2105 160 «2115 24 «2125 DNA «213» Artificial sequence «2205 «223» Synthetic 1 «4005 helix 24 "2105 161 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 161 si1u Rpe TPI Rpe Avp Avzp Tut A1a 1 5 "2105 162 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 162 assadeEddta dsddeddsas basa 24 "2105 163 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 163

I1ie Bek siu bek biu si1u TPk TiPg 1 5 "2105 164 "2115 33 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 164 dsdaaadaee staastdddu aaaestesdae ss 33 "2105 165 "2115 11 "2 "2125 "TPZ Artificial sequence "2205 "223" Synthetic "4005 165

A1ia guv Avr bBet Avp Tgtr s1u Avp RpPe Avr Iey

1 5 10 "2105 166 "2115 36 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 166 sachdadzsedeEsses gasasaddss saasaasadda aasses 36 "2105 167 "2115 12 "2125 PZT "213"220"5 "230 Synthetic "4005 167

Sip back Maii Ge Tut Akd bBet Avp Avp At Avp o RIe 1 5 10 "2105 168 "2115 9 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 168 gddasayesse Z "2105 169 "2115 Z "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 169

Ter A1ia 5eg 1 "2105 170 "2115 27 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 170 saasaastsaee agasstasess deasase 27

"2105 171 "2115 9 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 171 beep beep Tug Tut TPtg TPt Rkgo Tut TPg 1 5 "2105 172 "2115 354 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 172 chadadeEdsads sdddyddadeEs eddddddadds EEddsasads sedddddddES sseEDadasts 60

ЕссЕдеєдсад ссЕСтДдаєс сассеЕєсаас аастаєдсса Едаассддді ссдссаддсе 120 ссаддааадуд дасєддасвд ддЕСсСЕСааст асстадесддса дсддеЕддтас тгасааастас 180 дсадассссд Єдаадддссуд ЕЕссавсаве єСсСссдадаса деЕсссаааса сасдседнає 240 сеЕдсаааєда асадсстрдад адссдаддас асддссдеєає асстасеєдедс дааадаєстсе 300 ааседадддаа ассссдассе сЕддддссЧдЕ ддсассстдуд ЕсасеЕдссьс сеса 354

From "2105 173 "2115 118 "2125 PZT "213" Artificial sequence

З5 "2205 "223" Synthetic "4005 173 si Mai sb1ip Gets Mai sit becht siu s1u s1u Gay Uai bip Rgo sS1u 1U 40 1 5 19 15

Zzek Gay Agd Getsp bek Suv Aia Aia bek s1u Rpe TPI Rpe Avp Avp Tuk 20 25 30

A1ia Mes Avp TgIr Uai Akd sip Asia Rgo biu puv biu Gets Avr Tgr Uai 45 et TPt I1e bek siu Bek siu biu TPti TPI Avp Tut Aia Avr bek Uai1i zo bo puz s1u Agd Rore I1ie I1ie bBeg Agud Avzr betgt bek Kpuv Niz TPE Iets Tug 65 70 75 80 tep sip Mee Azp bet Gets Agd Aia bi Avr TPtI Aia Uai Tut Tut Suv 50 85 30 35

A1ia guv Avr bBet Avp Tgtr s1u Avp Rpe Avr Gay Tgr S1u Agd s1Uu TPg 1600 195 110 tep Ma1! TPg Uai beg 5beg 115 "2105 174 "2115 339 "2125 DNA

«213» Штучна послідовність «223» Синтетична «4005 174 часаєсудєда сдасссадеіс Ессадасісс стЕддсЕдЕдЕ сеЕСтдддсда дадддссасс 60 ассаасєсдса адеЕссадсса дадеЕднстєва сасаддесса асаагаддаа сЕЕСЄТтТадде 120 гддсассадс адааассадуд дсадссеісст ааєстасеса ЕсСтТассдддс астасссодуа 180 чааєссддадд Ессстдассу асеЕсадесддс адсодддЕссд ддасадарєс сасеЕстЕсасс 240 ассадсадсс сдсаддсєда адзаєдеЕддса чЧчЕЕсаєтасі дессаасаата ставастасе 300 ссдрбасасьє седдссаддуд дассаадсвд dadaesaaa 339 "2105 175 "2115 113 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 175

Avr Iie Maii Mesie TPtI sip bet Rgo Avr bBeit Hey Aia Uai! bet Ieets 01U 1 5 19 15 bi Agud Aza TPg o Iie Avp Suv Guz bek bBetg bip bek Maiyi Gets Tut Agd 20 25 30 zect Avp Avp Agud Avp o Rpe Tetsy S1u Ttr Tut sip sip puv Rgo sb1u bip

Rkto Rgo Avp Getsp getsp Iie Tukhk Tgtr Aia bBeg TPI Agd si bek s1i1u Uai

From 50 zo bo

Rgto Avr Agud Robe bek biu bek biu bek S1u TPiI Avr Rpe TPh Gets TiK 65 70 75 80

I1e Bek Bek Gets sip Ai1a si Avzr Uai Aia Uai Tut Tut Suv bip sip 85 90 35 35 Tut Tuk TPg TPt Rgo Tukhk TPg Rpe sSi1iu Sbip siu TPg Gpuv Gay bis Iie 1600 195 110

Ace 40 "2105 176 "2115 381 "2125 DNA "213" Artificial sequence 45 "2205 "223" Synthetic "4005 176 chachayedsaas sdddyddades edddddadds EEddESsSSsSads seddddoddES, sseEDadasis 60

Есссдсдсад ссЕСТддаре сассеЕєтаде адссассдда єдаадсдддає ссдссаддсе 120 ссадддаадд доассддадед ддсддссаас агааассаад асддаадесда дааасастає 180 чеЕддасестд сдаадддссу аєссассаєс ссададаса асдссаадаа сісаседеЕнс 240 сеЕдсаааєда асадсстрдад адссдаддас асддсеЕдеЕЧді асстасеєдедс дададатасєст 300 дсастааєсдда Єстаєдасає ддасвтастас гасстасддра єддасдеєсьд дддссааддуа 360 ассасддєса ссдустсстс а 381 «2105 177 «2115 127

"2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 177 si Mee sbip Gets UMaii sit becht siu s1u s1u Hey Uai bip Rgo sS1u 1U 1 5 10 15 bek tep Agd Tiets begt Suv A1ia A1ia bek sS1u Rbe TPti Rire 5 running back Level 20 25 30

Ter Mee gpuv Tktr Uai Agd bip Aza Rgo s1iu Ppuv s1u Gay si Tkr Uai1i 35 40 45

Aza Avp ch1I1e Avp o sip Avr siu bet bi guz Tut Tut Uai Avr bBet Uai 50 55 bo tuz s1u Akd RpPe TPI Iie bek Agud Avr Avp Aia Guv Avp Bek Gets RiGe 65 70 75 80 tep sip Mee Azp bet Gets Agd Aia bi Avr TPtI Aia Uai Tut Tuk Suv 85 Zo 95

Aza Agud Avr I1e Uaii ietsp Mee Ua1i Tug Avr Me Avr Tut Tut Tut Tug 100 105 110 siu Mesye Avr Uai Ttr siu sip siu TPti TPti Ua1 TPg Uai bek 5eg 115 120 125 "2105 178 "2115 24 "21325 Shtchna DNA "2 sequence

From "2205 "223" Synthetic "4005 178 chchayeesassya etadsadeEsa sedch 24

C5 "2105 179 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 179 cS1u Rre TPtI RiBe bet back News Tkgr 1 5 "2105 180 "2115 24 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 180 asaaaassaad ayesddaadyeda daaa 24

"2105 181 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 181

I1ie Aup sip Avr biu bek sbits Guv 1 5 "2105 182 "2115 60 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 182 dsdachachata seEdsastaae ddisvtaedae ayeddastasi asstasstasdch sayeddes 60 "2105 "152 "21 TPPZ2 "21 213" Artificial sequence "2205

From "223" Synthetic "4005 183

Aza Atud Avr chI1e Uaii Ggetsp Mee Ua1i! Tut Avr Mes Avr Tut Tut Tug 1 5 19 15 s1u Mee Avr Uai 20 "2105 184 "2115 336 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 184 chasaeedeeda sdasessades essaseEstss sEdsssdESa sssstddada dssssssssssssssssssssssssssssssssssssssssssssssssssssss savgadeaaesud daaaasasta EEeDChdayetvod 120 vassrdsada adssadddsa dESESsSsasad seESSEdaesst asstdddaeEs saayesdddes 180

EssdddadEss ssdasaddee sadsddsadi ddayessaddsa sadaeesstas asEdaaaayes 240 adsachadsdd addsedadda eDdEsddUCHdEE sayesastdsa gdsaaaaseis asaaastissu 300 seEsasEsssa dsddadddas saadddeddad ayesaaa 336 "2105 185 "2115 112 "2213 "2125

"223" Synthetic "4005 185

Avr Ii1e Ua1i Mes TPg beep bet Rkgo Gay beg Gay Rgo Ma1i TPg Rgo Sh1Uu 1 5 10 15

Sish Rgo Aia bek I1e bBeg Suv Agd bBet bek bip bek Gay Gets Niv 5beg 20 25 30

Avp o b1u Avp Avp Tut Gets Avr Tkgr Tut Gay beep uv Rgo siu sip beg 35 40 45

Rkto Sip Gay Ggep Iie Tukhk pHets siu bBeg Avp Agtud Aia Beh siu Uai1i Rgo 50 55 bo

Avr Atud Rpe bBet siu bBet siu bBet biu TPh Avr Rpe TPtI Gets Gpuv I1e 65 70 75 80 zet Akd Uai bi Aia si Avr Uaiii Siu Mai1i Tug Tuk Suv Mee sSip TPg 85 Zo 95 tep sip TPt Rko Gecp TPiI Rpe biu biu biu TPt puv Uaii sich I1e Guz 100 105 110 "2105 186 "2115 33 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 186 sadadsseEss gdsavtadsaa sbdaaaasas tae 33

From "2105 187 "2115 11 "2125 PZT "213" Artificial sequence

Z5 "2205 "223" Synthetic "4005 187 cSip back tep Petz Niv Back Avp s1u Avp Avp Tug 1 5 10 "2105 188 "2115 9 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 188

EsYADaESSE Z "2105 189 "2115 Z "2125 PZT "213" Artificial sequence

"223" Synthetic "4005 189

Tets o1u 5eg 1 "2105 190 "2115 27 "2125 DNA "213" Artificial sequence "2205 "223" Synthetic "4005 190 aesdsaaasis sasaaaess dsesase 27 "2105 191 "2115 9 "2125 PZT "213" Artificial sequence 2 "23"2205 Synthetic "4005 191

Meyese sip TPg Gay Sip TPt Rgo Gets TiPg 1 5

Зо «2105 192 «2115 2076 «2125 ДНК «213» Ношто зарієпз «4005 192 аєсдддсассд ссадсессад дсуддЕССТдд гЕддссдседс састдсеЕдсе дсеЕдстодст9 60 сЕдсЕСсССсСсдд дЕсСссдсдуд сдсссдедсд саддаддасу аддасддсда стасдаддад 120 сЕддЕдсвад ссЕсдсудєЕсС сдаддаддас ддсстддссуд аадсасссда дсасддаасс 180 асадссассе сссассдстд сдссааддає ссдєддадді єдсстддсас стасатєддед 240 чЧдЕдсЕдаадд аддадассса ссісЕСдсад сададсдса sedsssdssd sseEdsaddsess 300 saddsseEdsss dssddddava ssisassaad ayesstdsaedd ЕСЕЕсСсСауууд ссЕЕСЕtСсСсТ 360

ЧдасЕЕССсСтдд сЄдаачаєдад єддсдасстд седдадстдда ссеЕтдаадь дссссатдес 420 чассасаєсуд аддаддасес сессдЕсЕеЕ дсссададса Есссдєсддаа сстддадсоауд 480 аєсассссіс сасддрассу ддсддаєдаа бассадсссс ссдасддадд садсстддеєд 540 чаддедсаєс ссстадасас садсавасад адедассасс дддаааєсєсда дддсадддес 600 аєсддесассуд асЕссдадаа єдсдсссдад даддасддда сссдссссса садасаддсесс 660 адсаадсдед асадссаєдуд сасссасстд дсаддддєдда Есадсддсс ддаєдссдде 720 чдЕддссаадд деЕдссадсає дсдсадсстд сдсдЕдсеЕса ассдссаадд даадддсасу 780 дЕсадсддса ssseisavada ssiddadeEE arEsduddaaaa dssadstdddeE ssadstdnda 840 chdadadssasedd sddEdsEdsE dssssvddsud ddEdddrasa dssdsdEsse saasdssdss 900

Едссадсдсс єддсдадддс єддоддЕСЧЯвЕд сЕддЕсСассу седссддсаа стЕССДддас 960 чаєдсстдсс ссвасесссс адссеЕсадсе сссдаддеЕса Есасадессдда ддссассаає 1020 дсссаддасс адссддеєдас ссеЕддддасе Есдоаддасса асЕстддссу седеЕдесддас 1080 сСЕСЕЕєдссс саддддадда саєсаєсддЕ дссеЕссадсуд ассдсадсас стдсеЕттЧдєд 1140 сасададед ддасаєсаса ддседссдсс сасдеддстід дсаєтвдсадс саєдаєдстдд 1200

Есєдссдадс сддадсесас сстіддссдад ЕЕдаддсада дастдаєсса сЕЕсСЕСтдсс 1260 ааачаєдсса ссааєдаддс сеЕддЕЕсСсся даддассадс ддадтасєдас ссссаасстд 1320 дЕддссдссс гдссссссад сасссаєдду дсаддеЕєддс адсЕдесеєвда саддастдеєд 1380 гддЕсадсас асЕсддддсс басасддаєд дссасадсса Есдсссдствд сдссссадає 1440 чаддадсєдс Єдадседсес садеЕєЕСесс аддадсддда адсддсддадд сдадсдсавд 1500 чаддсссаад доаддсаадсеЕ дадЕсЕдссда дсссасаасуд СЕЕСтЯодоадЧда ЕдаддаєдеЕс 1560 васдссаєтд ссаддедсвд сседсвассс саддссаасі дсадсдеісса сасадстсса 1620 ссадсєдадд ссадсаєдудуд дасссдедеЕс сасєдссасс аасадддсса сдЕсСстсаса 1680 чдасЕдсадсеЕ сссасгддда ддсддаддас сЕгддсассс асаадссдсес ЕдЕДЯДСтдадд 1740 ссасдаддіс адсссаасса деЕдсдЕдддс сасадддадуд ссадсаєсса сдсЕссстдс 1800 гдссаєдссс саддеістдда асдсааадес ааддадсаєд дааєсссддас сссеЕсаддад 1860 саддедассд сддсстдсда ддадддседд ассстідастд дседсадедс ссСССТддуд 1920 ассесссасд Есссдддддс стасдссддва дасаасасудє дедтадессад дадссдддас 1980 дЕсадсаста саддсадсас садсдаадад дссд eEdasad ssdeEedssaye sedstdssdua 2040 adssddsass sddsdsadds seEsssaddad seEssad 2076 "2105 193 "2115 692 "2125 PZT "213" Noshto zariepz "4005 193

Mee so1u TPt MUMai bBet bBet Akud Akud bBeti Tgr Tgr Rgo iets Rgo Iets Iets 1 5 19 15

Te Ggets Ggets Ggetsp Gay Gay ge b1u Rgo Aiia SsS1u Aia Agd Aia sip si1ts 20 25 30

Avr bip Avr sb1u Avr Tut si si Ggets Uaii hets Asia Gets Agd bet b1ts

Si Avr sS1u tep Aza sS1c ATia Rgo Sits Niv biu TPg TPt Aia Trk Riye bo

Zo Niv Agd Suv Aia Puv Avr Rgo Tgr Agd tep Rgo SsS1u TPt Tuk Ua1 Uai 65 70 75 80 uai yeetsp puzv bitsh sits Tri Niv Te bBeht bip Beh Si Agd Trt Aia Agd 85 90 35

Atd Gets beep Aia sip Aia Aia Agud Agud Sbiu Tut Ggetsp TPt puv Iiee TGec

Z5 1600 195 110

Niz Uai Rbe Niv sS1u yets Giets Rgo b1u Robe Iyets Ua1! ace Meieser beh 01U 115 120 125

Avr tep yeets bish Tets ATia yeeetssh Guz yeets Rgo Niv Uai1i! Avr Tut I1e s1c 130 135 140 40 bi Avr bek bBegt Uai Rpe Aza sip bek Ii1e Rgo Tgr Avp Gets sit Agd 145 150 155 160

I1e TPkg Rkgo Rkgo Akud Tut Agd Aza Avr bi Tut bip Rgo Rgo Avr sh1U 165 170 175 siu bek Hey Uai sitz Maiii Tut Ggets Ggets Avr TPtI bet Iie bip 5Betg Avr 45 180 185 190

Niz Agd Si I1e si s1u Agd Ua1i Mee uUai Trg Avzr Do s1cp Azp Uai 195 200 205

Rgo Si bets Avr SsS1u TBt Agud Rbe Niz Agud Sip Aia bek Kpuv Suz Avr 210 215 220 50 bek Niv si1u TPk Niv yeets Ai1a sSi1u Uai Uai bek S1u Agd Avr Aia sS1Uu 225 230 235 240 uai Aia Aiia bet GeiBei Guv si1 Agud Uaii Gets Avp Suv sip 245 250 255 siu puv s1u TPt Maii bBet siu TPti Ge I1e sS1u yets bis Rpe Iie Agd 55 260 265 270 tuz bBet sip Gets Uaii bip Rgo Ua1 siu Rgo yets Uaii Uai Gets Gets Rgo2 Aaii 8 275 275 siu s1i1u Tut Bek Agd Uai Gay Aup Aia Aia Suv sip Agkd Gets

290 295 300

Aza Atu Aia siu Uai Uaiyi hets Uai TPi Aza Aza sb1iu Avp Rpbe Ag Avr 305 310 315 320

Avr Aia Suv Gets Tut bBet Rkgo Aia becht Aza Rgo bis Uai Iie TPt Uai 325 330 335 s1u Aia TPI Avp Aza sip Avr sip Rkgo Uai TPgt Iets biu TPg Ieets 01Uu 340 345 350

TPE Avp Rpe s1u Agd Suv Ua1i Avr Gay Rpe Aza Rgo s1u siy Avr She 355 360 365

Iie b1u Aia bek 5Bet Avr Suv bBetg TPg Suv Rpe Uaii bBet sip Beh SHU 370 375 380

TE back beep Aza ATa Aia Niz Uai Aia sSiu I1ie Ai1a Aia Mes Mee Ieets 385 390 395 400 zek Ai1a bij Rgo bi Ge TPg Hey Aia bi Gets Agd sip Akd Tecp I1e 405 410 415

Niz Rpe bek Aia Guz Avr Uai I1ie Avzp b1sh Aia Tgr Robe Rgo siy Avr 420 425 430 bip Akud Mai Getsp TPt Rgo Avp Gets Uai Aia Aza ep Rgo Rgo bet TPg 435 440 445

Niv sSi1u Aia sSiu Ter sSip Pec Rpe Suv Agd TPt Ua! Tgr bek A1ia Niv 450 455 460 bek bi1u Rgo TPh Agud Me Aza TPI Aza Ii1e Aza Agd Suv Aia Rgo Avr 465 470 475 480 sich bij Hey Gets bBet Suv bBet bet RPe bBeig Agud bet biu GPuv Agd Agd 485 490 495 Agd biye sSiu s1sh Aia sip s1u s1u puv Gets Ua1! Suv Agd Ayia Neves 500 505 510

Avp Aia Rpe siu siu sic siu Uai Tut Aza Iie Aia Agd Suv Suv TIets 515 520 525

Zo iets Rgo sbip Aia Avp Suv bek Ua1i Niv TPI AiTia Rgo Rgo Ai1a Ss1c A1a 530 535 540 bek Mee si1u TrI Agd Uai Niz Suv Niv sip sSip bi1u Niv Uai Gets TiK 545 5B5O 555 5b6O sSiu Suv beg Uzra bek Niv Gets s1u TPt Niz Kpuv Rgo

Z5 565 570 575

Wow! Gets Agd Rgo Agd S1iu Sip Rgo Avp Sip Suz Ua1i S1u Niz Agd 580 585 5ZO

Sish Asia bBeg I1ie Niv Aia bek Suv Suv Niv Aia Rgo SsS1u yeets Sich Suv 595 bo 605 puv Uai puv Sish Niv SsSiu I1e Rgo Aia Rgo s1p si s1p Ua1! TPkg Uai 610 615 620

A1ia Suv bis sit siu Ter TPt Gecp TPti sSi1u Suv Beh Aza Gets Rgo s1Uu 625 630 635 640

TE bek Niv Uai iets sS1u Ai1a Tut Aia Ua1i Avr Azp TPt Suv Uai Uai 645 650 655

Atud bBeg Agtud Awr Uai! bBet TPtI TPtI Siu bek TPtI bek bec sitc A1ia Uai bo 665 670

TE ATa Uai Aza I1e Suv Suv Agd bek Agud Niv tey Aia s1ip A1ia 5eg 675 680 685 sbip sich Ge sip 690 "2105 194 "2115 115 "2125 PZT "213" Artificial sequence "2205

"223" Synthetic "4005 194

Si Mai s1ip yets Uai SsSip back siu sS1u b1u Iiets Ua1! Niz Rgo сС1у 0Ш1уУ 1 5 10 15

Zzek Gay Agd Gets bek Suv Aia s1i1u Bek siu Rpe TPh Ribpe 5eg Agd Avp 20 25 30

A1ia Mesye Rpe Tgr Uai Akd sip Aia Rgo sbiu puv biu iets bi Tgr Uai 35 40 45 bek si1u I1ie siu TPpk si1u si1u Aia TPti Avp Tut Aia Avr bek Ua1i Guv 50 55 bo s1u Akud Rpe TPI Iie 5Begt Akuda Avr Avr Avp Api bet yeets Tut TGec 65 70 75 80 bip Mesye Avp o bBegt Gets Agud Aia si Avr Mee Aza Uai Tut Tut Suv A1a 85 Zo 95

Atud b1u Agud Tut Tut Rpe Avr Tut Tgr b1u bip biu TPt Gets Uai TPg 100 105 110

Ua1i Beck 5ecg 115 "2105 195 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 195

From s1u Rpe TPh Rpe beg Agd Avp Aia 1 5 "2105 196 "2115 7

C5 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 196

Iie s1u TPg siu s1u Aia TiK 1 5 "2105 197 "2115 9 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 197

Aza Akud b1u Agd Tut Tut Rpe Avr Tug 1 5 "2105 198

"2115 109 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 198 Jan. beep bet Uai1! bet bet 5beg 20 25 30

Tut ge Aia Tkr Tut beep beep uv Rgo b1iu beep Aza Rgo Agd Hey GIets 35 40 45

Iie Rpe si1u Aia bBet bBegt Agud Aza TPg sS1i1iu I1ie Rgo Avr Agd RPe 5eg 50 55 bo siu bek s1u Bek s1iu TPtI Avr RpPe TPti Gecp TPI Iie bBegt Agd Iets S1c 65 70 75 80

Rgo b1p Avr Rpe Aia Uai1 Tukhk Tut Suv beep sbip Tut siu bek bek Rgo 85 90 95

Rgo Tgr Tph Rpe siu bip s1i1u TPt puv Uai si I1e Guz 100 105 "2105 199 "2115 7 "2125 PZT "213" Artificial sequence "2205

From "223" Synthetic "4005 199

Sip becht UMaii bBet bek bek Tukg 1 5

Z5 "2105 200 "2115 Z "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 200 s1u Aia 5eg 1 "2105 201 "2115 10 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "4005 201 beep beep Tuk so1u Bek Bek Rgo Rkgo Tktr TPg 1 5 10

"2105 202 "2115 8 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "2205 "221" OPTION "2225 (1)...11) "223" Haa - s1Uu "2205 "221" OPTION "2225 (2)...(2) "223" Haa - Rije "2205 "221" OPTION "2225 (3)...(3) "223" Haa - TPg "2205 "221" OPTION "222" (4) ...(84) "223" Haa - RpPe or 5eg "2205 "221" OPTION "2225 (5)...(5) "223" Haa - back "2205 "221" OPTION "2225" (6). ..(6) "223" Haa - Bek or Avp "2205 "221" OPTION "2225 (7)...(7) "223" Haa - Tut or Rye "2205 "221" OPTION "2225" (8) ...(8) "223" Haa - Avr, Hey, am I "4005 202

Haa Haa Haa Haa Haa Haa Haa Haa 1 5 "2105 203 "2115 8 "2125 PZT "213" Artificial sequence

"223" Synthetic "2205 "221" OPTION "2225 (1)...11) "223" Haa - Te "2205 "221" OPTION "2225 (2)...(2) "223" Haa - bBet, sb1i1ip, Avr, or Ttgr "2205 "2215" OPTION "2225 (3)...(3) "2235" Haa - bBet, Bitz, TPg, or Rie "2205 "2215" OPTION "222" (4).. .(84) "223" Haa - Avr or Ai1a "2205 "2215" OPTION "2225 (5)...(5) "223" Haa - sbi1u or b1ts "2205 "2215" OPTION "2225" (6). ..(6) "223" Haa - Agud, I1e, is there no "2205 "2215" OPTION "2225 (7)...(7) "223" Haa - Avr or b1ts "2205 "2215" OPTION "2225" (8)...(8) "223" Haa - puv or TPg "4005 203

Haa Haa Haa Haa Haa Haa Haa Haa 1 5 "2105 204 "2115 21 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "2205 "221" OPTION "2225 (1)...11)

"223" Haa - Aiia or TPg

"221" OPTION "222" (2)...2) "223" Haa - Guz or Agd "2205 "221" OPTION "222" (3)...(3) "223" Haa - s1tsi "2205 " 2215" OPTION "222" (4)...(4) "223" Haa - Met, Rgo, biu, or Avr "2205 "2215" OPTION "222" (5)...(5) "223" Haa - Ma1i, bek, KGuv, Agd, is there no "2205 "2215" OPTION "222" (6)...(6) "223" Haa - Tut, Niv, Avp, is there no "2205 "2215" OPTION "222 » (7)...(7) "223" Haa - Tut "2205 "2215" OPTION "222" (8)...(8) "223" Haa - Avr or b1ts "2205 "2215" OPTION "222 » (9)...9) "223" Haa - I1e "2205 "2215" OPTION "222" (10)...(10) "223" Haa - Getsy "2205 "2215" OPTION "222" (11 )...(11) "223" Haa - TPg "2205 "2215" OPTION "222" (12)...(12) "223" Haa - s1Uu "2205

"2215" OPTION "222" (13)...(13) "223" Haa - Tut, Avr, or Niv "2205 "2215" OPTION "222" (14)...(14) "223" Haa - Niv, Azr, Tug, is there no "2205 "2215" OPTION "222" (15)...(15) "223" Haa - Avp, Tut, Niv, is there no "2205 "2215" OPTION "222" (16 )...(16) "223" Haa - Tut "2205 "2215" OPTION "222" (17)...(17) "223" Haa - Tut or Nivs "2205 "2215" OPTION "222" (18 )...(18) "223" Haa - C1u or Ai1a "2205 "2215" OPTION "222" (19)...(19) "223" Haa - Mei "2205 "2215" OPTION "222" (20 )...(20) "223" Haa - Awr "2205 "2215" OPTION "222" (21)...(21) "223" Haa - Uai or She "4005 204

Haa Haa Haa Haa Haa Haa Haa Haa Haa Haa Haa Haa Haa Haa Haa Haa 1 5 10 15

Haa Haa Haa Haa Haa 20 "2105 205 "2115 6 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic

"221" OPTION

"222" (1)...11)

"223" Haa - sip

"2205

"221" OPTION

"222" (2)...2)

"223" Haa - sb1u or Ai1a

"2205

"221" OPTION

"222" (3)...(3)

"223" Haa - I1e

"2205

"221" OPTION

"222" (4)...(4)

"223" Haa - Avp or Agd

"2205

"221" OPTION

"222" (5)...(5)

"223" Haa - Avp

"2205

"221" OPTION

"222" (6)...(6)

"223" Haa - Tut or Avr

"4005 205 xHhaa Haa Haa Haa Haa Haa

1 5

"2105 206

"2115 3

"2125 PZT

"213" Artificial sequence "2205

"223" Synthetic

"2205

"221" OPTION "222" (1)...11)

"223" Haa - TPh or Aia

"2205

"221" OPTION "222" (2)...2)

"223" Haa - Aiia or TPg

"2205

"221" OPTION "222" (3)...(3)

"223" Haa - back or Rieh

"4005 206

Haa Haa Haa "2105 207 "2115 9 "2125 PZT "213" Artificial sequence "2205 "223" Synthetic "2205 "221" OPTION "222" (1)...11) "223" Haa - sip or Ieets "2205 "221" OPTION "222" (2)...(2) "223" Haa - sip "2205 "221" OPTION "222" (3)...(3) "223" Haa - Tug, Niv, or Avr "2205 "221" OPTION "222" (4)...(84)

From "223" Haa - Avp or Tug "2205 "221" OPTION "222" (5)...(5) "223" Haa - TPg or beg "2205 "221" OPTION "222" (6)... (6) "223" Haa - Tut, Avp, or Niv "2205 "221" OPTION "222" (7)... (7) "223" Haa - RIgo "2205 "221" OPTION "222" (8) ...(8) "223" Haa - Petz, Ribe, Agd, is there no "2205 "221" OPTION "222" (9)...(9) "223" Haa - TPg "4005 207 xХhaa Haa Haa Haa Haa Haa Haa Haa Haa

Claims (7)

1 5 FORMULA OF THE INVENTION 1. Antibody or its antigen-binding fragment, which specifically binds the human glucagon receptor (NOSOC), where the antibody contains regions that determine the complementarity (SOK5) of the variable region of the heavy chain (NSUK), in which NSMUK has the amino acid sequence ZEBEO Ir MO: 86; ii COM5 of the variable region of the light chain (CM), in which the CM has the amino acid sequence zZEO IU MO: 88. 2. Isolated antibody or its antigen-binding fragment according to claim 1, containing a heavy chain variable region (HCHV) having the amino acid sequence zEO IU MO: 86. 3. The isolated antibody or its antigen-binding fragment according to claim 1, containing the variable region of the light chain (I SUK), which has the amino acid sequence ZEO IYUO MO: 88. 4. Isolated antibody or its antigen-binding fragment according to claim 1, containing: (a) NSMK, having the amino acid sequence 5EBEO 10 MO: 86; and (B) ISMK having the amino acid sequence ZEO IU MO: 88. 5. An antibody or its antigen-binding fragment that specifically binds the human glucagon receptor (POSOK), where the antibody contains: (a) the NSOKT domain, which has the amino acid sequence BEO IU MO: 72; (b) NSOK2 domain, having the amino acid sequence BEO IU MO: 74; (c) NSOKZ domain, which has the amino acid sequence zZEO IU MO: 76; (a) domain I SOKI, which has the amino acid sequence ZEO IU MO: 80; (c) domain Y SOK2, which has the amino acid sequence ХЕО ИХО МО: 82; and (9 domain ІСОКЗ, which has the amino acid sequence 5ЕО ЙХО МО: 84. 6. Antibody or its antigen-binding fragment according to claim 5, containing: (a) NSME, having the amino acid sequence BEO IU MO: 86; and (B) GSMK having the amino acid sequence ZEO IU MO: 88. Zo 7. The isolated antibody or its antigen-binding fragment according to claim 5, intended for use in order to reduce the levels of glucose or ketones in the blood or for the treatment of a patient having a disease or condition associated with or characterized, in part, by high levels of glucose or ketones in the blood, wherein the condition or disease is selected from the group consisting of diabetes, impaired glucose tolerance, obesity, nephropathy, neuropathy, retinopathy, cataract, stroke, atherosclerosis, delayed wound healing, diabetic ketoacidosis, hyperglycemia, hyperosmolar hyperglycemia syndrome, perioperative hyperglycemia, hyperglycemia in a patient in the intensive care unit, hyperinsulinemia, metabolic syndrome, insulin resistance syndrome, and impaired fasting glycemia. 8. Isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment of an antibody according to claim 5. 9. An expression vector containing a nucleic acid molecule according to claim 8. 10. The method of obtaining an antibody to human (ZSOM or its antigen-binding fragment, which includes the stages of introducing the expression vector according to claim 9 into an isolated host cell, growing this cell in conditions that favor the production of an antibody or its fragment, and isolating the produced in this way antibodies 11. A pharmaceutical composition containing an antibody or its antigen-binding fragment according to claim 5 and a pharmaceutically acceptable carrier or solvent. 12. Pharmaceutical composition according to claim 11, intended for use to reduce blood glucose or ketone levels or to treat a patient having a disease or condition associated with or characterized, in part, by high blood glucose or ketone levels, where the condition or diseases selected from the group consisting of diabetes, impaired glucose tolerance, obesity, nephropathy, neuropathy, retinopathy, cataract, stroke, atherosclerosis, delayed wound healing, diabetic ketoacidosis, hyperglycemia, hyperosmolar hyperglycemia syndrome, perioperative hyperglycemia, inpatient hyperglycemia intensive therapy, hyperinsulinemia, metabolic syndrome, insulin resistance syndrome, and impaired fasting glycemia. 13. A pharmaceutical composition containing an antibody or its antigen-binding fragment according to claim 5, and a second therapeutic agent containing an isolated antibody or its antigen-binding fragment that specifically binds to human ROBCO, with a pharmaceutically acceptable carrier 60 or solvent . 14. Pharmaceutical composition according to claim 13, intended for use to reduce blood glucose or ketone levels or to treat a patient having a disease or condition associated with or characterized, in part, by high blood glucose or ketone levels, where the condition or diseases selected from the group consisting of diabetes, impaired glucose tolerance, obesity, nephropathy, neuropathy, retinopathy, cataract, stroke, atherosclerosis, delayed wound healing, diabetic ketoacidosis, hyperglycemia, hyperosmolar hyperglycemia syndrome, perioperative hyperglycemia, inpatient hyperglycemia intensive therapy, hyperinsulinemia, metabolic syndrome, insulin resistance syndrome, and impaired fasting glycemia. 15. A method of reducing blood glucose or ketone levels or treating a condition or disease associated with or partially characterized by high blood glucose or ketone levels, or at least one symptom or complication associated with the condition or disease, and the method includes administering an antibody or the antigen-binding fragment of claim 5 to a patient in need thereof, whereby blood glucose or ketone levels are reduced or the condition or disease is affected, or the intensity or severity of at least one symptom or complication associated with the condition or disease is reduced. 16. The method according to claim 15, in which the condition or disease is selected from the group consisting of diabetes, impaired glucose tolerance, obesity, nephropathy, neuropathy, retinopathy, cataract, stroke, atherosclerosis, delayed wound healing, diabetic ketoacidosis, hyperglycemia, syndrome hyperosmolar hyperglycemia, perioperative hyperglycemia, hyperglycemia in a patient in the intensive care unit, hyperinsulinemia, metabolic syndrome, insulin resistance syndrome, and impaired fasting glycemia. 17. The method according to any one of claims 15 or 16, in which the antibody or antigen-binding fragment is administered to the patient in combination with a second therapeutic agent. 18. The method according to claim 17, in which the second therapeutic agent is selected from the group consisting of insulin, biguanide (metformin), sulfonylureas (such as glyburide, glipizide), PPAV gamma agonist (pioglitazone, rosiglitazone), alpha-glucosidase inhibitor (acarbose , voglibose), EHEMATIOEF (glucagon-like peptide 1), ZUMI IF (pramlintide), a glucagon antagonist and Z0, a second antagonist of ESAs. 19. The method according to claim 17, in which the second therapeutic agent is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMO-CoA reductase). 20. The method according to claim 19, in which the NMO-CoOA reductase inhibitor is a statin selected from the group consisting of atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. 21. The method according to claim 17, in which the second therapeutic agent is an isolated antibody or its antigen-binding fragment, which specifically binds to human proprotein convertase subtilisin-kexin type 9 (RSOZKO). 22. The use of the isolated antibody or its antigen-binding fragment according to claim 5 for the preparation of a medicinal product for reducing the levels of glucose or ketones in the blood or for the treatment of a patient having a disease or condition associated with or characterized, in part, by high levels of glucose or blood ketones in which the condition or disease is selected from the group consisting of diabetes, impaired glucose tolerance, obesity, nephropathy, neuropathy, retinopathy, cataract, stroke, atherosclerosis, delayed wound healing, diabetic ketoacidosis, hyperglycemia, hyperosmolar hyperglycemia syndrome, perioperative hyperglycemia, hyperglycemia in a patient in the intensive care unit, hyperinsulinemia, metabolic syndrome, insulin resistance syndrome, and impaired fasting glycemia. 23. A method of reducing blood glucose or ketone levels or treating a condition or disease associated or partially characterized by high blood glucose or ketone levels, or at least one symptom or complication associated with the condition or disease, and the method includes administration of a pharmaceutical composition according to claim 11 to a patient in need thereof, whereby blood glucose or ketone levels are reduced or the condition or disease is affected, or the intensity or severity of at least one symptom or complication associated with the condition or disease is reduced. 24. A method of reducing blood glucose or ketone levels or treating a condition or disease associated or partially characterized by high blood glucose or ketone levels, or at least one symptom or complication associated with the condition or disease, and the method includes administration of a pharmaceutical composition according to claim 13 to a patient in need thereof, whereby blood glucose or ketone levels are reduced or the condition or disease is affected, or the intensity or severity of at least one symptom or complication associated with the condition or disease is reduced. 204 th Gi z y t Zh KT» i s, z With h. U I zho Ko t - t | cher ee - zh "40 -- y still j y i Z z 2 is Bu 5y B Zh sa 5 Ki y Fk. 1 154 g | | є є є є ; are you Shyny, s St pi v i 2 Ko s 2? sh- g «g . yes I xx ! and yo "o SK Fig. 2 60. I to a ry ddfnnt TT 8 and EY ITTNInnya of the Pynitinyans 7 SHY -7 shu I Fe U o KU ke and FU you KU te of them Fig. 3 J yu r yk-- sia 8 vo. ETO PEK prtittloteet is (ov | | | ef g: "v. no o ! v- 204 s vu z i i i Ka F Z I i m Ka F k " Fig. 4