UA119539C2 - Bi-specific monovalent diabodies that are capable of binding cd123 and cd3, and uses therof - Google Patents

Abstract

The invention relates to bispecific monovalent diatyl that binds simultaneously to CD123 and CD3, a pharmaceutical composition containing such diatyl, and their use for the treatment of a disease or condition associated with overexpression of CD123.

Description

(54) BISPECIFIC MONOVALENT DIATHYLO, WHICH IS ABLE TO BIND WITH CO123 AND CO3, AND

ITS APPLICATION

(57) Abstract:

The invention relates to a bispecific monovalent diethyl, which simultaneously binds to CO123 and

CO3, pharmaceutical compositions containing such a dibody, and their use for the treatment of a disease or condition associated with overexpression of CO123.

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Links to related applications

This application claims priority under US patent applications

MoMo 61/869510 (filed Aug. 23, 2013; pending), 61/907749 (filed Nov. 22, 2013; pending), and 61/990,475 (filed May 8, 2014; pending), and European Patent Application No. 13198784 (filed on December 20, 2013), each of which is fully incorporated herein by reference.

Link to the list of sequences

This application contains one or more sequence listings according to 37

S.R.A. 1.821 of its ed., which are disclosed in both paper and machine-readable media, and whose paper and machine-readable disclosures are fully incorporated herein by reference.

The field of technology to which this invention belongs

This invention relates to СХО123 x POP bispecific monovalent diethyls, which are capable of simultaneously binding to СО123 and POP, and to the applications of such molecules in the treatment of hematological malignant tumors.

Prior art of this invention

I. 60123

CO0123 (interleukin 3 alpha receptor, I/-Z3Ka) is a molecule with a molecular weight of 40 kDa and is part of the interleukin C receptor complex (5iote5Ki, E.O. ei ai. (1996) "Nitap IpieieiKip-3 (1-3 ) Ipdyse5 Oivyide-ipKey 1/-3 AVeseriog AiIrna-Apa Veia-Snaiip

Neiegoditetgigayop, M/pisnp I5 Nedshcigei Rog Reseriog Asiimayop Vii Moi Nidp-Aiippu Vipaipd", Moi.

Sow Vio!. 16(6):3035-3046). Interleukin C (II -3) controls the early differentiation of multipotent stem cells into cells of erythroid, myeloid, and lymphoid progenitors. СО123 is expressed on СО34-- committed cells - precursors (Taiv55id, O.S. eyi ai. (2005) "Netaiiroiiiiiis Zieet Seiiie Ehrgez5 Miyirie Mueioid MaigkKkegv: Itriisayop5 Rog Te Ogidip Apa

Tagdeied Tnegaru OY Ascie Mueioid I eiketia", Viosa 106:4086-4092), but not on СО34х4/Сс038- normal hematopoietic stem cells. СО123 is expressed by basophils, mast cells, plasmacytoid dendritic cells, some expression is observed in monocytes, macrophages and eosinophils, and low expression or its absence is observed in

From neutrophils and megakaryocytes. Some that do not belong to hematopoietic tissue (placenta, Leydig cells in the testicles, certain cellular elements of the brain and some endothelial cells) express CO123; however, expression is primarily cytoplasmic.

It was reported that CO123 is expressed by leukoblasts and leukemic stem cells (I 5C) (Chogdap, S.T. Yei ai. (2000) "Tne IpiepetsyKip-3 Veseriog AIrna Saip Iv A Opidie

MaikKeg Rog Nytap Ascie MueiiIodepoiz I eiketia 5iet SeiIv", Yi eiketia 14:1777-1784; dip, MU. eyi ai. (2009) "Vedshaiiop OII TN17 Seii! Oimegepiiaiop Apa EAE Ipdisiop Wu MARZK MIC", Viosa 113:6603-6610) (figure 1). In normal human progenitor cell populations, CO123 is expressed by a subclass of hematopoietic progenitor cells (HPCs), but not by normal hematopoietic stem cells (NS). СО123 is also expressed by plasmacytoid dendritic cells (ros) and basophils and to a lesser extent by monocytes and eosinophils (I ore, A.R. ei ai. (1989) "Vesirgosai! Ippibyop OY Vipaipd Veimeep Ipiepetskip Z Apa Stapiosuiye-Masgorpade Soiopu-

Ziytshaipu Rasiog To Nytap Eoviporniiv", Rgos. Mai). Asad. bsi. (0O.5.A.) 86:7022-7026; Byp, O. eyi a. (1996) "Moposiopa! Apiirodu 723 Nesodpige5 Te M-Teptipa! Ootaip OI Te Nytap Ipiepeikip-

Z (1-3) Veseriog AIrna Spaip Apa Rypsiip5 Az A 5resiiiis I-3 Veseriog Apiadopivi", Viosa 87:83- 92; Miho, I. ei ai. (2001) "IpiepeiKip-3 Veseriog AiIrna SNaip (SO123) Iv Muideiu Ehrghezzei Ip

Netaiyodis Maiidpapsiev", Naetaiiyuodisa 86(12):1261-1269; Mabviep, V. ei ai. (2006) "Spagasiyegigayop OY Muyeioid Apa Riaztasuiy YuOepagyis Seiv ip o Nytap Gypd", u. Ittipoi. 177:7784-7793; Kogreiaipep, E.I. ei ai. (1995) "Ipepegop-Ssatta Orgeashiaiez IpiepestskKip-Z3 (1-3)

Veseriog Ehrgezviop p Nytap Epaoineia! Seijv Apa Zupegdige5 M/P 1-3 Ip Biitsiaippo Maiog

Niviosotraiibiyu Sotriech Siavz II Ehrgeziop Apa SuioKipe Rgodisiop", Viosa 86:176-182).

CO123 has been reported to be overexpressed on malignant cells in a wide range of hematological malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Milleau, GI. et al. (2001) "IpiegeiKip-3 Veseriog AIrna SNaip ( СО123) Iv M/idemu Ehrgezzei Ip Netaiiodis Maiidpapsiev", Naetaiodis 86(12):11261-1269).

Overexpression of CO123 is associated with a more unfavorable prognosis in AML (Tenatapiya,

M.5. her a. (2013).

Mpa A Mome! СО123-5resiis Spitetis Apiidep Veseriog", Vg. U. Naeetaio!. 161:389-401).

AML and MDS are thought to arise and be maintained by a small population of leukemia stem cells (15), which are typically dormant (i.e., cells that do not divide rapidly) and are therefore resistant to cell death (apoptosis) and conventional chemotherapeutic agents. means | 5C are characterized by high levels of expression of СО123, which are absent in the corresponding normal population of hematopoietic cells in normal human bone marrow (ip, MU. ei AI. (2009) "Nedshianyop ОЙ Ty17 Seii! ОйShegepiayop Apa EAE Ipdysiop Vu MARZK MIK", Vioiy 113:6603 -6610; Chogaap, O.T. (2000).

MaiKeg Rog Nytap Ascie MueiiIodepoiv I eiKetia biet SeiIv", YeiKetia 14:1777-1784). СО123 is expressed in 45 95-95 906 cases of AML, 85 96 cases of hairy cell leukemia (VCL) and 40 95 cases of acute B-lymphoblastic leukemia (B- Expression of ХО123 is also associated with numerous other malignancies/precancerous conditions: chronic myeloid leukemia (CML) progenitor cells (including CML with blast crisis); Ryd cells

Sternberg (K5) in Hodgkin's lymphoma; transformed non-Hodgkin's lymphoma (NHL); some types of chronic lymphocytic leukemia (CLL) (СО11сж); subclass of acute T-lymphoblastic leukemia (T-CLL) (16 95, the most immature subclass, mainly affects adults), malignant tumors from plasmacytoid dendritic cells (ROS) (0C2) and malignant tumors from ХО34-/С038-cells of the bone marrow in myelodysplastic syndrome (MDO).

AML is a clonal disease characterized by the proliferation and accumulation of transformed myeloid cells - precursors in the bone marrow, which ultimately leads to hematopoietic insufficiency. The incidence of AML increases with age, and older patients tend to have worse outcomes than younger patients (Kobak, T. eyi ai. (2009) "Sctepi Apa Etegdipd Tnegaryezye Rog Assye Muevioiy

Y enketia", Siip. TNeg. 2:2349-2370). Unfortunately, at present, the majority of adult patients with AML die as a result of their disease.

Treatment of AML initially focuses on inducing remission (induction therapy). Once induction is achieved, the focus of treatment shifts to consolidation of this remission (post-remission or consolidation therapy) and in some cases to maintenance therapy. The standard remission induction paradigm for AML is chemotherapy with an anthracycline and cytarabine combination followed by either consolidation chemotherapy (usually with increased doses of the same drugs used during the remission induction period) or human stem cell transplantation, depending on the patient's ability tolerate intensive treatment and the likelihood of recovery only with the help of chemotherapy (see,

Koo) for example, Kobog, S... (2012) "Sitepi Tgeaitepi OI Ascie Muevioiai GeiKetia", Sy. Orip. Opsoi 24:711-719).

Agents often used in induction therapy contain cytarabine and anthracycline.

Cytarabine, also known as Agas, kills malignant cells (and other normal, rapidly dividing cells) by interfering with DNA synthesis. Side effects associated with treatment with the help

Agas, contain reduced resistance to infection as a result of reduced production of leukocytes; bleeding as a result of reduced production of platelets; and anemia due to a potential decrease in the number of red blood cells. Other side effects include nausea and vomiting.

Anthracyclines (eg, daunorubicin, doxorubicin, and idarubicin) are characterized by several mechanisms of action, including inhibition of DNA and RNA synthesis, destruction of higher order DNA structures, and production of cell-damaging free oxygen radicals. The most significant side effect of anthracyclines is cardiotoxicity, which significantly limits the lifetime dose and, to some extent, the applicability of anthracyclines.

Thus, unfortunately, despite significant progress in the treatment of newly diagnosed AML, 20 96 - 40 95 patients do not achieve remission with standard induction chemotherapy, and 95 - 70 90 of patients who achieve an initial complete remission are estimated to relapse. not later than in 3 years. The optimal strategy during relapse or for patients with treatment-resistant disease remains unclear. It has been established that stem cell transplantation is the most effective form of anti-leukemia therapy in patients with AML 50 in the first or subsequent remission (Yorog, Sskh.). (2012) "Sctepi Tgeaitepi OG Asshie Mueioia

And eiketia", Sitt. Orip. Opsoi. 24:711-719).

I. SOZ

POP is a T-cell co-receptor consisting of four different chains (Uuispegrieppid, KMU. ей ай. (2010) "Zygistiga! Viioidou ОЙ ТНе T-СейЙ Neseriog: Ipvzidniv Ipio Neseriog

Avbzetbriu, Gidapa VNesodpyiop, Apa Ipyiayop OO Zidpayipud", Soyy brhipd Nagb. Regzresi. Vio). 2(4):а005140; radev5 1-14). In mammals, the SOZ complex contains a chain of SOZu, a chain of SOZb and two chains of SOZe. These chains bind to a molecule known as the T-cell receptor (T-cell receptor) to create an activation signal in T-lymphocytes. In the absence of TSCs, TSCs do not assemble properly and they disintegrate (Ppotav, 5. ейі айЙ. (2010) "MoiIesshag (510).

177). POPs are found bound to the membranes of all mature T cells and to almost no other cell type (see Chhapeuau, SA yei ai. (2005) Ip: Ittipobioiodu: Te Ittipe buviet Ip Neain

Apa Oivzease, " biy ey. Sapapa bsiepse Rybriizpipu, MM, pp. 214-216; Byp, 7. u. ei ai. (2001) "Mesnapiztv Sopigibshipa To T Seii! Veseriog Zidpaiiipud Apa Azzetriu Vehuyeaied Vu Te boishiop zigisishge OYI Ap Esiodotaiip Egadtepi OYI Te SOZe:u Neiegoditeg", Seyi 105(7):913-923;. Kippv,

M.5. her a. (2006) "Oesopvigysipa Te Ropt Apa Rypsiop OYI Te TSV/SOZ Sotrieh", Ittipyu. 2006 Rer.;24(2):133-139).

I. Bispecific diatoms

The ability of an intact, unmodified antibody (for example, Ids) to bind an antigen epitope depends on the presence of variable domains on the light and heavy immunoglobulin chains (ie, the MI and MN domains, respectively). The structure of the diatyl is based on the single-stranded structure Em (zsEm) (see, for example, Neuideg et al. (1993) "Oiarodiyev" Come on! Vimaiepi Apa

Vizresiiys Apiibodu Egadtepiv", Rgos. Mai!. Asai. Zsi. (0O.5.A.) 90:6444-6448; patent publication

United States Mo 2004/0058400 (New York Office of the Attorney General); 5 2004/0220388 (Mepepz ei ai.); Aiy ai ai. (1999) REVZ

And ekyK. 454(1-2):90-94; And y, 0. ей яй. (2005) "A shu Nitap Vesotrbipapi Ida-I iKe Visresiiis Apiirodu To

Woe to Te Eridepta! Stomly Raciog Veseriog Apa Tne Inzship-GiKe Stoulii Raciog Neseriog Rog

Epnapsed Apiishtog Asiimtsu", y). Vioi. Spet. 280(20):19665-19672; MO 02/02781 (Mepepz ei ai.);

OIaivep, T. ei ai. (2004).

Sopidayop Apa Vadioiabeiipa Rog Tytog Tagdeiipd Arriisaiopv", Rgoivip Epd. Oev5. Zei. 17(11):21- 27; My, A. eyii ai. (2001) "Miyitetgigayop OA SNitetis Apii-СО20 5ipdie Snip Em-Em Risiop Rgoivip

And Mediaied Tpgotsdp Magiarie botaip Ekhspapde", Rgoivip Epdipeetipu 14(2):1025-1033; Azapo ei ai. (2004) "A biarodu Rog Sapseg IttipoiNegaru Apa y5 Rypsiiopa! Epnapsetepi Wu Ryviop OY Nytap

Es Rotaip", ABvzigashi ZR-683, 9. Viospet. 76(8):992; Taketiga, 5. ei ai. (2000) "Sopvitisiop OTGA riarodu (Ztaii! Vesotrbipapi Vizresiiis Apiirodu) Ovipa A Veidipud Zuvziet", Rgoieip Epa. 13(8):583- 588; Vayetsenpie, R.A. ei a!. (2009) "Vizresiis T-SeiY Epdadipd Apiirodie5 Rog Sapseg Tegaru", Sapseg

Transportation 69(12):4941-4944).

The interaction of the light chain of the antibody and the heavy chain of the antibody and, in particular, the interaction of its MI and MN domains form one of the binding sites of the antibody epitope. In contrast, the 5cEm construct contains the Mi domain. and MH antibodies contained in one polypeptide chain, and in the domain are separated by a flexible linker, the length of which is sufficient to ensure the possibility of self-assembly of two domains into a functional epitope binding site. If the self-assembly of the MI and MH domains becomes impossible due to the insufficient length of the linker (less than about 12 amino acid residues), the two 5sem constructs interact with each other to form a bivalent molecule in which the Mi of one chain are associated with the MH of the other (reviewed in Magmiip ei a!. (2005).

Riaptasoi beep 26:649-658).

Natural antibodies are capable of binding to only one type of epitope (ie, are monospecific), although they can bind to multiple copies of that type (ie, exhibiting bivalency or multivalency). The prior art has reported the ability to make antibodies that differ from such natural antibodies in that they are capable of binding two or more different types of epitopes (ie, exhibiting bispecificity or multispecificity in addition to bivalency or multivalency) (see, e.g.,

Noiiiideg ei ai. (1993) "Oiarodie5" Come on! VimaIepi Apa Vizresiiys Apibodu Egaditepiv", Rgos. Ma).

Asad 5 (0.5.A.) 90:6444-6448; 05 2004/0058400 (Noipdeg eyi AI.); 05 2004/0220388 (Mepepvz eyi aI.); Hey, hey, hey. (1999) REV5 And hey. 454(1-2):90-94; 1 i, 0. ei a. (2005) "A Eshiu Nytap Vesotrbipapi Ida-

NKe Vizresiiis Apiirodu To Voip Te Eridegtai! StoolLy Rasiog Neseriog Apa Tpe Ipviiiip-GiKe Tables

Rasiog Reseriog Rog Eppapsey Apiiytog Asiimyu, " y. Vioi. Spet. 280(20):19665-19672; MO 02/02781 (Mepepz5 ei aiYi.); Mepep5, M. ei aiYi., "Memzh/ Nesotbipapi Vi-apa Tiizresiiys Apiibodu

Oegimaime5, " Ip: Mome! Egopiiet5 p Te Rhodisiop OYI Sotrocypa5z Rog Viotedis! O5e, A.

MapVgoekKromep ei a. (Ed5.), Kinsheg Asadetis Rybiizpetg5, Bogagesni, Tpe Meipepapavz (2001), radev5 195-208; We, A. ei ai. (2001).

Rgoivip I5 Mediaiea Tygotsoip Magiarie Ootaiip Ekhspapde, " Rgoivip Epipeegipd 14(2):1025-1033;

Azapo ei ai. (2004) "A biarodu Rog Sapseg Ittipoipegaru Apa 5 ERypsipa! Epnapsetepi Vu

Eiviop OY Nytap Es Rotaip, "Arzigashi ZR-683, U. Viospet. 76(8):992; TaKetiga, 5. ey ai. (2000) "Sopvigysiop OY A Oiarodu (Ztai! Vesotrbipapi Visresiiis Apiirodu) Ovipd A Neyidipd Buviet, "

Rhoivip Epd. 13(8):583-588; Vayetsenpie, R.A. oh yeah (2009) "Vizresiiis T-Sei! Epdadipa Apiirodiev Rog

Sapseg TNegaru, "Sapseg Res. 69(12):4941-4944).

The provision of non-monospecific diethyls provides a significant advantage: the ability to bind and colocalize cells that express different epitopes. Thus, bispecific antibodies are characterized by a wide range of applications, including therapy and immunodiagnostics. Bispecificity allows for great flexibility in the design and development of the diethyl in various applications, providing increased avidity for multimeric antigens, cross-linking of different antigens, and targeted targeting of specific cell types based on the presence of both target antigens. Due to their increased valence, low dissociation rates, and rapid clearance from the circulation (for small diethyls of -50 kDa or below), known art diethyl molecules have also demonstrated specific application in the field of tumor imaging ( (1997).

Risnia Raviogiv, " Rgoivip Epid. 10:1221). Of particular importance is the binding of different cells, for example, cross-linking of cytotoxic T-cells with tumor cells (5iaeg72 eyi aI. (1985) "Nurtiy Apiibodiez Sap Tagdei 5yez Bog ANask Vu T SeiII5, Mashte 314:628-631, apa

Noiiyideg heri a. (1996).

Visresiis Oiarsau, "Rgoivip Epa. 9:299-305).

Diethyl epitope binding domains can also be directed to the surface determinant of any immune effector cell, such as POP, CO16, CO32 or CObA4, which are expressed on T lymphocytes, natural killer cells (NK) or other mononuclear cells. Many studies have also found that diethyl binding to effector cell determinants, such as Esu (Esu) receptors, activates the effector cell (Noiidegh et al. (1996) Zresiiis Kpipud OGG utrpota Sei Wu Suishyuohis T-SeiIv Mediaiea Wu A Vizresiis Oiaboau, "Rhoieip Epd. 9:299-305; NoiYideg ei ai. (1999) "Sagsipoetrguopis Apiidep (SEA)-5resiis T-sei)

Asiimaynop Ip Soiop Sagsipota Ipdyseus Wu Apii-saz x Apii-sea Vizresiiis Oiarodies Apa B7 x Apii-sva

Visresiis Ryviop Rgoieip5, Sapseg Ve5. 59:2909-2916; international patent publications MoMo UMO 2006/113665; UMO 2008/157379; UMO 2010/080538; UMO 2012/018687; UMO 2012/162068).. As a rule, activation of effectors cell activation is triggered by binding of an antibody-bound antigen to an effector cell via the Eo-Esuc interaction, thus diethyl molecules can exhibit Id-like functionality regardless of whether they contain an Esuc domain (e.g., according to the results of any effector function assay known in the art or exemplified herein (e.g., AOSS assay)).By cross-linking tumor and effector

From the cells, the diabody not only brings the effector cell into spatial proximity to the tumor cells, but also leads to effective lysis of the tumor cells (see, for example, Sao y ai. (2003) "Vivresiis Apiirodu Sopitsdaiez Ip Tegaresiisv," Adu. Ogyd. Oeiim. Nem. 55:171-197).

However, the above benefits come at a significant cost. The formation of such non-monospecific diethyls requires the successful assembly of two or more separate and different polypeptides (ie, such formation requires diethyls to be formed by heterodimerization of different types of polypeptide chains). This fact contrasts with monospecific diethyls, which are formed by homodimerization of identical polypeptide chains. Since it is necessary to provide at least two dissimilar polypeptides (i.e., two types of polypeptides) for the formation of a non-monospecific diethyl, and since homodimerization of such polypeptides leads to inactive molecules (Taketiga, 5. ei ai. (2000) "Sopvigisiop OI A Oiarodu (Ztaii! Nesotrbipapi Visresiiis Apiirodu ) Ovipda A Veioidipd

Zuziet, " Pharmacology Epa. 13(8):583-588), the preparation of such polypeptides must be carried out in such a way that prevents the formation of covalent bonds between polypeptides of the same species (ie, so as to prevent homodimerization) (Taketitga, 5. (2000) "Sopvigosip OI A riarodu (Ztaii! Resotrbipapi Vizresiiis Apiirbodu) Ovipu A Veyidipdud bZuvziet, " Rgoivip Epd. 13(8):583-588). In this connection, a non-covalent bond was discovered in this field of technology tying of such polypeptides (see, for example, Oiaivep ei ai. (2004) "SomaIepi Rizinide-ikKe Apii-SEA Oiarodu IPomub 5Ce-zZresiiis Sopiydayop Apa NRadioiabeiipu Rog Titog Tagdeiipud Arriisaiope," Rgoi. Epog. Oev5. ze. 1721-27; Avapo ei ai. (2004) "A Oiarodu Rog Sapseg Ittipoiiyegaru Apa 5 Rypsiyopai

Eppnapsetepi Wu Ryviop OY Nytap Es botaip, "Arzigashi ZR-683, 9. Viospet. 76(8):992; TaKketiga,

Z. ei ai. (2000).

Veiadipa Zuviet, "Rhoyeip Epdu. 13(8):583-588; и, 0. yei a. (2005) "A Eshu Nytap Nesotrbipapi

Ids-GiKe Vizresiiis Apiirbodu To Voii Te Eridepta! Stouli Rasyug Reseriog Apa Tne Ipvyip-I ike

Stomy Rasiog Neseriog Gog Epnapsed Apiiytog Asiimpu, " u. Vioi. Snet. 280(20):19665-19672).

However, in this field of technology, it has been established that bispecific diethyls, which consist of non-covalently bound polypeptides, are unstable and easily dissociate into non-functional monomers (see, for example, G. and O. ei ai. (2005) "A Yeshu Nytap Nesotrbipapi

Ids-GiKe Vizresiiis Apiirbodu To Voii Te Eridepta! Stouli Rasyug Reseriog Apa Tne Ipziiiip-I ike

Stomy Rasiog Neseriog Gog Epnapsed Apiiytog Asiimpu, " U. Vioi. Spet. 280(20):19665-19672).

Due to such a problem, in this field, the techniques have achieved success in the development of stable, covalently bound heterodimeric non-monospecific diethyls (see, for example, international patent publications Momo MUO 2006/113665; UUO/2008/157379; MO 2010/080538; UMO 2012 /018687;

MO/2012/162068; UuChoppzop, 5. ей ай. (2010) "EpNesiog Seyi Neshyytepi M/y Mome!

Ayipyu Ve-Tagoeiipud Rgoieip I ead5 To Roiepi Tytog Suioiuviv Apa Ip Mimo V-Sei! Oeriyeiyop, " y.

Moyes. Vio! 399(3):436-449; Map, M.O. oh yeah (2010) "Tnegareshis Sopit! OI V Sei! Asiimayop Mia

Vestiytepi OI Esdatta Veseriog PIB (SOZ2B) Ippirbyogu Bipsiop M/yA A Momei! Review of Apiirodu zsanoid, " Apiigiiv Apeit. 62(7):1933-1943; Mooge, R.A. ei ai. (2011) "Aririisanyop OYI Otsa! Ayippus

Veiagoeiipd Moiesshevz To Aspieme Oriita! Nedigesiied T-Sei! Biosd 117(17)4542-4551). Such approaches involve the construction of one or more cysteine residues in each of the types of polypeptides used. For example, it has been shown that the addition of a cysteine residue to the C-terminus of such constructs ensures the formation of a disulfide bond between polypeptide chains, stabilizing the resulting heterodimer without creating obstacles to the binding characteristics of the bivalent molecule.

Despite this success, the production of stable, functional heterodimeric, non-monospecific diethyls can be further optimized by careful consideration and placement of cysteine residues in one or more of the polypeptide chains used. Such optimized diethyls can be obtained in higher yield and with increased activity than non-optimized diethyls. Thus, this invention relates to the problem of obtaining polypeptides that are specially designed and optimized for the formation of heterodimeric diethyls. The present invention solves this problem by providing illustrative, optimized CO123 x POP diethyl.

Brief disclosure of this invention

This invention relates to CO123 x POP bispecific diethyls, which are capable of simultaneously binding to CO123 and POPs, and to the applications of such molecules in the treatment of diseases, in particular hematological malignant tumors. сО0123 x POP bispecific diethyls according to the present invention contain at least two different polypeptide chains that associate with each other in a heterodimeric manner with the formation of one binding site specific for the CO123 epitope and one binding site specific for the POP3 epitope. Thus, the CO123 x POP dibody according to the present invention is monovalent in the sense that it is able to bind to only one copy of the CO123 epitope and only to one copy of the POP epitope, but is bispecific in the sense that a single dibody is capable of binding bind simultaneously with the CO123 epitope and with the POP epitope.

Individual diethyl polypeptide chains are covalently linked to each other, for example, by disulfide bonding of cysteine residues located within each polypeptide chain. According to specific embodiments of the diethyl according to the present invention, they additionally contain an immunoglobulin Es domain or an albumin-binding domain to increase the half-life of ip mimo.

More specifically, according to the present invention, a bispecific monovalent dibody characterized by an optimized sequence of СО123 x СОЯЗ is also provided, capable of specifically binding to the epitope of СО123 and to the epitope of COZ, and the dibody contains a first polypeptide chain and a second polypeptide chain covalently linked to each other , and:

A. the first polypeptide chain contains, in the direction from the M-end to the C-end: i. domain 1 containing: (1) subdomain (1A) containing the Mi domain. of a monoclonal antibody capable of binding to

SOZ (Mi soz) (ZEO IU MO:21); and (2) subdomain (18), which contains the UN domain of a monoclonal antibody capable of binding to CO123 (MNeoigz) (ZEO IU MO:26);

BO and subdomains 1A and 1B are separated from each other by a peptide linker (5EO IU MO:29); and. domain 2, and domain 2 is an E-helical domain (ZEO I MO:34) or a K-helical domain (ZEO IYU MO:35), and domain 2 is separated from domain 1 by a peptide linker (ZEO IYUO MO:30); and

B. the second polypeptide chain contains, in the direction from the M-end to the C-end: i. domain 1 containing: (1) a subdomain (TA) containing the Mi domain. of a monoclonal antibody capable of binding to

СО123 (Mi soigz) (ZEO IU MO:25); and (2) subdomain (18), which contains the UN domain of a monoclonal antibody capable of binding to SOZ (MNeoz) (ZEO IU MO:22); 60, and subdomains 1A and 1B are separated from each other by a peptide linker (5EO IU MO:29);

and. domain 2, and domain 2 is a K-helical domain (ZEO I MO:35) or an E-helical domain (ZEO IU MO:34), and domain 2 is separated from domain 1 by a peptide linker (5ZEO IU MO:30); and domain 2 of the first and second polypeptide chains are not both E-helical domains or both K-helical domains; and at the same time: (a) the specified MI domain. the specified first polypeptide chain and the specified domain

MN of the specified second polypeptide chain form an antigen-binding domain capable of specifically binding to the POP epitope; and (p) the specified MI domain. the specified second polypeptide chain and the specified domain

MNs of the specified first polypeptide chain form an antigen-binding domain capable of specifically binding to the CO123 epitope.

According to the present invention, a bispecific monovalent dibody characterized by an unoptimized sequence of СО123 x СОЯЗ is also provided, capable of specifically binding to the epitope of СО123 and to the epitope of SOZ, and the dibody contains the first polypeptide chain and the second polypeptide chain, covalently linked to each other, and:

A. the first polypeptide chain contains, in the direction from the M-end to the C-end: i. domain 1 containing: (1) a subdomain (TA) containing the Mi domain. of a monoclonal antibody capable of binding to

SOZ (Mi soz) (ZEO IYUO MO:23); and (2) subdomain (18), which contains the MH domain of a monoclonal antibody capable of binding to CO123 (MNeoigz) (ZEO IU MO:28); and subdomains 1A and 1B are separated from each other by a peptide linker (5EO IU MO:29); and. domain 2, and domain 2 is an E-helical domain (ZEO I MO:34) or a K-helical domain (ZEO IYU MO:35), and domain 2 is separated from domain 1 by a peptide linker (ZEO IYUO MO:30); and

B. the second polypeptide chain contains, in the direction from the M-end to the C-end: i. domain 1 containing: (1) subdomain (1A) containing the Mi domain. of a monoclonal antibody capable of binding to

СО123 (Ми сой2гз) (ZEO IU MO:27); and

Zo (2) subdomain (18), which contains the MH domain of a monoclonal antibody capable of binding to SOZ (MNeoz) (ZEO IU MO:24); and subdomains 1A and 1B are separated from each other by a peptide linker (5EO IU MO:29); and. domain 2, and domain 2 is a K-helical domain (ZEO I MO:35) or an E-helical domain (ZEO IU MO:34), and domain 2 is separated from domain 1 by a peptide linker (5ZEO IU MO:30); and domain 2 of the first and second polypeptide chains are not both E-helical domains or both K-helical domains; and at the same time: (a) the specified MI domain. the specified first polypeptide chain and the specified domain

MN of the specified second polypeptide chain form an antigen-binding domain capable of specifically binding to the POP epitope; and (p) the specified MI domain. the specified second polypeptide chain and the specified domain

MNs of the specified first polypeptide chain form an antigen-binding domain capable of specifically binding to the CO123 epitope.

In addition, according to the present invention, a variant of the implementation of the above-described bispecific monovalent diethyls is provided, in which the first or second polypeptide chain additionally contains an albumin-binding domain (ZEO IU MO:36), connected at the C-end to domain 2 or to M -ends with domain 1 using a peptide linker (ZEO IU MO:31).

In addition, according to the present invention, an embodiment of the bispecific monovalent diethyls described above is provided, in which the first or second polypeptide chain

BO additionally contains domain 3, which contains the CH2 domain and the CH3 domain of Es immunoglobulin Ide (ZEO IU

MO:37), and the C domain is connected at the M-end to the TA domain by means of a peptide linker (ZEO IO MO:33).

In addition, according to the present invention, a variant of the implementation of the above-described bispecific monovalent diethyls is provided, in which the first or second polypeptide chain additionally contains domain 3, containing the CH2 domain and the CH3 domain of the Es immunoglobulin to (ZEO IU

MO:37), and domain C is connected at the C-terminus to domain 2 by means of a peptide linker (ZEO IO MO:32).

In addition, according to the present invention, an embodiment of any of the above-described bispecific monovalent diethyls is provided, in which domain 2 of the first polypeptide chain is a K-helical domain (ZEO IU MO:35), and domain 2 of the second polypeptide chain is E -spiral domain (ZEO IU MO:34).

In addition, according to the present invention, an embodiment of any of the above-described bispecific monovalent diethyls is provided, in which domain 2 of the first polypeptide chain is an E-helical domain (ZEO IU MO:34), and domain 2 of the second polypeptide chain is K -spiral domain (ZEO IU MO:35).

In addition, according to the present invention, an embodiment of a bispecific monovalent dibody capable of specifically binding to the CO123 epitope and to the POP epitope is provided, and the dibody contains a first polypeptide chain and a second polypeptide chain covalently linked to each other, and: the specified bispecific the body contains the following:

A. the first polypeptide chain, which is characterized by an amino acid sequence

ZEO IYUO MO:1; and

B. the second polypeptide chain, which is characterized by the amino acid sequence 5EO

IO MO:Z; and the specified first and specified second polypeptide chains are covalently linked to each other by a disulfide bond.

Diabodies of the present invention exhibit unexpectedly enhanced functional activities, as further described below.

Diabodies according to the present invention are preferably able to cross-react with proteins CO123 and POPs, both human and primate, preferably proteins CO123 and POPs of the Javan macaque.

Diabodies according to the present invention are preferably capable of depleting plasmacytoid dendritic cells (ros) from the culture of primary PBMCs with an IC50 value of about 1 ng/ml or less, about 0.8 ng/ml or less, about 0, 6 ng/mL or less, about 0.4 ng/mL or less, about 0.2 ng/mL or less, about 0.1 ng/mL or less, about 0.05 ng/mL or less, about 0, 04 ng/mL or less, about 0.03 ng/mL or less, about 0.02 ng/mL or less, or about 0.01 ng/mL or less. The IC50 value is preferably about 0.01 ng/ml or less. In the analysis described above, the culture of primary PBMCs can be derived from the Javan macaque, in which case the specified depletion represents the depletion of plasmacytoid dendritic cells of the Javan macaque (ros). Optionally, the cells of the present invention may be capable of depleting plasmacytoid dendritic cells (PDCs) from a primary PBMC culture, as described above, and the assay is performed according to either the protocol of Example 14, as described herein, or a modification of such assay , which will be understood by those skilled in the art, or by other methods known to those skilled in the art.

Diabodies according to the present invention preferentially exhibit cytotoxicity in an IP migo assay

Casiti-3 with an EC50O value of approximately 0.05 ng/ml or less. The EC50 value is preferably about 0.04 ng/ml or less, about 0.03 ng/ml or less, about 0.02 ng/ml or less, or about 0.01 ng/ml or less. Optionally, diethyls according to the present invention may exhibit cytotoxicity as described above, and the assay is performed according to or in accordance with the protocol of Example 3 as described herein, or by modification of such assay as will be understood by those skilled in the art, or by others methods known to specialists in this field of technology.

Diabodies according to the present invention preferentially exhibit cytotoxicity in an IP migo assay

Mo!t-13 with an EC50 value of about 5 ng/ml or less. The EC500 value is preferably about 3 ng/ml or less, about 2 ng/ml or less, about 1 ng/ml or less, about 0.75 ng/ml or less, or about 0.2 ng/ml or less.

Optionally, diethyls according to the present invention may exhibit cytotoxicity as described above, and the assay is performed according to or in accordance with the protocol of Example 3 as described herein, or by modification of such assay as will be understood by those skilled in the art, or by others methods known to specialists in this field of technology.

Diatila according to the present invention is mainly capable of inhibiting the growth of tumor xenograft MY M-13 in mice. Diabodies according to the present invention can preferably be capable of inhibiting the growth of a tumor xenograft MY M-13 in a mouse at a concentration of at least about 20 μg/kg, at least about 4 μg/kg, at least about 0.8 μg/kg, at least about 0 .6 μg/kg or at least about 0.4 μg/kg.

Preferred antibodies of the present invention will inhibit the growth of a MY M-13 tumor xenograft in a mouse by at least 25595, but possibly by at least about 40 95 or more, at least about 50 95 or more, at least 60 by about 60 95 or more, at least about by 70 95 or more, at least about 8095 or more, at least about 9095 or more, or even completely inhibit the growth of MY M-13 tumor over a period of time or causing regression or disappearance of the tumor. This inhibition is observed at least for a line of mice

MS. Optionally, diethyls according to the present invention may be capable of inhibiting the growth of a MOI M-13 tumor xenograft in a mouse as described above according to or in accordance with the protocol of example b as described herein, or by modifying such an assay as will be understood by those skilled in the art technical field, or by other methods known to specialists in this technical field.

Diatila according to the present invention are mainly capable of inhibiting the growth of K54-11 tumor xenograft in mice. Diabodies of the present invention may preferably be capable of inhibiting the growth of tumor xenograft 2554-11 in a mouse at a concentration of at least about 0.5 mg/kg, at least about 0.2 mg/kg, at least about 0.1 mg/kg, at least about 0.02 mg/kg or at least about 0.004 mg/kg.

Preferred antibodies of the present invention will inhibit the growth of a K54-11 tumor xenograft in a mouse by at least about 25595, but possibly at least about 40 95, at least about 50 95, at least about 6096, at least about 7095, at least about 8095, at least about 90 95 or even completely inhibit the growth of the tumor 54-11 after a period of time or causing regression or disappearance of the tumor. This inhibition is observed at least for the MBO mouse line. Optionally, diethyls according to the present invention may be capable of inhibiting the growth of a K54-11 tumor xenograft in a mouse as described above according to or in accordance with the protocol of example b as described herein, or by modifying such an assay as will be understood by those skilled in the art techniques, or by other methods known to specialists in this field of technology.

Diabodies according to the present invention are mainly capable of depleting leukemic blast cells and migo in the primary culture of AML bone marrow cells. Diabodies of the present invention may preferably be capable of depleting leukemic myeloid blast cells in a primary culture of AML bone marrow cells at concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0, 04 ng/ml,

Of at least about 0.06 ng/ml, at least about 0.08 ng/ml, or at least about 0.1 ng/ml. Diabodies according to the present invention may preferably be able to deplete leukemic and myoblast cells in the primary culture of bone marrow cells

AML is less than 20 95 of the total population of primary leukemic blast cells at diethyl concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0, 06 ng/ml, at least about 0.08 ng/ml, or at least about 0.1 ng/ml, optionally after incubation of the primary culture with diethyl for about 120 hours. Leukemic blast cells are preferentially depleted of ip migo in primary culture of AML bone marrow cells to less than 2095 of the total population of primary leukemic blast cells at diethyl concentrations of approximately 0.01 ng/ml or 0.1 ng/ml, after incubation of primary cultures with diatyl for about 120 hours.

Diabodies according to the present invention are preferably capable of inducing the expansion of the T-cell population in the primary culture of AML bone marrow cells. Such expansion can preferably be about 70 95 or more of the maximum T-cell population that can be expanded in the assay. Diabodies according to the present invention preferably can be capable of inducing expansion of the T-cell population in a primary culture of AML bone marrow cells by about 70 95 or more of the maximal T-cell population that can be expanded in the assay at diabodies concentrations of at least about 0.01 ng/mL, at least about 0.02 ng/mL, at least about 0.04 ng/mL, at least about 0.06 ng/mL, at least about 0.08 ng/mL, or at least about 0.1 ng/ml, optionally after incubating the primary culture with diatyl for approximately 120 hours. The T-cell population preferentially expands in primary cultures of AML bone marrow cells by about 7095 or more of the maximal T-cell population that can be expanded in the assay at diethyl concentrations of about 0.01 ng/ml or about 0 ,1 ng/ml after incubation of the primary culture with diatyl for approximately 120 hours.

Diabodies according to the present invention are preferably capable of inducing the activation of the T-cell population of migo in the primary culture of AML bone marrow cells. Such activation can occur at diethyl concentrations of at least about 0.01 ng/ml, for at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0 .08 ng/ml or at least about 0.1 ng/ml, optionally after incubating the primary culture with diethyl for about 72 hours. Such activation can be measured by the expression of a marker of T-cell activation, such as CO25.

Activation of the ip migo T-cell population in a primary culture of AML bone marrow cells, as measured by expression of CO25, preferentially occurs at diethyl concentrations of about 0.01 ng/ml or about 0.1 ng/ml after incubation of the primary culture with diethyl within about 72 hours.

Diatila according to the present invention is preferably capable of depleting leukemic blast cells of ip migo in the primary culture of AML bone marrow cells to less than 20 95 of the total population of primary leukemic blast cells and at the same time inducing the expansion of the T cell population of ip migo in the primary cell culture bone marrow AML by about 70 95 or more of the maximum T cell population that can be expanded in the assay at diethyl concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml, or at least about 0.1 ng/ml, optionally after incubating the primary culture with diethyl for about 120 hours. Concentrations of diethyl are preferably about 0.01 ng/ml or about 0.1 ng/ml, and the primary culture is incubated with diethyl for about 120 hours.

Diabodies according to the present invention may be capable of depleting leukemic blast cells of glioma in a primary culture of AML bone marrow cells and/or inducing expansion of a glioma T-cell population in a primary culture of AML bone marrow cells and/or inducing activation of a glioma T-cell population myo in a primary culture of AML bone marrow cells as described above according to or in accordance with the protocol of Example 8 as described herein, or by modifying such an assay as will be understood by those skilled in the art, or by other methods known to those skilled in the art .

To avoid misinterpretation, diatoms of the present invention may exhibit one, two, three, more than three, or all of the functional features described herein.

Thus, dibodies of the present invention may exhibit any combination of the functional features described herein.

Diatila according to the present invention are suitable for use as a pharmaceutical agent. Diatila is preferably suitable for use in the treatment of a disease or condition associated with the expression of CO123 or characterized by the expression

CO123. The present invention also relates to the use of diethyl according to the present invention in the manufacture of a pharmaceutical composition, preferably for the treatment of a disease or condition associated with the expression of CO123 or characterized by the expression of CO123, as further defined herein.

A disease or condition associated with the expression of CO123 or characterized by the expression of CO123 may be a malignant tumor. For example, the malignancy may be selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis, and CML-associated Abelson oncogene (Vseg-AVI translocation). ) ), non-Hodgkin's lymphomas (NHL), including mantle cell lymphoma (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.

A disease or condition associated with the expression of CO123 or characterized by the expression of CO123 may be an inflammatory condition. For example, the inflammatory condition may be selected from the group consisting of systemic lupus erythematosus (SLE), allergy, bronchial asthma, and rheumatoid arthritis.

In addition, the present invention provides a pharmaceutical composition containing any of the diethyls described above and a physiologically acceptable carrier.

In addition, the present invention provides for the use of the pharmaceutical composition described above in the treatment of a disease or condition associated with the expression

C0123 or characterized by the expression of СО123.

In particular, according to the present invention, an embodiment of such application is provided, in which the disease or condition associated with the expression of CO123 or characterized by the expression of CO123 is a malignant tumor (especially a malignant tumor selected from the group consisting of the following: acute myeloid leukemia (CML), chronic myelogenous leukemia 60 (CML), including CML blast crisis and CML-associated Abelson oncogene

(Vseg-AVI translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-CLL), chronic lymphocytic leukemia (CLL), including CLL with Richter's syndrome or Richter's transformation, hairy cell leukemia (HCL), neoplasms with blast plasmacytoid dendritic cells (BPDC), non-Hodgkin lymphomas (NHL), including mantle cell lymphoma (MCL) and small cell lymphocytic lymphoma (SLL), lymphoma

Hodgkin's disease, systemic mastocytosis and Burkitt's lymphoma).

In particular, according to the present invention, an embodiment of such an application is provided, in which the disease or condition associated with the expression of CO123 or characterized by the expression of CO123 is an inflammatory condition (especially an inflammatory condition selected from the group consisting of the following: systemic lupus erythematosus (SLE), allergy, bronchial asthma and rheumatoid arthritis).

Terms such as "approximately" should be understood as an average within 10 95 , more preferably within 5 95 , of the stated value, unless the context dictates otherwise.

Brief description of graphic materials

Figure 1 shows that CO123 is known to be expressed on leukemic stem cells.

Figure 2 illustrates the structures of the first and second polypeptide chains of the two-chain CO123 x SOZ bispecific monovalent diethyl according to the present invention.

Figures 3A and 3B illustrate the structures of two versions of the first, second and third polypeptide chains of three-chain ХХО123 x SOZ bispecific monovalent Es-diatyl according to the present invention (version 1, figure 3A; version 2, figure 3B).

Figure 4 (panels A-E) shows the ability of different CO123 x POP bispecific diethyls to mediate T-cell-redirected cytolysis of target cells exhibiting varying amounts of CO123. The figure shows dose dependence curves, which indicate that a bispecific diabody characterized by an optimized sequence of СО123 x POPs ("SАКТ-

A"), containing the albumin-binding domain (OAKT-A with ABO "m/ABO") showed greater cytotoxicity than the control bispecific dibody (control SACT) or the bispecific dibody characterized by an unoptimized sequence of СО123 x POP ("SACT- B") in

Among the numerous types of target cells: K54-11 (panel A); TE-1 (panel B); Moit-13 (panel C);

Kaziti-3 (panel 0); of TNR-1 (panel E) in the E:T (effector:target) ratio of 107.

Figure 5 (panels A-O) shows the ability characterized by the optimized sequence of CO123 x CO3 bispecific diethyl (OACT-A), characterized by the optimized sequence of CO123 x COZ bispecific diethyl containing the albumin-binding domain (FACT-A with ABO " m/ABO"), which is characterized by an optimized sequence of СО123 x СОЗ of a bispecific diethyl containing the Es domain of immunoglobulin II (ОАВТ-А with Es "m/Bs"), to mediate T-cell activation in the process of redirected cytolysis of target cells. The figure shows dose-response curves showing cytotoxicity mediated by OAKT-A, SANT-A m/ABO and VACT-A m/Es in Kazyti-3 (panel A) and TNR-1 (panel B) cells and purified CO8 T cells at an ET ratio (effector cell: target cell) of 10:1 (18 h incubation). Panels C and O show dose-dependent curves of T-cell activation using the CO25 marker on CO8 T-cells in the presence (panel C) and in the absence (panel C) of target cells.

Figure 6 (panels A-B) shows the content of granzyme B and perforin in CO4 and CO8 T-cells after treatment with an optimized sequence of CO123 x POP bispecific diethyl (OAKT-A) (panel A) or control bispecific diethyl ( of control SACT) (panel B) in the presence of Kazity-3 target cells and dormant T cells at an E:T ratio of 10:1.

Figure 7 (panels A-B) shows the antitumor activity characterized by the optimized sequence of CO123 x COZ3 bispecific diethyl (YACT-A) at dosage levels in ng/kg. MOI M-13 cells (intermediate level of CO123 expression) were mixed with T-cells and implanted subcutaneously (TE 1:11) into M5O mice. Intravenous treatment was carried out once a day for 8 days (00x8), starting from the day of implantation. Different concentrations of OAKT-A were compared with the control bispecific diatyl (control SACT).

Panel A shows Moi!t-13 cells alone or with T cells, and the effect of different doses of OACT-A on tumor volume, even at time points beyond 30 days. Panel B shows the effect of increasing doses of UOACT-A on tumor volume observed in MO mice receiving MOG M-13 cells and T cells (TE 1:1) over a time period of 0-18 days.

Figure 8 shows the antitumor activity characterized by the optimized sequence of CO123 x POP bispecific diethyl (FACT-A) on K54-11 cells (ALL with monocyte features). The cells were mixed with T-cells and implanted subcutaneously (TE 1:1) into mice

Mo. Intravenous treatment was carried out once a day for 4 days (0Ох4), starting from the day of implantation. Different concentrations of UACT-A were compared with the control bispecific diethyl (control SACT).

Figure 9 (panels A-B) shows CO123 blast cells in bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) from patient 1 with AML (panel A) in comparison with the Kaziti-3 AML cell line ( panel B).

Figure 10 (panels A-C) shows the ability characterized by the optimized sequence of CO123 x POP bispecific diethyl (FACT-A) to mediate the reduction of blast cells in primary AML after 120 h. (panel A), to control the expansion of the T-cell population in primary AML after 120 h. (panel B) and induce T-cell activation at

GML after 48 hours. and 72 hours (panel C).

Figure 11 (Panels A-H) shows the identification of the CO123-4 population of blast cells in the primary sample of RVMO ALL. Panels A and E show the forward and lateral light scatter of a population of injected normal PBMCs (Panel A) and PBMC ALL (Panel E). In panels B and

E shows the identification of the lymphocyte population as mainly B cells (panel B) and leukemic blast cells (panel B). Panels C and b show the identification of the population of lymphocytes that are CO123g. Panels O and H show the identification of СО19 cells and Her СО123 cells.

Figure 12 (panels A-B) shows the identification of СО4 and СО8 populations of T cells in the primary sample of RVMSO ALL. Panel A shows forward and lateral light scattering of the injected RVMO ALL population. Panel B shows the CO4 or CO8 T cell populations present in the samples.

Quantities indicate that CO4 T cells represent approximately 0.5 95 of the total number of cells, and CO8 T cells represent approximately 0.495 of the total number of cells present in the PBMO ALL sample.

Figure 13 (panels A-H) shows the ability of a bispecific diabody characterized by an optimized sequence of CO123 x POP (OAKT-A) to mediate blast cell depletion

From cells in ALL with the help of autologous STIs. (cytotoxic T-lymphocytes). Panels A and E show forward and side light scattering of the population of normal PBMCs injected (panel

A) and RVMSO ALL (panel E). PBMCs were left untreated (panels B and E), treated with a control bispecific diethyl (control JUAKT) (panels C and b), or treated with JUACT-A (panels J and H) and incubated for 7 days, after which they were stained with ratios of SOZ34 and СО19.

Figure 14 (panels A-Y) shows the ability characterized by the optimized sequence of CO123 x POP bispecific diethyl (BACT-A) to mediate T-cell expansion (panels A, B, C, 0, H and I) and activation (panels 0 , E, E, U, K and ) in normal RVMS (panels A-E) and RVMS ALL (panels O-I). Cells were left untreated (panels A, 0, c, and Y) or treated with control bispecific diethyl (control UACT) (panels B, E, H, and K) or SACT-A (panels C, PE, I, and I) for 7 days.

Figure 15 (panels A-C) shows the identification of the AML blast population and T cells in a primary AML sample. Panel A shows the direct and lateral light scattering of the RBMS population

GML, which is introduced. Panel B shows the identification of the AML blast population in the AML sample.

Panel C shows the identification of the T-cell population in the AML sample.

Figure 16 (panels A-C) shows the ability of the optimized sequence of CO123 x POP bispecific diethyl (FACT-A) to mediate mast cell depletion in AML using autologous STIs. and T-cell expansion. Primary RVMS

AML from patient 2 was incubated with PB5, a control bispecific diethyl (control pACT) or SACT-A for 144 hours. Blast cells (panel A), CO4 T cells (panel B), and CO8 T cells (panel C) were counted.

Figure 17 (panels A-Y) shows the ability characterized by the optimized sequence of CO123 x POP bispecific diethyl (BACT-A) to mediate T-cell activation in AML. The expression of CO25 (panel A) and Ki-67 (panel B) was determined for CO4 and CO8 t-cells from patient 2 with AML after incubation with control bispecific diethyl (control pACT) or BACT-A with autologous PBMCs. The content of perforin (panel C) and granzyme B (panel 0) was determined for CO4 and CO8 T cells from patient 2 with AML after incubation with control SACT or pAVTA-A with autologous PBMCs.

Figure 18 (panels A-O) shows that a bispecific diabody characterized by an optimized sequence of CO123 x POP (OAKT-A) is capable of cross-reacting with proteins

СсО0123 and СО3 in both humans and primates. The panels show the curves of the VIASOVETM sensograms of the results of the analyzes carried out to evaluate the ability of OAKT-A to bind to the bileumes of POPs (panels A and B) and CO123 (panels C and Y) of humans (panels A and C) and non-human primates (panels

In 0). KO values are presented.

Figure 19 (panels A-B) shows the ability characterized by the optimized sequence of CO123 x POP bispecific diethyl (FACT-A) to mediate the depletion of autologous monocytes from human and cynomolgus PBMCs. The panels show the results of the dose-dependent curves of SACT-A-mediated cytotoxicity with primary ones

Human PBMC (panel A) or Javan macaque PBMC (panel B).

Figure 20 (panels A-M) shows the ability of the optimized sequence of CO123 x POP bispecific diethyl (AKT-A) to mediate ROS depletion in Javan macaques without systemic cytokine induction. Panels A-SU show the control results obtained after 4 h. and 4 days with inert vehicle and vehicle. Panels E-H show control results obtained after 4 h. and 4 days with a control bispecific diet (control SACT). Panels 1-M show the results obtained after 4 h. and 4 days at a dose of rAVT-A, which is 10 ng/kg/day, and after 4 days at a dose of POACT-A, which is 30 ng/kg/day.

Figure 21 (panels A-Y) shows the ability of the optimized sequence of CO123 x POP bispecific diethyl (FACT-A) to mediate dose-dependent depletion of ROS in cynomolgus macaques. Javan macaques were administered BABT-A doses of 0.1, 1, 10, 30, 100, 300, or 1000 ng/kg. PBMCs were evaluated at the indicated times and the total number of B cells (panel A), monocytes (panel B), MK cells (panel C), and ROS (panel

IN).

Figure 22 (panels A-Y) shows the ability of the optimized sequence of CO123 x POP bispecific diethyl (SACT-A) to periodically modulate T-cells in Javan macaques. Javan macaques were injected with doses of 0.1, 1, 10, 100, 300, or 1000 ng/kg of SACT-A. PBMCs were assessed at the indicated times and the total number of T cells (panel A), CO4 T cells (panel B), COb9 cells (panel C), and CO8 T cells (panel 0) were determined.

Figure 23 shows the 505-RACE analysis of purified YAKT-A protein under reducing (left) and non-reducing (right) conditions.

Figures 24A-24B show the physicochemical characteristics of purified OAKT-A. Figure 24A: 5ES profile of the SAKT-A protein on a calibrated column T5K Z30005UMUHkhi.. Figure 24B: mass spectrum of the OAKT-A protein.

Figures 25A-250 show the analysis of 5PE. binding of SAKT-A with immobilized CO123 and

CO3 of man or Javan macaque. Dashed lines represent the global approximation to 1:11 of the Langmuir model of experimental binding curves obtained at concentrations

RAVT-A of 0, 6.25, 12.5, 25, 50, or 100 nm (solid lines). Data are representative of three independent experiments.

Figures 26A-26E show that SACT-A is able to simultaneously bind to both POPs and

СО0123. Figure 26A-2688 presents the results of bifunctional analysis of EHI5A and demonstrated the simultaneous involvement of both target antigens of SACT-A. Human CO123 (figure 26A) or Javan macaque CO123 (figure 268) was applied to EGIZA tablets. The titration of the concentrations of SACT-A and the control POACT resulted in detection using

Human POP biotin. Figures 26C-26E demonstrate cell surface binding of pAVT-A on CO123-Moit-13 target cells (Figure 26C), human T cells (Figure 260) and cynomolgus macaque tT cells (Figure 26E). Binding was detected using the GAS5 assay using a monoclonal antibody specific for the E-helical and K-helical region of the OAKT-A molecule or control AKT.

Figures 27A-27H show the ability of POAKT-A to mediate redirected cytolysis of target cells by human or monkey effector cells against СО123-4-Casity-3 leukemic cell lines, and show the ability of molecules to bind to a subclass of normal leukocytes circulating in the bloodstream, including pOC and monocytes, and the ability of the molecules to deplete CO14-CO123 cells (pOC and basophils) without affecting monocytes (CO14- cells) is shown. Figure 27A shows the relative binding sites of the anti-CO123-PE 0937 antibody and leukemia cell Casiti-3 lines as determined by the OBAS5 assay Figure 27B shows a relatively low percentage of cytotoxicity mediated by SACT-A or control BACT in a AML cell line (0937 cells) which, as shown in Figure 27A, contain relatively few binding sites binding with CO123).Figure 27C shows the percentage cytotoxicity mediated by pAVT-A or control SACT in the presence of purified human T cells (as effector cells) on AML cell line (Kazipti-3 cells), which, as shown in figure 27A, contain a significant number of binding sites with CO123. In Figures 278-27C, the E:T ratio is 10:1. Figure 270 shows the vitelline cytotoxicity mediated by SAKT-A or control SOAKT in the presence of purified Javan macaque RBMSOS (as effector cells) on cells

Casiti-Z3 (E:T ratio 15:1), and demonstrated that OBACT-A can bind to Javan macaque T cells. Figure 27E shows the relative binding sites of the anti-CO123-PE antibody on Kazyti-3 cells, human monocytes, human plasmacytoid dendritic cells ("hPCs"), cynomolgus monocytes, and cynomolgus plasmacytoid dendritic cells as determined by the OBAS5 assay . Figure 27E shows the ability of rAVT-A to deplete cells of СО14-СО1239. Figure 275 shows the ability of OAET-A to undergo CO14-СО123Н depletion! human cells. Figure 27H shows the ability of OAVT-A to undergo depletion of СО14-СО12С3Н! cells of the Javan macaque. Cytotoxicity was determined by the release of HON, while the value of EC5O was determined using the SgarpRide RKIZMF software)

Figure 28 shows the application of the two-compartment model for estimating the pharmacokinetic parameters of YACT-A. The data show end-of-infusion serum concentrations of OACT-A in Javan macaques following a 96-hour infusion at 100 ng/kg/day, 300 ng/kg/day, 600 ng/kg/day, and 1000 ng/kg/day. Each dot represents a separate animal; horizontal lines represent the mean value for the dosage group.

Figures 29A-29C show the effect of OAKT-A infusions on cytokine production, 1-6. Serum II-b contents (the average of their ZEM) in monkeys that received OAKT-A infusions are shown by treatment groups. Javan macaques were injected with inert vehicle - control on day 1, followed by 4 weekly infusions of either inert vehicle (group 1) (Figure 29A) or OYUACT-A administered as 4-day weekly infusions starting on day 8, in 15, 22 and 29 days (groups 2-5) (figure 298) or as an infusion 7 days a week for 4 weeks, starting on day 8 (group b) (figure 29C). Treatment intervals are indicated by gray columns.

Figures ZOA-Z3OE show the effect of OAKT-A infusions on the depletion of СО14-/С0123- cells (figures ZОА-30С) and СОЗ303- cells (figures 300-ЗОЕ). The average xx ZEM of circulating in the bloodstream contents of СО14-/20123- (figures ЗОА-30С) or СО303- (figures 3000-30) by study days and by groups is shown. Javanese macaques were injected with an inert carrier - control on 1 day, after

From which 4 weekly infusions were administered, either an inert vehicle (group 1) (figures ZbA and 300), or OAKT-A administered as 4-day infusions every week, starting on day 8, on days 15, 22 and 29 (groups 2 -5) (figures Z0A and ZOE) or as an infusion 7 days a week for 4 weeks, starting on the 8th day (group 6) (figures ZOS and ZOE). Treatment intervals are indicated by gray columns.

Figure Z1A-31I shows the observed changes in T-cell populations (figures ZTA-31C),

Sra4 cell populations (figures 310-31KE) and CO8 cell populations (figures 310-311) receiving VACT-A, administered as 4-day infusions, starting on day 8, on day 15, 22 and 29. Legend: СО25 (gray squares); СОб9-- (gray triangles), RO-1 (white triangles); Titus-3-- (white squares). T-cells were counted using CO4 and CO8 markers, not the canonical one

SOZ3, to eliminate possible interaction with OAKT-A. Javan macaques were injected with inert vehicle - control on day 1, followed by 4 weekly infusions of either inert vehicle (group 1) or SACT-A administered as 4-day weekly infusions starting on day 8, at 15, 22 and day 29 (group 5) or as an infusion 7 days a week for 4 weeks starting on day 8 (group 6). Treatment intervals are indicated by gray columns. The average 5EM of the absolute number of T-cells circulating in the bloodstream by study days and by groups is shown (figures 31A-31C). The relative values (average percentage - BEM) of СО25ж, СОбой, РО-1 and

Tit-Z- on CO4 (figures 310-31E) or CO8 T-cells (figures C1E-31H) by study days and by groups.

Figures 32A-32E show the observed changes in CO4 T cell populations (Figures 32A-32) and CO8 T cell populations (Figures 3200-32) during and after a continuous 7-day infusion of pAVT-A. The average x 5EM is shown as a percentage of СО25.ж, СОбожь, РО-1.к and Tit-Z- on СО4 (figures

З2A-32C) or СО8 (figures 320-32E) T-cells by study days and by groups 2, 3 and 4.

Treatment intervals are indicated by gray columns. Legend: СО25-- (gray squares); СОб9-- (gray triangles), RO-1-- (white triangles); Tit-3- (white squares).

Figure ZZA-3ZE shows the observed changes in CO4 T-cell populations (Figures ZZA-33C) and CO8 T-cell populations (Figures 330-33E) during and after a continuous 7-day infusion of rAVT-A. The average x 5EM is shown as a percentage of СО4х naive cells (С095-/0028-), SMT (с0р95-/002в8ж) and EMT (2095-4/С2028-) T cells in СО4х population (figures ЗЗА-33С) or СО8 population (figures 330-3ZE) by study days and by groups 2, 3 and 4. Javan macaques were injected with an inert vehicle - control on day 1, after which 4 weekly infusions were administered or inert vehicle 60 or SACT-A, which is administered in the form of 4- daily infusions every week, starting from the 8th day, on the 15th, 22nd and 29th day (groups 2-4). Treatment intervals are indicated by gray columns. Legend: naïve cells (white triangles); SMT (black triangles), EMT (gray squares).

Figure 34 shows SACT-A mediated cytotoxicity against Kazyti-3 cells with

PBMCs from either unexposed monkeys or monkeys treated with multiple infusions of OACT-A.

Figures 35A-35E show that exposure to SACT-A increased the relative frequency of central

Spra4 of memory cells and effector CO8 in memory cells at the expense of the corresponding T-cell population not previously exposed to influences. The average of their ZEM is shown as a percentage of CO4- naive cells (сб0р95-2028я53, SMT (2095-2028) and EMT (2б095-/2028-) T cells in the СО4- population (figures

ZBA-35C) or in the СО8- population (figures 350-35E by study days and by groups. Javanese macaques were injected with an inert vehicle - control on day 1, after which 4 weekly infusions of either an inert vehicle (group 1) or SACT- A, administered as 4-day weekly infusions starting on day 8, on days 15, 22, and 29 (group 5) or as an infusion 7 days per week for 4 weeks starting on day 8 (group 6). treatments are indicated by gray columns.

Legend: naive (white triangles); SMT (black triangles), EMT (gray squares).

Figures 36A-36E show the effect of SACT-A on erythrocyte parameters in monkeys that received infusions of the molecules. Circulating erythrocyte (Figures 36A-36C) or reticulocyte (Figures 3600-36) contents (mean x 5EM) in samples collected at the indicated time points from monkeys treated with SACT-A are shown.

Figures 37A-37B show the frequency of occurrence (average percentage is ZEM) of СО123 cells (Figure 37A) or NSS (С034-7С2038-/26045-/26090-- cells) (Figure 37B) in the Hyp cell population in bone marrow samples, collected at the indicated time points from monkeys treated with SACT-A. Javan macaques were injected with inert vehicle - control on day 1, followed by 4 weekly infusions of either vehicle (group 1) or OAKT-A administered as 4-day infusions weekly starting on day 8, at 15, 22 and day 29 (groups 2-5) or as an infusion 7 days a week for 4 weeks starting on day 8 (group 6).

Detailed disclosure of this invention

This invention refers to bispecific monovalent diethyls characterized by an optimized sequence of СО123 x POPs, which are capable of simultaneously binding to СО123 and POPs.

From the applications of such molecules in the treatment of hematological malignant tumors. Although non-optimized CO123 x POP bispecific diethyls are fully functional, similar to the improvements obtained in gene expression by codon optimization (see, for example, Stgovieap, N. eyi ai. (1982) "Rgieiegepiia! Sodop Ozade Ip RgoKaguoiiis Sepevz : Te Oriitai!

Sodop-Apiisodop Ipiegasip Epegau Apa Tne Zeyesiime Sodop Ozade Ip ENisiepnu Ehrgezzei Sepev"

Sepe 18(3):199-209), it is possible to further enhance the stability and/or function of СО123 x SOYUZ bispecific diethyls by modifying or optimizing their sequences.

Preferred CO123 x POP bispecific diethyls according to the present invention consist of at least two polypeptide chains that associate with each other to form one binding site specific for the CO123 epitope and one binding site specific for the POP epitope (Fig. 2). Individual diethyl polypeptide chains are covalently linked to each other, for example, by disulfide bonding of cysteine residues located within each polypeptide chain. Each polypeptide chain contains the antigen-binding domain of the variable domain of the light chain, the antigen-binding domain of the variable domain of the heavy chain and the heterodimerization domain. The intermediate linker peptide (linker 1) separates the antigen-binding domain of the variable domain of the light chain from the antigen-binding domain of the variable domain of the heavy chain. The antigen-binding domain of the variable domain of the light chain of the first polypeptide chain interacts with the antigen-binding domain of the variable domain of the heavy chain of the second polypeptide chain to form a first functional antigen-binding site that is specific for the first antigen (ie, either CO123 or CO3). Similarly, the antigen-binding domain of the light chain variable domain of the second polypeptide chain interacts with the antigen-binding domain of the heavy chain variable domain of the first polypeptide chain to form a second functional antigen-binding site that is specific for the second antigen (ie, either CO123 or POPs, depending from the identity of the first antigen). Thus, the selection of the antigen-binding domain of the variable domain of the light chain and the antigen-binding domain of the variable domain of the heavy chain of the first and second polypeptide chains is coordinated so that the two polypeptide chains jointly contain the antigen-binding domains of the variable domains of the light and heavy chains capable of binding with СО123 and СО3.

The formation of heterodimers of the first and second polypeptide chains can be controlled by heterodimerization domains. Such domains contain ZMERKZS (5BO IYUO MO:50) (or

MERCZBS; 5EO IU MO:51) on one polypeptide chain and ZEMCOSES (5EO IU MO:52) (or

EMKOES; 5BEO IU MO:53) on another polypeptide chain (052007/0004909). Alternatively, such domains can be designed to contain helices of opposite charges. The heterodimerization domain of one of the polypeptide chains contains a sequence of at least six, at least seven, or at least eight positively charged amino acids, and the heterodimerization domain of the other polypeptide chain contains a sequence of at least six, at least seven, or at least eight negatively charged amino acids.

For example, the first or second heterodimerization domain will preferably contain a sequence of eight positively charged amino acids, and the other of the heterodimerization domains will preferably contain a sequence of eight negatively charged amino acids. A positively charged amino acid can be lysine, arginine, histidine, etc., or a negatively charged amino acid can be glutamic acid, aspartic acid, etc. A positively charged amino acid is preferably lysine and/or a negatively charged amino acid is preferably glutamine acid

СсО0123 x POP bispecific diethyls according to the present invention are designed so that such first and second polypeptide chains are covalently linked to each other by means of cysteine residues along their length. Such cysteine residues can be introduced into the intermediate linker that separates the Mi and MH domains of the polypeptides. Alternatively and more preferably, a second peptide (linker 2) is introduced into each of the polypeptide chains, for example, at the amino terminus of the polypeptide chains or in a position that places linker 2 between the heterodimerization domain and the antigen-binding domain of the variable domain of the light chain or the variable domain of the heavy chain.

According to specific implementation options, the bispecific monovalent diethyls according to the present invention, characterized by the optimized sequence of СО123 x POPs, additionally contain a domain

ES immunoglobulin or albumin-binding domain to increase the half-life of IP mimo.

СО123 x СОЗ3 bispecific monovalent diethyls according to the present invention, which contain the Es domain of immunoglobulin (i.e. СО123 x СОЗ bispecific monovalent Es - diethyls)

Zo consists of the first polypeptide chain, the second polypeptide chain and the third polypeptide chain. The first and second polypeptide chains are associated with each other to form one binding site specific for the CO123 epitope and one binding site specific for the SHOZ epitope. The first polypeptide chain and the third polypeptide chain are associated with each other to form the Es domain of the immunoglobulin (Figure 3A and Figure 3B). The first and second polypeptide chains of bispecific monovalent

Es - diethyl are covalently bound to each other, for example, by disulfide bonding of cysteine residues located within each polypeptide chain.

The first and third polypeptide chains are covalently linked to each other, for example, by disulfide bonding of cysteine residues located within each polypeptide chain. Each of the first and second polypeptide chains contains the antigen-binding domain of the variable domain of the light chain, the antigen-binding domain of the variable domain of the heavy chain and the heterodimerization domain. The intermediate linker peptide (linker 1) separates the antigen-binding domain of the variable domain of the light chain from the antigen-binding domain of the variable domain of the heavy chain. The antigen-binding domain of the variable domain of the light chain of the first polypeptide chain interacts with the antigen-binding domain of the variable domain of the heavy chain of the second polypeptide chain to form a first functional antigen-binding site that is specific for the first antigen (ie, either CO123 or SOYUZ). Similarly, the antigen-binding domain of the light chain variable domain of the second polypeptide chain interacts with the antigen-binding domain of the heavy chain variable domain of the first polypeptide chain to form a second functional antigen-binding site that is specific for the second antigen (ie, either POP or CO123, depending from the identity of the first antigen). Thus, the selection of the antigen-binding domain of the variable domain of the light chain and the antigen-binding domain of the variable domain of the heavy chain of the first and second polypeptide chains is coordinated so that the two polypeptide chains jointly contain the antigen-binding domains of the variable domains of the light and heavy chains capable of binding with CO123 and POPs. Each of the first and third polypeptide chains contain some part or all of the CH2 domain and/or some part or all of the CH3 domain of the complete Es immunoglobulin domain and a cysteine-containing peptide. Some part or all of the CHZ2 domain and/or some part of 60 or all of the CHZ domain are associated with the formation of the Es domain of the bispecific monovalent Es-diethyl immunoglobulin according to the present invention. The first and third polypeptide chains of bispecific monovalent Es-diethyl according to the present invention are covalently linked to each other, for example, by disulfide bonding of cysteine residues located within the cysteine-containing peptide of the polypeptide chains.

I. A bispecific diabody characterized by an optimized sequence of СО123 x SOZ, "VAVT-A"

According to the present invention, a bispecific diabody is provided, characterized by an optimized sequence, capable of simultaneously and specifically binding to the CO123 epitope and to the COS epitope ("СбО123 x СО3" bispecific diabody or SACT-A). As discussed below, YUBACT-A has been found to exhibit increased functional activity relative to others characterized by a non-optimized sequence of CO123 x POP bispecific diethyl-like compounds, and is therefore termed "sequence-optimized".

СО0123 x POP bispecific diabody.

The bispecific diabody characterized by the optimized sequence of СО123 x СОЗ (ОАВТ-А) contains the first polypeptide chain and the second polypeptide chain. The first polypeptide chain of the bispecific diethyl will contain, in the direction from the M-terminus to the C-terminus,

M-terminus, variable domain of the light chain (MI domain) of a monoclonal antibody capable of binding to POPs (MI soz), intermediate linker peptide (linker 1), variable domain of the heavy chain (UN domain) of a monoclonal antibody capable of binding to СО123 (МНеоигз), and С- the end. The predominant sequence of such a Mi soz domain is 5EO IU MO:21:

OAUMTOERBI TU5ROSTMUMMTI TSV5ZTOaAMTTZMUAMIMUMOOKRYasSARVY SS TMKAARMUTRAVN

EZOABI I! CHSOKAAI TITIAAOAEOEArUMSAI M UZMI MUURasaTtki tm a

Antigen-binding domain Mi soz contains SOKI 5EO IU MO:38: KZ55TOAMTTZMMUAM, SOC2

ZEO Ir MO:39: STMKEAR and SOCZ 5EO IU MO:40: A M/UZMI MM.

The preferred sequence of such linker 1 is 5EBEO ИЮ МО:29: СбОо5ООО0.

The preferred sequence of such domain МНсо:гз is 5EO IU MO:26:

EMO MOBOAEII KKRIABZBUKUZSKAZIAHUTETOUUMKUUVOARIOSII EMO OPROMYATEMMOKE

KavmtITMOKZTZTAUMEI 551 V5EOTAMUUSAVZNI AND VABMUBAUUSOSTI MTU

The antigen-binding domain of MNeoigz contains SOCI 5EO and MO:47: YMUMK, SOC2 5EO IU

From MO:48: OPROZMSOSATEUMOKEKO and SOCZ 5EO IYUO MO:49: ZNI AND KA.

The second polypeptide chain will contain, in the direction from the M-terminus to the C-terminus, the M-terminus, the Mi domain of a monoclonal antibody capable of binding to CO123 (Mi coygz3), an intermediate linker peptide (for example, linker 71), the MH domain of a monoclonal antibody antibodies capable of binding to POPs (MNeoz), and the C-end. The preferred sequence of such a domain Mi soigz is 5EO IU MO:25:

VEMMTO5ROBI AM5I SEVNUTMZOSKZOBY MZaMOKMUI PUMOOKROORRKOI TMM AZTVEZO

UekhROovezAzazatovTi tzi odeomauuusomomzurmtesostki bik

The antigen-binding domain of Mi soigz contains SOKI 5EO IU MO:44: K55OBI HER M5OMOKMTI t,

SOM2 ZEO IYU MO:45: MAZTKEZ and SOCZ 5EO IYU MO:46: OMOM5URUT.

The preferred sequence of such a MNsoz domain is 5EO IU MO:22:

EMO MESOSIA MOROIBI VI BSAAZOETEZTMAMMU UVO ROKY EUMavIVZKUMMUATUMA

OBMUMKOVETIZAVOBKM5I M OMM5I KTEOTAUUUSUVNOaMEeaMOuU UM EAUMUusOS,TI UTU55

The antigen-binding domain of MNsoz contains SOMK1T ZEO IU MO:41: TMAMM, SOC2 5EO and MO:42:

VIVZKUMMUATUMArZMKO and SOCZ 5EO IYU MO:43: NOMEOMZUUZMUBAH.

Bispecific diethyls characterized by an optimized sequence of CO123 x POP according to the present invention are designed so that the first and second polypeptides are covalently linked to each other by means of cysteine residues along their length. Such cysteine residues can be introduced into an intermediate linker (for example, linker 1), which separates the MI and MH domains of polypeptides. Alternatively and more preferably, the second peptide (linker 2) is introduced into each polypeptide chain, for example, in a position at the M-end relative to the Mi domain or at the C-end relative to the MN domain of such a polypeptide chain. The preferred sequence of such linker 2 is ZEO IU MO:30: ССО.

The formation of heterodimers can be controlled by further engineering such polypeptide chains to contain polypeptide helices of opposite charges. Thus, according to a preferred embodiment, one of the polypeptide chains will be designed to contain an "E-helical" domain (ZBO 10 /MO:34:

EMAAGEKEMAAGEKEMAAGEKEMAAGEK), whose residues form a negative charge at pH 7, while the other of the two polypeptide chains will be designed to contain a "K-helical" domain (5EBO IU MO:35: КМААГКЕКМААИКМАЙИIKEКМААИКЕ), whose residues form a positive charge at pH 7. The presence of such charged domains provides an association between the first and second polypeptides and thus promotes heterodimerization.

It is not essential which helix is provided by the first or second polypeptide chains.

However, preferably, the bispecific dibody characterized by the optimized sequence of sr123 x POP according to the present invention ("BACT-A") contains the first polypeptide chain, which is characterized by the following sequence (5EO IU MO':1):

OAUMTOERBI TU5ROSTMUMMTI TSV5ZTOaAMTTZMUAMIMUMOOKRYasSARVY SS TMKAARMUTRAVN

EZOABI I! CHSOKAAI TITAAOAEEOEARUMSAI M UZMI UUgssa7tki tm ssvazasasemoi MOaIAEI

KKROABUKUZSKABAMTETOUUMKUMVOAROOST EMISOPROMOATEUMOKeEKY VMT mok

AUMEG 551

IHEKEMAAG EC

Chain 1 SAKT-A consists of the following: BEO IU MO:21 - BEO IU MO:29 - BEO IU MO:C26 -

ZEO IU MO:30 - BEO IU MO:34. Coding chain 1 SACT-A polynucleotide is 5EO IU

МО:с2: саддсідюдідасісаддадсснсасідассдідіссссаддсддаасідідасссідасадсадаїссадсасаддсдсадщда ссасаїсіаасіасдссаайдддіясадсадаадссаддасаддсассааддддссідаїсдддддаіасааасаааадддсіссса ассссідсасдайнсіддаадісідсюдддсоддаааддссодсісідасіанассдддасасаддассдаддасдаадссданасіаняої дсісідюдіаіадсааєсідддансоаддададдсасаааасюдасідосідддаддададчоаїссддсдоасддаддсдаддаодса дсюддіадсадіссддддседадсідаадааасссддадсіссдідаадащісндсааадссадіддсіасасснсасадасіасіаїаї даадідддісаддасаддсіссаддасадддасіддааі(ддаісодсдаїаісанссиссаасдддассасцісіасааіїсадаадащтшаа аддсаддаюдасіанНассодюддасаааїсаасаадсасідснагадчдадсдадсісссідсдсісідаадаїасадссоаюіасіандю сісддісасассідсідададссадсідашоснандаддасаддоасасссіддідасадщіснссодадаашдіяддсдадодавчаасді дассдсасіддадааададданосідстодадааддаадаісдсдсасідааааддаддісдсадсссіддадааа

The second polypeptide chain of SANT-A is characterized by the following sequence (ZEO IU

MO:3):

VEMMTO5ROBI AM5I SEVNUTMZOSKZOZOBY MZaMOKMMI PUMOOKROORRK MU AZTVEZOa

УероовезаЗазатовТти ТЗ ОADEOMAUUUSOMZURUTRSOSTKI EIXSOSSOSOS, SEMOI MEZO sa MORAABI AND ZSAAZAETESTMAMMUMUUVOAOaroaKaIai EUUMaviVZKUMMMATUMAOSUKOVETIZA rOBKM5I M OMM5ZI KTEOTAUUUuSUVVNEOMEAMZUU UMA aOoS, sttIMAEKSaUsa KISzaa

From MAAI KEKMAAI KEKMAAI KE

Chain 2 SAKT-A consists of the following: BEO IU MO:25 - BEO IU MO:29 - BEO IU MO:22 -

ZEO IU MO:30 - BEO IU MO:35. Coding chain 2 SARBT-A polynucleotide is 5EO IU

МО:А: даснсдюдаїдасасацдісіссідаїадісіддссодадвісіддддаадсддадідасіаісндсаададсісссадісасідсда асадсддаааїсадааааасіаісідассіддіассадсадаадссаддссадсссссіааасідсідаїсіандддснссассадада асіддсддсссдасадансадсддсадсддсадсддсасаданнасссідасаацшсіадісюсаддссдаддасдюдсідщіа сіандісадааїдайцасадсіаїсссіасаспсддссадддадассаадсіддааайаааддаддсддаїссддсадсдааддсдад дідсадсіддіддадісюдаддаддснодіссадссіддадддісссідадасісіссіїдсадссісюдайсассіїсадсасаїасдс їаїдаанпдодіссдссаддсіссадддааддддсіддадідаднодааддаїсаддіссаадіасаасаанаїдсаассіасіаїдссо асісюі(дааддаіадайсассаїсісаадададаїйсааадаасісасіціаісідсаааїдаасадссідаааассдаддасасддсс діїанасюцідідадасасодіаасісддсаанснасощіснДдшщаснНапддадасаддддасасіддідасідщісноссдддадаа тіддсоадіддаааадіддссдсасідааддчадааадндсідсіндааададааддісдссдсаснааддааааддісдсадсссюда аадад

As discussed below, it was found that a bispecific diabody characterized by an optimized sequence of CO123 x POPs (OACT-A) is characterized by the ability to simultaneously bind CO123 and POPs in both human and monkey cells. It was found that provision of OAKT-A caused T-cell activation, mediated the reduction of blast cells, managed expansion of the T-cell population, induced T-cell activation, and caused redirected cytolysis of malignant target cells.

II. Bispecific diabody comparison characterized by non-optimized sequence

СОр123 x SOZ, "SAVT-V"

RAVT-B is a bispecific diabody, characterized by an unoptimized sequence of СО123 x СОЗ, characterized by a macrostructure similar to that of SАКТ-

A. The first polypeptide chain of SAKT-B will contain, in the direction from the M-terminus to the C-terminus, the M-terminus, the MI domain of a monoclonal antibody capable of binding to SOZ (Mi soz), an intermediate linker peptide (linker 1), MH domain of a monoclonal antibody capable of binding to

CO123 (MNeoigz), intermediate linker 2, E-helical domain and C-terminus. The Mi soz domain of the first polypeptide chain of SAKT-B is characterized by the following sequence (ZEO IYUO MO:23):

RIOGTO5RAIM5AZREIEKUTMTSVAZOZZUZUMMU UOOKatZRKAUMUOTZKMABOaURUVEZIa5 bo aBYatT5U5I TIZZMEAEVAATUUSOOMZ5ZMARI TEaASTKI EI K

The MNoeoigz domain of the first polypeptide chain of BACT-B is characterized by the following sequence (5EO IU MO:28):

OMOG MOBOAEI KKROAZUKUZSKABOAM TETOUUMKUUVOARIASAI EMISOPROMOATEMMOKE

KavmtITMOKZTZTAUMEI 551 V5EOTAMUUSAVZNI AND VABMUBAUUSOSTI MTU

Thus, chain 1 of SAKT-B consists of the following: BEO IYUO MO:23 - BEO IYU MO:29 -

ZEO IU MO:28 - BEO IU MO:30 - 5EO IU MO:34. The sequence of the first polypeptide chain of rAVT-B is as follows (5EO IYUO MO:5):

RIOGTO5RAIM5AZREIEKUTMTSVAZOZZUZUMMU USOKatZRKAUMLYUTZKMABAmMuRUVEbIA5 aBYatT5U5I TIZZMEAEVSAATUUSOOM5ZMRI TREOASTKGEI KsaSYaapasvomMoO! MOBSOAEI KKRO

ABUKUZSKABZIAACHUTETOUMKUMUVOARISII EMISOPROMOaATEMMOKEKKaTvVmMTtMOKZTZTAMME

I 55I A5EOTAUUUSAVZNI I VABM/ RAMU SPISTI UTUZAsSIasaEMAAEKEMAAI EKEMAAI EKE

MAAI EC

Кодуючий ланцюг 1 САКТ-В полінуклеотид являє собою 5ЕО ІЮ МО:6: дасайсадсідасссадісіссадсааїсаїдісідсаїсіссадддададааддісассадассідсададссадіїсаадчідіаацді іїіасадаасіддіассадсадаадісаддсассісссссаааададдащшаїдасасаїссаааддадснсіддадісссНаїсдснса ддасаддддісддчодассісаїасісісісасааісадсадсаддаддсідаадаїдсідссаснанасідссаасадюддацдіадіаа сссдсісасойсддіасідддассаадсіддадсідаааддадоасддаіїссддсддсаддаддссаддідсадсіддідсадієс9994 сюадсідаадааасссддадснссодідааддаідціснодсааадссадіддсіасассіїсасадасіасіаїадаадідддісаддсадад сіссаддасадддасіддааддаісддсодаїаїсанссіїссаасдддадссасшсіасааіїсадаадшаааддсадддадасіанас сдддасаааїісаасаадсасідснаіа(дчдадсюдадсісссідсдсісідаадаїасадссдідіасіанодідсісддісасассідсщда дадссадсіддашоснайддддасадддсасссіддідасадщіснссддададашіддсадіддадаадддссдсасіддадааа даддандасюстоддадааддаададісдсідсаспдааааддаддісдсадсссіддадааа

The second polypeptide chain of SAKT-B will contain, in the direction from the M-end to the C-end, the M-end, the Mi domain. of a monoclonal antibody capable of binding to CO123 (Mi soigz), Intermediate linker peptide (linker 1) and the MH domain of a monoclonal antibody capable of binding to SOZ (MNeoz), intermediate linker 2, K-helical domain and C-terminus.

The Mysoigz domain of the second polypeptide chain of OAKT-B is characterized by the following sequence (5EO IU MO:27):

VEMMTO5ROBI AM5I SEVNUTMZOKOZZOBY MZaMOKMM PUMOOKROORRKOI IMUUAZTVEZOa

From Ukraine

The MNsoz domain of the second polypeptide chain of OAKT-B is characterized by the following sequence (5EO IU MO:24):

RIKGIOO5SOSAEI AVROAZUKMZSKTZaMUTETAUTMNUUKOVRYaSai EUlamiMRZNam GTMUMOK

ECOKATI TTOKZZZTAUMOYI 551 T5EOBAUUUSAVNUMOONUSI OUMUuSsOSTT TU

Thus, chain 2 of SAKT-B consists of the following: BEO IYUO MO:27 - BEO IYU MO:29 -

ZEO IU MO:24 - 5EO IU MO:30 - BEO IU MO:35. The sequence of the second polypeptide chain of pAVT-B is as follows (5EO IU MO:7):

VEMMTO5ROBI AM5I SEVNUTMZOSKZOZOBY MZaMOKMMI PUMOOKROORRK MU AZTVEZOa

Ukraine

EGADREIAZUKMZSKTZamkhTETAMTMNUMKOARIOSIEMMOMIMRZVaMTMUMOKEKOKATI TOK

551

AAI KEKMAAIII KE

Coding chain 2 of the SAKT-B polynucleotide is 5EO IU MO:8:

Саснсаїдаїдасасадісіссідаіадісіддссуаюавдісіддадоааасоддаюдасіацісносаадаасісссадісастідсюда асадсддаааїсадааааасіаісідассіддіассадсадаадссаддссадсссссіааасідсідаїстандддсіссассаддда асіддсддсссдасаданйсадсдасадсддсадсддсасадайнНасссюасаанісіадісюсаддссдаддасаюдадсідщіа сіандісадааїдайасадсіаїсссіасаспсддссаддддассаадсідчааанйаааддаддсддаїссддсадсдаадоасодаїа ісааасідсадсадісаддддсдаасіддсаадассіддддссісадідаадащціссідсаадасісіддсіасасстасіадаїаса сдадсасідддіаааасададдссюдасадддісіддаадданддагасанааїссіадссудонНагасіаацасааісадааднс ааддасааддссасандасіасадасаааїссіссадсасадссіасаддсаасідадсадссдасаїсідаддасісюдсадісіана сюдсаадайанащдаюаїсанасідссідасіасіддддссааддсассасісісасадісіссіссддаддаціддсоддідавдаааад їддссадсасдааддадааадноасідстоааададааддісдссдсаснааддааааддісдсадсссідааадад

I. Modified variants of bispecific diethyl, characterized by an optimized sequence of СО123 x СОЗ (ОАКТ-А)

A. A bispecific diabody characterized by an optimized sequence of СО123 x POP containing an albumin-binding domain (FBACT-A with ABO "m/ABO")

According to the second embodiment of the present invention, a bispecific dibody characterized by an optimized sequence of CO123 x POP (OACT-A) will contain one or more albumin-binding domains (ABO" (FACT-A with ABO "m/ABO") on one or both 60 polypeptide chains diethyl.

As disclosed in international patent publication Me UMO 2012/018687, in order to improve the ip mimo pharmacokinetic properties of diethyls, diethyls can be modified so that they contain a polypeptide part of a protein that binds to serum proteins at one or more ends of the diethyl. Such a polypeptide part of the protein, which binds to serum proteins, will most preferably be embedded at the C-end of the diethyl molecule. A particularly preferred polypeptide part of the protein that binds to serum proteins, for this purpose, is the albumin-binding domain (ABO) from protein C of streptococcus. Albumin-binding domain C (AVOZ) of protein C of strain 5igeriosossi5 148 is particularly preferred.

Albumin-binding domain C (AVOZ) of protein C of strain 5igeriosossi5 5148 consists of 46 amino acid residues that form a stable three-helix structure and is characterized by a wide albumin-binding specificity (dopape5op, M.O. ei ai. (2002) "Bigisiige, Zresiisiku,

Apa Mode OI Ipiegasiiop Rog Vasiegia! AIritip-Vipaipd Modiyev, " U. Vioi. Speth. 277(10):8114-8120).

Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin contains several low-molecular-weight binding sites that allow it to bind noncovalently to other proteins and thereby extend their serum half-lives.

Thus, the first polypeptide chain of such a bispecific diethyl, characterized by an optimized sequence of СО123 x POP, containing an albumin-binding domain, contains a third linker (linker 3), which separates the E-helix (or K-helix) of such a polypeptide chain from albuminz' connecting domain. The preferred sequence of such linker C is ZEO IU MO:31: 50505. The preferred albumin-binding domain (ABO) is characterized by the following sequence (ZEO IU MO:36):

GAYEAKMI AMAEGOKMaUZOMUKMI IOMAKZAESUKAPIOEIIG AAI R.

Thus, the preferred first chain, characterized by the optimized sequence of СО123 x POP of the bispecific diethyl containing the albumin-binding domain, is characterized by the following sequence (5EO IU MO:9):

OAUMTOERBI TU5ROSTMUTI TAZ TAM TT ZMUAMUMUMOOKRIOOSARVIG IS TMKAARMUTRAN

EZOABI I! CHSOKAAI TITAAOAEEOEARUMSAI M UZMI UUgssa7tki tm ssvazasasemoi MOaIAEI

KKROABUKUZSKABAMTETOUUMKUMVOAROOST EMISOPROMOATEUMOKeEKY VMT mok

From AUMEG 551 A5EOTAMUUSAVZNI AND VABUURAUUuSsOS, TI MUTUZZPASSY, ASSSEMAAI EKEMAAI EKEMAA

HEKEMAAI ECOSII5I AEAKMI AMAEYI OKU UOMUKMYPOMAKZAESUKAPIOBEI GG AAI R

Diabody characterized by the optimized sequence of СО123 x СОЗ diabody containing albumin-binding domain consists of: BEO IU MO:21 - BEO IU MO:29 - BEO IU MOS26 - ZEBEO IV MO:30 - BEO IU MO:34 - 5EO 10 MO:31 - BEO IYUO MO:36. Полінуклеотид, що кодує таке диатіло, що характеризується оптимізованою послідовністю СО123 х СОЗ, що містить дериватив альбумінзв'язуючого домена, являє собою 5ЕО ІЮ МО:10: саддсідюдідасісаддадсснсасідассощдіссссаддсддаасідідасссідасаїддсадаїссадсасаддасдсадіда ссасаїсіаасіасдссаайдддіясадсадаадссаддасаддсассааддддссідаїсдддддаіасааасаааадддсіссса ассссідсасдайнсіддаадієідсдддсддаааддссдсісідасіанассддддсасаддссдаддасдаадссданастаної дсісідюдіаіадсааєсіаддансоаддададдсасаааасюдасідосідддаддддадчдаїссддсадсдааддсдадаюдса дсюддіадсадіссддддседадсідаадааасссддадсіссдідаадащісндсааадссадіддсіасасснсасадасіасіаїаї даадідддісаддсаддсіссаддасадддасіддааі(ддаісоддсдаїаісанссіссаасдаддссасінсіасааіїсадаадшаа аддсаддаюдасіанНассодюддасаааїсаасаадсасідснайадададсідадсісссідсдсісідаадаїасадссоіасіанодід сісддісасассідсідададссадсідашоснандаддасаддоасасссіддідасадщіснссодадаашдіяддсдадодавчаасді дассдсасіддадааадчадданосідстддадааддадаїсосідсасідааааддаддісдсадсссддадаааддсдасдадіс ісідссдаадсаааа diosiddssaassdsdaasiddaiaaaaiayudsuadadsdacnanNaiiiaadaaassidandasaasdsaaaaaissa sddaaddsdidaaadsasidaydaschdaaansiddssdsssidssi

The second polypeptide chain of such a diethyl, characterized by an optimized sequence of СО123 x СОЗ3, containing an albumin-binding domain, is characterized

BO by the sequence described above (5EBO IJU MO:3) and encoded by a polynucleotide characterized by the sequence 5EO IJU MO 4.

B. Bispecific dibodies, characterized by an optimized sequence of СО123 x POP, containing the Es Ide domain (BACT-A with Es "Mmy/gs")

According to the third variant of implementation according to the present invention, a bispecific dibody characterized by an optimized sequence of СО123 x СОЗ3, consisting of three polypeptide chains and containing the Es Id domain (version 1 and version 2 pAVTA-A with Es "m/Es") ( figure ZA-ZV).

For the formation of such an Es IdO domain, the first and third polypeptide chains of diethyl contain, in the direction from the M-terminus to the C-terminus, a cysteine-containing peptide, (most preferably peptide 1, characterized by the amino acid sequence (ZEO IU MO:55): OKTNTSRRSR), some part or all of the SNAI domain and/or some part or all of the SNZ domain of the complete Es immunoglobulin domain and the C-terminus. Some or all of the CH2 domain and/or some or all of the CH3 domain are associated with the formation of the Es domain of immunoglobulin bispecific monovalent retention

Es domain diethyl according to the present invention. The first and second polypeptide chains of bispecific monovalent Es-diethyl according to the present invention are covalently linked to each other, for example, by disulfide bonding of cysteine residues located within the cysteine-containing peptide of the polypeptide chains.

The CH2 and/or CH3 domains of the first and third polypeptides do not necessarily have to be identical and are preferably modified to facilitate the formation of a complex between the two polypeptides. For example, an amino acid substitution (preferably a substitution with an amino acid containing a bulky side group that forms a "protrusion", such as tryptophan) can be introduced into the CH2 or CH3 domain such that the steric effect prevents interaction with the similarly mutated domain and forces the mutated domain to pair with a domain in which a complementary or accommodating mutation, i.e., a "pocket" (for example, a glycine substitution), has been engineered. Such sets of mutations can be constructed in any pair of polypeptides containing a molecule of bispecific monovalent Es - diethyl, and, in addition, constructed in any part of the polypeptide chains of the indicated pair. Methods of protein engineering to promote heterodimerization instead of homodimerization are well known in the art, particularly with respect to the engineering of immunoglobulin-like molecules, and are contemplated herein (see, e.g., Kiddulau et al. (1996) Kporz-Ipio-Noyes' Epidep. OI Apiirbodu SNZ

Botaipe Rog Neamu SNaip Neiegoditegigayop, "Rgoieip Epadg. 9:617-621, Amueyi! yei ai. (1997) "Ztabiye

Neiegoditeg5 Egot Vetoaveiiipd Te YuOotaip Ipiepase OII A Notoditeg O5ipd A Riade Oizriau

Mrgagu, " U. Moi. Vioi. 270: 26-35, apa Hie ei ai. (2005) "A Mem Rogtai OYI Vizresiiis Apiibodu: Nidniu

ENisieepi Neiegoditegigayop, Ehrgeziop Apa Tytog Sei! Guviv, " U. Ittipoi. Meiyoav 296:95-101; each of which is fully incorporated herein by reference). Preferably, the "protrusion" is constructed in the CH2-CH3 domains of the first polypeptide chain, and the "recess" is constructed in the domains CH2-CH3 of the third polypeptide chain. Thus, the "protrusion" will assist in preventing homodimerization of the first polypeptide chain by means of its CH2 and/or CH3 domains. Since the third polypeptide chain preferably contains a substitution that

Zo ensures the formation of a "well", it will heterodimerize with the first polypeptide chain, as well as homodimerize with each other. The overhang is created by modifying the Es domain of the native Es dos domain so that it contains the T36bMU modification.

A preferred depression is created by modifying the Es domain of the native Es domain to contain the 73665, I Z368A, and U407M modifications. To assist in the purification of the homodimer of the third polypeptide chain from the terminal bispecific monovalent Es - diethyl containing the first, second and third polypeptide chains, the binding site with the protein A domains

СНН2г and СНЗ of the third polypeptide chain are preferably mutated by means of an amino acid substitution at position 435 (Н435К). Thus, the homodimer of the third polypeptide chain will not bind to protein A, while the bispecific monovalent Es-diabody will retain its ability to bind protein A through the binding site to protein A on the first polypeptide chain.

The preferred sequence for the CH2 and CH3 domains of the Es domain of the antibody, which is present in the first polypeptide chain, is (ZEO IU MO:56):

AREAASOIaRBZURBI ERRKRKOTI MIZVTREUTSMUMOUZNEOREUKEMU UMIMEMNMAKTKRAEEE

OMMOTUAUM5MI TM NOBUUL ME KEUKSKUSMKAI RARIEKTIZKAKYORAEROMMUTI RROVEEMTKM

OMBI MS UKOEMRZOIAMEZHM EZMSOREMMUKTTRRMI YUOBrazERI ZK TMOKZAUMOSMUEZOSBM

MNEAIII NMNUTOK5BI 5I YEAR

The preferred sequence for the CH2 and CH3 domains of the Es antibody domain, which is present in the third polypeptide chain, is (ZEO IU MO:11):

AREAASOIaRBZURBI ERRKRKOTI MIZVTREUTSMUMOUZNEOREUKEMU UMIMEMNMAKTKRAEEE

OMMOTUAUM5MI TM NOBUUL ME KEUKSKUSMKAI RARIEKTIZKAKSORAEROMUUTI RROVEEMTKM

OM5I ZSAUKagURZVIAMEMEZMSOREMMUKTTRRMI O5raZERI UK TUoKZAUMUOVAvaMUEZSOSVM

MNEAIII NMAUTOK5BI SI ROK

S. Construct version 1 OAKT-A m/gs

In order to illustrate such Es - diatila, according to this invention, the construct version 1 OAKT-A m/Es is provided. The first polypeptide of the construct version 1 OAKT-A m/Es contains, in the direction from the M-terminus to the C-terminus, the M-terminus, the MI domain. of a monoclonal antibody capable of binding to

CO123 (Mi soig2z), intermediate linker peptide (linker 1), MH domain of a monoclonal antibody capable of binding to SOZ (MHsoz), linker 2, E-helical domain, linker 5, peptide 1, 60 polypeptide containing CH2 domains and the NW of the Es domain, and the C-terminus. The preferred linker 5 is characterized by the sequence (5ЕСО IЮ MO:32): 5600. The preferred polypeptide, which contains the SNI and SНZ domains of the Es domain, is characterized by the following sequence (ZEO IЮ MO:37):

AREAASOIaRBZURBI ERRKRKOTI MIZVTREUTSMUMOUZNEOREUKEMU UMIMEMNMAKTKRAEEE

OMMOTUAUM5MI TM NOBUUL ME KEUKSKUSMKAI RARIEKTIZKAKYORAEROMMUTI RROVEEMTKM

OMBI MS UKOEMRZOIAMEM EZMSOREMMUKTTRRMI OBROWSERI ZK TMOKZAMmObaMUEOBM

MNEAIII NMNUTOK5BI SI ROK

Thus, the first polypeptide of this construct version 1 SACT-A m/s consists of the following: ZEO IU MO:25 - BEO IU MO:29 - BEO IU MO:22 - BEO IU MO:30 - BEO IU MO:34 - BEO

IO MO:Z32 - BEO IU MO:55 - BEO IU MO:37.

The preferred sequence of the first polypeptide of this construct version 1 of SAKT-A m/gs is characterized by the following sequence (5EO IU MO:13):

VEMMTO5ROBI AM5I SEVNUTMZOSKZOZOBY MZaMOKMMI PUMOOKROORRK MU AZTVEZOa

Uekhroovezzazatovti tzi oadeomauuusomzurutrsostki eikaosoabassassemoi mezo sa moreRaabi vi zsaazaeteztmammumuuuvoaroaKaIai EUUMaviVzkummmatumaoZukovetiza rOBKM5I M OMM5zi KTEOTAUUuuSUVvneOemeaMZUU ZaaAaSEM aOoS,s

MAAI EKEMAAI EKEMAAG ECOSSYASOKTNTTORRSORAREDASORBZUNBI ERRKRKOTI MIZAVTREMTSUUMO

MZNEOREUKEMUUUramEMNMAKTKRAEEOMMOZTUAMUMZMI TM NOBU/LI MEKEMKOSKMUMZMKAG RAR

IEKTIZKAKaORAEROMUTI RROVEEMTKMOM5BI M SI MKaEMRZOIAMEM EZMSEOREMMUKTTRRMI. Explanations of TMoKZAMOSS, MMEZSZUMNEAI NMNUTOKOBI 5TH YEAR

Переважний полінуклеотид кодуючий такий поліпептид, являє собою (ЗЕО ІЮ МО:14): даснсдюдаїдасасацдісіссідаїадісіддссодадвісіддддаадсддадідасіаісндсаададсісссадісасідсда асадсддаааїсадааааасіаісідассіддіассадсадаадссаддссадсссссіааасідсідаїсіандддснссассаддда асіддсддсссдасаданйсадсдасадсддсадсддсасадайнНасссюасаанісіадісюсаддссдаддасаюдадсідщіа сіандісадааїдайцасадсіаїсссіасаспсддссадддадассаадсіддааайаааддаддсддаїссддсадсдааддсдад дідсадсіддіддадісюдаддаддснодіссадссіддадддаісссідадасісіссіідсадссісіддайсассисадсасаїасус їаїдаанпдодіссдссаддсіссадддааддддсіддаяддаодааддаїсаддіссаадіасаасаацнаюдсаассіасіадхессд асісюі(дааддаіадайсассаїсісаадададаїйсааадаасісасіціаісідсаааїдаасадссідаааассдаддасасддсс діюїанасіцідідадасасодіаасіїсддсаанснасощдіснданоснанддде асаддоддасасіддаідасідідіснссддадда тіддсоадіддадаадіддссдсасіддадааададанддсідстддадааддаддісдсідсасндааааддаддісдсадсссіда

Зо адаааддсддсддддасаааасісасасаїдсссассдідсссадсассідаадссосддаадддассодісадісіссісносссссаа аасссааддасасссісаїдаїсісссддассссідаддісасадсдіддддодасддадссасдаадасссідаддісааднсаа сюдіасадаасдасдддваддідсаїааїдссаадасааадссосддааддадсадіасаасадсасотассдуідюддісадсаіїссі сассдіссюсассаддасіддсдааіддсааддадіасаадідсааддісіссаасааадсссісссадсссссаїсдадаааассаї сіссааадссазадддсадссссдадаассасаддідіасасссідсссссаїсссдддаддадаїдассаадаассаддісадссідчі даїдссіддісааадодсіїсіаїсссадсдасаіїсдссудіддадіяддададсаадддсадссддадаасаасіасаадассасдссіс ссдідсіддасіссдасддсісснсНссісіасадсаадсісассуіддасаададсаддіддсадсаддддаасодіснсісаїдсіссді даюсаїдаддсісідсасаассасіасасдсадаададссісісссідісіссдддіааа

The second chain of such a construct version 1 SAKT-A m/Es will contain, in the direction from the M-terminus to the C-terminus, the M-terminus, the Mi domain. of a monoclonal antibody capable of binding to SHUOZ (Mi soz), intermediate linker peptide (linker 71), MH domain of a monoclonal antibody capable of binding to SHUO123 (MNeoigz), linker 2, K-helical domain and C-terminus. Thus, the second polypeptide of this construct version 1 OAKT-A m/s consists of the following: BEO IU MO:21 -

ZEO IU MO:29 - 5BO IYUO MO26 - 5EBO I MO:30 - 5EBO IO MO:35. Такий поліпептид характеризується наступною послідовністю (5ЕО ІЮ МО:15): даумідерзм5радімств5ідамизпуапиудаКкрддаргдііддаіпКкгарулранздвзідокаанндадаєдеадуусаї жузпімладакимідддазададекцімаздавіккрдазукуизсКазаутауутКммтаарададїіемідайрзпданупакікаг міймакзівїауте!55іІїзеагамуусагзпІгазулаумддаіміи55дасддаКмааіКекмиааІКеКиааІКекмаа!Ке

Переважний полінуклеотид, що кодує такий поліпептид, характеризується наступною послідовністю (5ЕО ІЮО МО:16): саддсідюдідасісаддадсснсасідассдідіссссаддсддаасідідасссдасаїдсадаїссадсасаддсдсадщда ссасаїсіаасіасдссаайдддіясадсадаадссаддасаддсассааддддссідаїсдддддаіасааасаааадддсіссса ассссідсасдайнсіддаадісідсюдддсоддаааддссодсісідасіанассдддасасаддассдаддасдаадссданасіаняої дсісідюдіаіадсаафсідддаонсоаддададдсасаааасідасідідсідааддаддідаассддсддсоддадасдаддаоса дсюддіадсадіссддддседадсідаадааасссддадсіссдідаадащісндсааадссадіддсіасасснсасадасіасіаїаї даадідддісаддасаддсіссаддасадддасіддааі(ддаісодсдаїаісанссиссаасдддассасцісіасааіїсадаадащтшаа аддсаддаюдасіанНассоіддасаааїсаасаадсасідснагадаоадсідадсісссідсдсісідаадаїасадссоаютасіаноїд сісддісасассідсідададссадсідашоснандаддасаддоасасссіддідасадщіснссодадаадіяддсдадодаааацді дассдсасідааддадааауноасідстдааададааддісдссдсаснааддааааддісдсадсссдааадад

The third polypeptide chain of this version 1 of SAKT-A m/s will contain the CH2 and CH3 domains of the Es dO domain. A preferred polypeptide consists of peptide 1 (5EO IU MO:55) and domains CH2 and

The SNZ of the Es domain (5EO IYUO MO:11) and is characterized by the sequence 5EO IYU MO:54:

OKTNTSORRSORAREDASOIRBSUNBI ERRKRKOTI MIZVTREUTSUUMOMZNEOREUKEMU UMRYAMEM

NMAKTKRAEEEOUMZTUUVUUBMI TM NOSUMI MEKEUKSKUZMKAI RARIEKTIZKAKSORAEEROMMTI

RROVEEMTKMOMU5I BSAUKaeURZOIAMEM EZMSOREMMUKTTRRMI O5OrOazERI UZKITMOKZAMU

OOSMUMEZSSZUMMNEAII NMAUTOKOII 5I 5YEAR

Переважний полінуклеотид, який кодує такий поліпептид, характеризується наступною послідовністю (5ЕО ІЮ МО:12): дасаааасісасасаїдсссассудідсссадсассюаадссдсддаддддассадісадіснеосісносссссаааасссааддас асссісаїдаїсісссддассссдададісасадсддаюддіддасдададссасдаадасссідаддісаадісаасіддіасодчдас дасдіддаддідсаіааі|дссаадасааадссдсдддаддадсадіасаасадсасоаїассдщдіддісадсдіссісассдіссідсас саддасіддсіааддсааддадіасаадідсааддісіссаасааадсссісссадсссссаїсдадаааассаїсіссададссаа аддасадссссдадаассасаддідіасасссідсссссаїсссдддаддададассаадаассаддісадссідчадн дсдсадіса аадаснсіаїсссадсдасаїсдссудоаадодаоадададсаадддсадссддадаасаасіасаадассасдссісссоюдсіддасі ссдасддсісснсіїссісдісадсаадсісассдіддасаададсаддддсадсаддддаасдісіїсісаюдсіссдадаїдсащда9да сісідсасаассдсіасасдсадаададссісісссідісіссодддіааа

O. Construction version 2 OAKT-A m/gs

As a second example of such a diatyl YUAKT-A m/s, according to the present invention, a three-chain diatyl, "version 2 diatyl POAKT-A m/Es" is provided (figure 3B).

The first polypeptide of this construct version 2 OAKT-A m/Es contains, in the direction from the M-end to the C-end, the M-end, a peptide linker (peptide 1), a polypeptide that contains the CH2 and CH3 domains of the Es domain, connected (using linker 4) with the Mi domain of a monoclonal antibody capable of binding to CO123 (Mi soigz), an intermediate linker peptide (linker 1), domain. MN of a monoclonal antibody capable of binding to POPs (MNeoz), linker 2, K-helical domain and

C-end.

The preferred polypeptide, which contains the CH2 and CH3 domains of the Es domain, is characterized by the following sequence (5EO IU MO:37):

AREAASOIaRBZURBI ERRKRKOTI MIZVTREUTSMUMOUZNEOREUKEMU UMIMEMMMNMAKTKRAEEE 0) OMMOTUAUM5MI TM NOBUUL ME KEUKSKKUZMKAI RARIEKTIZKAKIORAEROMMUTI RROVEEMTKM

OMBI MS UKOEMRZOIAMEZHM EZMSOREMMUKTTRRMI YUOBrazERI ZK TMOKZAUMOSMUEZOSBM

MNEAIII NMNUTOK5BI ZI ROK "Linker 4" will preferably contain the amino acid sequence (ZEO IU MO:57): AR555.

The predominant "linker 4" is characterized by the sequence (ZEO IU MO:33): AROZZRME. Thus, the first polypeptide of this construct version 20AKT-A m/gs consists of the following:

ZEO IU MO:55 - BEO IU MO:37 - BEO I MO:3З3 - BEO IU MO:25 - BEO 10 MO:29 - BEO IU MO:22 -

ZEO IU MO:30 - BEO IU MO:35. The polypeptide characterized by this sequence is (ZEO IO MO:17):

OKTNTSORRSORAREDASOIRBSUNBI ERRKRKOTI MIZVTREUTSUUMOMZNEOREUKEMU UMRYAMEM

NMAKTKRAEEEOUMZTUVUUMI TM NOM MSKEUKSKUZMKAI RARIEKTIZKAKOORAEROMMTI

RROVEEMTKMOMU5BI MUSI UKEEMRZOIAMEM EZMSOREMMUKTTRRMI OBORAZERI KI TUOKVAMU

OOOSMMEZSSZUMMNEAII NMNUTOKOBI 5I 5ROKARZZZRMEVEMMTO5ROBI AM5I ENUTMZOSKZO

Z MBamMOKkmMmmI TUMOOKRYaORRKI PU AZTAEZaMURyerovezaZazatovTtI TZI OAEOMAMUUUS

OMOU5URUTRSOSTKI EIXOSOSZSSSIassemoi MEZO SOI MOROSBI V BSAABIETEZTMAMMUUV

OSAROKYa! EMUMAVIVZKUMMMATUMAOZUKOVETIZAVOBKM5I MI OMM5I KTEOTAUUUSUVNOIAME aM5UUUZUMUBgAUUsOstTI UTUZZaIasatsakmmMAAI KEKMUMAAI KEKUAAI KEKMAAI KE

Переважний полінуклеотид, що кодує такий поліпептид, характеризується наступною послідовністю (5ЕО ІЮ МО:18): дасаааасісасасаїдсссассудідсссадсассюаадссдсддаддддассадісадіснеосісносссссаааасссааддас асссісаїдаїсісссддассссдададісасадсуадаюддіддасоададссасдаадасссюададісааднсаасіддіасдадчдас дасдіддаддідсаіааі|дссаадасааадссдсдддаддадсадіасаасадсасоаїассдщдіддісадсдіссісассдіссідсас саддасіддсіааддсааддадіасаадідсааддісіссаасааадсссісссадсссссаїсдадаааассаїсіссададссаа аддасадссссдадаассасаддідіасасссідсссссаїсссдддаддадаїдассаадаассаддісадссідіадідссіддіса аадаснсіаїсссадсдасаїсдссудоаадодаоадададсаадддсадссддадаасаасіасаадассасдссісссоюдсіддасі ссдасддсісснсіїссісіасадсаадсісассдіддасаададсаддддсадсаддддаасдісіїсісаюдсіссдадаїдсащда9да сісідсасаассасіасасдсадаададссісісссідісіссдддіааадссссНссадсіссссіаюдаадасіїсдідаїдасасацдіс іссідаіадісіддссдідадісюддададсодддідасіащдісндсаададсісссадісасідсідаасадсуддаааїсадааааасіа ісдассіддіассадсадаадссаддссадсссссіааасідсідаісіандддаснссассадд дааісіддсдідсссдасадайсад сддсадсддасадсддсасадаціасссідасаацісіадісідсаддссудаддасдіддсідідіасіандісадааюдайнасадсіаїс бо ссіасасісддссаддддассаадсіддааанаааддадоасддаїссддсддасоддадоасдаддаідсадсюдіддадісюдадда даснддаіссадссіддадудісссюдадасісіссіддсадссісіддансассисадсасаїасдсіадаанддаїссдссаддсісс аддааадддасіддадчіадонадааддаїсаддіссаадіасаасаанНаїдсаассіасіаїдссдасісюідааддагадайсассаї сісаададаїдансааадаасісасідіаіїсісааадаасадссдаааассдаддасасддссдідіанасдідідадасасоддіа асісддсаанйснасущісндашндснанддаддасаддддасасіддідасідщіснссаяададчадіддсддіддаааадіддссус асідааддадаааандсдс і ндааададааддісдссдсасіааддааааддісдсадсссідааадад

The second polypeptide chain of this construct version 2 of OAKT-A m/s contains, in the direction from the M-terminus to the C-terminus, the Mi domain of a monoclonal antibody capable of binding to SOZ (Mi soz), an intermediate linker peptide (linker 1) and the MH domain of a monoclonal antibody capable of binding to ХХО123 (МНео12з). The specified part of the molecule is connected (using linker 2) by 3 E-helical domains. Thus, the third polypeptide of this construct version 2 of OAKT-A m/s consists of the following: BZEO IU MO:21 - BEO 10 MO:29 - BEO IU MO:26 - BEO IU MO:30 -

ZEO IU MO:34. A polypeptide characterized by such a sequence is (ZEO IU

MO:1), and is preferably encoded by a polynucleotide characterized by the sequence ZEO IU

MO:2.

The third polypeptide chain will contain the CH2 and CH3 domains of the Es domain. The preferred polypeptide consists of peptide 1 (ZEO IO MO:55) and the CH2 and CZ domains of the Es domain (ZEO IO

MO:11) and is characterized by the sequence ZEO IU MO:54.

To evaluate the activity of the above-mentioned CO123 x POP bispecific diatoms (ACT-A, OAVT-A m/AvO, RAVT-A m/Rs, OAVT-B) a control bispecific diatom (control SACT) was obtained.

Control SAVT is able to simultaneously communicate with RITS and SOZ. Its two polypeptide chains are characterized by the following corresponding sequences:

Chain 1 of the control OAKT (5EO IU MO:19):

OMUMMTOTREBI RUB aiSOABIZSAZBZOBBI UNZMOaMT MI V/M OKREOZBRKMIUKUZMAEBamMR ragEzazazatorETI KIZAMEAEOY aUMESZOZTNURUTEROSSIatTtKI EIKaOSOZa,vaASSEMOYI MEZOvSS

HMORISBI VI BSAAZSOETEMT MAMMUUVOAROKATI EUUMAVIVZKUMMMATUMAOBUKOVETIZAOO

ZKM5I M OMM5I KTEOTAUU US UVNMEAMZUUBMEAUMmaOa, tI UtTUZOIasY, sSEMAAGEKEMA

AI EKEMAAI EKEMAAI EK

Chain 2 of the control OAKT (5EO IU MO:20):

OAUMTOERBI TU5ROSTMUTI TAZ TAM TT ZMUAMUMUMOOKRIOOSARVIG IS TMKAARMUTRAN

From EZOaBI I! ЧСаКААЙ TITIAOАЕОЕАРУМСАЙ M UZMI UUgssa7tki tm svssvsazasasEMUKI RETOSOYI.

MOREIVvARMKI BSMAZbaYETEZOMMU MMUUUKOZREKSOG EM MAO AMKRUMMETUUZOBUKOaVETIZNAOO zZKkZZUM OMMM AMEOoMaimUstazUuamruUmsoateUtuvZaasatsiakmMAAI KEKMAAI KEKMA

AI KEKMAAI KE

IM. Pharmaceutical compositions

The compositions of the present invention comprise bulk drug compositions suitable for the manufacture of pharmaceutical compositions (eg, crude or non-sterile compositions) and pharmaceutical compositions (ie, compositions suitable for administration to a subject or patient) that can be used in the preparation standard dosage forms. Such compositions contain a prophylactically or therapeutically effective amount characterized by an optimized sequence of CO123 x POPs of bispecific diethyls according to the present invention or a combination of such agents and a pharmaceutically acceptable carrier. Compositions according to the present invention preferably contain a prophylactically or therapeutically effective amount characterized by an optimized sequence of CO123 x POPs of the bispecific diethyl according to the present invention and a pharmaceutically acceptable carrier.

The present invention also provides pharmaceutical compositions containing bispecific diethyls characterized by an optimized CO123 x POP sequence according to the present invention and a second therapeutic antibody (e.g., a tumor-specific monoclonal antibody) that is specific for a particular malignant tumor antigen, and a pharmaceutically acceptable carrier .

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in

US Pharmacopoeia or other generally accepted Pharmacopoeia for use in animals and more specifically in humans. The term "carrier" refers to the diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete), excipient, or inert vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils , including oils of mineral, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred vehicle if the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose solutions and solutions glycerol can also be used as liquid carriers, in particular for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate , talc, sodium chloride, skimmed milk powder, glycerin, propylene, glycol, water, ethanol and the like.

The composition, if necessary, may also contain small amounts of wetting agents or emulsifiers, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, dragees, capsules, powders, slow-release compounds, and the like.

Typically, the ingredients of the compositions according to the present invention are supplied either individually or mixed together in a standard dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet, with an indication of the amount of active agent. If the composition is intended for administration by infusion, it may be sold with an infusion bottle containing sterile water or pharmaceutical grade saline. If the composition is intended for administration by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

Compositions according to the present invention can be included in the composition in the form of neutral or salt forms. Pharmaceutically acceptable salts include, without limitation, salts formed by anions such as anions derived from hydrochloric, orthophosphoric, acetic, oxalic, tartaric acids, etc., salts thereof formed by cations such as cations derived from sodium , potassium, ammonium, calcium, iron hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The present invention also provides a pharmaceutical package or kit containing one or more containers filled with bispecific diethyls characterized by an optimized sequence of СО123 x SOYUZ, according to the present invention alone or in such a pharmaceutically acceptable carrier. In addition, one or more other prophylactic or therapeutic agents suitable for the treatment of the disease may also be included in the pharmaceutical package or kit. The present invention also provides a pharmaceutical package or kit containing one or more containers filled with one or more ingredients of the pharmaceutical compositions according to the present invention.

Optionally, such container(s) may be accompanied by a notice in the form prescribed by the governmental authority regulating the manufacture, use or sale of pharmaceuticals or biological products, and said notice reflects the approval by the relevant authority of the manufacture, use or sale for administration to humans.

The present invention provides kits that can be used in the methods described above. The kit may contain bispecific diethyls characterized by an optimized sequence of CO123 x POPs according to the present invention. The kit may additionally contain one or more other prophylactic and/or therapeutic agents suitable for the treatment of a malignant tumor in one or more containers; and/or the kit may additionally contain one or more cytotoxic antibodies that bind to one or more malignant tumor antigens associated with a malignant tumor. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.

M. Methods of introduction

The compositions of the present invention may be administered to treat, prevent, and reduce the intensity of one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of a fusion protein or conjugate molecule of the present invention or a pharmaceutical a composition containing a fusion protein or a conjugated molecule according to the present invention. According to a preferred aspect, such compositions are substantially purified (ie, substantially free of substances that limit its effect or cause undesirable side effects). According to a specific embodiment, the subject is an animal, preferably a non-primate mammal, a mammal (eg, a cow, horse, cat, dog, rodent, etc.) or a primate (eg, a monkey, such as, Javan macaque, humans, etc.). According to the preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer the compositions of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing an antibody or fusion protein, bo receptor-mediated endocytosis (see, e.g., MU et al. (1987) "Keseriog-Mediaivd p Mio Sepe

Tkapetogptaiyop Wu A bBoishrie OMA Sattieg Zuvziet, " U. Vioi. Spet. 262:4429-4432), a nucleic acid construct in the form of a part of a retroviral or other vector, etc.

Methods of administration of a molecule according to the present invention include, but are not limited to, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal administration (eg, intranasal and oral routes). According to a specific variant of implementation, bispecific diethyls characterized by an optimized sequence of CO123 x POPs according to the present invention are administered intramuscularly, intravenously, or subcutaneously. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or skin-mucosal membranes (eg, oral mucosa, rectal and intestinal mucosa, etc.) It can be administered together with other biologically active agents. Introduction can be systemic or local. In addition, pulmonary administration can also be used, for example, using an inhaler or nebulizer and a compound with an aerosol agent. See, e.g., U.S. Pat

MoMo 6019968; 5985320; 5985309; 5934272; 5874064; 5855913; 5290540 and 4880078 and international patent publications according to PCT MoMo UMO 92/19244; UMO 97/32572; MO 97/44013; UMO 98/31346 and

UMO 99/66903, each of which is fully incorporated herein by reference.

According to the present invention, it is also provided that bispecific diethyls characterized by an optimized sequence of CO123 x POPs according to the present invention are packaged in a hermetically sealed container, such as an ampoule or a sachet with an indication of the number of molecules. According to one embodiment, bispecific diethyls characterized by an optimized sequence of CO123 x POPs according to the present invention are supplied in the form of a dry sterilized lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be diluted, for example, with water or saline to a suitable concentration for administration sub to the object Bispecific diethyls characterized by an optimized sequence of СО123 x POPs according to the present invention are preferably supplied in the form of a dry sterile lyophilized powder in a hermetically sealed container in a standard dosage amounting to at least 5 μg, more preferably at least 10 μg, at least 15 μg, at least 25 μg, at least 50 mcg, at least 100 mcg or

Of at least 200 mcg.

Lyophilized, bispecific diethyls characterized by an optimized sequence

СсО0123 x СО3 according to the present invention must be stored at a temperature of 2 to 8"C in their original container and the molecules must be administered no later than 12 hours, preferably no later than 6 hours, no later than 5 hours, no later than in 3 hours or no later than 1 hour after their dilution According to an alternative embodiment, bispecific diabodies characterized by an optimized sequence

СОр123 x POPs according to the present invention are supplied in liquid form in a hermetically sealed container with an indication of the amount and concentration of the molecule, fusion protein or conjugated molecule. A liquid form characterized by an optimized sequence of СО123 x СОЮЗ bispecific diethyls according to the present invention is preferably supplied in a hermetically sealed container, in which the molecules are at a concentration of at least 1 μg/ml, more preferably at least 2.5 μg/ml, at least 5 µg/ml, at least 10 µg/ml, at least 50 µg/ml or at least 100 µg/ml.

The amount of a composition according to the present invention that will be effective in treating, preventing or reducing the intensity of one or more symptoms associated with the disorder can be determined using standard clinical techniques. The exact dosage used in the composition will also depend on the route of administration and the severity of the condition, and should be determined according to the judgment of the physician and all relevant circumstances of the patient. Effective doses can be extrapolated from dose-response curves from IP test systems or animal model test systems.

For the bispecific diethyls provided by this invention, characterized by an optimized sequence of CO123 x POPs, the dosage administered to the patient is preferably determined based on the body weight (kg) of the recipient subject. The dosage administered is typically from at least about 0.3 ng/kg per day to about 0.9 ng/kg per day, from at least about 1 ng/kg per day to about 3 ng/kg per day, from at least about 3 ng/kg per day to about 9 ng/kg per day, from at least about 10 ng/kg per day to about 30 ng/kg per day, from at least about 30 ng/kg per day to about 90 ng/kg per day, from at least about 100 ng/kg per day to about 300 ng/kg per day, from at least about 200 ng/kg per day to about 600 ng/kg per day, from at least 60 to about 300 ng/kg per day to about 900 ng/kg per day, from at least about 400 ng/kg per day to about 800 ng/kg per day, from at least about 500 ng/kg per day to about 1000 ng/kg per day, from at least about 600 ng/ kg per day to about 1000 ng/kg per day, from at least about 700 ng/kg per day to about 1000 ng/kg per day, from at least about 800 ng/kg per day to about 100 0 ng/kg per day, from at least about 900 ng/kg per day to about 1000 ng/kg per day, or at least about 1000 ng/kg per day.

According to another embodiment, the patient is administered a treatment regimen that provides one or more doses of such a prophylactically or therapeutically effective amount, characterized by an optimized sequence of СО123 x СОЗ3 bispecific diethyls provided according to the present invention, and the treatment regimen is administered for 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. According to certain variants of implementation, the treatment scheme involves the periodic administration of doses of a prophylactically or therapeutically effective amount, characterized by an optimized sequence of СО123 x СОЗ3 bispecific diethyls provided according to the present invention (for example, administration of a dose on day 1, day 2, day C and day 4 of this week and lack of administration of prophylactically or therapeutically effective doses characterized by an optimized sequence of CO123 x POPs of bispecific diethyls provided according to the present invention on day 5, day 6 and day 7 of the same week). As a rule, 1, 2, 3, 4, 5 or more courses of treatment are carried out. Each course can follow the same scheme or different schemes.

According to another embodiment, the administered dose is increased over the first quarter, first half, or first two-thirds or three-quarters of the regimen(s) (eg, the first, second, or third treatment regimen of 4 courses) until the daily prophylactic dose is reached. or a therapeutically effective amount characterized by an optimized sequence of СО123 x

POPs of bispecific diethyls.

Table 1 presents 5 examples of the different dosage regimens described above for a typical course of treatment.

Table 1

Scheme | Day | Diatyl dosage (ng diatyl per kg body weight of subjects per day)

Dosage and frequency of administration, characterized by an optimized sequence of CO123 x

CO3Z of bispecific diethyls according to the present invention can be reduced or changed by increasing absorption and penetration, characterized by an optimized sequence of CO123 x POPs of bispecific diethyls in the tissue by means of such modifications as, for example, lipidization.

Dosages administered to the patient characterized by an optimized sequence

СО0123 x POPs of bispecific diethyls according to the present invention can be calculated for use as monotherapy. Alternatively, bispecific diethyls characterized by an optimized sequence of CO123 x POPs according to the present invention are used in combination with other therapeutic compositions, and the dosage administered to the patient is less than when using these molecules as monotherapy.

Pharmaceutical compositions according to the present invention can be administered locally to the area required for treatment; this can be achieved, for example, without limitation by means of a local infusion, injection or by means of an implant, and said implant is made of a porous, non-porous or gelatinous material, including membranes such as biaiawiis membranes or fibers. When introducing a molecule according to this invention, it is mainly necessary to pay attention to the use of materials that do not absorb the molecule.

Compositions according to the present invention can be delivered in a vesicle, in particular a liposome (see Hapdeg (1990) "Mem Meijoaz OYI Ogid Oeiimegu," Zsiepse 249:1527-1533); Tgeai yei ai., v

Prosotez ip eate TPegaru oi Ipyesiioiv Rizease apa Sapsetg, I ore2-Vegevivip apa Riaeg (ed5.), I ivv,

Mem Mogk, pp. 353-365 (1989); I ore2-Vegezivip, iria., year Z 17-327; see mainly ibid.).

The compositions of the present invention can be delivered in a controlled or sustained release system. Any technique known to a person skilled in the art can be used to prepare sustained release compounds containing one or more bispecific diethyls characterized by an optimized sequence of СО123x

POPs according to the present invention. See, for example, US Pat. No. 4,526,938; international patent publications according to PCT MoMo UUO 91/05548; UMO 96/20698; Mipa ey aiYi. (1996) "Ipgashtogai!

"

Vadioyegaru 5 Opsoiodu 39:179-189, 5opd eyi ai. (1995) "Apiirodu Mediaiied I pa Tagodeiipud OI!

Gopa-Siksciaipud Etyiviope, " ROA Shoshgpa! oi Rpaptaseshiisa! 5siepse is Tesppoiodu 50:372-397;

Siyeek ei ai. (1997) "Viodedgadabrie Roiutegis Saptietv Rog A BERSE Apiirodu Rog Sagaiomazsshag

Arriisayop, " Ro. Ipg!. Butr. SopigoYi. Nei. Vioasi. Maieg. 24:853-854; iat ei ai. (1997) "Misgtoepsarztsiaiop OYI Vesotbipapi Nitapiled Moposiopa! Apiibodu Eog I wasa! Oyeiimegu, " Rgos. ii! Zutr. Sopigo! Nei. Vioasi. Maiieeg. 24:759-760, each of which is fully incorporated herein by reference. According to one embodiment, the pump can be used in a controlled release system (See I apdeg, earlier; beyop, (1987) "Itriapiabie Ritrz", " SVO Si. Vem. Viotea. Epd. 14:201-240; Wyspmaia ei ai. (1980) "Hopd-Tegt,

Sopiiipysiv Ipigamepoi5 Neraiip Aadtipivigayop Vu Ap Itriapiabiie Iptivsiop Rytr Ip Atriiayugu

Raiepiv M/Yi Vesshitepi Mepoib5 Tygotrovziv, "Zygdegu 88:507-516; and Zatsdek ei ai. (1989) "A

Rgyeiitipagu Ttia! OII TNe Rgodgattabie Itriapiarie Medisayop Zuziet Rog Ipstsiip Oeiiimegu, " M. Epadi.

U. Mea. 321:574-579). In another embodiment, polymeric materials can be used to achieve controlled release of molecules (see, e.g.,

Meadis! Arriisaiop5 oi Sopigo!Iey Veiyease, I apdeg apa M/ize (edz.), SVO Rgez., Vosa Vip, Riogida (1974); SopitoPeia Abomination Vioamayarishu, Abomination Rgodisi Oezidp apa Repogptapse, Ztoiep apa Vai! (ed.), MPeu, Mem Mogk (1984); And emu her a)y. (1985).

Maimez Wu and wasp! SopigoPei-NeiIease Oirpozrpopaiie, " Zsiepse 228:190-192; VBippa eyi ai. (1989) "Sopigoiei Veivaze OI Juoratipe got A Roiutetis Vgaip Itriapi: ip Mimo Spagasivgigayop, " App.

Mesztsgoi. 25:351-356; Nomaga ei ai. (1989).

Metogu Oeiisiyiv, "U. Meshgozygu. 7(1):105-112); US patents MoMeo 5679377; 5916597; 5912015; 5989463; 5128326; international patent publications according to PCT MoMo UMO 99/15154 and UMO 99/20253).

Examples of polymers used in sustained release compounds include, but are not limited to, 2-hydroxyethyl methacrylate polymer, methyl methacrylate polymer, acrylic acid polymer, ethylene vinyl acetate copolymer, methacrylic acid polymer, polyglycolides (PIC), polyanhydrides, M-vinylpyrrolidone polymer, vinyl alcohol, polyacrylamide, polyethylene glycol, polylactides (Ri A), copolymer of lactides and glycolides (RI BA) and polyorthoesters.

A controlled-release system can be placed close to the therapeutic target (e.g., the lungs) so that only a fraction of the systemic dose is required (see, e.g., Soodzop, in Meaisa! Arriisaiop5 oi SopigoPei Keiiaze, formerly, my. 2, pp. 115- 138 (1984)). Compositions suitable as controlled release implants can be used according to Juipp et al. (see US patent Me 5945155). This specific method is based on the therapeutic effect of the controlled release of the bioactive material from the polymer system. Implantation usually occurs anywhere in the patient's body that needs therapeutic treatment. A non-polymeric delayed delivery system can be used, whereby a non-polymeric implant in the subject's body is used as a drug delivery system. When implanted in the body, the organic solvent of the implant will disperse, disperse, or leach from the composition into the surrounding tissue fluid, and the non-polymeric material will gradually coagulate or precipitate to form a solid, microporous matrix (see US Pat. No. 5,888,533).

Controlled-release systems are discussed in the review I apdeg (1990, "Mem Meshod

OG Ota Owiimegu, " Zsiepse 249:1527-1533). Any technique known to one skilled in the art can be used to prepare sustained release compounds containing one or more therapeutic agents of the present invention. See, e.g., patent

USA Me4526938; international patent publications of MoMo UMO 91/05548 and UMO 96/20698; Mipo ei ai. (1996) "Iptashtoga! Radioittipoiyegarnu OG A Nytap SoYop Sapseg Hepoagai Ovipud A 5iviaipead-

Wiese C, "Wadionegaru 5 Opsiodu 39:179-189, 5opd eei a!. (1995) "Apiirodu Mediaied I pd

Tagdeipud OY Gopd- Sigsshaipd Ethiiiviop5, " ROA hilddaii oi Rnaptaseciisa! Zsiepse e TesppoYodu 50:372- 397; SieeK ei ai. (1997) "Viodedgadabrie Roiutegs Sagpiet5 Boh A ryu Apiirodu Rog

Sagaiomazsshiag Arriisayop, " Rgo. IpgI. 5utr. Sopigoi. Vei. Vioasi. Maieg. 24:853- 854; ii at ei ai. (1997) "Mistoepsarzshtianiop OYI Vesotbipapi Nitapiged Moposiopa! Apiibodu Horn and wasp! Oeviyimegu, "

Rgos. IPgI. 5 a.m. Sopigoi! Wow! Vioasi. Maieg. 24:759-760, each of which is incorporated herein by reference in its entirety.

If the composition of the present invention is a nucleic acid encoding a bispecific dibody characterized by an optimized CO123 x POP sequence according to the present invention, the nucleic acid can be administered i.p. to stimulate expression of the bispecific dibody encoded by it characterized by an optimized CO123 x POP sequence by constructing it as part of an appropriate nucleic acid expression vector and introducing it so that it becomes intracellular, for example by using a retroviral vector (see US Patent No. 4,980,286), or by direct injection, or by using microparticle bombardment (e.g. , gene cannon; Vioiivis,

Biropi), or coating with lipids, or cell surface receptors, or by means of transfection, or by introducing It, linked to a homeobox-like peptide, which is known to penetrate into the nucleus (see, for example, chChoioi ei aI. (1991 ) "Apieepparedia Noteoroch Reriide Nedsiaies Meigkai

Mogrpodepev, "Rhos. Mai!. Asaid. bsi. (0O.5.A.) 88:1864-1868) etc. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression by homologous recombination

Treatment of a subject with a therapeutically or prophylactically effective amount characterized by an optimized sequence of CO123 x POP bispecific diethyls according to the present invention may involve a single treatment or may preferably include a series of treatments. According to a preferred example, the subject receives treatment with characterized by an optimized sequence of CO123 x POP bispecific diethyls according to the present invention once a week for about 1-10 weeks, preferably for 2-8 weeks, more preferably for about 3-7 weeks and even more preferably for about 4, 5 or 6 weeks. Pharmaceutical compositions according to the present invention can be administered once daily, twice daily, or three times daily. Alternatively, the pharmaceutical compositions can be administered once a week, twice a week, once every two weeks, once a month, every six weeks, once every two months, twice a year, or once a year. In addition, it should be understood that the effective dosage of molecules used for treatment may increase or decrease over the course of a particular treatment.

E. Application of compositions according to this invention

Bispecific diabodies characterized by an optimized sequence of CO123 x POPs according to the present invention are characterized by the ability to provide treatment for any disease or condition associated with the expression of CO123 or characterized by the expression

СО0123. Thus, without limitation, such molecules may be used in the diagnosis or treatment of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), including CML blast crisis and CML-associated Abelson oncogene (Vsg-AVI translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-CLL), chronic lymphocytic leukemia (CLL), including CLL with Richter's syndrome or Richter's transformation, hairy cell leukemia (CLL), neoplasm from blast plasmacytoid dendritic cells (BPDC), non-Hodgkin's lymphomas (NHL), including mantle cell lymphoma (MCL) and small cell lymphocytic lymphoma (MLL), lymphomas

Hodgkin, systemic mastocytosis and Burkitt's lymphoma (see example 2); autoimmune lupus (SLE), allergies, bronchial asthma and rheumatoid arthritis. In addition, the bispecific diethyls according to the present invention can be used in the manufacture of drugs for the treatment of the conditions described above.

Having presented the description of the present invention in general, it will become more clear by reference to the following examples, which are presented by way of illustration and are not intended to limit the present invention unless otherwise indicated.

Example 1

Construction of CO123 x POP bispecific diethyl and control protein

Table 2 presents a list of bispecific diethyls that were expressed and purified.

A bispecific diabody characterized by an optimized sequence of СО123 x POP (OAKT-

A) and a bispecific diabody characterized by an unoptimized sequence of СО123 x POPs (САВТ-В) are capable of simultaneously binding to СО123 and POP3. The control bispecific diabody (control SACT) is capable of simultaneously binding to RITS and SOZ. Bispecific diethyls are heterodimers or heterotrimers with the listed amino acid sequences.

Methods of formation of bispecific diethyls are presented in international patent publications

MoMo UMO 2006/113665, UMO 2008/157379, UMO 2010/080538, UMO 2012/018687, UMO 2012/162068 and

MO 2012/162067. (510)

Table 2 of the tower KLKTTKYA. at. . : and Nucleic acids

Bispecific diatyl sequences y and y . coding sequences of the polypeptide chain

Bispecific diabody characterized by optimized | 5EO AND MO ZEO AND MOSg2 sequence СО123 x POP (BACT-A) ZEO IU MO:Z ZEO AND MO (binds with POP on epitope 1)

Bispecific diabody characterized by non-optimized | 5EO IU MO:5 ZEO IU MO:6 sequence CO123 x POP (BACT-B) ZEO IU MO:7 ZEO I MO:8 (binds with POP on epitope 2)

A bispecific diabody characterized by an optimized sequence of CO123 x POPs containing

AB binding domain (AKTA 5rO Ir MO EO Y MOM (binds with POPs on epitope 1), ZEO o MOZ ZE o MO contains an albumin-binding domain (ABO) to increase the half-life of ip mimo

Version 1, characterized by an optimized sequence of СО123 x

POPs of bispecific diethyl, containing | 5EO I MO:54 ZEO Ir MOl2 domain Es da (version 1 OAVT-A m/Es) ZEO I MOZ ZEO IU MO:14 (binds with POPs on epitope 1), ZEO IU MO5 ZEO I MO:16 contains a domain bs to increase the half-life period of ip mimo

Version 2, characterized by an optimized sequence of СО123 x

POPs of bispecific diethyl, containing | 5EO I MO:54 ZEO Ir MOl2 Es Ids domain (version 2 OAKT-A m/Es) ZEO IU MO:17 ZEO I MO:18 (binds with POPs on epitope 1), ZEO I MO ZEO I MOSg2 contains a domain bs to increase the half-life period of ip mimo

Control bispecific diabody (control SACT) , (binds to POPs on epitope 1) another 5 MO oo (binds to an irrelevant target - I

ITS

Example 2

Antibody labeling of target cells for quantitative RASZ (OBAS5)

A total of 105 target cells were collected from the culture, resuspended in 10 95 serum

Human AB in buffer for EAS5Z (RVZ-1 95 ВБ5АчО, 1 95 MaA?liade) and incubated for 5 min. to block Es receptors. Antibody labeling of microspheres with different abilities to bind to the antibody (Schapisht" MM Bitriu SeiPshamkv (0552), Vapoz I Arogayogiev5, Ips., Rizpeg5, IM) and target cells were labeled using RE antibodies to CO123 (VO Viozsiepse5) according to the instructions manufacturer. Briefly, one drop of each O5C microsphere was added to a 5 ml polypropylene tube and PE-labeled antibody to CO123 was added at a concentration of 1 μg/ml to both target cells and microspheres. The tubes were incubated in the dark for 30 minutes at 4 °C. Cells and microspheres were washed by adding 2 ml of buffer for GASZ and centrifugation at 2500 x b for 5 minutes. One drop of the population of empty microspheres was added after washing.

The microspheres were analyzed first on a flow cytometer to set the instrument settings specific to the analysis (RMT voltages and compensation). Using the same device settings, the geometric mean of fluorescence values of microspheres and target cells was recorded. A standard curve of antibody binding sites on populations of microspheres was obtained from the geometric mean of fluorescence values of populations of microspheres. Binding sites of 3 antibodies on target cells were calculated on the basis of the geometric mean of the fluorescence values of target cells using the standard curve obtained for microspheres in the OtsiskSai spreadsheet (Vapoz I Arogayogiev5).

To determine suitable cell lines - targets for evaluation of СО123 x СО3 bispecific diethyls, levels of surface expression of СО123 on lines - targets of Kaziti-3 (GML),

Moit13 (AML), TNR-1 (AML), TE-1 (erythroleukemia) and K54-11 (ALL) were evaluated using the quantitative analysis of EASZ (OBAS5). The absolute number of binding sites with the CO123 antibody on the cell surface was calculated using the OBAS5 kit. As shown in Table 3, the absolute number of binding sites with the CO123 antibody on the cell was of the order of Kaziti-

Z (high) » Moit!3 (medium) » TNR-1 (medium) » TE-1 (moderately low) » K54-11 (low). The three cell lines with the highest expression levels were AML cell lines: Kazyti-3, MY! M1Z and TNR-1. Those unrelated to AML cell lines: TE-1 and K54-11, were characterized by moderately low/low expression of CO123, respectively.

Table C

Cell line - Surface expression of ХО123 targets (antibody binding sites)

Example C

Analysis of STI cytotoxicity. (analysis of IOH release)

Attached tumor cells - targets were detached with the help of 0.25 95 solution of trypsin-

EOTA and collected by centrifugation at 1000 rpm. within 5 min. The suspension of target cell lines was collected from the culture, washed with the medium for analysis.

Cell concentration and viability were measured by trypan blue extrusion using a VesKtap SoshKeg Mi-SeiY cell counter. Target cells were diluted to 4x105 cells/ml in medium for analysis. 50 μl of the diluted cell suspension was added to a 96-well O-bottom plate treated with cell culture (Mo by cat. VO RaYsop 353077).

Three sets of controls were set up to measure maximal release from the target (ME), antibody-independent T-cell cytotoxicity (AICT) and spontaneous release from the target cell (TC) as follows: 1) ME: 200 μl of assay medium without SHO123 x POPs of bispecific diethyls and 50 μl of target cells; detergent was added at the end of the experiment to determine the maximum release of GI OH. 2) AISS: 50 μl medium for analysis without CO123 x POP bispecific diethyls, 50 μl

From target cells and 100 μl of T cells. 3) ZE: 150 μl of medium without CO123 x SOYUZ bispecific diethyls and 50 μl of target cells.

СсО0123 x СО3Z bispecific diethyls (ACT-A, OAVT-A m/ABO and BACT-B) and controls were first diluted to a concentration of 4 μg/ml and then serially diluted to a final concentration of 0.00004 ng /ml (ie 40 Ffmg/ml). 50 μl of dilutions were added to a plate containing 50 μl of target cells/well.

Purified T cells were washed once with assay medium and resuspended in assay medium at a T cell density of 2 x 109 cells/ml. 2 x 105 T cells in 100 μl were added to each well to a final ratio of effector to target cell (E:T) of 10:1. Tablets were incubated for about 18 hours. at 37 "С at 5 95 СО".

After incubation, 25 μl of 10x lysing solution (Rgoteda, MO under cat. C182A) or 1 mg/ml of digitonin was added to the maximum release control wells, mixed with a pipette 3 times, and the tablets were incubated for 10 min. until complete lysis of target cells. Tablets were centrifuged at 1200 rpm. for 5 minutes, and 50 μl of the supernatant was transferred from each well of the analytical tablet to a flat-bottomed tablet for EIG IZA, and 50 μl of the SON substrate solution (Rgoteda, Mo. under cat. 51780) was added to each well. Tablets were incubated for 10-20 min at room temperature (RT) in the dark, then 50 μl of stop solution was added. The optical density (0.0.) was measured at 490 nm within 1 hour. on a tablet-

From riders Misog2 Myschiabei (Regkip EIPteg, Mo under cat. 420-014). Cytotoxicity in 96 was calculated as described below, and dose-response curves were obtained using SgarpRai RKIBM5F software)

The lysis of specific cells was calculated from the data 0.0. using the following formula:

Cytotoxicity (95) - 100 x (00 sample - 00 AISS) EAR MA-ООО 58)

Redirected cytolysis of target cell lines using different levels of surface CO123

СО0123 x POP bispecific diethyls showed a pronounced ability for redirected cytolysis with concentrations that should reach 50 95 of the maximum activity (EC505) in the sub-ng/ml range, regardless of the specificity of binding to the POP epitope (OAKT-A compared to OAKT -B) in cell lines - targets with a high level of expression of CO123, Kazity-Z (EC50O-0.01 ng/ml) (figure 4, panel 0), medium expression level of CO123, Moit!Z3 (EC50O-0O,18 ng /ml) and TNR-1 (EC5O-0.24 ng/ml) (Figure 4, panel C and E, respectively) and moderately low or low level of expression of CO123, TE-1 (EC50-0.46 ng/ml) and 54-11 (EC50O-0.5 ng/ml) (Figure 4, panels B and A, respectively). Similarly, CO123 x POP bispecific molecule-mediated redirected cytolysis was also observed with numerous cell lines - targets with T cells from different donors and no activity in the form of redirected cytolysis was observed in cell lines that do not express CO123. The results are summarized in Table 4.

Table 4

Cell line - Surface expression of EC50O, which Max. 95 cytolysis of CO123 targets (sites characterized by binding to an optimized antibody) sequence of CO123 x POP bispecific diethyls (ng/ml)

E:T-10: vali 7 / Ю7777171717957.......Х.Ш.|.ЮЮЮЮЮ.05.....БЙ 1 |. YuYMdlov village

If there is a need to reproduce the specified example, it should be understood that a specialist in this field of technology will be able, within reasonable and acceptable limits, to modify the protocol described above in a suitable way to reproduce the described results. Thus, it is not implied that the illustrated protocol must be followed in a precisely defined manner.

Example 4

T-cell activation during redirected cytolysis with the help of bispecific diethyls (OAVT-A, OANT-A m/ABO and SACT-A m/s) characterized by an optimized sequence

СОр123 x SOZ

З Bispecific dibodies characterized by the optimized sequence of СО123 x POPs showed a pronounced ability for redirected cytolysis regardless of the presence or absence of half-life extension technology (SACT-A compared to OAKT-A m/ABO compared to OAKT-A m/Es) in cell lines - targets with high expression of СО123, Casyti-3, and medium expression of СО123, TNR-1 (figure 5, panels A and B, respectively). In order to obtain characteristics of T-cell activation during the process of mediated, characterized by an optimized sequence of СО123 x POPs of bispecific diethyl redirected cytolysis,

T cells from the redirected cytolysis assays were stained for the T cell activation marker CO25 and analyzed with BAS5. As shown in Figure 5, panel OY, C025 was upregulated in C08 T cells in a dose-dependent manner, indicating that bispecific diethyls characterized by an optimized C0123 x C03 sequence induced T cell activation in the process of redirected cytolysis. In contrast, in the absence of target cells, activation of CO8 T cells was not observed (Figure 5, panel C), indicating that bispecific diacetyl characterized by an optimized sequence

СОр123 x POPs do not activate T-cells in the absence of target cells. Similarly, СО8 T-

cells were not activated when incubated with target cells and a control bispecific dialyte (control UAKT) (Figure 5, panel Y), indicating the need for cross-linking of T cells and target cells with bispecific dialytes characterized by the optimized sequence of СО123 x СОЗ3.

Example 5

Intracellular staining for granzyme B and perforin

Analysis to determine the intracellular contents of granzyme B and perforin in T cells

The STI was configured as described above. After about 18 hours. cells from the analytical tablet were stained with antibodies to CO4 and to CO8 by incubation for 30 minutes at 4 "C. After surface staining, the cells were incubated in 100 μl of buffer for fixation and permeabilization (VO Viobsiepse5) for 20 minutes at 4 "C. Cells were washed with permeabilization/washing buffer (VO Viobsiepsev) and incubated in 50 μl of a mixture of antibodies to granzyme B and perforin (prepared in 1X permeabilization/washing buffer) at 4 °C for 30 minutes. Then cells were washed with 250 μl of buffer for permeabilization/washing and resuspended in permeabilization/washing buffer to obtain EAC5 data.

Positive regulation of granzyme B and perforin characterized by an optimized sequence of СО123 x СОЯЗ bispecific diethyl (0ОАКТ-А) in T cells during redirected cytolysis

To study the possible mechanism of cytotoxicity mediated by the optimized sequence of CO123 x POP bispecific diethyl (YACT-A)

T cells, intracellular contents of granzyme B and perforin were measured in T cells after redirected cytolysis. A dose-dependent up-regulation of granzyme B and perforin contents in both CO8 and CO4 T cells was observed after incubation of T cells and Kaziti-Z cells with SACT-A (Figure 6, panel A). Interestingly, positive regulation was almost twice as high in

CO8 T-cells compared to CO4 T-cells (Figure 6, panel A). If the analysis was performed in the presence of inhibitors of granzyme B and perforin, cell lysis was not observed. Up-regulation of granzyme B or perforin was absent in CO8 or CO4 T cells when T cells were incubated with Cavity-3 target cells and control bispecific diethyl (OBACT control) (Figure 5, panel B). The given data indicate that the mediated

From rAVTA-A, the lysis of target cells can be mediated through mechanisms involving granzyme

In her perforin.

Example 6

IP mmo antitumor activity characterized by the optimized sequence of CO123 x POP bispecific diethyl (AKT-A)

Isolation of PBMCs and T-cells from human whole blood

PBMCs from healthy human donors were isolated from whole blood using centrifugation in a Ficol gradient. Briefly, whole blood was diluted 1:1 with sterile PB5. 35 ml of diluted blood was applied to 15 ml of RisoP-Radie"M Riya5 in 50 ml test tubes and the tubes were centrifuged at 1400 rpm for 20 min. with deceleration. The leukocyte film between the two phases was collected in a 50 ml test tube with 45 ml of РВ5 by the tubes were centrifuged at 600 x d (1620 rpm) for 5 min. The supernatant was removed and the cell pellet was washed once with RVZ and the number of viable cells was determined by trypan blue dye extrusion. The RVMS was resuspended to a final concentration of 2, 5 x 105 cells/ml in complete medium (KRMI 1640, 10 95EV5, 2 mM glutamine, 10 mM HERE5, 100

MKM OOμM/ml of penicillin/streptomycin (P/5).

Isolation of T cells. Unexposed T-cells were isolated by negative selection from

PBMC from human whole blood using a human T-cell isolation kit

Bupareada5 Opioispei (Gite TesppoYiIodie5) according to the manufacturer's instructions. After isolation, T-cells were cultured overnight in KRMI medium with 1095 EU5, 195 penicillin/streptomycin.

Tumor model

Human T-cells and tumor cells (Moit13 or 54-11) were combined in a ratio of 1:5 (1 x 1051 5 x 106, respectively) and suspended in 200 μl of sterile saline and injected by subcutaneous injection (5C) into 0 research day (500). A bispecific dibody characterized by an optimized sequence of CO123 x POP (OAKT-A) or a control bispecific dibody (OAKT control) was administered intravenously (IM) using 100 µl tail vein injections as presented in Table 5 (MY M113) and Table 6 (54-11).

Table 5

Scheme of research for the MYIM13 model

Treatment group Dose (mg/kg)

Control - inert carrier (cells

MY M-13 are separately implanted or 50O,1,2, 3 - T-cells)

RAVT-A 5rO,1,.2,23 |. 8 2 2sh(-

RAVT-A 5rO,1,.2,23 |. 8 2 2sh(-

RAVT-A 5rO,1,.2,23 |. 8 2 2sh(-

RAVT-A 5rO,1,.2,23 |. 8 2 2sh(-

OAVT-A 0.004 500,1,2,3 | YDshch 8

OAVT-A 0.0008 500,1,2,3 | YDshch 8

RAVT-A 0.00016 50r0.1.223 | щХХХ((8щж

Table 6

Study scheme for the K54-11 model

Treatment group Dose (mg/kg)

Control - inert carrier (cells 8554-11 separately implanted) ZO, 1,2, Z

Control - inert carrier (K54-11-4T cells implanted) ZO, 1,2, Z

Control OAVT 500,1,2,3 | 8 2 zhKfE

RAVT-A 500,1,2,3 | 8 2 zhKfE

RAVT-A 500,1,2,3 | 8 2 zhKfE

RAVT-A 500,1,2,3 | 8 2 zhKfE

RAVT-A 500,1,2,3 | 8 2 zhKfE

RAVT-A 0.004 5r0,1,2,3 | 8

Data collection and statistical analysis:

Animal weights - The weights of individual animals were recorded twice a week until the end of the study, starting from the moment of injection of tumor cells.

State of agony/mortality - Animals were observed twice a week for general state of agony and daily for mortality. Animal deaths were assessed as drug-related or technical based on factors including macroscopic observations and body weight loss; animal deaths were recorded daily. length (mm) x width?

Tumor volume - Volumes of individual tumors were recorded twice a week, starting no later than one week after tumor implantation and continuing until the end of the study.

Tumor volume (mm)

And epdium (tt) x midi"

Tytog Moishte (tt) - ZPSM, 9 (5 uh ah

Animals that died due to technical or drug-related death were excluded from data calculations.

Tumor Growth Inhibition - Tumor growth inhibition (TGI) values were calculated for each group containing treated animals using the following formula:

Middle school the volume of the tumor (which was subjected to treatment) is the average beginning. tumor volume (which was treated)

Middle school tumor volume (control) - average beginning tumor volume (control) 1. Meap Ripa! Titog MoYishte (Tgeaied) - Meap Ipiya! Tytog Moishte (Tteaied) x100

Meap Ripa! Titog Moishte (Sopigoi) - Meap Ipiya! Titog Moishte (Sopigoi)

Animals in which partial or complete remission was observed, or animals that died due to technical or drug-related death, were excluded from TOI calculations. Criteria

of the National Cancer Institute for the activity of the compound is ТО1»58 95 (Sogreii ei ai. (2004)

Apiisapseg Ogia YuOemeIortepi Siide; Toyozha, Mu: Nytapa 99-123).

Partial/complete tumor response - Individual mice characterized by the presence of tumors less than 1 mm in size? on day 1, were classified as those characterized by partial remission (RP) and the value of tumor regression in 9o (901) was determined using the following formula:

Initial tumor volume (mm)

Final tumor volume (mm)

N Z

1- Nila! Tytog Moishte (tt 4009,

Yup! Tytog Moishte (tt")

Individual mice that did not have palpable tumors were classified as having undergone complete remission (CR).

Tumor Volume Statistics - Statistical analysis was performed between treated and control groups comparing tumor volumes. For these analyses, two-way analyzes of variance with a posteriori criterion were used

Bonferroni. All analyzes were performed using SgarpRay software

RKIBMO (version 5.02). Weight-to-tumor data from individual animals that died due to technical or drug-related death were excluded from analysis. However, tumor data from animals that demonstrated a partial or complete response were included in these calculations.

The results are MINE! M1Z3

The AML cell line, MOGM13, was premixed with activated T cells and implanted with 5C MOB)/ZSIUO gamma (M5O) knockout mice (M-v/group) on day 500, as detailed below. MYM13 tumors in the vehicle-treated group (MYM13 cells alone or together with T cells) showed an aggressive growth profile and mimo (Figure 7, panels A and B). On the 5th day, the average volume of tumors in the group treated with an inert carrier was 129.8x429.5 mm, and by the 5015th day, the tumors reached an average volume of 786.4-156.7 mm3. By the end of the experiment on day 5018, the tumors reached an average volume of 1398.82236.9

MM.

Treatment with UACT-A was started on the same day as the mixture of tumor cells and T cells (KZOO) was implanted. and continued with daily injections for an additional 7 days to reach a total of 8 daily injections. Animals were treated with VACT-A at 9 dose levels (0.5, 0.2, 0.1, 0.02 and 0.004 mg/kg, 20, 4, 0.8 and 0.16 μg/kg).

The results are shown in Figure 7, panel A (0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg) and Figure 7, panel B (20, 4, 0.8, and 0.16 μg/ kg). On study day 11, MY M13 tumor growth was significantly inhibited at dose levels of 0.16, 0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg (p < 0.001). Moreover, treatment of MY M13 tumor-bearing mice at dose levels of 20 and 4 μg/kg dose resulted in 8/8 and 7/8 CE, respectively. By the end of the experiment on day 5018, the average volume of tumors treated with VACT-A at a dose of 0.8 - 20 μg/kg was in the range from 713.6.05267.4 to 0 mm3, and all tumors were significantly smaller than tumors in the control group treated with an inert carrier. TO values were 100, 94, and 49,95 for groups receiving doses of 20, 4, and 0.8 μg/kg, respectively. Compared with the vehicle-treated group with MOG M13 tumor cells, the OACT-A dose-level groups of 20 and 4 μg/kg reached statistical significance by day 5015, whereas the group that received treatment with 0.8 μg/kg, reached statistical significance by day 5018.

Results K54-11

The ALL cell line, K54-11, was premixed with activated T cells and implanted with 5C MOB)/ZSIUO gamma (M5O) knockout mice (M-v/group) on day 500, as detailed below. K54-11 tumors in the vehicle-treated group (K54-11 cells alone or in combination with T cells) demonstrated an aggressive growth profile (Figure 8).

Treatment with UACT-A was started on the same day as the mixture of tumor cells and T cells (KZOO) was implanted. and continued with daily injections for an additional 3 days to reach a total of 4 daily injections. Animals were treated with YAVT-A at 5 dose levels (0.5, 0.2, 0.1, 0.02 and 0.004 mg/kg). The results are shown in Figure 8.

A bispecific diabody characterized by an optimized sequence of CO123 x POP (RAVRT-A) effectively inhibited the growth of both MY M1Z3 AML and K54-110LL tumors implanted

ZS MOB/5SIO mice in the context of the M/ipp model, if the administration of doses was started on the day of implantation and continued for 3 or more consecutive days. Based on the established criteria

According to the National Cancer Institute, AKT-A at a dose level of 0.1 mg/kg and above (TOI »58) is considered active in the K54-11 model, and an OACT-A dose of 0.004 mg/kg and above was active in MY M13 model. The lower doses of BACT-A associated with tumor growth inhibition in the MY M13 model compared to the K54-11 model would be consistent with the ip migo data demonstrating that MY M13 cells express higher levels of CO123 than K54-11 cells , which correlates with increased sensitivity to OAKT-A-mediated cytotoxicity of migo in MY M13 cells.

If there is a need to reproduce the specified example, it should be understood that a specialist in this field of technology will be able, within reasonable and acceptable limits, to modify the protocol described above in a suitable way to reproduce the described results. Thus, it is not implied that the illustrated protocol must be followed in a precisely defined manner.

Example 7

Surface expression of CO123 on leukemic blast cells and stem cells in a primary tissue sample from patient 1 with AML

To determine the expression pattern of CO123 in primary samples from patient 1, cryopreserved primary samples of bone marrow and PBMC of a patient with AML were evaluated for surface expression of CO123 on leukemic blast cells.

GML bone marrow sample - clinical report

Age: 42

Female

GML subtype: M2

Percentage ratio of malignant cells based on morphology: 67.5 90

Bone marrow immunophenotyping:

СбОр15-19 95, СО33-98.5 96, СО38-28.8 uyu, С045-81.8 96, СО64-39.7 U, СО0117-42.9 Fo, NO A-

OrA-1795, Сб0р2-1.895, Со5-0.5395, 0007-0295, С0О10-0.41 95, СО19-1.1 95, СбОр20-1.4 Ob,

Зо bОр22-0.71 У6 6О034-0.82 95

Expression of ХУО123 in leukemic blast cells in bone marrow mononuclear cells (BM MMC)

A total of 0.5 x 106 bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) from patient 1 with AML were evaluated for CO123 expression. The Kaziti-Z cell line was included as a control. Leukemic blast cells were identified using the myeloid marker SOZ33. As shown in figure 9, panel A, 87 90 cells from the bone marrow of AML from patient 1 expressed CO123 and CO33. CO123 expression levels were slightly lower than in the highly CO123-expressing AML cell line, Kazity-3 (Figure 9, panel B).

Example 8

Analysis of autologous STI cytolysis. using primary samples from a patient with AML

A cryopreserved primary AML sample (bone marrow mononuclear cells (BMNCs) and peripheral blood mononuclear cells (PBMCs)) from patient 1 with AML was thawed in KRMI 1640 with 10 96

ЕВ5 and left to recover overnight at 37 "С in 5595 СО". cells were washed with analysis medium (KRMI 1640--10 95ЕВ5) and the number of viable cells was determined by trypan blue extrusion. 150,000 cells/well in 150 μl of analysis medium were added to a 9b-well O-bottom plate (VO Viozsiepse5).The bispecific diabody characterized by the optimized sequence of CO123 x POP (OACT-A) was diluted to 0.1 and 0.01 ng/ml and 50 μl of each dilution was added to each well ( final volume - 200 μl).

Control bispecific diabody (control OAKT) was diluted to 0.1 ng/ml and 50 μl of each dilution was added to each well (final volume - 200 μl). A separate analytical plate was set up for each time point (48, 72, 120 and 144 hours) and the plates were incubated at 37 "C in an incubator with 595 CO". At each time point, cells were stained with antibodies to CO4, CO8, С025, С0р45, 2033 ii СО123. Labeled cells were analyzed in a flow cytometer of RASZ Sairig, equipped with software for data collection

SepOitsevzi Rgo, version 5.2.1 (VO ViozZsiepse5). Data analysis was performed using RiIoullo m9.3.3 software (Tgeeviag, Ipswich). T-cell expansion was measured by gating on the СО4 and СО8 populations, and activation was determined by measuring the average value of the fluorescence intensity of СО25 (ME) on the gated in relation to сра- and Сов populations. The population of leukemic blast cells was identified using CO45-CO33 gating.

Autologous depletion of tumor cells, T-cell expansion and activation using a bispecific dialyzer characterized by an optimized sequence of СО123 x СОЗ (ОАВТ-А) in primary samples from patient 1 with AML

To determine mediated bispecific diethyl, characterized by an optimized sequence of CO123 x POP (BAVT-A) activity in patient 1 with AML, samples from the patient were incubated with 0.1 ng/ml or 0.01 ng/ml OABT-A and the percentage ratio of leukemic blast cells and T cells were measured at different time points after treatment. Leukemic blast cells were identified by CO45-/CO033- gating. Incubation of primary AML bone marrow samples with POACT-A resulted in depletion of the leukemic cell population over time (Figure 10, panel A), accompanied by concomitant expansion of residual T cells (Figure 10, panel B) and induction of markers of T cell activation ( figure 10, panel C). In rAVTA-A treated samples, T cells expanded from about 7 95 to about 80 95 to 120 h. T-cell activation, measured by the expression of СО25 on С04 and СО8 cells, reached its peak after 72 hours. and decreased to the time point of 120 h.

If there is a need to reproduce the specified example, it should be understood that a specialist in this field of technology will be able, within reasonable and acceptable limits, to modify the protocol described above in a suitable way to reproduce the described results. Thus, it is not implied that the illustrated protocol must be followed in a precisely defined manner.

Example 9

Surface expression of SUO123 on leukemic blast cells and stem cells in a primary tissue sample from a patient with ALL

To determine the expression pattern of CO123 in primary samples from a patient with ALL, a cryopreserved primary sample of PBMC from a patient with ALL was evaluated for surface expression of CO123 on leukemic blast cells.

Expression of CO123 in leukemic blast cells in peripheral blood mononuclear cells (PBMO)

A total of 0.5 x 106 peripheral blood mononuclear cells (PBMC)) from a healthy donor and a patient with ALL were evaluated for CO123 expression. As shown in Figure 11, panels B-H, the vast majority of cells from the bone marrow of ALL expressed CO123. On the contrary, how

As expected, in a normal donor, B-cells are negative for CO123 and cells and monocytes are positive for CO123 (Figure 11, panel 0).

The T-cell population was identified in a sample of a patient with ALL by staining cells in relation to CO4 and CO8. As shown in figure 12, panel B, only a small fraction of the total

PBMC in a sample from a patient with ALL were T cells (approximately 0.5 95 were CA T cells and approximately 0.4 95 were SOB8 T cells).

Example 10

Analysis of autologous STI cytolysis. using primary samples from a patient with ALL

A cryopreserved primary sample of ALL (peripheral blood mononuclear cells (PBMC)) was thawed in KRM 11640 with 10 95 ЕВ5 and left to recover overnight at 37 "C in 5 95

SO". Cells were washed with analysis medium (KRMI 1640-10 9585) and the number of viable cells was determined by trypan blue exclusion. 150,000 cells/well in 150 μl medium for analysis were added to a 96-well O-bottom plate (VO Viobsiepsev).

A bispecific diabody characterized by an optimized sequence of СО123 x POP (OANT-

A) was diluted to 10.1 ng/ml and 50 μl of each dilution was added to each well (final volume - 200 μl). A separate analytical tablet was installed for each time point (48, 72, 120 and 144 hours) and the tablets were incubated at 37 "С in an incubator with 5 95 СО". At each time point, cells were stained with antibodies to СО4, СО8, С0р25, 2045 , СО33 and СО123.

Labeled cells were analyzed in an EAS5 Saiirbig flow cytometer equipped with SePoitsevi Rgo data collection software, version 5.2.1 (VO Viozsieepse5). Data analysis was performed using Riom/o m9.3.3 software (Tgeeziag, Ipswich). T-cell expansion was measured by gating on the СО4- and СО8-- populations, and activation was determined by measuring ME! СО25 on the gated in relation to СО4- and СО8- populations.

The population of leukemic blast cells was identified using gating

Sr45-C033-n.

Autologous depletion of tumor cells, T-cell expansion and activation with the help of a bispecific diatyl characterized by an optimized sequence of СО123 x СОЮЗ (ОАВТ-А) in primary samples from patients with ALL

To determine the activity mediated by a bispecific diethyl characterized by an optimized sequence of CO123 x POP (OACT-A) in primary samples from a patient with ALL, patient samples were incubated with 1 ng/ml BACT-A and the percentage ratio of leukemic blast cells and T cells was measured in different time points after treatment. Leukemic blast cells were identified by CO45:/20337 gating. Incubation of primary ALL bone marrow samples with OAKT-A resulted in depletion of the leukemic cell population over time compared to untreated control or control BACT (Figure 13, panel H compared to panels E and b). When T cells were counted (CO8 and CO4 staining) and activation (CO25 staining) analyzed, T cells were expanded and activated in the SACT-A sample (Figure 14, panels I and I, respectively) compared to untreated samples or treated with the control YAKT samples (Figure 14, panels H, b, K and y, respectively).

Example 11

Surface expression of CO123 on leukemic blast cells and stem cells in a primary tissue sample from patient 2 with AML

To determine the expression pattern of CO123 in primary samples of patient 2 with AML, cryopreserved primary samples of bone marrow and PBMC of a patient with AML were evaluated for surface expression of CO123 on leukemic blast cells.

Expression of ХУО123 in leukemic blast cells in bone marrow mononuclear cells (BMMS)

A total of 0.5 x 105 bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs)) from patient 2 with AML were evaluated to identify leukemic blast cells.

Leukemic blast cells were identified using myeloid markers SOZZ and СО45. As shown in figure 15, panel B, 94 95 cells from bone marrow from AML were leukemic blast cells. The T-cell population was identified by the expression of SOC3. As shown in Figure 15, panel C, approximately 15 95 cells from the AML and PBMC bone marrow sample were T cells.

Example 12

Analysis of autologous STI cytolysis. using primary samples from patient 2 with AML

A cryopreserved primary AML sample (bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs)) from patient 2 with AML was thawed in KRM 11640 with 10 Fo

EB5 and left to recover overnight at 37 "С in 595 СО". cells were washed with analysis medium (KRMI 1640--10 96 EB5) and the number of viable cells was determined

From the extrusion of trypan blue. 150,000 cells/well in 150 μl medium for analysis were added to a 9b-well O-bottom tablet (VO Viozsiepse5). A bispecific dibody characterized by an optimized sequence of CO123 x POP (OACT-A) and a control bispecific dibody (control UAKT) were diluted to 0.1 and 0.01 ng/ml and 50 μl of each dilution was added to each well (final volume - 200 μl). A separate analytical plate was installed for each time point (48, 72, 120 and 144 hours) and the plates were incubated at 37 "С in an incubator with 5 95 СО". At each time point, cells were stained with antibodies to СО4, СО8, С025, С0р45 .

SepOitsevzi Rgo, version 5.2.1 (VO ViozZsiepse5z). Data analysis was performed using RiIoullo m9.3.3 software (Tgeeviag, Ipswich). T-cell expansion was measured by gating on the СО4- and СО8 populations, and activation was determined by measuring

MEASURES of С0О25 on gated in relation to СО4- and СОвж- populations. The population of leukemic blast cells was identified using gating СО45-4СО33-.

Autologous tumor cell depletion, T-cell expansion and activation in primary samples from patient 2 with AML

To determine mediated bispecific diatyl characterized by an optimized sequence of CO123 x POP (OAKT-A) activity in primary samples from patient 2 with AML, patient samples were incubated with 0.1 or 0.01 ng/ml BACT-A and percentage ratios of leukemic blast cells and T cells were measured at various time points after treatment. Incubation of primary AML bone marrow samples with OAKT-A resulted in depletion of the leukemic cell population over time (Figure 16, panel A), accompanied by a concomitant expansion of residual T cells (both CO4 and SOB8) (Figure 16, panel B and figure 16, panel C, respectively).

To determine whether T cells were activated, cells were stained for CO25 or Ki-67, both markers of T cell activation. As shown in Figure 17, panels A and B, incubation of primary AML bone marrow samples with POAKT-A resulted in T cell activation.

The indicated data represented a time point of 144 hours.

Intracellular staining for granzyme B and perforin.

To determine the intracellular contents of granzyme B and perforin in T cells, STI analysis was performed. After about 18 hours. cells from the analytical tablet were stained with antibodies to CO4 and to СХО8 by incubation for 30 minutes at 4 °C. After surface staining, the cells were incubated in 100 μl of buffer for fixation and permeabilization for 20 min at 4 °C.

Cells were washed with permeabilization buffer and incubated in 50 μl of a mixture of anti-granzyme B and perforin antibodies prepared in 1X permeabilization/washing buffer at 4 °C for 30 minutes. Cells were then washed with 250 μl of permeabilization/washing buffer and resuspended in buffers for permeabilization/washing to obtain EAS5 data

Positive regulation of granzyme B and perforin characterized by the optimized sequence of СО123 x СОЯЗ bispecific diethyl (OAKT-A) in T cells during redirected cytolysis

To investigate the possible mechanism of T-cell cytotoxicity mediated by a bispecific diethyl characterized by an optimized sequence of CO123 x POPs (OAKT-A), the intracellular contents of granzyme B and perforin were measured in T-cells after redirected cytolysis. If the T-cells were incubated with the control bispecific diatil (control SACT), the positive regulation of granzyme B and perforin was not observed. Positive regulation of the content of granzyme B and perforin in both COB8 and CO4 T-cells was observed under the action of a bispecific diethyl characterized by an optimized sequence of CO123 x POP (PART-A) (Figure 17, panels C and B). Interestingly, the upregulation was nearly twofold higher in CO8 T cells compared to CO4 T cells (Figure 17, panel C and Figure 17, panel 0).

These data indicate that OAKT-A-mediated lysis of target cells is mediated through a pathway involving granzyme B and perforin.

Example 13

A bispecific diabody characterized by an optimized sequence of CO123 x POPs cross-reacts with nonhuman primate CO123 and POPs proteins

To quantitatively determine the degree of binding between a bispecific diethyl characterized by an optimized sequence of СО123 x POP (ОАВТ-А) and POPs of a person or a Javan macaque, VIASORE "M" analyzes were performed. In VIASOKE"M analyzes the dissociation rate constant, Ka, is measured. The binding affinity (CO) between the antibody and its target is a function of the kinetic constants for association (association rate constant, Ka) and dissociation (dissociation rate constant, Ka) according to the formula: KO - IkaKaiY. In the analysis of VIASOBE "M

Surface plasmon resonance is used to measure directly indicated kinetic parameters. Recombinant human or Javan macaque POPs were directly immobilized on the substrate. Purified CO123 from humans or Javan macaques was captured and immobilized on the substrate. The duration of dissociation was measured and bivalent approximation of the data was performed. Binding and affinity constants were obtained using a 1:1 binding approximation. The results of the VIASOVE"M assays, which compared binding to human CO123 and POP proteins in comparison with CO123 pain and Javanese macaque POPs, are shown in Figure 18. Affinities of binding to Javanese macaque CO123 proteins (Figure 180) and Javanese macaque POPs (Figure 188) are comparable to binding affinities for human CO123 proteins (Figure 182) and human POPs (Figure 184A).

Example 14

Autologous depletion of myoblast monocytes from PBMCs of humans and Javan macaques

RVMOs from human or cynomolgus macaque whole blood samples were added to O-bottom plates at a T-cell density of 200,000 cells/well in 150 μl assay medium.

Dilutions characterized by an optimized sequence of СО123 x POP bispecific diethyls (FACT-A or OACT-A m/AVI)) were obtained in the medium for analysis. 50 μl of each dilution of OAVT-A or SAKT-A m/ABO was added to the plate containing PBMC in the wells in two parallels. Tablets were incubated for 18-24 hours. at 37 "C. Supernatants were used to determine cytotoxicity as described above. As shown in Figure 19 (panels A and B), depletion of ROS cells was observed both in human PBMCs (Figure 19, panel A), such

RBMS of the Javan macaque (Figure 19, panel B). These results indicate that ROS circulating in the bloodstream can be used as a pharmacodynamic marker for preclinical toxicological tests in Javan macaques.

If there is a need to reproduce the specified example, it should be understood that a specialist in this field of technology will be able, within reasonable and acceptable limits, to modify the protocol described above in a suitable way to reproduce the described results. Thus, it is not implied that the illustrated protocol must be followed in a precisely defined manner.

Example 15

Depletion of plasmacytoid dendritic cells in Javan macaques treated with a bispecific diatyl characterized by an optimized sequence of СО123x

SOZ (SAVT-A)

As part of a toxicological study to find a dose range, Javan macaques were injected with a bispecific diabody characterized by an optimized sequence of СО123 x SUOZ (РАВРТ-А) in the form of 4-day infusions in doses of 0.1, 1, 10, 30, 100, 300 or 1000 ng/kg. Control SACT was administered at a dose of 100 ng/kg. To identify ROS and monocyte populations

RBMS of the Javan macaque, cells were labeled with an antibody to CO14-RITS. Monocytes were identified as the CO14- population and pOS were identified as the CO14-200123: population. As shown in Figure 20, panels K and I, ROS were depleted no later than 4 days after infusion at a dose not higher than 10 ng/kg OACT-A. No depletion of ROS was observed in monkeys treated with control bispecific diethyl (control SUACT) or treated with inert vehicle together with monkey filler at the 4-day time point (Figure 20, panels C, H, C and 0, respectively ). The content of cytokines: interferon-gamma, TME-alpha, 11 6, II 5, 11 4 and 1/2, was determined 4 hours after the infusion. There was little or no increase in cytokine levels in animals treated with pAVTA-A compared to control POACT or vehicle-treated animals.

Figure 21 and Figure 22 present the results of EAS5 analysis for B cells (C020) (Figure 21, panel A), monocytes (CO14") (Figure 21, panel B), MK cells (СО0О159-5016) (Figure 21, panel C), ros (Sr12zni, 20143) (Figure 21, panel 0) and T cells (generally, СО4» and СО8») (Figure 22, panel A, figure 22, panel B and figure 22, panel 0, respectively ).

Treatment of monkeys with the control OAKT did not lead to noticeable effects on T- or B-lymphocytes, MK-cells, monocytes and ROS. Treatment of monkeys with BACT-A at doses of 10 ng/kg/day or higher resulted in elimination of ROS (Figure 21, panel 0).

PO depletion was complete and sustained, returning to pre-treatment levels several weeks after dosing was completed. The content of circulating T-lymphocytes in the bloodstream decreased with the introduction of OAKT-A, but returned to pre-treatment levels by the end of each

Out of a weekly cycle, indicating changes in migration rather than true depletion. Both CO4 and CO8 T-lymphocytes were characterized by the same pattern. A marker of T-lymphocyte activation, COb69 (Figure 22, panel C), was only marginally positive among circulating cells and did not change following the administration of a dose of UACT-A. The content of B-lymphocytes, monocytes and MK-cells fluctuated during the course of administration of doses of SUACT-A, with significant variability observed among monkeys. A trend toward elevated levels of circulating B-lymphocytes and monocytes was observed in both monkeys at the highest doses.

Therefore, the above results demonstrate the therapeutic efficacy characterized by the optimized sequence of СО123 x POP bispecific diethyl (AKT-A).

A bispecific diabody characterized by an optimized sequence of СО123 x POP (SАКТ-

A) can be used as a therapeutic agent for the treatment of numerous diseases and conditions, including the following AML, AVI (ALL), CLL, MDS, rossi, mantle cell lymphoma, hairy cell leukemia, CLL with Richter's transformation, blast crisis in CML, HIV. (subclass is СХО123. (see example 2); systemic lupus erythematosus (SLE), allergy (basophils are СО123.), bronchial asthma, etc.

Example 16

Comparative properties of a bispecific diethyl characterized by an optimized sequence of CO123 x POP3 (OAKT-A) and a bispecific diethyl characterized by an unoptimized sequence of CO123 x POP (OAKT-B)

An unexpected advantage and properties characterized by an optimized sequence

С0123 x POPs of bispecific diethyls

As discussed above, SAKT-A and SAKT-B were constructed in a similar manner, and the first polypeptide of both constructs contain, from the M-terminus to the C-terminus, the M-terminus, the Mi domain. monoclonal antibody capable of binding to SOZ (Mi soz), intermediate linker peptide (linker 1), MH domain of monoclonal antibody capable of binding to SHO123 (MNeoigz), linker 2, E-helical domain and C-terminus. Similarly, the second polypeptide of both constructs contain, in the direction from the M-terminus to the C-terminus, the M-terminus, the MI domain of a monoclonal antibody capable of binding to CO123 (Mi surigs), an intermediate linker peptide (linker 1), the Mn domain of a monoclonal antibodies capable of binding to POPs (MNeoz), linker 2, K-helical domain and

C-end.

As shown in example 1, it was found that both CO123 x POPs of bispecific diethyls are capable of simultaneously binding to POPs and CO123. In addition, as disclosed in Example C and Figure 4, panels C and O, the two CO123 x COZ3 bispecific diethyls showed a marked capacity for redirected cytolysis with concentrations that should reach 50 95 of maximal activity (EC50) in the sub-ng/range. ml, regardless of the specificity of binding to the epitope

POPs (SAVT-A compared to VAKT-B) in target cell lines with high CO123 expression.

Thus, small variations in specific sequences of СО123 x POPs of bispecific diethyls do not completely nullify biological activity.

However, in all cell lines examined, SACT-A was found to be more active and more efficient in redirected cytolysis than SACT-B (see, for example, Figure 4, panels

A, C and Y). Thus, SACT-A showed an unexpected advantage compared to similar OACT-B.

Example 17

Pharmacology of BACT-A in a nonhuman primate for the treatment of hematologic malignancies

The alpha chain of the interleukin C (1-3) receptor, CO123, is overexpressed on malignant cells in a wide range of hematological malignancies (Mypog, Y. Yei ai. (2001) "Ipepecykip-3 Veseriog AIrpa SNaiip (CO123) I5 MUidemu Ehrgezzed Ip Netayiodis

Mayidpapsie5, " Naethaioiodisa 86:1261-1269; Tevia, SHO. ei aI. (2014) "C0123 Ie A Metrgbape

Viotazheg Apa A Pegaretsshiis Tagdei Ip Netaiioiodis Maiidpapsiev, " Viota!K. Nev. 2:4) and is associated with an unfavorable prognosis (Megde, EE. ei aiYi. (2011) "Nidn Hemeiei OG SbO34-СО3810/-

SO123--VIAv5iv Age Rgyedisiime OYI Ap Admegze Otsisote Ip Ascie Mueyiyoia I eoKetia: A Stgoire O!tsevi-

EI Oez I eisetiez Aiidsez EI MaiYadiez bi Zapa (OBG AM5) 5iiau, " Najetaiyuodisa 96:1792- 1798). Moreover, it has been reported that CO123 is expressed by leukemic stem cells (I 5C) (Chogdap, ST. ei ai. ( 2000) "Tne IpiepetsyKip-3 Veseriog AIrna Saip Yv A Opidie

MaikKeg Rog Nytap Ascie Muveiodepoizv I eiketia biet SeiYi5, " eiketia 14:1777-1784; ip, I... viii ai. (2009) "Moposiopa! Apiibodu-Meadaiaivea Tagdeipud Oi CO123, 1-3 NReseriog AIrna SpNaip, EiYitipaiev

Nytap Asshiye Mueioiy IeyKetis 5yet Sei, " Se biet SeyiY 5:31-42), which is a promising feature that allows to target the main cause of such diseases. According to this finding, SUO123 is also involved in the autocrine loop of II/-3, that supports the onset and development of leukemia, as shown by the ability of a blocking CO123 monoclonal antibody to reduce engraftment of leukemic stem cells and improve survival in a mouse model of acute myeloid leukemia (AML) (ip, Y. ei ai. (2009) "Moposiopa! Apiroau-

Mediateid Tagdeinpud ОЙ СО123, I/-3 Veseriog AIrna Spaip, EItipayez Nytap Ascie Mueioia

Yeniketis S1et SeiiIv, " Seii biet Seii! 5:31-42). However, in a phase 1 study in patients with high risk of AML, the monoclonal antibody did not show anti-leukemic activity (Nobegpiv5, A. MU. ei a. (2010) "A Rnaze I 5ishau OG Apii-sa123 Moposiopa! Apiibodu (tab) S5I 360 Tagayeiipu I eoKetia 5iet

Seiyiv (I5C) Ip AMI,, " U. Siip. Opsoi. 28(Z, irri):e13012). Thus, alternative approaches to the targeted impact on СО123, including depletion strategies, are needed. Although СО123 is expressed by a subclass of normal hematopoietic cells -progenitors (HSCs), hematopoietic stem cells (HSCs) express CO123 in small amounts or do not express it at all (Chogdap, S.T. ei a. (2000) "TNe IpiepeiKkip-3 Veseriog AIrpa Spaip I5 A Opidoe MaiKeg Rog Nytap

Ascie MuyeIodeposhz IGeiKetia 5iet SeiYiv, " GeiKetia 14:1777-1784; ip, MU. ei a. (2009) "Vedshiayop

OG Tn17 Sei! OiNegepiianyop Apa EAE Ipdysip Vu MARZK MIC, " Viosa 113:6603-6610), indicating that strategies based on depletion of cells with CO123 provide recovery through normal hematopoiesis.

Targeting the patient's own T-lymphocytes to leukemic target cells is a promising immunotherapeutic strategy for the treatment of hematological malignancies.

An attempt to apply the therapeutic potential of this approach was made using blinatumomab (ViTte based on a bispecific antibody characterized by the ability to bind to POPs and B-cell antigen CO19) in patients with B-cell lymphomas and pre-

B-cell acute lymphoblastic leukemia

Vezropze OI Raiiiepiv M/yp V-I ipeade Asshe I utrpobiaviis I eIKetia To Sopiipysiv Iptiviop OI T

SeiI-Epdadipda СО19/С03-Vivzresiis VITE Apiirodu Viipaistotar, " Viosa 119:6226-6233; Torr,

M.5. oh oh (2012).

ОЙ Viipaishtotab Ip Raiepiv M/yp MAO Ip V-I ipeade AG, "Viosa 120:5185-5187; Torr, M.5. ei a). (2011) "Tagdeiied Tnegaru MUyN TNne T-SeiI-Epodadipa Apiibodu Viipaiishtotab oi SpetoiNegaru -

Veitgasiogu Mipita! Nezidypa! Oizease Ip V-I ipeade Ascie I utrpobiaviiis I eiKetia Raiepiv Neziiv Ip

Nidyp Nezropze Naie Apa Rgoiopdei I eiketia-Egeye Zyguimai, " U. Siip. Opsoi. 29:2493-2498).

Bispecific diethyl CO123 x POP molecules according to the present invention, such as OAKT-A, because they contain an alternative bispecific, antibody-based modality that provides improved stability and more reliable properties for production performance (doip5op, 5. ei a!i. ( (2010)

To Roiepi Titog Suyuiuviv Apa Ip Mimo V-Sei! Oeriyeop, " U. Moi. Vioi. 399:436-449; Mooge, RA. Ayipyu Neiagdeipu MoiYesshev To Aspieme Oriita! Neadigesiyed T-Sei

Kipa oi V-SeiI Gutrpota, "Viooi 117:4542-4551).

In order to demonstrate the advantage and effectiveness of CO123 x POP molecules of the bispecific diethyl according to this invention, the biological activity of the above-described SACT-

And in IP miigo and preclinical models of leukemia, as well as its pharmacokinetics, pharmacodynamics and safety pharmacology in Javan macaques (Masasa gazsisgiagie) were evaluated in relation to either the control VBACT described above (bispecific in relation to POPs and fluorescein), or the "control OAKT-2" , which is bispecific for CO123 and fluorescein).

Amino acid sequence of the first polypeptide chain of "control OAVT-2" (СО123ЗМИ - linker - 4-4420МУН - linker - E-helix; linkers are underlined) (ZEO IO MO:58):

REMMTO5ROB GAM5I YAEVMT MZOKZZOBI MBAMOKMMST MUMOOKRYaORR

TO WHOM ABZTA EZEMROVEZOa ZIAZAIAITOGTI t I55I OAEOMA MUUSOMOUBU

RUTROOSTKI EIXSO)aBaSO SEMUKI OETaSs; aa MORSVRMC AND 5SMABOETE

ZVUMMMUMUMVO ZREKSOGEM MA OTIAVMKRUMUUE TUUZOBUKOaV ETIZNOOZK

ZU OMMMI A MEOMSIMUST aBUMaMOUMUss batvUtUZZa aSOaaEMAA.

EKEMAAI EKE MAAI EKEMAA AND EK

Amino acid sequence of the second polypeptide chain of "control OANT-2" (4420MI - linker - СО12ЗМН - linker - K-helix) (ZEO IU MO:59):

OMUMTOTREB GRUBI aROABISSAZBZOVI M NIMAMTMiAM M OKROOBRK

MIMKU5MAE 5aMROVEBZa5 a5SaTOETIC BAMEAEOII YAM MES5OBTNUR mtRaastTkKGE IKasaasais EMO MOBOAE and KKRIABUKM BSKABaMTET

OUUMKUMUVOA RIASAGEMISO PROMSOATEM MOKEKaVMTtI TMOKOTOTAU

ME! 55I A5EO TAUUUSAV5N GI VABM/ GAMU SOSTI MTU SSSO, aakmMAA

YIKEKMAAI KE KMAAI KEKMA AI KE

Bifunctional analysis of EI IZA

Makhizogr EGIZA tablet (Mips), on which soluble IZ K-alpha was applied during the night

From human or Javan macaque (0.5 μg/ml) in bicarbonate buffer, blocked with 0.5 96 VA; 0.1 95 Gueep-20 in RV5 (RVZT/VZA) for 30 minutes at room temperature.

SUAKT-A molecules were applied, followed by the sequential addition of the POP conjugate human biotin and streptavidin-NCR (daskbzop IttypoKezeagsi). NKR activity was detected by the transformation of tetramethylbenzidine (ViobH) as a substrate within 5 min.; the reaction was stopped with 40 μl/well of 1 95 H25O" and the absorbance was read at 450 nm.

Analysis of surface plasmon resonance

The ability of OAKT-A to bind to POP or CO123 beads of humans and Javan macaques was analyzed using the ViAsoge 3000 biosensor (CE, Neaisage) as described in doppzop,

Z. ei a). (2010).

Yi eado5 To Roiepi Titog Suyuiuvziv Apa Ip Mimo V-Sei! Oeriyeop, " .). Moi. Vio!. 399:436-449) and Mooge,

R.A. oh oh (2011).

T-Sei! Kiipud SO V-Se! Gutrpota, Biol 117:4542-4551). Soluble POP or CO123 (1 μg/mL) was injected onto the surface of activated CM5 in 10 mM sodium acetate, pH 5.0, at a volumetric flow rate of 5 μL/min, followed by the addition of 1 M ethanolamine for deactivation. Binding experiments were performed in 10 mM NEREZ5, pH 7.4, 150 mM Mass, 3 mm EOTA, and 0.005 95 surfactant P20. Regeneration of immobilized receptor surfaces was performed by pulse injection of 10 mM glycine, pH 1 ,5. The value of KO was determined by the global approximation of the binding curves to the 1:1 Langmuir binding model (ViAem software|chayop

U4.1).

Analysis of cytolysis

Cell lines used for cell lysis assays were obtained from the American Type Culture Collection (ATSC) (Mapazza5, MA). RBMS was isolated from the blood of healthy donors using a RisoiI-Radie Riiv kit (SE NeiNsage); T cells were purified using a negative selection kit (Iie Tespoiodieb5). The density of CO123 on the cell surface was determined using Otsapisht zitriu SeiIshmiag granules (Vapoz I Arogayogiev, Ips., Rizpegv, IM).

Cytotoxicity assays were performed as described by Mooge, R.A. oh oh (2011) ("Arriisanyop OG Oiai

Anippu Neiagodeiipd Moiesshtcevz To Aspieme Oriita! Nedigesied T-Sei! Kiipou OY V-Seiyi Gutrpota, " 60 Biood 117:4542-4551). Briefly, target cell lines (105 cells/ml) were treated with serial dilutions of proteins such as UACT-A or control UACT in the presence of T cells in the indicated ratios of effector cells to target cells and incubated at 37 C overnight. Cell lysis was determined by the release of lactate dehydrogenase (ION, Rgoteda) in the culture supernatant. To analyze cytolysis using flow cytometry, target cells were labeled using SMTMEK (Se Tesppoiodie5) and cytolysis was monitored using an EAScSZSaiiirig flow cytometer.Data were analyzed using RKIZMO 5 software (sgarpRad) and presented as cytotoxicity in 95.

Pharmacology in relation to the Javan macaque

Experiments on non-human primates were carried out in Spagiy Rimeg Iarogayogie5 (ANepo, MM), in accordance with the requirements of the local institutional committee for the maintenance and use of animals (ASIS). Specially bred, previously unexposed Javan macaques (Masasa gazsisciagi5) of Chinese origin (age range 2.5-9 years, weight range 2.7-5 kg) were administered an inert vehicle or OAKT-A by intravenous infusion through a port system in the femoral and jugular vein using programmable infusion pumps powered by a battery (SAOO-IedasufF, 5IM5 Oepces, Ips., ZI. Rush, MM). Peripheral blood or bone marrow samples were collected in tubes containing anticoagulant at the indicated time points. Analyzes of the cell surface phenotype were carried out using the Y5K Rogiezza analyzer (VO ViozZsiepse5), equipped with lasers with wavelengths of 488 nm, 640 nm, and 405 nm, and using the following antibodies: C0O4-M450, C08-M450,

СО123-РЕ-Су7, СО45-РегбР, СО4-АРОС-Н7У, СО8-RITS, СО25-РЕ-Су7, СОб69-РегСР, РО-1-РЕ,

TIM3-ARS, SOZ-Rasiis Vigne, C095-ARS, Srgv-vu421, SO16-RITS, SOZ3-AIekha488, SOZ38-RE,

СОр123-РЕ-Су7, СО117-РегСбР-Суб5,5, СО34-АРС, 2090-ВУ421, СО45ВА-АРО-Н?7 and СО33-АРС (ВО

Viobsiepse5). The absolute number of cells was determined using TtSOOMT (VO

Wiozsiepse5). The content of cytokines 1-2, 1/-4, 1-5, 1-6, TME-a and IRM-u in the serum was measured using a set of cytometric beads with monoclonal antibodies to the cytokine

TI1/Tp2 of a non-human primate (VO Viozsiepse). The concentration of OAKT-A in monkey serum samples was measured using a sandwich immunoassay with electroluminescence detection (Mesozsaili Oiadpovziis5, MBO, KosKmiye, MO). Briefly, recombinant human I/-3 Ka (K8UO bouviet) was applied to an analytical plate (M5O) and the plate was blocked with 5 95 VB5A. Calibrated standards or diluted test samples were applied, after which a biotinylated monoclonal antibody was added, which shows specific binding to the E-helical (5EO IU MO:34) and K-helical (5EO IU MO:35) domains of the molecule described above. Labeled ZYOGRO-TAStTM conjugate of streptavidin (M5O) was added and the formation of complexes was analyzed in the image forming device M5O 5ESTOKF) Concentrations of OAKT-

A was determined from standard curves created by approximating light radiation intensity data in a five-parameter logistic model.

Determination of the physicochemical characteristics of purified OAKT-A demonstrated a homogeneous heterodimer with a molecular weight of 58.9 kDa (Figure 23; Figures 24A-248), which is stable at 2-8"C for up to 12 months in RV5. Analysis 5PE showed almost identical binding affinities of OAKT-A to the corresponding soluble human and Javan macaque antigens POP and CO123 (Figures 25A-250 and Table 7). In addition, pAVT-A simultaneously bound to both antigens in the EHI5A format, in which was used

human or monkey CO0123 for capture and human POP for detection (figures 26A-26B), and demonstrated similar binding to human and monkey T lymphocytes (figures 26C-26E). The data in Table 7 represent average values from 3 independent experiments, each of which was conducted in two parallels.

Table 7

Equilibrium dissociation constants (Ko) for binding

RAVT-A with POPs and CO123 of humans and Javan macaques

Ka (I 50) Ka (I 50) Ko (5 50)

pAVT-A mediates redirected cytolysis by human or cynomolgus T-lymphocytes pAVT-A mediated redirected cytolysis of target cells by human or monkey effector cells against CO123- leukemic Casipti-3 cell lines (Figure 27A-270)), which was accompanied by induction of activation markers. No activity was observed against CO 123-negative targets (0937 cells) or with control SACT, indicating that T cell activation is strictly dependent on target cell engagement and that monovalent engagement of POPs by UACT-A was insufficient to trigger T-cell activation. Since CO123 is expressed on a subclass of normal circulating leukocytes, including white blood cells and monocytes (Figure 27E), the effect of OAKT-A was further investigated in normal PBMCs of humans and monkeys.

A step-change effect was observed among human PBMC with a dose-dependent rapid depletion of CO14-CO123gog cells (ros and basophils) observed no later than 3 hours after the start of treatment, while monocytes (CO14- cells) were not affected at this time point (figures 27-27). The depletion of СО14-СО123"9" cells increased over time at all concentrations of the SUAKT-A molecule, while the number of monocytes slightly decreased up to 6 h. and was depleted after 18 hours and at concentrations higher than 1 ng/ml. Incubation of monkey RVMS with

RAVT-A resulted in a comparable dose-dependent depletion of СО14-СО123"9! cells (Figure 27H), further confirming the relevance of this species for OAKT-A pharmacology (СО14- monkey cells express little or no СО123, and they do not undergo depletion).

Pharmacokinetics of SACT-A in Javan macaques

The cynomolgus macaque was chosen as an acceptable pharmacological model for the analysis of OAKT-A based on the equivalent distribution of both target antigens in this species compared to humans based on immunohistochemistry with pAbr precursors, according to published information (Mypo;, ІІ. еі аі. (2001) "IpiepneiiKkip-3 Veseriog AIrpa SNaiip (СО123) Iv U/iaeiu

Ehrgezeid Ip Netaiiodis Maiidpapsie5, " Naietaiiodis 86:1261-1269; Kogreiaipep, E.I. ei ai. (1996) "P/-3 Veseriog Ehrgeziop, Neashayop Apa Eipsiip Ip SeiYiiz OYI Tne Mazsshaishge, " Ittipo).

Koo) Sei! Vioi 74:1-7).

A study conducted according to the present invention would include treatment groups consisting of 8 cynomolgus monkeys per group (4 males, 4 females) (Table 8). All groups received an inert vehicle - control for the first infusion; then the inert vehicle or SUACT-A was administered intravenously for 4 weekly cycles. Animal group 1 received an inert vehicle control for all 4 subsequent infusions, while groups 2-5 received weekly increasing doses of SACT-A for 4 days per week for all subsequent infusions. Animal groups 6 were treated with 7-day continuous weekly escalating doses of OACT-A for all infusions. The 4-day administration/3-day no-administration and 7-day administration schedules were designed to distinguish between long-term and transient effects associated with SACT-A administration.

Two males and 2 females per group were killed at the end of the treatment phase (day 36), while the remaining monkeys were killed after a 4-week recovery (day 65). A subset of monkeys developed anti-drug antibodies (ABA) directed against humanized Em to both POPs and CO123, and data after the occurrence of ABA were excluded from LC analysis. All monkeys were exposed to rAVT-A during the study period.

Table 8

Inert (4 days of administration/C days without administration) (7 days

Me infu- Days of carrier ng/kg/day of administration) sion of experiment- Ing/kg/4 days| ng/kg/day Ing/kg/7 days 1 2 Z 4 5 (5) carrier carrier carrier carrier carrier carrier with n carrier 400 400 400 400 700 carrier 400 1200 1200 1200 2100 carrier 400 1200 2400 2400 4200 carrier 400 1200 2400 4000 000

After the hay, we went to the north of the field

The two-compartment model was used to estimate the parameters of the RK (table 9 and figure 28). Tiga was short (4-5 min.), reflecting rapid binding to circulating targets;

Ti28 was also rapid, as expected for a molecule of this size that undergoes renal elimination. Analysis of serum samples collected at the end of each infusion from group 6 monkeys showed a dose-dependent increase in Stach OAVT-A. In Table 9, the inert medium was

РВ5, pH 6.0, containing 0.1 mg/ml recombinant human albumin, 0.1 mg/ml P5B-80, and 0.24 95 benzyl alcohol was used for all inert vehicle infusions during the first 4 days of each infusion week, followed by administration of the same compound without benzyl alcohol during the remaining 3 days of each weekly infusion. OAKT-A was administered at the same time points as a continuous intravenous infusion of PB5 solution, pH 6.0, containing 0.1 mg/ml recombinant human albumin, 0.1 mg/ml RZ-80, and 0.24 95 benzyl alcohol in the required concentration.

Table 9

Two-compartment analysis of SACT-A RK parameters in Javan macaques (mean 50) (mean 50)

Cytokine release in Wag-A-treated Javan macaques

Given the properties of OAKT-A in terms of T cell activation, an increase in circulating cytokines accompanying the infusion was anticipated and therefore a low initial dose was used as a "desensitization" strategy based on previous data obtained with similar compounds (see, e.g. , Torr, M.5.

Se I-Epdadipu Apiirodu Viipaiitotab OI SpetoiNpegaru-Veitasiogu Mipita! Star! Bizease Ip B-

Ipeade Asshie I utrpobiaviis I eiKetia Raiiiepiye Neziyv5 Ip Nidp Rezropze Naiee Apa Rgoiopdea

Y enketia-Egeye Zyguimayi, " in. SiIip. Opsoi. 29:2493-2498; Vagooi, V. evi a!. (2008) "Titog VRedgezviop

Ip Sapseg Raiepiv Vu Megu Gom/ Rozez OII A T SeiI-Epdadipud Apiirodu, "Zsieepse 321:974-977). Of the studied cytokines, I/-6 demonstrated the most significant changes during infusion, even if temporary in nature, of minimal magnitude and with with greater inter-animal and inter-group variability (Figures 29A-29C).Small, transient changes in II/-6 were also observed after inert vehicle infusions (all infusions in group 1 and all infusions on day 1), indicating the sensitivity of this cytokine to manipulation stress However, OACT-A-dependent increases (80 pg/ml) in serum I-6 were observed in some monkeys after the first infusion of OACT-A (100 ng/kg/day), which returned to baseline to 72 h. Interestingly, the magnitude of release of 1-6 decreased with each successive infusion of BACT-A, even as the dose level was increased up to 1000 ng/kg/day.Also, minimal and transient OACT-A-related increases were observed

TME-a in serum ("10 pg/ml); as in the case of I/-6, the highest amount of TME release was observed after the first infusion. No SACT-A-related changes in levels were observed

I -5, IP -4, I/- 2 or IRM-u during the entire study in comparison with controls. It can be concluded that the release of cytokines in response to treatment of monkeys with OAKT-A was minimal, transient, and was a first-dose effect controlled by increasing the dose within a single subject.

SAVT-A-mediated depletion of circulating СО147/С0123: leukocytes ip mimo

The absolute contents of circulating ХОО14-/20123-ж- cells were measured during the study as a pharmacodynamic end result. While the number of CO123" cells in control group 1 remained stable over time, treatment with SACT-A was associated with a large depletion of circulating CO14-/C0123- cells (94-100% from baseline before the study), observed starting at the first measured time point (72 hours) after the start of the first OACT-A infusion (100 ng/kg/day) in all animals in all active treatment groups (Figures ZOA-30C).

C-day weekly dose break in group 2-5, returning to baseline levels only during a prolonged recovery period. To eliminate the possibility of rAVT-A masking or CO123 modulation (an unlikely scenario resulting in low levels of circulating

BAVT-A), the amount of ROS was calculated using an orthogonal marker, SOZ303.

According to the data for СО123, SOZOZ-ros were similarly depleted in monkeys treated with SACT-A (figures 300-ZOR).

From the content of circulating T-lymphocytes, activation and analysis of subclasses

In contrast to the sustained depletion of circulating CO123 cells, OAKT-A administered in a 4-day on/3-day off schedule (groups 2–5) was associated with weekly fluctuations in circulating T cell counts, with administration in the form of continuous 7-day infusions led to a similar decrease in circulating T-cells after the first administration, which slowly recovered without fluctuation even during the period of administration of doses (Figures 31A-31C). The difference between the two dosing strategies indicates that the effect of UACT-A on T lymphocytes would be consistent with cell migration and/or clustering at the edge of the inflammatory site, rather than depletion. After cessation of dosing, T cell content unexpectedly rose to levels approximately 2-fold higher than baseline for the duration of the recovery period. UACT-A infusion was associated with an exposure-dependent, progressive increase in the incidence of T cells expressing the late activation marker, PO-1, particularly on CO4- cells, with dose group 6 showing the highest total counts (Figures 310-311 figures 32A-32E and figures ZZA-ZZE). Tit-3, a marker associated with T-cell exhaustion, was not detected on CO4.T-cells and only at a low frequency among

Sov of cells (5.5-9.7 95) and contained 20.5-35.5 95 СО8-/РО-1- double-positive cells. There was no consistent change in the marker of early T-cell activation, COb9, and only small changes in CO25 expression among circulating cells.

To rule out exhaustion after ip mimo exposure, ex mimo cytotoxic potential of effector cells isolated from Javan macaques receiving multiple infusions of OAKT-A was compared with this parameter in naïve monkeys. As shown in Figure 34, PBMCs isolated from monkeys treated with OBACT-A showed cytotoxicity comparable to that of cells isolated from previously naïve monkeys, indicating that the effect of SACT-A is not had a negative effect on the ability of T cells to destroy target cells.

The influence of SACT-A increased the relative frequency of occurrence of central CO memory cells and effector CO8 memory cells at the expense of the corresponding T-cell population not previously exposed to the effects (figures ZBA-35E and figures 32A-32E and figures ZZA-Z3ZE), indicating that exposure to OAKT-A stimulated the expansion and/or mobilization of these cells.

Effects on hematopoiesis and bone marrow progenitors rAVT-A was well tolerated by monkeys at all tested doses; however, reversible decreases in erythrocyte parameters were observed at the highest doses (figures ZbA-36C). Rapid blood sampling could potentially contribute to this, as animals treated with an inert vehicle showed a slight decrease in erythrocyte counts.

A reticulocyte response was observed in all animals; at the highest level of exposure (group 6), however, the response was somewhat less pronounced for a similar decrease in the number of erythrocytes (Figures 360-36E). Morphological analysis of bone marrow smears during the study was unremarkable. Flow cytometry, however, revealed that the frequency of CO123 cells within the immature cell line-negative (Hyp-) populations of bone marrow decreased in SACT-A-treated animals at the end of the dosing period, returning to baseline at the end of the dosing period. reconstruction period (figures 37A-37B). H5C (defined as I ip-/2034-/2038-/2045BA-/С090-- cells (Rapa,

MU MU her a. (2011).

Mueioiid-Viazei MUyi Ade, "Rhos. Mai. Asad. si. (0O.5.A.) 108:20012-20017)) showed greater variability between groups; monkeys treated with BACT-A, group 4- 6 showed some apparent reduction from the respective pre-dose levels, however, no reduction was observed in all treatment groups compared to vehicle-treated animals.These data indicate that

NOS were less sensitive to targeting with SUACT-A and would be consistent with the observed reversibility of the negative effects of SUACT-A treatment on hematopoiesis.

As demonstrated above, in terms of infusions over 4 weeks on a weekly schedule of 4 days of administration/3 days off or on a schedule of 7 days of administration every week from initial doses of 100 ng/kg/day, which are increased in stepwise fashion every week to 300, 600 IU 1000 ng/kg/day administration of OACT-A to Javan macaques was well tolerated. Depletion of circulating CO123 cells, including ROS, was observed after the first administration and continued throughout the study at all doses and regimens. A reversible decrease in the bone marrow precursor СО123 was also observed. Cytokine release, as a significant safety hazard in POP-targeted therapies, was reversible and consistent with the effect of the first dose. Slight reversible anemia was noted at the highest doses, but not noted

No other (related to the purpose of exposure or not related to the purpose of exposure) adverse effects.

The Javan macaque is an acceptable animal model for pharmacological evaluation

RAVT-A, given the high degree of homology between the orthologs and the ability of RAVT-A to bind with similar affinity to antigens and mediate redirected T-cell cytolysis in both species. In addition, both antigens are consistently expressed in monkeys and humans, including similar expression by hematopoietic progenitors and in the cytoplasm of the endothelium of numerous tissues. Minor exceptions are expression in Leydig cells in humans rather than monkeys and low or absent CO123 in monkey monocytes compared to humans.

The primary focus of therapeutic strategies involving T-cell activation involves the release of cytokines and is not related to cytotoxic effects. A recent study with POP x CO123 bispecific immune construct fusion of 5Cem with bivalent recognition of POP demonstrated anti-leukemic activity of ip miigo, but caused non-specific activation of T cells and secretion of IEM-u (Kyo, 5.K. ei ai. (2012) "Epdipetipa A СО123хХСО3 Vivresiis vsEm Ittipoiibviop Rog Te Tgeaitepi OI GeshKetia Apa

The monovalent nature of each of the binding arms and the highly homogeneous form of SACT-A ensure that

T-cell activation depends exclusively on the involvement of target cells: T-cell activation was not observed in the absence of target cells or when using the control rAKT molecule, which contained only the arm of targeted influence on POPs. In addition, high doses (up to 100 μg/kg/day) of the control OAKT molecule did not trigger cytokine release in Javan macaques.

An initial dose of 100 ng/kg/day of the SACT-A molecule was well tolerated with minimal cytokine release. However, the cytokine storm did start at a high starting dose (5 μg/kg/day); however, this dose could be reached safely with stepwise weekly dose increases, indicating that VACT-A-mediated cytokine release is likely to be primarily an effect of the first dose. Depletion of CO123- target cells, thereby eliminating the source of ligation with POPs, may explain the effect of the first dose: almost complete depletion of CO123- cells was observed at doses not exceeding 3-10 ng/kg/day, indicating that that in the release of cytokines, a shifted dose-dependent effect is followed in comparison with cytotoxicity. The dose-response profiles for cytotoxicity and cytokine release by human T cells are also consistent with the presented observation.

T-cell desensitization, in which BACT-A-mediated upregulation of ROT may play a role, also appears to contribute to the limitation of cytokine release after the first SOACT-A infusion. Recent studies show that increased expression of PO-1 after antigen-induced arrest of T cells at sites of inflammation contributes, through interactions with PO-I 1, to the termination of the stop signal, thus releasing and desensitizing the cells (Nopaa, T . ei ai. (2014) "Titipud OYI Apiidep Zepzyimku Vu T Seii!I Keseriog-

ЮОеерепадепи Медаийме Реедраск Зопигоие T Se! Empesyug Rypsiip Ip Ipyatei Tivvtsev, " Ittipyu 40:235-247; MMei, RE. ei ai. (2013) "Zmepdiy OYI RO-1 Zidpaipu OiyegepiaPu Anesiv T-Sei! EPesiog

Eipsiyop5, "Rhos. Mai!. Asai. 5si. (0O.5.A.) 110:E2480-E2489). The antidote of RO-1 signal strength

TSC is not informative: in addition to the fact that proliferation and cytokine production are most sensitive to PO-1 inhibition, the effect on cytotoxicity is minimal (Umei, E. ei ai. (2013) "Zygepaii OII RO-1 5idpaiipud OiiiyegepiiaPyu Anesiv T- Seyi EpNesiug Eipsiyop5, " Rgos. Mai!. Asad. zZsi. (0O.5.A.) 110:E2480-E2489). Consistently, the cytotoxic potential of T-cells from monkeys exposed to multiple infusions of SACT-A was comparable to that of T-cells from monkeys previously not exposed to experiments, despite the increased levels of PO-1 in the former. In addition, the upregulation of PO-1 was not accompanied by the expression of TIMZ, a hallmark of T cell exhaustion, as shown for T cells subjected to long-term antibody stimulation

POPs or chronic infections (sSebei, N.M. ei a). (1989) "T SeiI5 Egot Raiepiv Bissez5ishu Tgeaiei

Mun OKTZ bo Moi Veasi M/N Tne T-SeiI Veseriog Apiirodu, " Nit. Ittipoi. 26:123-129; MNe!tu,

E.9. (2011) "T Sei! Ekhnaiviyop, " Mai. And so on. 12:492-499).

Depletion of circulating CO123- cells in SACT-A-treated monkeys was rapid and continued during the weekly dosing break in a 4-day on/3-day off schedule, consistent with elimination of target cells. In contrast, temporal fluctuations in the number of circulating T cells were likely the result of migration from/into tissues and lymphoid organs in a VOAKT-A-dependent manner. The effect of SACT-A stimulates the expansion and/or mobilization of antigen-exposed T-lymphocytes, cells that are mainly located in tissues and faster

Zo exhibit a cytotoxic effector function (Migepaa, UM. ei a. (2007) "Rpuzioiodis Apa Abeitapi

Vedshiaynyop OI Metogu T-Sei! Ttapiskipd Wu Te Soviitshayugtu MoiIesshe CO28," Viosa 109:2968- 2977; Magee-Wegu, R.M. ei ai. (2010) "Metogu T-Sei Tgtaiiskipd: Mem Oigesiope Rog Vivu

Sottiegv, " Ittypoiodu 130:158-165).

Depletion of СО123.- normal cells can predict potential risks. ros and basophils express high contents of CO123 in comparison with reduced contents in monocytes and eosinophils (oreg, A.R. Yei ai. (1989) "Vesirgosai! Ippibyiop OY Vipaipu Veimeep IpiepecyKkip Z Apa Stapiiosuie-

Mastornade Soiopu-5iitiiaipoa Rasiog To Nytap EozipornNiie, " Rgos. May). Asad. osi. (0.5.A.) 86:7022-7026; Mipo, Y. ei ai. (2001) "IpiepeshiKip-3 Veseriog AIrpa Spaip (SO123 ) Iv Mideiu

Ehrghezzei Ip Netaiiodis Maiidpapsiev5, " Naetayuiodis 86:1261-1269; Mabviep, V.. yei ai. (2006) "SNagasiyegigayop OY Muveioii Apa Riaztasuyuy YuOepagtyis Seyiiv and Nytap Gypd, " U. Ittipo). 177:7784-7793; Kogreiiep, Kogreia E.I. ei ai. (1995) "Ipepegop-Ssatta Orgeashiaiez IpiepestskKip-Z3 (1-3)

Veseriog Ehrgezviop p Nytap Epaoineia! Seijv Apa Zupegdige5 M/P 1-3 Ip Biitsiaippo Maiog

Niviosotraiiibiyu Sotrieh Siavz II Ehrgeziop Apa SuyukKipe Rgodisip, " Viosa 86:176-182). Ros has been shown to play a role in the control of certain viruses in mouse or monkey models of infection, although they have not been shown to be critical for controlling the immune response in influenza ( Soioppa, M. evi ai. (1997) "Zresiisnu Apa Ripsiiop OI Ittipodiobiiip Zirepatiu MK Seii Ippiriogu

Apa biiytiiashyugu Veserioghe5, " IttypoiY. Vem. 155:127-133; Ztii, U.u. ei a. (2006) "Riaztasuyia

Oepagtyis Seiyiv Ippirii Ritopagu Ittiporaioiodu Apa Rgotoiee Sieaagapse OI Rezrigayugu Zupsuiai

Migi5, " U. Ehr. Mei. 203:1153-1159). ROS tumor models can stimulate tumor growth and metastasis, while depletion of ROS resulted in tumor inhibition (Zauapi, A. ei ai. (2012) " Seyiyv Ipirye Tytog StoulLY Apa Rgemepiv Vope

Meiausziazie OY Vgeabzi Sapseg Seiv, " U. Ittipoi. 189:4258-4265). Transient, minor, dose-dependent facial swelling was observed in some monkeys treated with UAKT-A; however, these monkeys did not have elevated levels histamine or in the case when human basophils were lysed by VACT-A-mediated T-cell lysis.

Monocyte depletion may predict potential risks of infection; consequence of ROS, basophil or eosinophil depletion in humans should therefore be monitored.

Committed hematopoietic progenitors that express CO123, such as common myeloid progenitor (CMP) (Chogaap, S.T. eyi ai. (2000) "Tne IpiepecshKip-3 Veseriog AIrna Spaip Iv A Opidie 60 MaiKeg Bog Nytap Asshe Mueveiodepoiv I eikEtia biet SeiIv , " | eoKetia 14:1777-1784; Wiedeg,

MA. oh oh (2012) "Netaioroiyev, " Soyd Zrgipd Na. Regresi Vio!. 4:24008250), may be targeted by UACT-A, which is a possible explanation for the slight anemia observed after administration of the highest dose of UACT-A. Presumably, the erythropoietic reticulocyte response operates at all levels of the dose of UAKT-A; however, relative to adequate reductions in erythrocyte parameters, animals exposed to the highest doses of SACT-A (group 6, 7-day infusion) showed a reduced reticulocyte response, indicating possible cytotoxic activity on progenitors (eg, CMP). The effect was reversible after cessation of SACT-A treatment, consistent with repopulation from preserved NOS with low or no CO123.

Alternative approaches for the depletion of CO123 cells contain a specific aspect

CO0123 is a second-generation monoclonal antibody with an enhanced Es region (ip, Y. ey ai. (2009) "Moposiopa! Apiirodu-Meadieda Tagoeiiipd OY CO123, 1-3 Veseriog AIrpa Spaiip, Eytipa(iez Nytap

Ascie Mueioia I eiKetis 5iet Seiyiv, " SeiYi biet Seii! 5:31-42; Vobegiv, A. MU. ei ai. (2010) "A RNaze I zishau OG Apiii-sa1i23 Moposiopa! Apiibodu (tar) C5I 360 Tagdeiipud GeiKetia 5iet SeiYiv (I 5C) Ip

AMI, "U. SiIip. Opsoi. 28(Z,irri):e13012), related to 1-3 diphtheria toxin (Rhapkei, A. ei ai. (2008) "Rpavze I Siipisa! idu OI Oirpipetgia Tohip-IpiepeitsykKip From Ryviop Rgoivip Ip Rayepiv M/N Asshe

Muye!oia Geschketia Apa Mue!Ioduzriavia, " Geik. Gutrpota 49:543-553), cytokine-induced killer cells (KIK) expressing CO123-specific chimeric antigen receptors (SAC) (Tepatagpii, 5. ei ai. (2013) "Tagdeiipu Oi Ascie Mueioia I eikaetia Wu SuyukKipe-Ipdysed KiPeg SeiiYiv

Vedigesived M/yN A Mome! СО123-5resiis Spitetis Apiidep Reseriog, " Vg. U. Nayetatio)!. 161:389-401) and 60123 SAC T-cells (ACTIONS, 5. ei a. (2014) "ENisasu Adaipvi Nytap Ascshie Mueyiyoia I eiKetia Apa

Mueioabiayop OY Mogpta! Netaiyoroieziz p A Moise Mode!i O5ipd Spiteps Apiidep Veseriog-

Moaiiea T. Seyiv, "Viosa 123(15): 2343-2354; Magaigov, A. Yei ai. (2013) "T SeyI5 Ehrgezzipd CO123- zresiis SPitegis Apiidep VNeseriogv Ehpirii Zresiis Suyuoiyus EpPesiog Eipsiop5 Apa Apiitog

Enesiv Adayipvi Nytap Asshiye Mueiyoiia I eiKetia, " Viosa 122:3138-3148. SAV T-cells revealed pronounced cytolysis of leukemic blast cells of the myoblast and anti-leukemic activity in a xenogeneic model of disseminated GML (Magaigo5, A. Yei aI. (2013)) " Se Ehrgezzipd СО123-5resiiis

Spitegis Apiidep Neseriog5 Ehpirii Zresiis Sushuiuis EpPesiog Rhypsiopbe Apa Apiishtog ENesiv

Adaipzi Nytap Asshie Mueioi IGeiKetia, " Viosa 122:3138-3148). In a recent study

Ablation of normal hematopoiesis in M5O mice transplanted with human SOZ34h cells after transfer of СО123 SAC T cells was reported (Si, 5. ей а). (2014) "EmPisasu Adaipwi

Nytap Ascie Muevioid I eiKetia Apa MueiYoabiayop OY Mopta! Netaiyoroiievziz p And Moize Moavbvi!

Ovipa Spitetis Apiidep Neseriog-Moaditiea T Seiyiv, " Viosa 123(15): 2343-2354), although other authors did not observe similar effects of ip migo or ip mimo (Tenatapii, 5. ei ai. (2013) " Tagdeiipud OY Ascie

MueyeiYoid IeshKaetia Wu SuiyoKipe-Ipdysed Kieg SeiI5 Vedicgesiei M/yN A MomeI СО123-5resiis

Spitegis Apiidep Neseriog, " Vg. U. Nayetaiyoi!. 161:389-401; Riggiuia, I. ei ai. (2014) "Spitegis

Apiidep Neseriogyi5 Adainsii SOZ3Z/СО0123 Apiidep5 EPisiepnu Tagdei Rgitagu Ascie Mueioid I eiKketia

Seiv ip mimo, "GeshKetia adoii:10.1038/Ley.2014.62). In the experiments discussed above, depletion of CO123- populations of bone marrow progenitors was observed, but it was reversible during the recovery period; moreover, depletion of this small population did not lead to changes in saturation bone marrow cells or in the erythroid to myeloid (E:M) ratio at all SAKT-A dose levels tested. These differences highlight the potential advantages of SAKT-A over cell-based therapies as it provides a titratable system that is based on autologous T -cells as opposed to "overefficient" ex mimo-transduced cells, which are more difficult to control. CO123 is overexpressed in some hematological malignancies, including AML, hairy cell leukemia, blast plasmacytoid dendritic cell neoplasms (BPDC), pre-B-cell acute lymphoblastic leukemia (B-CLL) and chronic lymphocytic leukemia, Hodgkin's disease with the presence of cells Reed-Sternberg, as well as in myelodysplastic syndrome and systemic mastocytosis (Kpapap-Yuraba), M.A. oh oh (2013) "Oiadpoviis Apa Tnegareshis Admapsez Ip Viaviis

Riaztasuioidy Oepaiiis SeiY Meoriazt: And Rosyv Op Netayoroiviis Seiy! Tgtaperiapiayop, " Vioi. Viosa

Maitom Tgaperiapi. 19:1006-1012; Riopap, 5. yei a. (2006). Gutrpota 47:207-222; Mipo, Yi.. yei ai. (2001).

Netaiodis Maiidpapsie5, " Naeetaiodis 86:11261-1269; Egott, 9.8. (2011) "Rome Suiteiis

Apaiuziz OT СО123 И5 Ovzetsi God Ittyporpepoiuripd Siazzis! NodokKip Guprpota, " Suyuoteigu V

That's it. Suiot. 80:91-99). The predicted pharmacodynamic activity and controlled safety profile observed in nonhuman primates further supports the clinical applicability and efficacy of SACT-A as an immunotherapy for these disorders.

In conclusion, it should be noted that SACT-A is an antibody-based molecule that engages the TSE subunit. to redirect T-lymphocytes against cells expressing

СОр123, an antigen that undergoes positive regulation in some hematological malignant tumors.

RAVT-A binds both human and Javan macaque antigens with similar affinities and redirects T-cells of both species to destroy CO123 cells. Monkeys infused 4 or 7 days per week with weekly escalating doses of UACT-A demonstrated depletion of circulating CO123- cells after 72 h. after the start of treatment, which lasted for 4 weeks of treatment, regardless of the dosing regimens. A decrease in circulating T cells also occurred, but recovered to baseline by the next infusion in monkeys treated with a 4-day dosing schedule, consistent with YACT-A mediated mobilization. Administration of SAKT-A increased circulating ROT, but not TIM-Z, T-cells; in addition, ex vivo analysis of T cells from treated monkeys revealed unchanged redirected lysis of target cells, indicating no depletion. Toxicity was limited by a minimal temporal release of cytokines after the first COACT-A infusion, but not after subsequent administrations, even as the dose was increased, and a minimal decrease in the number of erythrocytes with a concomitant decrease in CO123h-bone marrow progenitors. A clinical trial of SACT-A in hematological malignancies is justified.

All publications and patents mentioned in this description of the invention are incorporated herein to the same extent as if each individual publication or patent application were specifically and individually incorporated by reference in their entirety. While the present invention has been described using specific embodiments thereof, it should be understood that additional modifications are possible, and it is intended that this application cover any changes, applications, or adaptations of the present invention, adhering generally to the principles of the present invention and including contain such deviations from this disclosure as are consistent with known or generally accepted practice in the art to which the present invention belongs and which may be applied to the essential features set forth herein above.

LIST OF SEQUENCES

"Those MACROGENICS,. Inc.,

Bonvini, Eziz

Johnson, Leslie

Zhudn, Lin

Moore, Pozh

Chichili, Gurunadh Dyaderson, Ral "IA" BISPENIFIED MONOVALENT DKATILYAA THAT ARE ABLE TO DISTRIBUTE

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СЕКТ ССсКТвІ СЕСссЕребЗа зЕссссТКссС ЗвСтсССсостТа ївЕВВсВстТІ сЕТЕЗІваса КЕ сВЕТСТССЕш ВІОБІСТЕВЕС СЕТЕВБССТШ БЕЕРБаБІврЕ їрастВТЕїС тірсвадаес Уа тсесарієвс твсІваасав СЕБаваїісваЕ зававстаїтсє ГїввсстІррІв ссавгаравв Ве кевшЕСсвЕс сссотаВвВсу шстТЕВІсСтаї їрБЕКСІКСЄСВ ССаЕрЕВАТС ТЕБСЕКрЕСЄ Ук васяЕВсІса ЕСЕРІВРІвв сСаЕсовсасВ вВАТТіВссс Єсвовасстс тарісїссав ще

VISEVZREVSE TEESSTsteta StvitiEisyaad zaivaisasv detvissstv Sas Ede Tada savEEBassa VESSirEVavi TyavavBBaBES BEZTSSEVESV BSBEBaBESEa BEBESavBste iZ55

ЕКЕЕВЕТСТр ЕвЕБасВСТХ врІссавсст БЕВБЕЕКССІ Теврастстс стІвТЕсавсЕ ї1158 їстрЕВІїсВз ссїТїсавсВс абасЕСТаТЕ ЗАТЕ ЕЇСС ЄССаББСЕСС ВЕРЕС ЕБЕ т2а сТЕвВЕтрЕвс ТтІЄєБавеває саврріскаве Тасвасзаїтї взікгавсста статвЕствах ї25о їсте ваврЕ ВвіВврастсяас саїсісвава пзідвіїсаа аравстсвсї вдтаїстосав Іа азівавсавсс їраваассва сшасасврІс ше Еасї ЄТЕТРарВсВ сЕБТаВТТє їз

BEEsSaVVIESET All EKS SVI TERREVsaRE EsVsasIDEH nast iz43 issteEBanEVI BIBBssvshtivv zazawnivris vsaossisvane zrazavttvs svBOTTTtvava Ia daEjasETSE SirdSVIiiVa Esavavavvt rivRSSSira VaBvash i545 whales" from "SHI1i" du "guide" RNU "213

Juices ok:

"3235 First Polypeptide Chain of the Control PAT cha" 15 dev Uvi Uvi Mei TVgobBiv TVgoBgo Bve ego iv Bga Uvi ek iv shchiu i 5 Iia ii5 dor pip dia 5eg Iii zeg osSe5 dDgE o beg back dinner bZegoivez Uvi Niya bag a de 3 dep bit A5p ogo itK ish AkBsh Tgroiub yiv Bip iu5 Rgo Bi iya Bag 35 yav 45

Rgo from Uvi iv ii Tur ie Uvi bEeeE dhp o yGE Be bek bi Udi ge za V dav VgE VNe bZeg Bi bBeg o bi Ber obi TBg od5o vBe TBgoiev i95 1

Ua t wa ---g ygEB Uvi BIS: biz Bim der iv piu Mai TUugb Be Su5 Bebg biv bag a still pa

TVe nih Fai Rgo Tr otr ovNe Bi b b b TB o iiuz iv 53 ii 155 shcha i8a 115 syn Shchi Ziu Beg Shchi shchiuU bi ZI Ziz Uvi biy ish Uvi bib bek i yije15 ia 195 bi Bi iz Uvi bip Rho bi BR bBeb yvi brE evts bZeg obSuv Viz from 138 Ii5 Ia

Bek pi RBe Tag Re dm TVg oTUuk dia Mei dl Tho Uvi dra biv Aiz 155 155 i55 158

RO bIiU ik5 BI gen Bim Ter o Uvi Atia AbE 118 AgEb bZeg ofuh Tuk vp y55 ET 195

Asp o tTUg div TK o yUR TuUub Aia A5a beg Uvi ih Ar dk o vBBe Tvg o tiv y5a yd5 ia ek BGE Ak yhr bek i y5 Ahp bek yiv Tugk ivgv Biy MeE dov bey oi vu y55 zva V yUu Tak o biv y5o TVg o yiz Maii TUuk Tuye Suz Udi AgE Ni5 Bi dp RBe

IV 25 va

Z b5p Bek BUG Khvi Bek Tgr obme diz Tug iga di bij ZA iVBg o yiva

IB in 35 AH chai TE Uzi Zeg ozZag shchi Bi huk Bi Bi BI piv Uvi biz Viz ate; 45 and 55

Ziv i5 biv Uvi diz biz iv Bib iUu5 bib Udi iv diz iv" bii yuuh 2858 5 a

Ziz Uvi div Viz iv piz ik sich "Ii" and "ii" in "Tt12U RET "2133 Artificial Puslidoannost "BU "223" Friend Peptide Chain of Control BVET "IN" ZV

Zip yid Uvi vi Tng piz bij Bho zer oyer Tk Uvi Zek Rgo siu Bi ii ih IV i5

Te uv te oivgu TB osu5 dev bek obee TV Bi bid UVDE Te tTVvg o bek eV shi BI dep otUg div Or tvo Uvi bib bij Geo Re Bi biv yiz Rg AgsE Bi 35 V 5 yen; Tie bi vu TBg dai is yGE Diya Rg Tr oiir o Bgo Aiva Age WBe i 23 vag obi zZvgoivu iva zi bi Uh biv viv iec im Ti Te Z div 5 KE U5 da biv Aza biz vb5v biv action DV oTuk TUug Sub Aia iv TO otTuk zve v5a shcha 5 iets igb5 Udi Re biu pi bi TT oi 5 iv Te Uvi iv zi Zi bi dav 185 118 bik zZek Zi Bi biu pi Bi: Uvi iuUu5 iv Av oshiz Te Bi schi iu iza Ii iev Uai biv Rgo bi agavy Rga Met ii iv bee SUu5 Udi biz bek 15 ih ku ia

TVe tvg o Bbe 5eg od5r tTuk Tgo Mei yb5ap Tag oUvi be bij Zek Rgo Bim z35 yash yi55 IB ich pi vu BI: igr Uvi Aiva biv Ti db Ahp oi Be tugo Dhv tuye ii155 IT Tt5 siv tag therefore Tag begoiboo Bek Uvi ie5 bi dB Re TK Tiz zve two id 155 98

V5v Ah Zag oiuUuh ego zZeg Uvi TU o ivn biv Met yhp iv iv AgE Udi

IZ zva 285 pis dv BEH piU Be Tuk Buk Se Tvgobib bek Tek te Zi Mei dav

MKEsYA 215 ha

Tk tega vi BIB BI tve bek Uvi tBBoUai ev bek bi Bi Suye biu

ZV 235 tav

SiUu Bi gu5 Mai biv iz iv i bin iuz Uvi 518 biz ge GUukh5 Bits ge BV sha5 izhe Uvi iz biz iv iz Bi" ie Uvi div Div iv i" Bi

Ba VU g "iz gi "shchiev 118 eiiih RT "21i3z Artificial Pulse Sequence se "223 Light Chain of SOZ-Binding Domain of PACT-A. cha" 21

Bib At Uvi Udi ivg obi bi: Ro Zeg oiev TK ovi 5Beye ho Shchi s1u i 5 Из их

Tng o Uaiii Tvgoi vo Te oSukh5 dv back zago TK obi iv di TK o TVKE ve and TV ZV

Vp otUug Div Aza oigvr uv bip viv gu5 Reo bib ziv Aza Rgo AgЕЖ schu

З5 вк «вх ив; ієе Z si Tbg o ykhp i AB Div bka tgb TB o Rgo DEa dr BBe pa wa ek obBiu Zag oigcs ivcs Zi» siu EUu5 Viz viz iets o iBb gi Tv Bi dia still TV ta va

Z aiv Bin Vr biv biz dB o TUk TUg o Yeuz biv iv o TkKrotiuk Bek ep a SYA 5 ivm tKrouUyai Ree si shiu bib TE og e5 iv BT Uvi ivets BITu for 185 it "EI" 25 "zhiyiz 135 "kiz" RT " 2135 Artificial Sequence of "823 Nazhkhmy Chain of CO3-Binding Domain of PAKT-B. "ah 25 ziv Uvi bip oiso Uvi biv zZeg o Zi shchiu BI iv iv: Uvi Biy God shchi ih i Ki IV 15 zvk iv o ADgvoivgu Bek iz Div Aa bek o bi Be Te ve o beg tk tuyu za 5 ZV liz Mei yza treb o Uvi agE biz Wiz Bko bi iuz Zi i: Bib TgB Uvi

ZiU AgE Ge ygr Bek oi5 Bug Aa ysap Teg Aza yBg o tuk TUe Ava Abo 55 B ve Uvi uh ykhv AgE RnNe TK Ife ego AgBBE v5r o y5r bek Ye u5 dr beg 65 and 75 still ev Tuk igen biv Mei v5y Zeg ben Buch TR OBES Aqua TV o Aha Uvi tuye 5 IsEoya 5

Tuk KSukh Uvi dzhsh Ni shchiu yzp Be BiU yzpobeg o tTug Chai beg TKr o Rve ha Ii it

Viz yishk yiv Bib BI Bi TK yiv Uvi TBg ovi Zvko5Zeg 5 iz I "yiv 23 i21iz B kiy RE "213" Vtuchna Pyusodlnost "loh 223 DEgknn Dantsyug KO3Z- Binding Doven PAKT-V aa" V

Vhr Yyye biv iv o ivgb biv bek RKO iv Bie KMei 5eg iv Zeb o rgo bu ii a IV i5 gub Uvi tTve Mei Teg o Seh dgB ia Zeg bBeg Beg Uvi Beg Tugb MEYE i Mk: za

Zav iKkv oTUuk biv biv iiUu5 bek BZfu TBgeoyeg Bgo gu dgsh o Tgo o Tiv Tugb i5 45 45

Aha TK bag oiuh Udi Az bBeg Bi Udi Bko tugo YabE BBe beb ie bek 5 for a

Zi bek yiU tre bek o Tub bek ate TV TIV Zee oves MEZH 15 AR shi 5 U 5 shcha

Av At div iBK oTuk Tag Se Bi Bii Tcrobeg Zeg dhy RKO iv otTvg yaz za Za

Be ih Aia Bi yivpg oiuh geo ish gecz yuh iv 15 "VivVk va "ISHE TEZ "2325 RET "213z Artificial Persistence "OVU "23" Heavy Chain of SOZ-Za"jasing Lumen PDAET-V "408 for ihv YIT2e iU5 iv biv Shy Bek BI biz bis ivou yiv AgE RgGO Pih div ii 5 iv Ii bEgoUvi ie5 Mei ego osSukh ie5 TB obeeB bi Tu TR Be tvg odgd o tuk o; 5 BI:

Te Me Ni Ter oMai ge5 bip As Rko Bi Bip pi ivc Biz Ter otivye 35 2 Z pib Tag Ti yhp o Rkoa Bek vge bZiu TZHB TK o b5y TUK dhk biv gu Ne vi ich b5a i Via TBg oi TBg o iVg od5o i Beg o bek ego ZV Ava TtTug

Mei ziv iv Zeg ozek iv TVg obeg pich dov Beg viv Uvi Tub TUYE Su yah a z iz Agsh TuUuk TUg ber OR Ni TUuk buh im Ah» Tug Tev pi in bi

Ii i85 116

Tk o tVgoiev TvAg o Uvi zeg back 115 "its 95 "11 113 "its RT "yiZ" Artificial Consistency

KV

"23" ДЛегжный Ланцег СБИ23-Знязюючога дома PANT-A kaYayabh 35 dkv vve Uvi Mei IZg otspiv zeg gg Aa Zeg oivgu Aza Uvi BZeg yiv o biuU i E a Ih biz YaE Uvi Tvg oMei bBeg osbub5 iz Bek 5eg biy Beg iv iv A5a o 5eg 2 5 EI

Si yhp Zip o iuh yhp tTkgoivy tTVe o tTgOo Tug Biy civ zub Rg pf biv 35 V "5 go Bgo iu5 iv ev fi Tug tTgroyiz 5eeb TBg AgE biz ego Z Uvi 8 ko Java AgE be bek 51 beg Bi zZvg bit TVg dev o BvVe Tr oivts Tae 55 V and a

IIiv bZvgo ben oivu biv Za bib A5v o bai AtTa Uvi TU Tu Gu Biv Av ko i 5

Art tek back Tug Bgo TK TVgb obBe z bij bi TVg oi Uhiv biv Thie zva ev ihe85 it "a V "ii i13y "hii RET "213" Artificial Sequence ah "333" VBzhkai Lantsvshutie SBIZ3-Binding Domain PANT-A "OV 28 bi: Uvi bib iso Uvi ziv dec bi iz Bib yivu yt5 fu Bgto bi dia y 5 Ta IV

Met Uvi Guz Udi beg KUuz 15 viz zv obi TUg Te o Bbe TVg o Yahro tuye ha Zh B it Met ih ygB Uvi ge biv dia Rgo bib bib biu igr biv TkBr o ti iy5 Bi 5

Z 5 118 Ki BgoO Zeg Ahp Z biv TK RN o Tuk vzy bij it5 Be az Z iz pi dgE Udi tyag o Tie TK Uvi y5y iuh bBeg TygoBeye TVB AR tu g KE 75 Ve

Met Bin ivy obeb bek Evu Av vk tsiu ver Tng o iv Uvi tb o tUK guh v shcha 5 13 YagE bek Ni ieu ivu Ag Did begb Tev RBe Via TUuB tev shchi biv

ЭЯКЗ ии155 118 цих Твг оивц Уди улик Уви Бек БЕК

K15 128 "left in "ii" Z "under RT "213" Artificial Sequence ex "223" DegkkYA Chain SBI23-Binding Dozen PANT-B "az 2U

Av Rre tav Met tivak Biy 5Zek Rge va beg oyi ve: yiz Uvi begoiivu BSHchu i Z IV Their

Z: GE UaiE KTBg Met ZegoYeh Her 5 ber obegr bib bek yin ier; dep beg a 5 B sh-iu vh biv ih y5a TB ie TK o Ter oTug Ship biv i5 Rko Z biv i5 a 5

Bho bho yuuz iv ben Ii Tuk tigroyiz Beg To Ag biy zeg o pbiu Uvi

BV 55 5

Gka y5r ge ge beee biu beg Bi beg obi TVg des KBBe Troi tk 55 KE and more

Ii bek o zZvgoivv tsiv Aiz beat a5r Uzi beat Uvi TU tugo Se5 beat dep shcha 5

Art tugo BEK o Tuk RKE TEg o IBg RNe bib biv ZI Te oiiU5 iv bin Iig

Iva 15 EV ich "vii 28 "kaiiv 158 vii VKT "313" Artificial Sequence ve" "23" Weight Chain of SBIZZ-Binding domain PAVI-B "88 28 ip Uvi bil ivc Uvi biv ego bi biz Bib vu gu i5 Rg Shiu diz 3 5 IV ih ceg Uvi iz Uvi bek GUu5 15 dia ego obi Tug fiirgroKBe To YAZ tuk dya 25 Ku

Tu MI yif5 TgB Uvi Aga sip div Bho biv iv Z web BV pr oti ko Ya5

Bi Yar Ti ti Bgo bek b bi iya TE Re TUg y" bij it5 Be shcha 55 Bo izhe Bib AkE Chai TBkg ot1ie TB Uai Av iuh Beg te ze TVgoVbiv tuk

KE and more

Mei pizza iv ego obek i en dkw beg Bib yo TK Aza Uvi Tek Tuk uh 5 a 55 div dgE Zeg Nih ev ivy dgv o Aiv bek Theo Re Aia Tug tTgbvbv obiu biv aa і55 118 i Tab iv Mdi tvg o Udi bveg obBEetg і15 ia "18" 35 "ZAEkh y 2135 BRV "Viz" Artificial gpeoslIDIVITY 248" "223" Linker 1 Polypentide ai" 25

Z schik zhbiu Zeg Bi Bi pi I 4 there are teas" Za "TE K "313" RET "PPE" Artificial Sequence "Cattle "823" binkeyuv 2 Polypeptide "zah za

Bi iU in bi vi Bi i 5 "tah ZI h21i" y "From RNA "Z135 Artificial penlidity is 23yU "hy3" Link with polypeptide cha" ZE zyu schu BiuUu Bag 1 218" 33 "ai" 35 vav "yy" 33

"1" 5 "2125 RET "13" Artificial Sequence se "33 DLink and Pdyazpaett kah questionnaires From BelipEzhitiya "and" From

Aiv rRga Zeg bek Beh Rgo Met Bib i 5 stand for "her" 8 "12" CT "213" Artificial Sequence

J

"23" E-epiralnik Domen chenny for

Si Uai ia div iv Bib i5 Bivu Uschi 8iz Ah iv bi uz pizza Uvi i 5 and 15

Wiz Lizie Bi: ih Bio Uvi dia div ev bib i u sei 5 "sii" 8 "13" CT "2i13" Artificial Sequence

Horror "23 k-epiral Domain "aa 5 iU5 Uvi from virgins i5 bis ich schi from ABa ivts and Bi Bu Uvi 1 5 and 15

Viz liv with iuu5 Bin ih Fai Afa dia ien BU 51 za y "tiv ZB "her" av "313" RT "213" Artificial Sequence i" "223 Predominant Albumin-Binding Domain "Vih de im Had bin div 5 Uvi 12" Aza Azlp ygv bi ivo ber ov uv Tug bish i k yev I5

Udi bek y5oO Tur tur ih vp iv Ti yo v5v div iUu5 Zek Afa 515 a 25 ZV

Z Uvi gU5 Aia Yev) Ti b5r Bin 112 iv ARa div iv o bge 35 shcha i "Ia" 5 115 du "3125 REK "2135 Noschsho zavivph "VaVh "gai WEB REVTSKE "sva2v 1... 237 23" SNI-SNZ to the women of Lyudska B Oblast

I" 8U

Atia Rgo biv Viz ta siu biu Bho bZee Mai Re ieo Re RgOo Br i 1 5 ha I5

Rg iv and TvKoEvy Me BE bek Age Tg o RKO biv Uvi PE ssukh UVE a 25 ZV

Uvi Uvi d5v uUdiy bek Ni bib vzr Re Bib Uvi ih Re da teo otug 35 scha dB chi Ahr biu Uai Bib Uvi Ni Ahv Aiz iuh TVgG oi u5 Reo ygdsh i 015 -E 55 ve ip o TuUug o bop bek TK o TUug oigsh Uvi Mi beg Mai ivy tik o Uvi ivn Ni 55 Ua t shcha

Zip y5r o ikoroivy wn shiu ih Bin TUg o fUe Sub ie Uvi Zet AHA yi 5 jak ia ge RgOo Aza Rgo Ti bib buh tpg liv Zeg oiu5 Wiz Bu5 shchi ip

More And For

TgRO YaGE pizza Rgo biv tai TK TvVgoivu Rgo Rgo beb dr viv p: Be 115 128 175

TnNg gu dk bip Uvi ego iv ygr os Uuh iv Uvi Rukh Bi Re izhk VO 13 135 yishcha bek Av tiv Diz Uvi shi Tgb Bib Zeb yep o Biye bij bgo Bi» d5zh Av і145 iso 155 ide

Tek oichuz TVg OTVeb RBgoa Rgo baif i2en dv ego Ar o iu bBeyeb Be ve yiv 155 I 175

TUg ober oi ie TVg o Uvi yoo u 5erg dev Tgv o bip biv bi hv Uvi

IB i155 cha vne bek Su5 bZeg Uai Met Ni ie biz iets Ni yo Niya tTUK o TVkK biv

IZ zva ha

YiUui BEg izn Zeg ate bek obgo Bib tu

EV gi "ii" 38 "eiih i4 "Ti" RT "vii Mu veestiyh "MYH "T2i8 MI BEATOKE "ER SER. "23" SOY ZARZIABLE DOMAIN LEGKEG PLANTSYUG YNTI-SV3 DNTITILA "VAZ Z

AgE Bek o bBeg Tig Bi iv di TK o tTng o Beg yo Tug ATV bla i Z IV «tiv Z «tii 7

"32 RET "133 BMui5 yaviesnice "Re "2 VIBS BEDTOKE "23" 5 BarEIZVbelnogoa and Domain of Light Lannyug bYanti-S 3 Antibodies "355 For the side of іZg bha from к Div bro і 5 "із 4 іх Z "12" RT "іZh My vyhschice «eV «ee VIV BEATTSNE

VIV SEC

"273 "03 Variable Precipitated Light Chain dnty-CO3 dDntitila "aa" from ivy tk o BUYE Zek i iiv o iigv o Udi i i i i i i i 5 "i i i i i 5 "213 RT -ei3" You yavosmine

BEH

"2IZ VI BEATZKE "2235 "No Variable Domain of the desired Chain dnty-- 3 yntitila "aa 1 tTVg otUue Viva Met Dev

With is "Viv" 427 "PPE 18 "Vida RT "13" Mykh fvyZsnine a" "ali" MES REDTUKE "aayh IC. "223" KB of the Varisable Domain of the VZazhgoa Chain of Vnty-СО3 dDntitila "ЯВВах 4

Ag Tiv dgd obeg oi Uh TUug dv dha TUg biz Te tuye Bug div 5 o beg y 5 IV 15

Uvi uh y5Yu "vii" a "Zi 34 "yiv RET 135 Myh viesmine "but" "aaih MISS BEATOKE "dovi y1..K15 "223" KP Variable Dyomen Heavy Laniyug dnty-SOZ Antibodies "ashchyh ah

Ni bi Dyak BvVe bi dep bZeg Tug Uvi beg o Tgv Re div Tuk i 5 Iv chaiv 44 "ii" 17 "215x RVC "13 Muh yaiascine a" "ali" MES REDTUKE "vah (I... I15i "223" KO ВBarizbelny yoven Light Chain ynty-S0i23 dntitila "40" ad ih Beg Zeg bBip beg ver oie; y5v Zeg Bi A5p piv i uh 5 o tiugB iv i iv iv 15

That

"1 yah "3135 7 "812z RET "ei3U Muye shi tsinkh "eh "a" MES BEBTOKE "2283 SEC "223" SONO of the Variable Domain of the Light Chain dnty-СО133 ynititila "ai" 45

Tra viv beg TP AgE viv bBeg

In Bach "iy ay "her" Z "Ta At "iz" Mu shvyzsmine o" ev" WEB BEDTCHNE "ah WED. "some SOY of the Yazdarziable domain of the light chain of yntai-S OZ dntitida "WHO 45 ziv vp y Bug Bek o Tug Bro tTub TK y 5 1" 4 2115 5 "TA RyaT ii" My5 zhihscits "ah "2235 MIS REACTSNE "gas (ECO " 223" SBVIi Varivbelny Domain of the Bazhsky Chain ynty-0123 yntvtila "aaah 4u vo o TUk Tub Mei i 55

From the

"-5і85Бх 45 ka У "832У REK "kaz My vok nicheh "ad" "VEU VI BEDTYKE "aa" 53.05 "223 СО of the Boiling domain of the Heavy Yaantsyug ynty-С0123 ynteitila "ah am az 1і1e Tiiv o bgo zZeg dep obi dia tb Be tag without BIAYA iUu5 Re i 5 IV iz -shiu "3165 8 "EV U "5325 RT goats" My5 shuihsnishe "2 "aa VI REAROKE "vadu y1..K75 "iz ЗИZ VarkhZbelnoy Demen VajkogOo Lentsyuga dnty-3B1Z3 dnititila "OV 4 bek Ni ich ivz age biv bek y 5 "tb 5 "Her 7 "VI VI "213" Stechna Sequence "ex "223 Heterodynamevization Domain coffee x 58

With Uvi Bi6 Rg te Bek SB i 5 kaivyz" 5

"zii B "ai2 RT "813" Ttuchna Sequence "VaVU "23" Heterodimerization Dogen "aa Bi

UaPai ish Rgo and uU5 Bek buh and 5 eh B "Vii "213" vit "813" Artificial Peslidavnist

OV

"223" Yeeteroduzhevnyzaniya Dozhena "438 52

From Re and Ven Bi from SU5 4 " "zion 53 "1EU in "vi2z RT "Z135 Brechna Poslilovnost shi liquid cm leadivity "vav "823" Doben heterodynerization "ah 53

Tvge bl Yahweh obi Bib su z v "shchi" Ba -e21Ex 25U "ia RA "iz" Artificial Consistency

VK

"ha3z Third Poplipentidny Lantseg PAVET-th verS Bersia 31 Construct ah 58 y vu5 Te No TVK obUu5 Bgo Bko uz Rga biv Rgo biz div dia Shchi y 5 IV i15

With Bgo bag Udi Re ivc bBe RBga go ih Rgo gUuh y yVg iz Me ha TE ZV ii2 Chek AgB o Ig Rgo biz Mai TvgoSukh Uvi Uvi Mi y5v Uai bek No 35 I "5

He vv Rgo beat Uvi uz Be zv o Tho te Uvi hv te Uvi called Uvi. 5 a

Ni5 dhp div iue TVg ovU5 Rgo be bib bib ZiB TUg dey begb TK otee

KE and a

Ag Uvi Uyai Zegk Uvi ivy Tk uv iets Nih Biy De TeB ie Ahv Bi ih A 5 i BE TU 15 su Bez Uvi 5eg Ah ih Div be Bho viz BBo і18

Io I55 i115 biv izhe ipg oliv ego oiu5 viv Gaia 51 siy Bho AgE bin Rg biv Uvi ije15 zv i125

ТЖг ТК оти бра Rgo beg o вбE biv bib MOH Ti Her 55 dp biv Uvi beg 138 55 Ia ivy Bek SU5 Aiz Uvi i5 Bi Be ig Ko Zeg ybBi Bi ABa Uvi biv 145 158 155 i155

Tgv BIB Zeg dep viye bij Mgo biz ba Aa Tuk ge Te OTVe go ha 1855 I158 175

Uvi in ar 5eg Azr obi 5eg ba RBe ivu Uvi Begoiue yiv ivg Uvi y58 ik a az uv zegoAgE Tr otsip bBip Bi Aza Uvi Re bZve oSU5 beg o Udi KE 155 28 zah

Ni5 bib biz iveh Nih Aa dgshy tuye TvK biv g 5 bek oivy begoi vu 5eg

IV 15 Ks go Bu iu"

Ka "eiiiih 5 "vtyh i8 "1 RT "Z13z Artificial Sequence "ih "23" Peptide Her "aa" 55

Ayavoit: TTG No TBg oSUu5 Bgo Rgo Sya Rgo 1 5 Ii "Phys 55 "Vii ZE "hii RET "213" Tuchna Sequence "Va" "23" Predominant SNO 1 SNI Doveny Bo Oblasti "aa 56

Viv Rge shiv Aza Via bi biu Bgo beb Udi Re be RBBe Bgo Rgo 15 th 5 3 15

Rga ih ZY TVgoivy Met Iiv beg ygsh TO o RgOo bib Uvi Teg osbuh Udi ha Kk: Za

Uvi Udi dv Mai bek Ni bij b5v Rgo Bib Uvi ie BBe yhap Ter o TUg i5 8 d

Uvi 5 Zi Mai biv tazi Ni y5p did ib5 TVg oi Uh Bk ygrsh iv BI shcha ip TUg Dhv Beg Tvg oTUg r Uvi vi Beg Mi Ziezz Te Uvi iv Ni 55 Ua 75 Va bij vv tiKooivv dv pi uh Bish yTg oiiu5 su Gu Uvi Bek Up and uv 85 a ze

Viz iv ho viz God i12e Bib iz iBg oti ek iz From their su biv to iiz5 zo

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Claims (5)

FORMULA OF THE INVENTION 1. Diabody, characterized by an optimized sequence, capable of specifically binding to the CO123 epitope and to the POP epitope, and the diabody contains the first polypeptide chain and the second polypeptide chain, covalently linked to each other, and: A) the first polypeptide chain contains, in the direction from the M-end to the C-end: i) domain 1 containing (1) subdomain (1A), which contains the MI domain of a monoclonal antibody capable of binding to SOZ (Mi soz) (ZEO IU MO:C21) and (2) subdomain (18), which contains the MH domain of a monoclonal antibody capable of binding to bind to СО123 (MNeoigz) (ZEO IU MO 26), and the mentioned subdomains TA and 1B are separated from each other by a peptide linker (ZEO IU MO:29); i) domain 2, and the specified domain 2 is an E-helical domain (5EO IJU MO:34) or a K-helical domain (5EO 10 MO:35), and the specified domain 2 is separated from the specified domain 1 by a peptide linker (ZEO IJU MO :30); and B) the second polypeptide chain contains, in the direction from the M-terminus to the C-terminus: i) domain 1, which contains (1) subdomain (1A), which contains the MI domain of a monoclonal antibody capable of binding to CO123 (Mi soi2gz ) (ZEO IU MO C25) ; and (2) subdomain (18), which contains the MH domain of a monoclonal antibody capable of binding to SOZ (МНесоз) (ZEO I MO:22), and the specified TA and 1B subdomains are separated from each other by a peptide linker (ZEO IJ MO: 29); ii) domain 2, and the specified domain 2 is a K-helical domain (5EO 10 MO:35) or an E-helical domain (5EO 10 MO:34), and the specified domain 2 is separated from the specified domain 1 by a peptide linker (5EO ИХО MO :30); and the specified domain 2 of the specified first and specified second polypeptide chains are not both E-helical domains or both K-helical domains; and at the same time: (a) the specified M domain of the specified first polypeptide chain and the specified UN domain of the specified second polypeptide chain form an antigen-binding domain capable of specifically binding to the POP epitope; and (p) the specified MI domain. of the specified second polypeptide chain and the specified UN domain of the specified first polypeptide chain form an antigen-binding domain capable of specifically binding to the CO123 epitope. 2. Diatilo according to claim 1, in which the specified first polypeptide chain additionally contains an albumin-binding domain (5EO IU MO:36), connected to the specified domain 2 by means of a peptide linker (ZEO I MO':Z31). Z. Diatylo according to claim 1, in which the specified second polypeptide chain additionally contains domain 3, which contains the CH2 and CH3 domains of the Es immunoglobulin domain (5EO IU MO:37), and the specified domain C is connected to the specified domain 1 by means of a peptide linker (ZEO IO MO:33). 4. Diatyl according to claim 1, in which the specified first polypeptide chain additionally contains domain 3, which contains the CH2 and CH3 domains of the Es immunoglobulin domain (5EO IU MO:37), and the specified domain C is connected to the specified domain 1 by means of a peptide linker (ZEO IO MO:33). 5. Diatyl according to claim 1, in which the specified second polypeptide chain additionally contains domain 3, which contains the CH2 domain and the CH3 domain of the Es immunoglobulin (5EO IU MO:37), and the specified domain C is connected to the specified domain 2 by means of a peptide linker (ZEO and MO:32). 6. Diatilo according to claim 1, in which the specified first polypeptide chain additionally contains domain 3, which contains the CH2 and CH3 domain of the immunoglobulin E domain (5EO IU MO:37), and the specified domain C is connected to the specified domain 2 by means of a peptide linker (ZEO and MO:32). 7. Diabody according to any one of claims 3-6, and said diabody additionally contains a third polypeptide chain containing the SNEI2 domain and the SNZ domain of Es immunoglobulin (ZEO IU MO 11). 8. Diabody according to any of claims 3-7, and said diabody additionally contains a cysteine-containing peptide (5EO IU MO:55) of the M-end to the specified CH2 domain and the CH3 of the specified EC domain of immunoglobulin. 9. Diatyl according to any of claims 1-8, in which said domain 2 of said first polypeptide chain is a K-helical domain (5EO IO MO:35), and said domain 2 of said second polypeptide chain is an E-helical domain (ZEO IU MO:Z34). 10. Diatyl according to any of claims 1-8, in which said domain 2 of said first polypeptide chain is an E-helical domain (5EO IO MO:Z4), and said domain 2 of said second polypeptide chain is a K-helical domain (ZEO IU MO:Z5). 11. Diabody according to any of the previous items, and the diabody is able to cross-react with proteins of CO123 and POPs of both humans and primates. 12. A bispecific dibody capable of specifically binding to the CO123 epitope and to the POP epitope, and the dibody contains the first polypeptide chain and the second polypeptide chain, because they are covalently linked to each other, and the said bispecific dibody contains the following: A) the first polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:1; and B) the second polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:Z; and the specified first and specified second polypeptide chains are covalently linked to each other by a disulfide bond. 13. A bispecific dibody capable of specifically binding to the CO123 epitope and to the POP epitope, and the dibody contains a first polypeptide chain and a second polypeptide chain covalently linked to each other, and additionally contains a third polypeptide chain, and said bispecific dibody contains the following : A) the first polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:13; and B) the second polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:15; C) the third polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:54; and the specified first and specified second polypeptide chains are covalently linked to each other by a disulfide bond. 14. A bispecific dibody capable of specifically binding to the CO123 epitope and to the POP epitope, and the dibody contains a first polypeptide chain and a second polypeptide chain covalently linked to each other, and additionally contains a third polypeptide chain, and said bispecific dibody contains the following : A) the first polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:17; and B) the second polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:1; C) the third polypeptide chain, which is characterized by the amino acid sequence 5EO 10 MO:54; and the specified first and specified second polypeptide chains are covalently linked to each other by a disulfide bond. 15. Diatilo according to any of the previous items for use as a pharmaceutical agent. 16. Diatilo according to any one of claims 1-14 for use in the treatment of a disease or condition associated with expression of CO123 or characterized by expression of CO123. 17. The body according to claim 16, and the specified disease or condition associated with the expression of CO123 or characterized by the expression of CO123 is a malignant tumor. 18. The method of claim 17, wherein said malignant tumor is selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis, and CML-associated Abelson oncogene (translocation All-AVI), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-CLL), chronic lymphocytic leukemia (CLL), including CLL with Richter's syndrome or Richter's transformation, hairy cell leukemia (HCL), plasmacytoid dendritic cell neoplasms (UBPDK), non-Hodgkin's lymphomas (NHL), including mantle cell lymphoma (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma. 19. Diet according to claim 16, and the specified disease or condition associated with the expression of CO123 or characterized by the expression of CO123 is an inflammatory condition. 20. Diatilo according to claim 19, and said inflammatory condition is selected from the group consisting of the following: systemic lupus erythematosus (SLE), allergy and bronchial asthma, rheumatoid arthritis. 21. A pharmaceutical composition containing a diabody according to any one of claims 1-14 and a physiologically acceptable carrier. 22. Use of the pharmaceutical composition according to claim 21 in the treatment of a disease or condition associated with the expression of CO123 or characterized by the expression of CO123. 23. Use according to claim 22, in which said disease or condition associated with CO123 expression or characterized by CO123 expression is a malignant tumor. 24. The use of claim 23, wherein said malignant tumor is selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis, and CML-associated Abelson oncogene (translocation AllI-AVI), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-CLL), chronic lymphocytic leukemia (CLL), including CLL with Richter's syndrome or Richter's transformation, hairy cell leukemia (HCL), plasmacytoid dendritic blast neoplasms cells (UBPDK), non-Hodgkin's lymphomas (NHL), including mantle cell lymphoma (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma. 25. The application according to claim 22, in which said disease or condition associated with expression of CO123 or characterized by expression of CO123 is an inflammatory condition. 26. 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SE tn, BE be I -- vazht-a Zh ot Zak -- st Fo 6 zazhtov BO ke -- AVI z NE В shvat z Ka Zh s SZ -- yad ZY 5 EI ШЧ Шк шче sa Я З з 7 Counter . BACT would 5 5 deContr. AVT iy che KZ still 7 Zhe 4 - Kk, s Ka a E CUSTOMS Mk hnnv Ei vi v zn Now -E osot I smoke "I zaz zha yah tk Sai za? what? z sh t inv bzhto vel s utoteva shltiny y y p concenIMation NINI Concentration of sntbmli Mav OUN me V z moakt-38 sce Z yod zht-ya Z : yak so vah Z-e - PACT zZANI y on I -b- BEN t- a Ж de Y Menya t |i rat ANE I. pi I her "Zhe vadt-v d h ke -h Y o vaz y Ko he - Ha Z -Z one t YAKU x 2 koe A I Y dova Zhe Mk 4 Kont BAKE I - and what KZ and what VH! KE E; I'm married to ME aka de ro nh -Юe и Kol ;. ххх Would you like it? вх зах зах та х "в в к шх m - . - Her h yv y o Sho ze Ezncentration (ng/ml) Kannentration i ngoyali un tTnE-4 o ne gou NI g I Z tv What ro -an o rakt-a BU I schozha i - і і х щ I che kov - k- Contre. PAKT Shk H y: pen y ki zo i identi on as cha yad chat zah Conpentration intoml) Figtra Z Perceived STI activity. in relation to the cell EI A. Cavity-3 V. TTN up to 130 Э вы чи ШЕ Е ве К - мил шы пы Р ШХ В ; - t i- SCHE p 494 «i i r I-y SE Z ve E dova A sh - MK : s E SCHE mae same; Ше че че чаны всуч Чи т. нн нина прогноз Мни яая ша ган н Вещ ве Concentration (who) Concentration (NM) Absence of 1-cell activation in the absence of simultaneous involvement S. Absence of target cells "Vaomzklitnnamn TNV-Z GML that KEEEEso that is still co ЖЖ with VinU! K e nu i vo. ze Zhe u ve soossKo ooo oo oo o o KK soo. EV t cha zak oh ko still maenoclonal antitivz i ngoml) Misnoclokal antibody gngliali - ypactTt-A z. 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MONA PP she neck Ki and N EN Bu and OSTI sit shcha still ov po sho I z ia NN te TU 5 zh SIK Z zh i Z sho zh bi sha - she she schi in a dream. 5 I KOHZ p. 0 KBU a pi o it ! 4 and | Ozheta: oh - oh ZHI | shki y I DEC o net nnnnnrnnnnk TO s Zeozeek X non kan ny rennia : ; gene 8 po shchyu I OO YAN oz Neon eV IN kovo conk VN: RES-Nevnye EN: VEZH NaavE YAN: VAN BAS M I-Nabavi MORE. kegekknInne RESN VUS duklvie RES-N, BK iukai ES YAVNI VK nn FENIZATRMEO ORZ aVVI YAZVIZANRANI AND SKKNIo 0 btZ51ZABSVKSYE 101934Ю0U put tBIZASTVYA KoachavOV za" In kir di KOYU Bilkiv leads In keksk 19 how many liv MMZAT gav RAM19 - control DAT PAKT-D y MK Uma I vekche MDOU tet still M Tr NI o -E | I a: I EE eat KVoEE SHE M gave E. in a VN battle VN pri roda s p, E she ONY YAN Sho BZH OO ss MY oo cit, "iiy KOZH r An NI zo VYN S o U Goty I-y SSO Bey 0 OO. Z u Ya vy yah VO zhi ssh SHYN NI NN ! " zh pn shen shin shin zh zmo Artek ! g PO KK what PE EN WE POI LI Z OTAK: her Datekoeichnny nn peltettristtt 1 ekeetkktrnst sno nnnnnnny 00 MK systnin ryhnyetyy 00 M Mini AK riki sne nava IN I V S i V NN ON ZV V KE A M M Z KS KUS-NevnE EBYA-N : V A-Naidi KANA An aku A-N: I I-Nezav Zdatni ES AOM age of RUSSIV pi ese si ne ke I tviza VV NOO FENIZAMN TM POEMS 0 bizBIZATSVEVEV ONA Soya vyakzayem V i ozaoyuya kilkintk polov Obpv kilkit pozhiv I how many is UM number of ZM events Normal REMA V control AVT S OAVKT-A I Quechui ypei y zm y Unformed Shegmya Enemy KE OK ibn B NY : SCHA she m POV po VI Z sha Yoan y "rya M S zi NE i UK y- t She p which y me she y; more: shin she NI AN NN NA i NN F shin sia SHIA o SHIN na NE Ha o OKU she 5 more M s - ee she she ia ; she 5 shin her shin SraA-ARS ti tako Щ May OK these 10 FILL pi OP i pi chk o ily KENE why they ie Neo oyu NO: pe not this otse RAN: EKa-NeidnE RYAN: RKYA-Neidnh RAN: ANeyuny ES-N, SAN di dkljae EBS-N. From year EZKAN, ZaS-N dk IB ZAKETOOMVO SVOI fee era TLO ZANI-TSVYAVZ TO number of oxen 2482 number of drivers 203 kilkkitktso in 16 , hontrolne PACT ACTA B, veobrosyuyu E EV attempt E Y vglmd zn ked: Kk ; And 2003. 150th IV sia in Z SHE: A kh. ! y I 5. 150 Ku y a OA II E GU i he zay i i im KOHJ i oo i ne 15 KAL sh E i zh EMO 4 X, N N i SOM kyu g i syu ki I o, U Z V I V in Z V, V x KOSOVI V Ya i BZh ev i x De Yu Ya I-I still V i ShChN x N i still U y yin ka x x E Y et N U Ye x g i masha her zh. y x, y M schi te Sche a Ost 00 Ferettveettetttyunt 00 Orest ce OH on ce re poshi zh RESNIK S Oz Be 1 RN: RI 2-Nedne R-NI Z-Nezdna RANI EE2-Neitsne "Viru va 14 pen, shcha n idvlyae, and I, You Nibakazkh SCHO gozhN, that "No way SHY ia n TET ZATOK OVO nog TET FOR AKSENIOH MT ZATOK WE 5 FROM NISCHK 5 BIV pischiicha pi RBIZ number of cattle 1915O number of oxen TM number of species of Ishcheti РЕМ вра А жмут рольне АКТ ВБАНК-А (3 ЗУ ЖЕ НН , х І - : З необробвні (ЖЕ в'яла І Ентлнт ш с БИ шу що Еш ще І а й Вл Ж с Ша а ле «7 Коосюсююююююююююьюк я у осююююююююєюкужкююьсо for 4 du udosko t z 8 10. tsa 19 I she ma 1 She Pe Noya I that LEVA M- 00 OU : ш , I zav I I la KE I dela KO. i OO KON : F: OKO : IH SOZARS a o -e ZOSH Shin B SHE en Zoni nty Gr, TO TAKE U noz nonninnnizhazh and REK, vocal yutetiyuust , Moy ne pe one ZO pe ee ce onko ne ne ve EEYAANE EN SEN: V ANEAN ON: EYA-NEChenE EBUN, ek -N pl E ZK-N. An ED Zh5-N, 55-n klKh OUN ATSSK PET ZAST M VNU. MVA TSEVOIVOZOOH oxenability TO the number of BOM number of OO VIV kayerolne ZDET RAVT-A 41. peyuorobleni Kshch veovrovleni Io velyal And nem SNY zennnvnns zvennnnnnn NI nn guilty NOW mn Z and PE o, ENYNSHN E and NV dya SCHE h V Ol i sravmv 8 shi shshnsh nshshe And racial and GG c. AND HAD with. with Kaya yad Mah i u GK y" V v nya A NU iy V Pk U Sheche i KI 1 i os aa y shi KE a SYA Zh sne 1 ne vo ry also ke Ge Ts Ne ov o ve ne zu SAN EO-Neyuti SPRING EZ-Sunday ESNTEO-Nei dn Chhura 14 RON LAN idkaze RELATIVES BUT -Nesukahe VA-N, RITA vinkike m SH VM ZATV a VO ES YAMI STOCK OVAYua TATI Events) kiziki podi I kil polis 185 number of pi zh: e zvo Go Ks NK How ZY p sha KE KI va I zmekiobnnu1 VI she nn p nn p. and | scabies! Z Olastni klitEnEe nn ee nn I a gv| | "NK! "thanks to AYU I. more and more VAL i sho and 00 ery SHIA peyashsh shk as Sh ve o. and in ! 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POI ngoml b i BE ML shgomlo b 1 ng/ml Fe i emitM t WAKTA BALLET OCANKe Zap Ye Figure 16 (prolapse) СсОп25 000 Soy x 3000 x и- х x каА 2000 x Х 000 Х x - KV dO fatigue i ngbmlob ОИ ng Й ng/ml e A control VAKTA bak Ki-b7 СО 2000 I и X ш- I ing ml i BI mmloi shcmlo 0 ngma: Fe in the control ACTA by Figtra17 Pezforin zvo. som ST» r sha sha THEY called WHO ELSE WHO I. fri ntimlod Her ngoml ON ng nMml S yet | SVT A zhadtrOolno Graznznm V Zoo spa Sov 2yu t i i skh x . t000- SCHI vi: shi shOshih m ngimlob i shglyal VE ngomab i) ntumo I) m proranh bai Figure 17 (continued) SIYU duchyny SB mavny VO ve y "p kp- 12 "ZNM I K p-a 1 b y DAM y pas Y 5 en VIM chas a chas 5 kaz Ze5 KCha.Yae-3 Kaa1ve5 Ka. 7e-3 C123 pyulynn SI mavyh and KO 1405 KO-O.1vnM behind KO-0a7yM and: And two x VE in digging 1vp. nn chn 4 vk sesekennstnnkh nan GI ss sskvss for more with. yo I E. I IZ V - VI s i E s zo opovo ov what 0 0 50 00 00200 time 5 sho 7 zd 5 kai1e5 KO1, Bei Kai dev KZ ei Figure 185 a, Attelologic leplenia of human REMS menocytes eContr. PAM I I, - PACKAGE Zh ok zeva a: PACKAGE ZHAV m Fk z pe a te te i she sa tanya Z v; ! zo te still t Zhencentration ngimli V. Autologous Denletion of the Mennonite RBMIS of the Javan macaque w 8 Ko sho |; Z "y x re -v Kch -o rAKTA a Seezho-a. SAKTA m/AVo i - Ch - 1 for 17 and Isii Kanientraniya s ngomli Figure IZ inert carrier Z filler 0 F4izz» 05 ointment B she 5 ME B o neck B NN: and sh. PKU, CIRCLE, Z ee u piz z ME He sa is YN Hm 1 ee CO14 ARS SOLYA ARS SE OKO zeil o OVU Ok 5. | y shchi shch Tons 106 ee bo zn СО014 АРС СС14 АРС «а ma: Z soma Figure 20 what shchi . Cantral ANT TObkkgoktilen aka millet ooo ooo ooo ooo t I SHYMKY s Y ee ; and I la CSO aZ. : Y 5. 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Ш E і ШЧО К БЖ БЖ Z Г5, ЭХ КБ зх З ЗЕ SU - Беж об пих хи Ma pe х КВ Ку ХУ ЖЖ y po - SK, з жи : -k Ozhe x poh - schy Re hv ж VE e - R «z - BE KU he KI B EK Uh ION oh pe she vosh om shov on im kimikom - SHOE ShshOovVaAV BEN YOU sh - na o Zh sh oh se M-e ho - - 5 - - still what jan - rea хиш шу SUSHU и КЗ деожеожай Figure ZI (panels SAT T-klinny Sat SO SHO szhets : х дхжт t M female OO Il U -E E su i a I ru ! - And i- I je ih Y OBO chi x J i x i | . X x! ц х х х х Her: : in EN I | Shi SHE. : | her SHON y: З х Зах ЖК Ей ssh У ХХ х З х З: KK TB х вк ve GE s "A (1 KI EE li ye yan ВХ he U MU zh za teo meh eh VI om I zdoh mech t- I -No Zh M v pe i KA a OK Oshchi a a ХК pah oou U ke Uh - re ee WE OM ku he he KO U MM OHH JAH UK kA Ukku v she She She Which mo sh- chy ha ho zhoren ooyak yaksho EK od ee osn rn i sho v she ZBE EE Zh 5 Zh - oon pl Fe g VE that V a - - YaVoh -x zey A «E zhk ш ш - Sak ШЯ male и х GU ш I la | 5 vi. MM slaeka I KU KU M KU х хх х X 5 их Z 5 Х х хх У na ех х х Х хх Х ш З КИ: ЖИ Z ne Х х х х 5 : х : - Х KU KU KU xx KU KU х i X KU KU KU With their KU KU x m X KU KU KU With their KU KU xx Х 5 MORE Х Х EZ - Х х х З х х х х х N -- Х KU х х хх KU KU х х ве Х У х хх х х х Мк х х х х х и х х х ххххх ГУ 5 MORE Х хх х Х х Z KE KE Gaya chih KU KU KU Z their KU ХК. их х KKU х - х х х х х Хх х хх хх х КУ х че ЗИ: ВИЗ ШИ: и: Z 5 SU и 3. х GU х х х 5 х х х х ХЕ ИЗ "har EU х Х Z 5 х х GU х хх E З х KU х х хх KU х х ККУ E Ч КУ КУ КУ Z их КУ КУ КУ ХККУ x X KU KU KU With their KU KU KU xx x KKU x X KU KU KU With their KU KU KU xx KU x X KU KU KU With their KU KU KU xx KKU x X KU KU KU With their KU KU KU xx M KU x X KU KU KU With their KU KU KU x ah M KU x X KU KU KU With their KU KU KU xx M KU x X KU KU KU With their KU KU KU With M KU x X KU KU KU With their KU KU KU x ih M KU x H KU KU KU Z ih KU KU KU x ih M KU x X KU KU KU Z their KU KU KU x ih M KU x shi i I SHA NN : de Ih B BY KN I BE FROM BACK Kk S B ICH zh sh--o ME OH du zho "o U xx koh "EI M u KE Y - - her MO ev so Zh. 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ЧЕ и- EE BEK S 5 EN: EN E EE her t KU x X x Z x x X X y: x X x X X x U KKU x X X o x X X X X x x KKU KU X x how X х х х х х хх гу х з т х х х ку х х ККУ х ку ре х х Х Х х х Э ГУ З х их ЗБЕ БЕК з EN ШЧЕ; ХХХХ ХХХХ ХХХ ХХ ХХХ ХХ ХХ ХХ ХХ ХХХ ХХ ХХ Х КУ 5 х Х Х Х Х х Х х KU Х Х КУ Х Х х х Х КУ Х Х КУ Х Х х х Х КУ Х КУ х КУ Х х KU KU x KU KU КИ KZ - Щхх КУ хх КУ КУ x КУ КУ КИ КЗ 5 х х КУ х х х х х х КУ Z Шх Ж x KU KU KU х х х KU KU Ж KU х KU Х х х х х х КУ From the X X Ku Ku X X X X X EU 5 of X X X X X X X X X X X X X X X XIM CHE BUC X X X D; х В х -х o Mn day ek KO le eyes che M I I py ny NS S NN Z sa nn ss s NN s p Z - - uh spro yh. х и - Mod et oshi - s. leg. Оу - 2 л ШИ - ак - шш -К штон-к Me - ШЭ Шш ЗЕ хх -e M - ш ж ме - й - 2 5 й Ж я Я - more t-- С о- -U o ж - t yarizhch: , , zda ich tk Figure 223 (neli C-IU) restoration of the activation of the bank account - "Figure 23 0.8 and 06th « 04 0.2 Her 00 5.0 10.0 0.15.0 200 Х250 300 evnlyniI Figure Z4A zvvOnya Z oh1oe 50x105 tya 59500 59000 59500 Maszridy z i Figtra ZAV SAT ОЗ lyuchina STO Yanan macaque still SHY mo Shk SHY wed 0040 chnih Chasis! hChasie) «Figtra 25A «Figure 2368 Eagles of SPeblyavanska makakn i shk shi - ІЙ et Ash i a Den vera 0080 Day «BO I 50300 5 -B o 50100000 Chasisi Chas isis) Figtra C Figure 230 Capturing: 213125 people BVnyavleniya: SI people -z -- PANT-A iTo-- Contre BAV , h - Kantr. BANIT-schi: with ««- I and her SI I Anna 08 5.84 in 4 pavt (imkghimli Figure ZA Zahnat: SI Yanan macaque ! BNyavleniya SIIA lyulinia Z -- OANT-A I -- Contr. pAnt M - 84 -- ZONES . rANTt-z : se ram a. od v s PACT imkgoml) Figure 268 There is no BACT account in OOVAVT, Moit-13 and in Figtra Z 100000 LOANTGA EK 0000 UContr, BACT SHE with i. . she will still throw T-cells of lyudnna 3 B cells OO x her person is not there: And live to x h AUTO | Ffigtra 20 s CHOVAVT-A I B a-EK I Kovetr. BA KT g. Y CHE and T-cells of yavenoki her and shk sh | There is no figure 16E! и и и ши ше ше a-EK sg 100,000 shk | I - zak antibody to SB IZ o voobo SHI Zotyaipnyi control I o! zvayah xxx y; x B v i KZ che - ns Figure TA Tse57 ETO: 1004 F 80 | -- ORANTA - 404 105 404 402 4060 Concentranichingomli original TV Cavitsti-3 - T-cell tumors as ET: Z oo. 8 S PAT A ESza-o vi NML in 80 syu Kontu PAVT KE; ; So 20 and dad 18 and Concentration (ng/ml Figure S Kazshti-3 - REM of a Javan macaque ET -4 51: la - t her ESE - 5.2 vnglyal be -- pAVT-A x «v. e pavt E -- Control. BAKT 10403 02 409 382 Concentration (ngomli Figtra 27 HER. created to STENOSIS ! . Call! 2! | i -: o i p chavovo4 ro E . and I'm 54. E |. . same I. i B x |. . o I KZ Ka V z KZ KU EK Z ovo shsh sh no - ON Ko BE Z I EZ t. I CalypiZmionopytni s 00 Monocytes VES of Javanese people Javanese SVKaka Figure TE Manetsnty (sptaleso123 7, Ш Z hour jan. dey ybgeliv i 1553 | 5 Z E shi she she more. Shen shk Ya. in GE he 2B5chYASH EE SHE N shE TO E-Z IC her SHE SHE SCHE) AND MORE like OK SIV 5 KZ EE xx : SHE Z SIV KZ HU EI - х k m k e : V ee with F zh Ky y e sh in VAVT-A (nNgiMl Figtra TE plo . tr tota zh hu EV le sota svo1237 at 125 Shi ostodnny m Y U bgodni V SO 15 o'dnia ma what ! I, Kt is. shcha g i "v u I u it SHI t sh SCHE E Her i Shkz i st I st vk them still g ee "Z i k she e: Y pAVtT-A ingmli Figure 7 RVMS of the Javanese macaw screma (BAS sh YO o 2 zh . ni Sho ne KE i MG. DN o, 24 t ve still vecho in She ze ach sch V.B t zh nn And p. )! ; Concentration ng/ml Figure PN - | o - 150 - ac | VOoo- o to ee "| v», va ooo "r ka sh o sm o o xz i xx Bai Lozaini'kgidenvi Figure 28 inert nosin a shi schi that Z I 15 and 23 ЗB day of research Figure 70A raDkt-A 501 00000 pears: Ж групиз -y pi o g SHE SHE E x B B in VO r ki EE i o ENN K MA y MD i in KZ aa still se ki sabn ova ay z Z 8 15 ke 23 ki daendospizzhennya Figtra 2 vVadit- AND 1. В ЕЗ thing KK DK sense de EK 1 B 15 22 28 From the day of research Figure 29E inert carrier - 0 - group - 5 shsh shsh td ZO shi sh sh t her sh what daily 00 BE 00 B 0 NO - in go a Fo she SUCH SUCH , about us. still I a : y pen ' snkanrn, : enny ze sily zanu day of research Figure ZA rdait-A - 0 she AS Group? 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I za s schinnyi i i i Z 7500 i E sti di WHAT i : xx k «m E 2500 g i i i v t WRITE OOSKE fen MY i i 1 at 1155:5:28 5845508 5754 research day Figtra 514 pact da - - Group with her neck Lush Write and Eschez UTA Shen Ie Scar 30000345 It 8 525555348 455855 84 The Day of Fagitra Iv W. AAVT-A IN 12568 00000000-Group B is «APA 0! I 1 y y y y y y y : vNnya yy - I 5 Х 5 ov A a 2580: go" 6 h y 1532293 3643 50 57 84 research day Figure C Group 1 inert seed in porridge zy ishi I o TYSI NEK I B 4532 29 36 43 50 B? on the 4th day of the study Figure C Group Z paAavt-A in Kos i zho Z SHYNS OSHSHISHIIO OOISHIINY PISHE in i SHENEE Zo OSI OV a I ra i OK. OM - B ai B: sk I Ba SHE I ss: Ka: DY va U KO SOYA oosyu. sok Y v ve EE VV 1 4 Z5 22 25 35 45 50 657 55 Figtra LE Tula pAnT-a Oh | 5555555. VOD OVO I and sch with M KANAV 1 and chzhi5 295 58 35 45 5 BT Every day of research Figure ZIK Goabpai Inert medium shi zhi m shi shi fe Tut. Tettt U --y scha I Zauk A. people, ge A a ryn nya nya I her Z 5 25 25 35 45 5 BI a day of research Fig. 316: Group 5 rAaAt-A Zoi she sishny sho vya shi i i sho Z JSC eYshe 203 yr et i "viny re o" VILETSI Che AN Kn toriina ch a y 5 22 29 36 43 50 57 64 lendy research Fittra ZIN Grotuna b PAKT A zo" pe YANIS OIV OYI :- oj he M V I BOM" m VO . i Po OV KA t -X | B in Ky in VE VN a 1 y 15222556 53550 5755 Figtra ZY paAnt-a Group Z de, SHENISHY BNO Zo MNE och i ON IN NYA OO s -k t yav EE. as Bom nya ni shin and avya dnu -E Z | t sh- S zh pp Ya ren, - "ex sov o CC vein In KZ given Zh 1 zh Ya5 35 29 For 43 BO BT on the day of research Fntra MA pACT-A Group Z now and EE 1 mom vo» y. Yam oo. oh wow A A Her - woman A t nsh y Vshuoe p ENN NK an rennya t 8 15259225 35435057 54 day of research Figure ZV rAvt-A Group 4 ladies and shi vin y. oo actions - in . And A ; t. 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" KKKKKI PYSHNYI PP SHYN ram 0100 АХ Ж in I t to what ZY ee zho o ziv o ny her and 1527" 29 3543 50 57 5 day of research Fgtra ZAE rAVt-A Gotpa 2 in write i and i. eooshsh, "SHI SCHE Ше х я и тов 9 ЦИ 1 ш и ши и 8 1522 95 36 43 50 57 84 data of the study Figure C) PACT-A Group Z 1983 no no ne shi ury ni zh vad y (g shin il ya shir r OO 55 BO Mke BO YAM rdshnnym i Vi B kN Shi ih y di ley i y y PYSHY EE KO ren y y 5 15252 29335 43 58 5U Ba day of research Figure LE PANT A Group 4 1905 5 no no no ours OO SHE M ANU p u un un un rennie is; and - her I 152272 79 36 43 50 55 Ba day of blinding Figure ZLE Those who didn't know Those who were affected by the 100th act of the Act in the World War II! s a i: o v 50 (y o m : y s: i y o (y o Z : (y o a x Y V: 1 ! E. uki Control ACT BAKT-A Control BAVT-A Ffigtra Group of Inert media that PE EE PEN BNVO sha Kn usu uk an - She EE y. and in zajzhiyyydya same t. Z hu PISHE OSHESHESHE OMYSHSHNE PYSHNE 1 in t5z ZV ЗB Figure ХА Gruna 5 ін ш я жк "ій 5. NUT our shi. shsh. wedges and her a і5 22 29 36 43 50 BU 84 day of research Figure 5 PAKT A Group b 1063 s d I 1 00 in OA TO I BO bik VE, naik you MOU oi zhk 8 20. p. - s fri W sen INT t 8 1523223 35435057 54 day of research Figure E Group Inertnny carrier yav v/m iV : y i she ny t V i A A M - ee She SHY i i UM i, SHA t i V m SCHE be ke zen nny V 1529 29 36 43 50 57 54 day of research Figure ZE BANT-A Goutsa 5 lnshe Oi a - - o va V 00000000 NN to shi g V V AAU om. By. a and re de re Sho: , her in 1522 25365 43 50 57 64 jan research "FVigtra Z5ZE PACT. 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And VO 0000 Hope? e | tI4 t zno Group Z sho Zoo OKA I 0 Gopa4 - I M to I goupa 5 in OO rupa z NI vk o shi 2» h Y y shE. ate shi tyny she yes and shi gods her shh rsasnisns, and . a bin geRVeeya m vi, pr, in ROBNNVYA z: V so g z rninininnyy ss, -48 z her 8 1559 25 36 43 Ba V; on the day of the study Figtra According to PACT A t 40 g I 00000070 Brupab call. what did I do: JE g nshikh ie; Ше шимы Мне Др фрняяннняу з |янн нн вукун лк вв золоти уковоко іно ковуккиккккуоку -ы 3 из 8 из 22 25356 43 0 57 64 лен геасы Figtra OK I ШЙ Group and inertnin kosi "a y tr y I Group? rAVT-A m nie SHHI, 4 sne s Z Y GroupZ rAnt-A si ne Oi vo, I sh I o. yE SU Erupad acta EK nei SHE KA GroupZ ravt-d g a SCHI KY: tya y teye Me A She M: p. also Trupab rakt-A z ZE dir SHEU I SHE See ep sh How ency E BACH MO Melon IB izhe - M o. 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