BIPARATOPNYE POLYPEPTIDES - ANTAGONISTS OF SIGNAL TRANSMISSION OF WNT IN TUMOR CELLS

28-12-2018 дата публикации
Номер:
EA0201891331A1
Контакты:
Номер заявки: 33-91-20181
Дата заявки: 02-12-2016

[1]

BIPARATOPNYE POLYPEPTIDES - ANTAGONISTS OF SIGNAL TRANSMISSION OF WNT IN TUMOR CELLS

TECHNICAL FIELD

[2]

The present invention relates to new polypeptides, binding like receptor low density lipoprotein protein 5 (LRP5) and like receptor low density lipoprotein protein 6 (LRP6).

[3]

The invention also relates to nucleic acids, encoding such polypeptides; methods of manufacture of such polypeptides; cells - host, which express or are able to express such polypeptides;

[4]

compositions, containing such polypeptides; and also applications of such polypeptides or such compositions, in particular, in therapeutic purposes in the field of cancer diseases.

BACKGROUND OF THE INVENTION

[5]

Activation signal path of WNT requires binding extracellular of WNT-and-ligands with receptor Frizzled and byby LRP5 (number of access:

[6]

UniProtKB -075197/lrp5_human) or its close related homologue LRP6 (number of access: UniProtKB - 075581/lrp6 human being). In cells of mammals there is 19 proteins of WNT and 10 receptors Frizzled. In the absence of of WNT ligand cytoplasmic beta - catenin phosphorylated protein complex, consisting of frame proteins Axin and activated protein C, as well as kinases gsk3 - 6eta and sk1a. Followed by recognition of beta - tgsr - ubikvitinligazoi leads to ubiquitin - mediated degradation of beta - katenina. In the presence of of WNT ligand binding of WNT with Frizzled and LRP5 or LRP6 leads to to fish cytoplasmic of the effector protein Dvl and phosphorylation cytoplasmic tail LRP5 or LRP6, that provides site for connection Axin. Sequestration Axin by means of LRP5 or LRP6 leads to inactivation of complex Axin-and-American Power-and-GSK3-and-6eta, and it, to stabilization and accumulation of intracellular beta - katenina. So, level of beta - katenina in cytoplasm grows, and beta - catenin moves to nucleus, forming complexes with terms of family transcription factors tkletochnogo factor (Taverage)/factor, binds lymphoid enhancer (LEF). Then mechanism are basal transcription and transcription the coactivators, including protein (CREB-and-optional binding protein, SZRU), which binds with protein, binding with cAMP - sensitive element (cAMP of Response the Element-and-optional binding protein, CREB), or its homologue rzoo, which leads to expression of different target genes, including Axin2, cyclin d1 and with - the ICC.

[7]

Additional level of ligand - dependent regulation of track of WNT mediated ez - by the ligase RNF43 and its close related homologue ZNRF3, and also secreted proteins of r-spondinami (the de Lauand DP. "Of The of R-spondin/lgr5/rnf43 the Module: Regulator to care of WNT the Signal strength". The dev Genes. 2014;

[8]

28 (4): 305 - 16). RNF43 is intermediary in ubikvitinirovanii receptor complex Frizzled/LRP5 or LRP6 cell surface, results its degradation and so suppressing ligand - dependent activity track of WNT.

[9]

Activity of RNF43 counteract family members of r-spondinov (Rspondinovye ligands 1 - 4). In the presence of R spondinovogo ligand it eliminates RNF43 with cell surface, it possible accumulation of complex Frizzled/LRP5 or LRP6 and amplification of WNT-and-signalinga in the presence of of WNT-and-ligands.

[10]

LRP5 and LRP6 act as gatekeepers ligand - dependent activation of signal path of WNT and, consequently, can considered targets for achievement of full block track, all mediated 19 - th ligands of WNT and 10 - second receptors Frizzled, and also amplified of r-spondinovymi ligands. In particular, of WNT-and-ligands can be divide into classes and WntlWnt3a, each of which for signal transmission binds with different epitopes/sections LRP5 and LRP6. An extracellular domain LRP5 and LRP6 has four repeating beta - propeller element, connected with EGFR-and-similar domain, behind them follow three LDLR-and-repetition type. integration structural and functional analysis of LRP5 and LRP6 makes assume, that Wntl (ligand class Wntl) binds with fragment of LRP6, which contains beta - the propellers 1 and 2, a of Wnt3a binds with fragment, which contains beta - the propellers 3 and 4. present in known moment only image extracellular domain of LRP6, containing beta - propeller sections 1 - 4, in low solving (Ahnand DP."Structural BASIS team care of WNT signalinginhibition By the Dickkopf optional binding t lrp5/6". The dev the Cell. 2011; 21 (5): 862 - 73). However inaccuracy compensator of these reconstructions in low solving (40 a°) and absence of data about the structure of extracellular domain of LRP6 in complex with of WNT-and-ligands do not allow accurately determine epitopes, receiving participate in binding ligands Wntl or Wnt3a.

[11]

Giperaktivatsiya signal path of WNT receives participate in pathogenesis of various types of cancer. In some types of cancer frequent mutations in molecules signal descending tract promote constitutive activation of track of WNT (nair ., mutations activated protein C at of colorectal cancer; activated beta - catenin mutation at pechenochnokletochnoi carcinoma). Opposite, at three times negative breast cancer (tnrmzh), nemelko cellular cancer light (nmkrl), to the adenocarcinoma pancreatic, and also subset colorectal crayfish (operation switch) and endometrialnykh crayfish activation signal path of WNT is actuated ligand - dependent mechanism (T. e ., autokrinnoi/parakrinnoi activation of WNT), detectable by endocellular accumulation of beta - katenina. At nmkrl, tnrmzh and to the adenocarcinoma pancreatic ligand - dependent activation of WNT mediated multiple mechanisms, including to increase the nnuyu expression of WNT-and-ligands and/or receptors LRP5 and LRP6, or same silencing negative regulator LRP5 and lrp6 - dkk1 (tnrmzh: Liuand DP "LRP6overexpressiondefines a of the class care breastcancersubtype and sort Li a of the target for Laptops therapy ". The proc NatlAcad SCI to the USA 2010; 107 (11): 5136 - 41;

[12]

Khramtsovand DP. "Of WNT/of BETA-and-cateninpathway the activation Li enriched in vitro basal-and-like in their places breastcancers and sort predictspooroutcome". AW JPathol. 2010; 176 (6): 2911 - 20; NMKRL:

[13]

Nakashimaand DP. "Wntloverexpression stating Associated Hotel With tumorproliferation and sort of a poorprognosis of in vitro the non-the Small the Cell lung-cancerpatients". Oncol AGE. 2008; 19 (1): 203 - 9 ; pancreatic cancer: the Zhang and DP. "The Canonical of WNT signaling Li the Required for Laptops pancreaticcarcinogenesis". Cancer resolution RES. 2013; 73 (15): 4909 - 22). In particular, published data show, that in healthy tissues (nair ., epithelium breast and lung) beta - catenin lokalizirovan strictly in plasma membrane. In contrast from this, in most primary clinical specimens tnrmzh, nmkrl and adenocarcinoma pancreatic was intracellular beta - katenina (T. e ., in cytoplasm/core; biomarker activation of WNT-and-signalinga) from - the disturbed of WNT-and-signalinga. In recent the publications was shown, that ligand - dependent activation of signal path of WNT mediated mutated/inactivated RNF43 (Giannakisand DP. "RNF43 Li frequentlymutated in vitro colorectal and sort endometrialcancers". Address Genet. 2014; 46 (12): 1264 - 6) or activation of r-spondinovykh fused transcripts (encoding proteins Rspondin2 or of r-spondinz, controlled constitutively active strong promoters; Seshagiriand DP. "Recurrent of R-spondinfusions of in vitro the colon cancer". Nature 2012; 488 (7413): 660 - 4) Was shown, that inactivation of mutations as RNF43, so and R spondinovykh fused transcripts enhances ligand - dependent of WNT-and-signalingin vitro of, increasing relative content of Frizzled on the cell surface. Was it is shown, that ligand - dependent activation of WNT in tumors stimulates the growth and its tumor resistance to chemotherapy or immunotherapy, and also is connected with recurrence in pre-clinical models.

[14]

From level of technology are known some molecules, binding LRP5 or LRP6 and capable to modulate signal path of WNT:

[15]

Dickkopf-and-1 (DKK1) - this inhibitor LRP5 and LRP6. DKK1 binds with both byby of WNT, LRP5 and 6, and also transmembrane protein Kremen, suppresses of WNT-and-signaling and leads to rapid internalization LRP5 and LRP6. Was it is shown, that DKK1 suppresses as Wntl -, and Wnt3aoposredovannuyu signal transmission. Examination by structural modeling have shown, that separate molecule DKK1 cooperative and binds with elongated section extracellular domain of LRP6 (from 1 - th to 3 - of beta - propeller). Structural analysis predict interaction DKK1 and LRP6 by principle of cooperative binding, with initial binding with 3 - m beta - propeller section, which facilitates the interaction/bonding with 1 - m and 2 m - beta - propeller sections, since changes conformation extracellular domain of LRP6. However, as already mentioned, specific epitopes in domains 1 - of, 2 - of and 3 - of beta - propellers, receiving participate in binding DKK1 and LRP6, not clarified from - the low-resolution reconstructions structure complete extracellular domain of LRP6, coupled with DKK1.

[16]

Was it is shown, that treatment DKK1in vivo of leads to serious toxic reactions of gastro - intestinal tract. In particular, was shown, that indirect adenovirus expression DKK1 in adults mice considerably it inhibited proliferation in thin and intestine, that accompanied by progressive degeneration of its architecture, large loss of body weight and mortality from colitis and generalized infection. In particular, LRP5 and LRP6 expression in proliferating epithelial cells of small intestine and necessary for proliferation of intestinal epithelium, that induces on suggested, that inhibition of LRP5 and LRP6 may be toxic for this and other normal tissues (Zhongand DP. "Lrp5 and sort Lrp6 the Play compensatory Roles list of in vitro the Mouse intestinal their Development". The Cell of j Biochem.

[17]

2012; 113 (1): 31 - 8). Therefore doubts arise in, that inhibitors of LRP5 and LRP6 or do which - or inhibitors of signal path of WNT (Wntl and Wnt3a) can be used in therapeutic purposes, nair ., may go in development as agent against cancer.

[18]

W02009/056634 relates to molecules, binding with LRP6, which may antagonistically or agonisticheski interact with signal by Wntl or with signal by wnt3/3a, and which may be used in diagnosis or for treatment of "disorders, associated with signal by of WNT", such as osteoarthritis, polycystic kidney or cancer. In this document not mentioned which - or specific examples similar binding molecules, certain by their amino acid sequences.

[19]

In wo2011/138391 and wo2011/138392 invention describes multivalent antibodies, binding LRP6. In wo2011/138391 invention claims antibodies, blocking one of signal tracts of WNT (Wntl or Wnt3), not enhancing at that other path (respectively Wnt3 or Wntl). In wo2011/138392 among other things presented antibodies or fragments of antibodies, reinforcing signal of WNT by formation of receptor clusters LRP6.

[20]

In wo2011/138391 explained, that for desired effect molecules, binding LRP6, should be adjusted to format full of IgG antibodies. Examples are given biparatopnykh molecules LRP6, including molecule of IgG with the first specificity binding, connected with single-chain chain Fv-and-section with the second specificity binding.

[21]

Some formats are described as having considerably WiFi client continuously nnoi thermal stability (Tiel from 50 to 52 theoretically). Fn-and-section can to molecule of IgG effector function, such as complement - dependent cytotoxicity of (kzts) or antitelozavisimayakletochnooposredovannaya cytotoxicity of (azkts).

[22]

Β-wo2013/067355 invention describes binding LRP6biparatopnye immunoglobulin scFv antibody-and-constructs with prolonged period half-, obtained from molecules of IgG, described in wo2011/138391.

[23]

In wo2011/119661 there described are antibodies, binding with LRP6 and suppress signal transmission through first to the isoform of WNT, especially through Wnt3 or Wnt3a, but reinforcing transmission signal through the second to the isoform of WNT, which can be isoform Wntl, 2, 2, 4, 6, 7a, 7 [7 [], 8a, 9a, 9 [9 [], 10a or 10 [10 [].

[24]

Invention describes bispecific molecules, binding with E1 - E2 - section LRP6, as well as with ez - E4 - section LRP6. For production of bispecific antibodies used technology "projections - in - cavity".

[25]

Epitopes binding (defined amino acid residues within the extracellular domain of/beta - propeller sections LRP6), receiving participate in binding LRP6 with antibodies, not identified neither in w02009/056634, neither in wo2011/138391 or wo2013/067355, and only partially identified in wo2011/119661. In particular, binding LRP6 antibodies are able to suppress signal transmission of WNT by means of alternative mechanisms, corresponding binding with different sections LRP6, including direct competition with Wnts or inhibition of formation of triple receptor complexes (of WNT-and-LRP6-and-Frizzled), as other amplified signal transmission, it is possible, by formation of receptor clusters (Ahnand DP "Structural BASIS team care of WNT signalinginhibition By the Dickkopf optional binding t lrp5/6".

[26]

The dev the Cell. 2011; 21 (5): 862 - 73).

[27]

Nevertheless, neither one of described in the level of equipment binding molecules until that has not been committed members health care to use as a medicinal agent for treatment of any of - or disease. Speaking more specifically, like application requires very special binding properties of, correct specificity, to such molecules not attributed, have not activated or not inhibited other target (Eg ., results of undesirable activation or inhibition of other signal tracts, or same insufficient activation or inhibition of relative to target to the isoform), in case of biilimultispetsifichnykh agents - overlying balance of two or more the specificities binding, proper pharmacokinetic and pharmacodynamic properties of, acceptable toxicological profile and, end same, efficiency of in vivo of.

[28]

In light of above contained there is demand for new therapeutic agents, providing effective treatment of various types of cancer diseases and tumors. So, the invention is provision similar pharmacologically active agents, which may be used in treatment of row cancer diseases, including nmkrl and tnrmzh.

[29]

In particular, the invention is provision similar pharmacologically active agents, compositions and/or methods of treatment, which have certain advantages as compared with used and/or known from the equipment on moment agents, compositions and/or methods. These advantages include efficiency in vivo of, improved therapeutic and pharmacological properties, lesser amount of side effects, as well as other favorable properties, such as simplified manufacture of or WiFi client continuously nnaya cost of goods, in particular comparison with already known potential of medicinal preparations.

BRIEF DESCRIPTION OF THE INVENTION

[30]

According to the first aspect, present invention polypeptides are provided, which specifically bound with LRP5 or LRP6, like the polypeptide according to the invention has first separate immunoglobulin variable domain (and), selected from the group of separate up immunoglobulin variable domains from (1) to (of III), determined the presence of the following CDR--sequences:

[31]

(I):

[32]

CDR1: TYTVG (=SEQ N0:1)

[33]

CDR2: AIRRRGSSTYYADSVKG (=SEQ N0:2)

[34]

CDR3: DTRTVALLQYRYDY (=SEQ N0:3)

[35]

(II):

[36]

CDR1: SYAMG (=SEQ N0:4)

[37]

CDR2: AIRRSGRRTYYADSVKG (=SEQ N0:5)

[38]

CDR3: ARRVRSSTRYNTGTWWWEY (=SEQ N0:6)

[39]

(III):

[40]

CDR1: RYTMG (=SEQ N0:7)

[41]

CDR2: AIVRSGGSTYYADSVKG (=SEQ N0:8)

[42]

CDR3: DRRGRGENYILLΥSSGRYEΥ (=SEQ N0:9),

[43]

and second immunoglobulin separate variable domain (b), selected from the group of separate up immunoglobulin variable domains (of IV) and (V-), determined the presence of the following CDR--sequences:

[44]

(IV):

[45]

CDR1: SYAMG (=SEQ N0:10)

[46]

CDR2: AISWSGGSTYYADSVKG (=SEQ N0:11)

[47]

CDR3: SPIPΥGSLLRRRNNYDΥ (=SEQ N0:12)

[48]

(V):

[49]

CDR1: SYAMG (=SEQ N0:13)

[50]

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

[51]

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15).

[52]

Terms "first" and "second" relative to this separate immunoglobulin variable domains are intended only for the designations, that these domains are two different domains (as they at least contain different CDR--sequence) .data therefore terms should not be understood as indicating accurate order or sequence domains in similar polypeptide chain.

[53]

Polypeptides according to the invention optionally contain third immunoglobulin separate variable domain, especially such, as albuminsvyazyvayushchii separate immunoglobulin variable domain, as is - domain Alb 11, containing the following CDR-:

[54]

CDRl (Albll): SFGMS (=SEQ n0:16)

[55]

CDR2 (Albl 1): SISGSGSDTLYADSVKG (=SEQ n0:17)

[56]

CDR3 (Albl 1): GGSLSR (=SEQ n0:18).

[57]

According to more specific version of polypeptides according to the invention include separate immunoglobulin variable domains, which are domains VHH, preferably humanized domains VHH.

[58]

According to more specific version of polypeptides according to the invention contain the first separate immunoglobulin variable domain (and), selected from the group of separate up immunoglobulin variable domains from (1) to (of III), which have the following sequence:

[59]

AVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTVGWFRQAPGKEREFV AAIRRRGSSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTRTVALLQYRYDYWGQGTLVTVSS

[60]

(=SEQ N0:19)

[61]

(II):

[62]

A OF OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-S-S-GGTFΥAMGWFRQ Α-P GKEREF OF V AAIRRSGRRTΥΥ AD OF S-VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYY OF C AAARRVRSSTRYNTGTWWWEYWGQGTL VT CIRCUITS VS OF S-

[63]

(=SEQ N0:20)

[64]

(III):

[65]

AVQLVESGGGLVQPGGSLRLSCAASGLTFSRYTMGWFRQAPGKEREF VAAIVRSGGSTYYADSVKGRFTISRDNSKNTVYFQMNSFRPEDTAVYYCAADRRGRGENYIFFYSSGRYEYWGQGTF VT CIRCUITS VS OF S-

[66]

(=SEQ N0:21),

[67]

and second immunoglobulin separate variable domain (b), selected from the group, consisting of separate up immunoglobulin variable domains (of IV) and (V-), which have the following sequence:

[68]

(IV):

[69]

E OF V QF THE VE S-GGGE OF V QUANTIZATION GGS THE FRF S WITH A-A-S-S-S-GRTFΥAMGWFRQ AP GKEREF OF V A OF THE AIS OF W SGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASPIPYGSLLRRRNNYDYWGQGTLVTVS S-

[70]

(=SEQ n0:22), and

[71]

(V):

[72]

E OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-S-S-GGTFΥAMGWFRQ Α-P GKEREFVAAISWRSGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADPRGYGVAYVS HBSAG AY YEYWGQGTLVTVS S-

[73]

(=SEQ N0:23).

[74]

According to especially preferable alternative polypeptides involves additionally fragment, drives enhanced half, said fragment, drives enhanced half, is covalently bound with said polypeptide and optionally is selected from the group, consisting of albuminsvyazyvayushchego fragment, such as albuminsvyazyvayushchii peptide or albuminsvyazyvayushchii immunoglobulin domain, preferably albuminsvyazyvayushchii separate immunoglobulin variable domain, more preferably domain Alb 11, transferrinsvyazyvayugtsego fragment, such as antitransferrinovyi immunoglobulin domain, molecules of polyethylene glycol, human serum albumin, and also fragment of human serum albumin.

[75]

Especially preferable are polypeptides, which in addition to two separate immunoglobulin variable domains (and) and (b) according to the description above contain domain a1y1, having the following sequence:

[76]

EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSS ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS

[77]

(=SEQ N0:24)

[78]

According to the next version of the invention specific polypeptides, containing or consisting of any of the following three polypeptide chains:

[79]

F13500575 with sequence SEQ ID n0:25,

[80]

F13500571 with sequence SEQ ID n0:26, and

[81]

F13500720 with sequence SEQ ID n0:27.

[82]

According to further aspects the invention concerns nucleic acid molecules, expression vectors, cells - host, and also methods of production of, used for manufacture of invented polypeptide.

[83]

Nucleic acid molecules, encoding polypeptides according to the invention, in separated form can be used for construction of corresponding expression vectors, which are then can be transferred by transfection into cells - hosts, used for biopharmaceutical production of polypeptides according to the invention. Similar methods of manufacturing, as a rule, include stages culturing cells - host in the conditions, expression of polypeptide, isolation of polypeptide and its cleaning known in the field of methods.

[84]

Further aspects, versions, application and methods, are used in which polypeptides according to the invention, will be clear from below of the following detailed description of the invention and of the formula applied.

[85]

Invention presented new molecules, which make possible more efficient treatment of various types of cancer, such as tnrmzh, operation switch and nmkrl, with smaller number of side effects. Polypeptides according to the invention provide unexpected therapeutic effect (T. e ., the validity) in treatment of cancer patients patients, consists in, that they are able to cause regression of tumor, which to the full patomorfologicheskomu response (CSPs). Expected, that this in its turn leads to considerable improvement of survival rate without progressing and common survival rate, especially in case of large unsold medical demand, such, as, nair ., at breast cancer. So, polypeptides according to the invention provide new therapeutic possibility for treatment of row types of cancer, in particular the, where detected razregulirovannyi signal path of WNT and beta - katenina.

[86]

Moreover, polypeptides according to the invention easily to, they have high stability and low antigenicity, and also is opened a number of capabilities relative to ways of introduction, apart from injections and infusions.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[87]

On Figure 1 it is shown schematic image of biparatopnykh polypeptides - antagonists of signal transmission Wntl and Wnt3a. They consist of three domains, two of which are with certain epitopes LRP5 and LRP6 (blocking agent and WntlWnt3a) and one serves for extending half-time (domain, binding with human serum albumin).

[88]

On Figure 2 it is shown absence of correlation between analyses binding by methods FACS and is preferable for representative amount of LRP6-binding VHH, obtained in immunization Lamas. Panel VHH under number "1" is characterised by high affinity to cells, expressing LRP6 on plasma membrane, the detected by means of analysis of binding FACS (along axis shown in value MCF). Panel VHH under number "2" is characterised by high affinity to recombinant extracellular domain of human LRP6 (rhLRP6-and-fn), the detected by means of analysis of binding is preferable (X axis shown value od405).

[89]

On Figure 3 it is shown binding three biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged period with half-LRP5 (figure behind) and LRP6 (figure of members), which they are supered-express in cell lines [nek 293, as compared with negative control, consisting of nenatselennogosvyazyvatelya (VHH-construct, binding with bacterial protein, which in cells [nek 293 not is expressed).

[90]

On Figure 4 is indicated complete competition three biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged period with half-DKK1 behind binding as with human LRP5 (Figure 4 and), and human LRP6 (Figure 4 in), which they are supered-express in cell lines [nek 293, detected in analysis DKK1-and-competition of based on FACS.

[91]

On Figure 5 it is shown complete inhibition track Wntl and Wnt3a three biparatopnymiperekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs with prolonged period half-(Figure 5 and) and comparison with other molecules, coupling LRP6 (Figure 5 β-: Knob ns yw2 10.09 and MOR08168IgGlLALA 6475 scFv antibody; Figure 5 with: 802t) in combined analysis of gene - to the reporter and WntlWnt3a.

[92]

On Figure 6 demonstrated is inhibition of WNT-and-signalinga in cancer cells, detectable inhibition of relative mRNA expression Axin2 (Figure 6 and), and also cell proliferation (Figure 6 in), used for the WiFi client continuously by agreed to percent (%) viable cells, after treatment three biparatopnymiperekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs with prolonged half-period (in final concentration of 1 mm) and after treatment of 802t, as compared with f013500571 and untreated (control) cells (of left and right sides figure 6B); for treatment of one biparatopnym lrp5/lrp6 construct with prolonged period half-(Figure 6 with) and 802t (Figure 6 d of) shown curves dependence response from dose.

[93]

On Figure 7 is indicated efficiency biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged half-period in vivo of (F013500571 on figure 7a and f013500720 on figure 7B), and also Knob ns yw2 10.09 (Figure 7 with), on models tumors, caused by of WNT (ksenotransplantatnaya model MMTV-and-Wntl).

[94]

On Figure 8 demonstrated is inhibition track of WNT in tumors, treated biparatopnymiperekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs with prolonged period half-f013500571 and f013500720, was that WiFi client continuously detectable level of expression of mRNA Axin2 in the control group.

[95]

On Figure 9 it is shown the effect of suppression of the \ up13a - signalinga on release of generating TNF-alpha - dendritic cells (Figure 9 and), and influence on activation of T - cells, revealed release interferon - gamma, after treatment biparatopnymperekrestnoreaktivnym by lrp5/lrp6 VHH-and-construct with prolonged half-period. Each symbol means separate donor dendritic cells (DC). Given data standardised to level of TNF-alpha - in shape of the control (Figure 9 and), and each symbol means unique donor pair for DC and T - cells (panel in).

OVERHEAT DESCRIPTION OF INVENTION

Definitions

[96]

Set out above and other aspects of and versions of the invention will be clear from given then description, in which:

[97]

and) if not as indicated or not is determined or other, all used terms have usual for the given area value, which obviously skilled. For example, present refer on standard aid, such as Sambrook and others ., "MolecularCloning: a of Laboratory and manuallymanual" (2 - E of 2 yr.), T. 1 - 3, the Cold of Spring Harbor framework Laboratory and Press Books (1989); Lewin, "Genes of IV", the Oxford University of Press Books, herpex - York, (1990), Roittand ∂ρ ., "Immunology" (2 - E of 2 yr.), the Medical Gower Publishing by, London, herpex - York (1989), as well as on here cited documents total level of technology. In addition, if not as indicated or other, all methods, stages, procedures and manipulations, specific part of which do not invention describes, may be made known and implemented of per of Se by method, that will be clearly testify skilled. For coded again - still refer on standard aid, here cited documents total level of equipment and further characteristic documents.

[98]

b) if not as indicated or other, terms "immunoglobulin" and "immunoglobulin sequence" - are used designations for whether they are heavily chaining antibodies same or common 4 - chaining antibodies used as common terms, embracing full-length antibodies, their separate circuit, as well as any their parts, domains or fragments of (including, but not by them, antigen-binding domains or fragments, such as VHH-and-domains or the VH/VL-and-domains, respectively). In addition, here used term "sequence" (for example, in composition of such terms, as "sequence of immunoglobulin", "sequence antibodies", "sequence (separate) of the variable domain of", "VHH sequence" or "sequence of protein") should be as a whole understand as embracing and corresponding to amino acid sequence, and encoding its nucleic acid sequence or nucleotide sequences, if only context of not implied more limited interpretation;

[99]

in) term "domain" (polypeptide or protein) hereinafter means folded protein structure, which is capable to retain its tertiary structure independently from the rest of protein. As a rule, domains meet the separate functional properties of proteins, and in many cases may be added, removed or transferred to other protein without loss of function residue protein and/or domain.

[100]

g) term " immunoglobulin domain" hereinafter used for designations globular section in chain of antibody (such as, Eg ., circuit common 4 - chaining antibody or heavily chaining antibodies) or same polypeptide, which in fact consists of such globular section.

[101]

Immunoglobulin domains characterized in that is stored characteristic for molecules antibodies immunoglobulin laying, representing 2 - sloinyi "sandwich" from approximately seven antiparallel beta - threads, laid in two beta - sheet, optionally stabilized conservative disulfide bond.

[102]

d) term " immunoglobulin variable domain"hereinafter means an immunoglobulin domain, consisting in essence of four" frame sections ", which here and in documents level of technology designated as" frame section 1 "or" fr1 ";" frame section 2 "or" fr2 ";" frame section 3 "or" fr3 "; and" frame section 4 "or" fr4 ", respectively; these carcass sites are alternated three" sections, determining complementarity "or" CDR-", which here and in documents level of technology designated as" complementarity determining section 1 "or" CDR1 ";" complementarity determining section 2 "or" CDR2 "; and" complementarity determining section 3 "or" CDR3 ", respectively.

[103]

So, common structure or sequence of the variable domain of immunoglobulin may be displayed to in the following form: fr1 - cdr1 - fr2 - cdr2 - fr3 - cdr3 - fr4. namely immunoglobulin variable domain (s -) gives antibody specificity by antigen, since on it is antigen-binding section.

[104]

E) term "separate immunoglobulin variable domain" hereinafter means immunoglobulin variable domain, which is capable of specifically binding with epitope of the antigen, not paired with additional variable immunoglobulin domain. One of examples of separate up immunoglobulin variable domains in a context of invention are "blast antibodies", such as immunoglobulin separate variable domains the VH and VL (the VH-and-domains and VL-and-domains). Other important example of separate up immunoglobulin variable domains are " VHH-and-domains " (or simply VHH) camelids, determination of which description specifies below.

[105]

In light of above contained of determining antigen-binding domain common 4 - chaining antibodies (such, as molecule of IgG, of IgM, of IgA, IgD or IgE; are known from level of technology) or of Fab-and-fragment, P (a ') 2 - fragment, fv [fragmentafv, such as it is disulfide cross-linked fv [ilifv scFv antibody-fragment, or diatela (all are known from level of technology), from functioning like common 4 - chaining antibodies, as a rule, will not be considered separate immunoglobulin variable domain, as in these cases binding with corresponding epitope of the antigen usually is not at use of only one (separate) immunoglobulin domain, but requires pair (combined) up immunoglobulin domains, such as variable domains of light and heavy chain, T. e ., pairs of the VH-to other State up immunoglobulin domains, which jointly bound with epitope corresponding antigen.

[106]

the El) "VHH-and-domains", known as well as VHH, inH H - domains, VHHfragmenty antibodies and VHH-antibody, initially have been described as antigen-binding immunoglobulin (variable) domains "heavily chained antibodies" (T. e ., "antibodies, not having light chains of";

[107]

Hamers-and-Casterman with, Atarhouch T, Muyldermans s-, the Robinson of G, Hamers of c, Songa preservation of the, Bendahmanν, Hamers R: "Naturallyoccurringantibodies devoid ones care Emitting chains"; Nature 363, 446 - 448 (1993)). Term "VHH-and-domain" was selected, to distinguish such variable domains from variable domains of heavy chain, available in ordinary 4 - chained antibodies (which hereinafter marked as "OS - domains" or "the VH-and-domains") and from variable light chain domains, available in ordinary 4 - chained antibodies (which hereinafter marked as "u - domains" or "VL-and-domains"). VHH-and-domains are capable of specifically binding with epitope without additional antigen-binding domain (as distinct from vh [ilivh VL-and-domains common 4 - chaining antibodies, in case of which vl [ivl the VH-and-domains recognize epitope together). VHH-and-domains are small, stable and effective antigenraspoznayushchimi units, formed by separate immunoglobulin domain.

[108]

In a context of the present invention terms VHH-and-domain, VHH, VHH-and-domain, VHH-fragment antibody, VHH-antibody, and also "Nanobody®" and "Nanobody® - flOMeH" ("Nanobody" is trade mark company AblynxN.V.; Ghent; Belgium) are used interchangeably and designated representatives separate up immunoglobulin variable domains (having structure fr1 - cdr1 - fr2 - cdr2 - fr3 - cdr3 - fr4 and binding specifically with epitope, without dependence for this purpose in the presence of the second immunoglobulin variable domain of), which also can be discrimination between from the VH-and-domains by so-"distinctive remnants of" according to the determination of, nair ., w02009/109635, Figure 1.

[109]

Amino acid residues of VHH-and-domain numbered on general system numbering OS - domains, developed Rabatand DP. ("Sequence care proteins care immunologicalinterest", the US the Public the Health the Services, NIH Bethesda, Maryland, publication № 91), according to its use for the VHH-and-domains camelids, shown, nair ., on figure 2 from Riechmann and Muyldermans, J immunol.

[110]

Methods from 231, 25 - 38 (1999). According to this system numbering

[111]

- Fr1 consists of amino acid residues in positions 1 - 30,

[112]

- CDR1 consists of amino acid residues in positions 31 - 35,

[113]

- Fr2 consists of amino acid residues in positions 36 - 49,

[114]

- CDR2 consists of amino acid residues in positions 50 - 65,

[115]

- Fr3 consists of amino acid residues in positions 66 - 94,

[116]

- CDR3 consists of amino acid residues in positions 95 - 102,

[117]

- Fr4 consists of amino acid residues in positions 103 - 113.

[118]

However it should be mentioned - and this well known for OS - domains and for VHH-and-domains - that total number of amino acid residues in each CDR-can vary and may not correspond to the total amount of the amino acid residues, numbering carbonator by Rabat (namely, one or more positions according to numbering by Rabat in real sequences may not be busy, or same real sequence can more than amino acid residues, than is system Rabat). This it, that as a whole numeration by Rabat may correspond to or not correspond to real numbering amino acid residues in real sequence.

[119]

From level of technology are known alternative methods of numbering amino acid residues OS - domains, which may similar manner be used and to VHH-and-domains. Nevertheless, if not as indicated or other, the present description, formula and on shapes is used numeration by Rabat, applied to VHH-and-domains according to the description above.

[120]

The total number of amino acid residues in VHH-and-domain, as a rule, is in the range from 110 to 120, frequently between 112 and 115. Should be, however, noted, that for described here targets can approach and more short and long sequence.

[121]

Further structural characteristics and functional properties VHH-and-domains and containing their polypeptides can be summarized as follows:

[122]

VHH-and-domains ("designed" nature for functional binding with the antigen without presence of variable domain of the light chain and without any - or interaction with it) are able function as separate, relative to small functional antigen-binding structural unit, domain or polypeptide. This differs VHH-and-domains from vh [ivh VL-and-domains common 4 - chaining antibodies, which alone, as a rule, not are suitable to be practical use as separate of antigen-binding proteins or separate up immunoglobulin variable domains, but require the combination of the in the or other form of, to provide functional antigen-binding unit (as, for example, in ordinary fragments of antibodies, such as of Fab fragments; in scFv antibody, consisting of the VH-and-domain, covalently connected with VL-and-domain).

[123]

Due to this unique properties field VHH-and-domains separately or as part of larger polypeptide - has a row of considerable advantages as compared with using normal vh [ivh VL-and-domains, scFv or normal antibody fragments (such, as fab [ilifab p (a ') 2 - fragments):

[124]

- for high affinity and high-selective binding antigen is required only one domain, so that there is no necessity in presence of two separate domain, and also certified in correct spatial conformation and configuration of these two domains (T. e ., by means of special linkers, as in case of scFv antibody).

[125]

- VHH-and-domains can expressed one gene and do not require posttranslational laying or modifications;

[126]

- VHH-and-domains can be easily built in multivalent and multispecific formats (about than states then);

[127]

- VHH-and-domains soluble and do not show tendency to aggregation (as murine antigen-binding domains, the described dollpage and DP., Nature 341:

[128]

544 - 546 (1989));

[129]

- VHH-and-domains very resistant to effect of temperature, pH of, proteases and other denaturating agents or conditions, and therefore may be made of, be store or without using refrigerating equipment, thereby saving finance, time and preventing consequences for the environment;

[130]

- VHH-and-domains relative to simple and .5 cl to be made, even in industrial scale. For example, VHH-and-domains and containing their polypeptides can be produced by culturing microbial (nair ., according to description below), not using expression systems mammals, required, for example, for normal antibody fragments;

[131]

- VHH-and-domains as compared with conventional 4 - chain antibodies and their antigensvyazyvayushchimi fragments relative to small (approximately 15 kDa, or 10 times less than, than usual of IgG), and therefore

[132]

- better penetrate tissue and

[133]

- may be introduced in more high doses

[134]

than common 4 - chain antibodies and binding fragments thereof;

[135]

- VHH-and-domains can an so called properties of the mandarin binding (among other things due to elongated as compared to ordinary the VH-and-domains loop their CDR3), and therefore are accessible to them also target and epitopes, inaccessible for normal 4 - chained antibodies and their antigen-binding fragments.

[136]

Methods for preparing VHH-and-domains, binding with specific antigen or epitope, have been described previously, Eg ., in w02006/040153 and wo2006/122786. Also in this document detail described is, that VHH-and-domains camelids can be "humanize", replacing one or more amino acid residues in amino acid sequence original VHH-sequence one or more amino acid residues, which are in the same positions the VH-and-domain common 4 - chaining human antibody. Humanized VHH-and-domain can contain one or more fully human sequences frame sections, and in more specific version of realisation can contain human sequence frame sections, obtained from of DP-29, of DP-47, of DP-51, or their parts, optionally combined with sequences JH, such as jh5.

[137]

℮2) " Blast antibodies", also known as" Dab "," the Domain Antibodies ", and" dAbs "(where terms" the Domain Antibodies "and" dAbs " are used as trade marks group companies GlaxoSmithKline), have been described, Eg ., in dollpage E.S. and ∂ρ. : "Optional binding activities care of a repertoire care the Single immunoglobulin the variable Domains list secreted the From of Escherichia coli"; Nature 341:544 - 546 (1989); HoltL.J. and ∂ρ. : "The Domain antibodies: proteins for Laptops therapy"; TRENDS in vitro Biotechnology 21 (11): 484 - 490 (2003); and also in w02003/002609.

[138]

Blast antibodies in fact correspond to vh [ilivh VL-and-domains mammals, that are not verblyudovymi, in particular, human 4 - chained antibodies. That it may be to bind epitope as separate antigen-binding domain, T. e ., not in pair with corresponding vl [ilivl the VH-and-domain, is required special withdrawal by such portions properties, Eg ., by means of libraries sequences separate human vh [ilivh VL-and-domains.

[139]

Blast antibodies, as VHH, have molecular mass from approximately 13 to approximately 16 kDa, and if obtained from fully human sequences, then do not require for humanization, nair ., therapeutic application article on. As in case of VHH-and-domains, they well expression of in prokariotnykh systems expression, that provides considerable reduction common production costs.

[140]

Blast antibodies, as VHH-and-domains, can be subjected affinnomu ageing, introducing in amino acid sequence of one or more CDR-change, which lead to improved affinity of the obtained separate immunoglobulin variable domain to its corresponding antigen as compared with corresponding parent molecule. Subjected affinnomu ageing molecules separate up immunoglobulin variable domains according to the invention can be produced from known level of technology methods, for example, described in the Marks and other ., 1992, Biotechnology 10:779 - 783, or Barbasand other ., 1994, The proc. Address. Acad. SCI to, the USA 91:3809 - 3813. ;Shierand ∂ρ ., 1995, Gene 169:147 - 155; Yeltonand ∂ρ ., 1995, Immunol. 155:1994 - 2004; Jacksonand ∂ρ ., 1995, J Immunol. 154 (7): 3310 - 9; and also Hawkinsand ∂ρ ., 1992, J Mol. Biol. 226 (3): 889,896; Ks variance at Johnson and sort Re of Hawkins, "Affinitymaturation care the Using phageantibodies the Display", the Oxford University of Press Books 1996.

[141]

℮3) in addition, skilled in this area also will be clearly testify, which can be "transplants" one or more of above mentioned CDR-to other "frames", including, but not by them, frames of human origin or nonimmunoglobulin frames. Suitable frames and procedures for similar transplantation CDR-are known from level of technology.

[142]

solid) terms "epitope"and"antigenic determinant", which may be used interchangeably, designate part of macromolecules, such as polypeptide, being recognized antigensvyazyvayushchimi molecules, such as common antibodies or polypeptides according to the invention, and more specifically section portions of these molecules. Epitopes is determined minimum section binding of immunoglobulin and therefore are target specificity of immunoglobulin.

[143]

Part of binding molecules (such, as usual antibody or polypeptide according to the invention), which recognizes epitope, called byby.

[144]

3) term "biparatopnaya " (antigen -) binding molecule or "biparatopnyi" polypeptide hereinafter means polypeptide, comprising the first separate immunoglobulin variable domain and the second immunoglobulin separate variable domain according to this here determination of, the these two variable domain are able to bind with two different epitopes of one antigen, one monospetsifichnyi immunoglobulin, such as, nair ., usual antibody or one separate immunoglobulin variable domain, in normal conditions not binds with both these epitopes simultaneously. Biparatopnye polypeptides according to the invention consist of variable domains with different by the specificities by epitopes and not contain mutually complementary pairs of variable domains, binding with one and the same epitope. Therefore they not competed with each other the binding with LRP5 or LRP6.

[145]

and) polypeptide (such, as immunoglobulin, antibody, immunoglobulin separate variable domain, polypeptide according to the invention or antigen-binding molecule or its fragment in common understanding), capable of "to bind", " to bind with ","specifically to bind"or"specifically to bind with ", showing"affinity to" and/or "specificity" a certain epitope, antigen or protein (or at least one its part, fragment or epitope), called "polypeptide to" or "polypeptide, directed against"said epitope, antigen or protein, or same it is"binding" molecule with respect to such epitope, antigen or protein.

[146]

to) as a whole term "specificity relates to row different types of antigens or epitopes, with which can to bind specific antigen-binding molecule or antigen-binding protein (such, as immunoglobulin, antibody, immunoglobulin separate variable domain or polypeptide according to the invention). Specificity of antigen-binding protein can be determined on the basis of its affinity and/or avidity. Affinity, displayed equilibrium dissociation constant antigen and antigen-binding protein (to), is measure of force binding epitope and antigen-binding section antigen-binding protein: the less value to, the greater is the force binding epitope and binding molecules (alternatively affinity can be express as constant affinity (kDa), which is 1/to). As will be understandable skilled (for example, on the basis of further information from this document), affinity can be determined known of per of Se by, depending on particular antigen of interest. The avidity - this measure force binding binding molecules (such, as immunoglobulin, antibody, immunoglobulin separate variable domain or polypeptide according to the invention) with the specific discussion antigen. Depends on the avidity as from affinity epitope and its antigen-binding section binding molecule, and from amount of corresponding acceptable sites of binding on binding molecule.

[147]

As a rule, antigen-binding proteins (such, as polypeptides according to the invention) have binding dissociation constant (Kd of) from 10E - 5 to 10E - 14 mole/liter (m) or less, preferably from 10E - 7 to 10E - 14 mole/liter (m) or less, more preferably from 10E - 8 to 10E - 14 mole/liter, more preferably from 10E - 11 to 10E - 13 (measured, nair ., analysis Kinexa; is known from level of technology), and/or association constant (kDa) at least 10e7 IU - 1, preferably at least 10 [e10 8 IU - 1, more preferably at least 10 [e10 9 IU - 1, such as at least 10e11 IU - 1. Any value Kd of the above 10E - 4 m, as a rule, accepted pointing at nonspecific binding. Preferably polypeptide according to the invention binds with desired antigen with Kd of at, than 500 nm, preferably at, than 200 nm, more preferably at, than 10 nm, such, as less than 500 PM. Specificity binding antigen-binding protein with antigen or epitope can be determined by any suitable known of per of Se by method, including, for example, the described here analysis, analysis of sketcharda and/or analysis competitive binding, such, as the radioimmunoassays (RIA), Automatic analysis (IEA) and sandwich - competition of analysis, as well as various from versions, known of per of Se from level of technology.

[148]

l) term "perekrestnoreaktivnyi" relative to binding molecules, capable of binding and with LRP5, and with LRP6(" perekrestnoreaktivnye by lrp5/lrp6") should be understood as then, that such binding molecules may specifically to bind with epitope, contained in molecule LRP5, and also alternatively can specifically to bind with epitope, contained in molecule LRP6. Usually such cross reactivity can arise in case, when epitopes different proteins, with which binds such binding molecule, have similar structure and/or sequence, nair ., are conservative epitopes, nair ., which there is in included in a certain family of proteins (nair ., LRP5 and LRP6, which belong to family proteins LRP).

[149]

m) amino acid residues of designated standard trigraphic or single-letter codes amino acids, know to and generally accepted in this area. At comparison of two amino acid sequences term " aminokislotnayaraznitsa" means insertion, deletion or replacement of the number of amino acid residues in position reference sequence as compared with the second sequence. In case of substituting (- th) it (- and) is preferably (- went) conservative (- and) amino acid (- and) substitution (- business units), that is amino acid residue replaced by on the other amino acid residue with similar chemical structure, and this does not influence or does not influence significantly on function, activity or other biological properties of polypeptide. Similar conservative amino acid replacement well are known in this area, for example, from of clayey 98/49185, where conservative amino acid substitution are preferably substitutions, at which one amino acid from below reduced groups (1) - (of V) substituted other amino acid residue from the same group: (1) small aliphatic, polar or slightly polar residues:-Ala, Ser-, of Thr, Pro and-Gly; (of II) polar, negatively charged residues and their (uncharged) amides:

[150]

-Asp, Asn-, GIUs and Gin; (of III) polar, positively charged residues: His-, of Arg and Lys-; (of IV) large aliphatic, polar residues: met polypeptide, -Leu, not, -Val Cys;

[151]

and (of V) aromatic residues: Phe-, tug and flows of gas solutions. Especially preferable are the following conservative amino acid replacement:

[152]

On-Ala-Gly or on Ser-;

[153]

Of Arg Lys-on;

[154]

Asn-on Gin or on-His;

[155]

Asp-on GIUs;

[156]

Cys at Ser-;

[157]

Gin on Asn-;

[158]

GIUs Asp;

[159]

On-Gly Ala or on of Pro;

[160]

On His-Asn-or on Gin;

[161]

lie on-Leu or on-Val;

[162]

-Leu on lie or on-Val;

[163]

Lys-on of Arg, on Gin or on GIUs;

[164]

Met polypeptide on-Leu, on tug or on not;

[165]

On Phe-met polypeptide, on-Leu or on tug;

[166]

Ser-on of Thr;

[167]

Of Thr on Ser-;

[168]

Of flows of gas solutions on tug;

[169]

Tug on flows of gas solutions or on Phe-;

[170]

On-Val not or on-Leu.

[171]

and) nucleic acid molecule or polypeptide is considered to be "(in) in fact isolated (form)" - for example, as compared with its natural biological source and/or reaction medium or culture medium, from which (- th) is obtained - after separation of this molecule from at least one other component, with which it usually is connected in designated (- th) source or medium, such, as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one polluter, impurity or secondary component. In particular, nucleic acid molecule or polypeptide is considered to be "in fact separated" after the, as its cleaned at least 2 - fold, in particular, at least 10 - fold, more specifically at least 100 - is a multiple of and up to 1000 - fold or more. Nucleic acid molecule or polypeptide, which is in "in fact isolated form", preferably in essence it is homogeneous, that is determined by means of suitable method, such as suitable chromatographic method, such as electrophoresis in polyacrylamide gel;

[172]

about)"Identity of sequences", eg ., of two sequences of separate up immunoglobulin variable domains, reflects percentage share of amino acids, which in these two sequences coincide. It may be to calculate or determine by description in paragraph of f) on pages 49 and 50 w02008/020079. "Similarity sequences" reflects percentage share of amino acids, which either are identical, or are amino acid substitutions.

Specificity by target

[173]

Polypeptides according to the invention are specific by LRP5 and LRP6, in sense, that contain immunoglobulin separate variable domains, specifically binding with epitopes, nalichestvuyushchimi in both of these molecules (molecules, perekrestnoreaktivno binding lrp5/lrp6).

[174]

Molecules according to the invention bound with human forms of LRP5 and LRP6, and preferably also with their analogs in other types of, having value for development of drugs, τ. ℮ ., LRP5 and LRP6 Javanese macaque and mouse.

Polypeptides according to the invention

[175]

In the same wide sense invention are provided new pharmacologically active agents for treatment of cancer diseases. Agents according to the invention to belong to a new class of binding molecules, namely to biparatopnymperekrestnoreaktivnym by lrp5/lrp6 polypeptides, which contain two or more separate up immunoglobulin variable domains, binding with LRP5 and/or LRP6 by different epitopes. Terms "perekrestnoreaktivnyi" and "biparatopnyi" explained above, so that biparatopnyeperekrestnoreaktivnye by lrp5/lrp6 molecules can be determined as molecules, capable to bind with LRP5 along two different epitopes, contained in protein LRP5, and also capable to bind with LRP6 along two corresponding epitopes, contained in protein LRP6.

[176]

More specifically polypeptides according to the invention contain:

[177]

- the first immunoglobulin separate variable domain, capable of specifically to bind and with LRP5, and with LRP6 (perekrestnoreaktivnyi by lrp5/lrp6) by epitope/so, which leads to suppression signal path Wntl, suppressing activated Wntl transcription gene - target, and

[178]

- the second immunoglobulin separate variable domain, capable of specifically to bind and with LRP5, and with LRP6 (perekrestnoreaktivnyi by lrp5/lrp6) by epitope/so, which leads to suppression signal path Wnt3a, suppressing activated Wnt3a transcription gene - target.

[179]

Due to two separate immunoglobulin variable domains in the similar polypeptide, the two domain are with different epitopes (having ratio to transfer signal Wntl/Wnt3a), these molecules are biparatopnymi coupling molecules. This mode biparatopnogo binding schematised shown on figure 1.

[180]

In connection with this it should be mentioned, that supposed, that polypeptides according to the invention can be connected to a separate one molecule LRP5 or LRP6 both its 1l^r5/1l^r6 - binding domains, as shown on Figure 1 (mode intramolecular binding). Nevertheless, may have place and other modes of binding.

[181]

Finally, supposed, that polypeptides according to the invention are able compete with DKK1 - natural ligand LRP5 and LRP6, preventing Wntl - and wntza-and-cignalingy - the binding with LRP5 and LRP6, thus by inhibiting signal track Wntl and Wnt3a. Nevertheless, this theory also should not be understood as limits volume of invention.

[182]

More specifically polypeptides according to the invention specifically bound with LRP5 or LRP6, the like polypeptides contain the first separate immunoglobulin variable domain (and), selected from the group of separate up immunoglobulin variable domains from (1) to (of III), determined the presence of the following CDR--sequences:

[183]

(I):

[184]

CDR1: TYTVG (=SEQ N0:1)

[185]

CDR2: AIRRRGSSTYYADSVKG (=SEQ N0:2)

[186]

CDR3: DTRTVALLQYRYDY (=SEQ N0:3)

[187]

[=CDR-domain Wntl-and-333E06mod]

[188]

(II):

[189]

CDR1: SYAMG (=SEQ N0:4)

[190]

CDR2: AIRRSGRRTYYADSVKG (=SEQ N0:5)

[191]

CDR3: ARRVRSSTRYNTGTWWWEY (=SEQ N0:6)

[192]

[=CDR-domain Wntl-and-333g06]

[193]

(III):

[194]

CDR1: RYTMG (=SEQ N0:7)

[195]

CDR2: AIVRSGGSTYYADSVKG (=SEQ N0:8)

[196]

CDR3:DRRGRGENYILLΥS-SGRYEΥ (= SEQ N0:9)

[197]

[=CDR-domain Wntl-and-332D03mod],

[198]

and second immunoglobulin separate variable domain (b), selected from the group of separate up immunoglobulin variable domains (of IV) and (V-), determined the presence of the following CDR--sequences:

[199]

(IV):

[200]

CDR1: SYAMG (=SEQ N0:10)

[201]

CDR2: AISWSGGSTYYADSVKG (=SEQ N0:11)

[202]

CDR3: SPIPΥGSLLRRRNNYDΥ (=SEQ N0:12)

[203]

[=CDR-domain wnt3a - 093a01]

[204]

(V):

[205]

CDR1: SYAMG (=SEQ N0:13)

[206]

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

[207]

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15)

[208]

[=CDR-domain wnt3a - 367b10],

[209]

Field of terms "first" and "second" relative to this separate immunoglobulin variable domains is intended only for the designations, that these domains are different domains, as they contain different CDR--sequence and are with different epitopes. However data terms should not be understood as indicating accurate order or sequence domains in similar polypeptide chain.

[210]

Other words, above of the mandate separate immunoglobulin variable domains (and) and (b) in polypeptide according to the invention can be arranged in order of (and)- (b) or in order of (b)- (and).

[211]

Term "specifically bound with LRP5 or LRP6" should be understood as then, that separate immunoglobulin variable domains (and) and (b) are perekrestnoreaktivnymi relative to LRP5 and LRP6. A itself, binding properties of such molecules are determined their (CDR-) sequences, so that outlined above and in the formula of the feature of "specifically bound with LRP5 or LRP6" is intended only for illustration practical application of the invention, but not for limiting its volume.

[212]

Separate immunoglobulin variable domains, as a rule, have in fact of four frame sections (fr1-and-fr4, respectively) and of three sections, complementarity determining (CDR1-and-CDR3, respectively). To reside in one polypeptide or polypeptide chain, said first and second separate immunoglobulin variable domains should be covalently are connected, or directly, or through peptide - linker.

[213]

Therefore common structure molecules according to the invention may also be portray as:

[214]

Of Fr (a of) - L-CDR-(of a) - L-of Fr (of a) 2 - CDR-(of a) 2 - of Fr (of a) 3 - CDR-(of a) 3 - of Fr (of a) 4 - [peptide - linker] - of Fr (6) 1 - CDR-(6) 1 - of Fr (6) 2 - CDR-(6) 2 - of Fr (6) 3 - CDR-(6) 3 - of Fr (6) 4

[215]

where

[216]

Of Fr (of a) means frame section of the first separate immunoglobulin variable domain,

[217]

Of Fr (6) means frame section of the second separate immunoglobulin variable domain,

[218]

CDR-(of a) means of the first CDR-separate immunoglobulin variable domain,

[219]

CDR-(6) means of the second CDR-separate immunoglobulin variable domain,

[220]

[peptide - linker] means optionally nalichestvuyushchii peptide - linker,

[221]

the CDR-have defined above sequence.

[222]

Again - still should be understand, that (and) and (b) can vary points, T. e ., molecules with common structure

[223]

Of Fr (6) 1 - CDR-(6) 1 - of Fr (6) 2 - CDR-(6) 2 - of Fr (6) 3 - CDR-(6) 3 - of Fr (6) 4 - [peptide - linker] - of Fr (of a) - L-CDR-(of a) - L-of Fr (of a) 2 - CDR-(of a) 2 - of Fr (of a) 3 - CDR-(of a) 3 - of Fr (of a) 4

[224]

also are embraced by present invention.

[225]

Peptide - linker optionally contains third domain or consists of it, such, as albuminsvyazyvayushchii separate immunoglobulin variable domain, as is - domain a1y1, containing the following CDR-:

[226]

CDR-(Albl 1) 1: SFGMS (=SEQIDNO: 16)

[227]

CDR-(Albl 1) 2: SISGSGSDTLYADSVKG (=SEQ n0:17)

[228]

CDR-(Alb 11) 3: GGSLSR (=SEQ n0:18)

[229]

Hence group of polypeptides according to the invention with the general structure:

[230]

Of Fr (a of) - L-CDR-(of a) - L-of Fr (of a) 2 - CDR-(of a) 2 - of Fr (of a) 3 - CDR-(of a) 3 - of Fr (of a) 4 - [peptide - linker] - of Fr (Alb 11) 1cdr (Alb 11) 1fr (Alb 11) 2cdr (Alb 11) 2fr (Alb 11) 3-and-CDR-(Albll) 3 - of Fr (Albll) 4 - [peptide - linker] - of Fr (6) 1 - CDR-(6) 1 - of Fr (6) 2 CDR-(6) 2 - of Fr (6) 3 - CDR-(6) 3 - of Fr (6) 4.

[231]

Again - still, order of three separate up immunoglobulin variable domains (and), (b) and Alb 11 is not fixed, opposite to, polypeptides with the following sequence above said domains:

[232]

(b)- Alb 11 - (and)

[233]

also are embraced.

[234]

Moreover, invention also are embraced by polypeptides, in which domain Alb 11 is HaNili with - end of polypeptide (Eg ., Alb 11 - (and)- (b), Alb 11

[235]

- (b)- (and), (and)- (b)- Alb 11, or (b)- (and)- Alb 11).

[236]

In three preferable versions realization polypeptides according to the invention contain separate immunoglobulin variable domains, certain as follows:

[237]

The first preferable version of realisation: polypeptides, containing the first separate immunoglobulin variable domain with the following sequences of CDR-:

[238]

CDR1: TYTVG (=SEQ N0:1)

[239]

CDR2: AIRRRGSSTYYADSVKG (=SEQ N0:2)

[240]

CDR3: DTRTVALLQYRYDY (=SEQ N0:3)

[241]

and second immunoglobulin separate variable domain with the following sequences of CDR-:

[242]

CDR1: SYAMG (=SEQ N0:10)

[243]

CDR2: AISWSGGSTYYADSVKG (=SEQ N0:11)

[244]

CDR3: SPIPΥGSLLRRRNNYDΥ (=SEQ N0:12).

[245]

The second preferable version of realisation: polypeptides, containing the first separate immunoglobulin variable domain with the following sequences of CDR-:

[246]

CDR1: SYAMG (=SEQ N0:4)

[247]

CDR2: AIRRSGRRTYYADSVKG (=SEQ N0:5)

[248]

CDR3: ARRVRSSTRYNTGTWWWEY (=SEQ N0:6)

[249]

and second immunoglobulin separate variable domain with the following sequences of CDR-:

[250]

CDR1: SYAMG (=SEQ N0:13)

[251]

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

[252]

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15).

[253]

Third preferable version of realisation: polypeptides, containing the first separate immunoglobulin variable domain with the following sequences of CDR-:

[254]

CDR1: RYTMG (=SEQ N0:7)

[255]

CDR2: AIVRSGGSTYYADSVKG (=SEQ N0:8)

[256]

CDR3: DRRGRGENYILLΥSSGRYEΥ (=SEQ N0:9)

[257]

and second immunoglobulin separate variable domain with the following sequences of CDR-:

[258]

CDR1: SYAMG (=SEQ N0:13)

[259]

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

[260]

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15).

[261]

Of course, outlined above versions - τ. ℮ ., optionally containing peptides - linkers and/or additional domains, in particular domain a1y1, with different sequence of separate up immunoglobulin variable domains can be used and to these three preferable versions of realization.

[262]

In especially preferable version albuminsvyazyvayushchii separate immunoglobulin variable domain is located between two eer5yaler6 - coupling separate immunoglobulin variable domains. Therefore three especially preferred alternatives of can be imagine as follows:

[263]

The first especially preferable version of realisation:

[264]

Polypeptides, containing the first (eer5yaler6 - binding) separate immunoglobulin variable domain with the following sequences of CDR-:

[265]

CDR1: TYTVG (=SEQ N0:1)

[266]

CDR2: AIRRRGSSTYYADSVKG (=SEQ N0:2)

[267]

CDR3: DTRTVALLQYRYDY (=SEQ n0:3) albuminsvyazyvayushchii separate immunoglobulin variable domain with the following sequences of CDR-:

[268]

CDR1: SFGMS (=SEQ N0:16)

[269]

CDR2: SISGSGSDTLYADSVKG (=SEQ N0:17)

[270]

CDR3: GGSLSR (=SEQ N0:18);

[271]

and second (eer5yaler6 - binding) separate immunoglobulin variable domain with the following sequences of CDR-:

[272]

CDR1: SYAMG (=SEQ N0:10)

[273]

CDR2: AISWSGGSTYYADSVKG (=SEQ N0:11)

[274]

CDR3: SPIPΥGSLLRRRNNYDΥ (=SEQ N0:12);

[275]

or in set out order, or with altered sequence above said domains.

[276]

The second especially preferable version of realisation: polypeptides, containing the first (eer5yaler6 - binding) separate immunoglobulin variable domain with the following sequences of CDR-:

[277]

CDR1: SYAMG (=SEQ N0:4)

[278]

CDR2: AIRRSGRRTYYADSVKG (=SEQ N0:5)

[279]

CDR3: ARRVRSSTRYNTGTWWWEY (=SEQ n0:6); albuminsvyazyvayushchii separate immunoglobulin variable domain with the following sequences of CDR-:

[280]

CDR1: SFGMS (=SEQ N0:16)

[281]

CDR2: SISGSGSDTLYADSVKG (=SEQ N0:17)

[282]

CDR3: GGSLSR (=SEQ N0:18);

[283]

and second (eer5yaler6 - binding) separate immunoglobulin variable domain with the following sequences of CDR-:

[284]

CDR1: SYAMG (=SEQ N0:13)

[285]

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

[286]

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15);

[287]

or in set out order, or with altered sequence above said domains.

[288]

Third especially preferable version of realisation: polypeptides, containing the first (eer5yaler6 - binding) separate immunoglobulin variable domain with the following sequences of CDR-:

[289]

CDR1: RYTMG (=SEQ N0:7)

[290]

CDR2: AIVRSGGSTYYADSVKG (=SEQ N0:8)

[291]

CDR3: drrgrgenyillυssgryeυ (=SEQ n0:9); albuminsvyazyvayushchii separate immunoglobulin variable domain with the following sequences of CDR-:

[292]

CDR1: SFGMS (=SEQ N0:16)

[293]

CDR2: SISGSGSDTLYADSVKG (=SEQ N0:17)

[294]

CDR3: GGSLSR (=SEQ N0:18);

[295]

and second (eer5yaler6 - binding) separate immunoglobulin variable domain with the following sequences of CDR-:

[296]

CDR1: SYAMG (=SEQ N0:13)

[297]

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

[298]

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15);

[299]

or in set out order, or with altered sequence above said domains.

[300]

Above said sequence CDR-are brought in Tables la, 1b and Ib table la: sequence CDR-separate up immunoglobulin variable domains, preventing transmission of signal Wntl:

[301]

Wntl-and-333E06modWntl-and-333g06Wntl-and-332d03 mode
CDR1TYTVGSYAMGRYTMG
(SEQ N0:1)(SEQ N0:4)(SEQ N0:7)
CDR2AIRRRGS S-ΤΥΥ AD OF S-AIRRSGRRTYYADSVAIVRSGGSTYYADS
VKGTHE KGVKG
(SEQ N0:2)(SEQ N0:5)(SEQ N0:8)
CDR3DTRTVALLQYRYDARRVRSSTRYNTGTWDRRGRGENYILLΥ S-
ΥWWEYSGRYEY
(SEQ N0:3)(SEQ N0:6)(SEQ N0:9)

[302]

Table head module: sequence CDR-separate up immunoglobulin variable domains, preventing transmission of signal Wnt3a:

[303]

Wnt3a - 093a01Wnt3a - 367b10
CDR1SYAMG (SEQ N0:10)SYAMG (SEQ N0:13)
CDR2AISWSGGSTYYADSVKG (SEQ N0:11)AISWRSGSTYYADSVKG (SEQ N0:14)

[304]

CDR3SPIPΥGSLLRRRNNYD OF Y DPRGY OF G OF V OF A Υ V-S-A-ΥYEΥ

[305]

(SEQ N0:12) (SEQ N0:15)

[306]

TABLE LB: Sequence CDR-separate up immunoglobulin variable domains, binding with serum albumin (domain A1Sh:

[307]

Albl 1 domain
CDR1SFGMS (SEQ N0:16)
CDR2SISGSGSDTLYADSVKG (SEQ N0:17)
CDR3GGSLSR (SEQ N0:18)

[308]

In addition to above installed sequences CDR-separate immunoglobulin variable domains, contained in polypeptides according to the invention, have sequences up immunoglobulin frame sections (of Fr). These sequences preferably are not immunogenic for people, and therefore are preferably human or humanized FRposledovatelnostyami. acceptable human or humanized FRposledovatelnosti are known from level of technology. Particularly preferred sequence of Fr can be find in shown below versions, complete describing separate immunoglobulin variable domains, and it, and sequence CDR-and fr.

[309]

According to more specific version of polypeptides according to the invention include separate immunoglobulin variable domains, which are domains VHH, preferably humanized domains VHH.

[310]

According to more specific version of polypeptides according to the invention contain the first separate immunoglobulin variable domain (and), selected from the group of separate up immunoglobulin variable domains from (1) to (of III), which have the following sequence:

[311]

AVOLVESGGGLVOPGGSLRLSCAASGRTFSTYTVGWFROAPGKEREFVAAIRRRGSSTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDTAVYYCAADTRTVALLOYRYD ROTATIONGOGTL VT CIRCUITS VS OF S-

[312]

[=domain Wntl-and-333E06mod;=SEQ n0:19]

[313]

of mAVOLVESGGGLVOPGGSLRLSCAASGGTFSSYAMGWFROAPGKEREFVAAIRRSGRRTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDTAVYYCAAARRYRSSTRYNTGTWWWEYWGOGTL VT CIRCUITS OF V S-S-

[314]

[= domain Wntl-and-333g06;=SEQ n0:20], and

[315]

(III) AVOLVESGGGLVOPGGSLRLSCAASGLTFSRYTMGWFROAPGKEREFVAAIVRSGGSTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDTAVYYCAADRRGRGENYILLYSSGRYE OF YWGOGTLVTVSS

[316]

[= domain Wntl-and-332D03mod;=SEQ n0:21],

[317]

and second immunoglobulin separate variable domain (b), selected from the group, consisting of separate up immunoglobulin variable domains (of IV) and (V-), which have the following sequence:

[318]

(IV) EVOLVESGGGLVOPGGSLRLSCAASGRTFSSYAMGWFROAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDTAVYYCAASPIPYGSLLRRRNNYD OF YWGOGTLVTVS S-

[319]

[=domain Wnt3a-and-093α01;=SEQ n0:22], and

[320]

SH

[321]

E OF V OL THE VE S-GGGL V OF THE OP GGSLRL S WITH A-A-S-S-GGTFS-ΥAMGWFRO AP GKEREF OF V AND AISWRSGSTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDTAVYYCAADPRGYGVAYVS HBSAG AY YEYWGOGTL VT CIRCUITS VS OF S-

[322]

[=domain wnt3a - 367b10;=SEQ n0:23].

[323]

Preferable of the methods are polypeptides, containing

[324]

- the first immunoglobulin separate variable domain with amino acid sequence according to SEQ n0:19 and second immunoglobulin separate variable domain with amino acid sequence according to SEQ n0:22; or

[325]

- the first immunoglobulin separate variable domain with amino acid sequence according to yet SEQ of n0:20 and second immunoglobulin separate variable domain with amino acid sequence according to SEQ n0:23; or

[326]

- the first immunoglobulin separate variable domain with amino acid sequence according to yet SEQ of n0:21 and second immunoglobulin separate variable domain with amino acid sequence according to yet SEQ of n0:23.

[327]

So, above described versions can be schematised portray as

[328]

iovd (and)- [peptide - linker] - iovd (b),

[329]

where "iovd" means corresponding separate immunoglobulin variable domain, and the rest of determining and versions remain the same, and above that, especially with respect to presence of optional peptides - linkers and/or additional domains, especially domain a1y1, and also with respect to different orders of separate up immunoglobulin variable domains.

[330]

According to specific versions above realization said polypeptides can additionally contain fragment, drives enhanced half, said fragment, drives enhanced half, is covalently bound with said polypeptide and optionally is selected from the group, consisting of albuminsvyazyvayushchego fragment, such as albuminsvyazyvayushchii peptide or albuminsvyazyvayushchii immunoglobulin domain, preferably albuminsvyazyvayushchii separate immunoglobulin variable domain, more preferably domain Alb 11, transferrinsvyazyvayushchego fragment, such as antitransferrinovyi immunoglobulin domain, molecules of polyethylene glycol, serum albumin, preferably human serum albumin, and also fragment (human) serum albumin.

[331]

Sequence above of said separate immunoglobulin variable domain of the Alb 11 next:

[332]

EVOLVESGGGLVOPGNSLRLSCAASGFTFSSFGMSWVROAPGKGLEWVSSISGSGSDTbYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSOGTLVTVSS

[333]

(=domain Albll;=SEQ n0:24)

[334]

Further examples of separate up immunoglobulin variable domains, binding with human serum albumin, are known from level of technology and are described in detail, Eg ., international patent publications wo2006/122787 and w02008/028977. Other peptides, binding with human serum albumin, are described, nair ., in w02008/068280, wo2009/127691 and wo2011/095545.

[335]

So, three preferable specific of the methods of invention are:

[336]

The first preferable a specific version of realisation:

[337]

Polypeptides, containing

[338]

- the first (eer5yaler6 - binding) separate immunoglobulin variable domain with amino acid sequence according to SEQ n0:19;

[339]

- albuminsvyazyvayushchii separate immunoglobulin variable domain with amino acid sequence according to SEQ n0:24;

[340]

- second (eer5yaler6 - binding) separate immunoglobulin variable domain with amino acid sequence according to SEQ n0:22;

[341]

or in set out order, or with altered sequence above said three domains.

[342]

The second preferable a specific version of realisation:

[343]

Polypeptides, containing

[344]

- the first (eer5yaler6 - binding) separate immunoglobulin variable domain with amino acid sequence according to SEQ n0:20;

[345]

- albuminsvyazyvayushchii separate immunoglobulin variable domain with amino acid sequence according to SEQ n0:24;

[346]

- second (yarb/yarb - binding) separate immunoglobulin variable domain with amino acid sequence according to yet SEQ of n0:23;

[347]

or in set out order, or with altered sequence above said three domains.

[348]

Third preferable a specific version of realisation:

[349]

Polypeptides, containing

[350]

- the first (yarb/yarb - binding) separate immunoglobulin variable domain with amino acid sequence according to yet SEQ of n0:21;

[351]

- albuminsvyazyvayushchii separate immunoglobulin variable domain with amino acid sequence according to SEQ n0:24;

[352]

- second (eyarbyalerb - binding) separate immunoglobulin variable domain with amino acid sequence according to yet SEQ of n0:23;

[353]

or in set out order, or with altered sequence above said three domains.

[354]

In more preferable specific versions of the albuminsvyazyvayushchii separate immunoglobulin variable domain is located between two eyarbyalerb - coupling separate immunoglobulin variable domains.

[355]

Sequence mentioned above separate up immunoglobulin variable domains are brought in Tables Pa, PB and cooler:

[356]

Table Pa: sequence of separate up immunoglobulin variable domains, preventing transmission of signal Wntl:

[357]

VHH OF ID SEQ ID N0:FR1CDR1FR2CDR2FR3CDR3FR4
Wntl-and-333ε06 mode SEQ N0:19AVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTVGWFRQAPGKEREFV A OFAIRRRGSSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTRTVALLQYRYDYWGQGTLVTVSS
Wntl-and-333g06 SEQ N0:20AVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAM OF GWFRQAPGKEREFV A OFAIRRSGRRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAARRVRSSTRYNTGTWWWEΥWGQGTLVTVSS

[358]

VHH OF ID SEQ ID N0:FR1CDR1FR2CDR2FR3CDR3FR4
Wntl-and-332d03 mode SEQ N0:21AVQLVESGGGLVQPGGSLRLSCAASGLTFSRYTM OF GWFRQAPGKEREFV A OFAIVRSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGRGENYILLYSSGRYEYWGQGTLVTVSS

[359]

Table PB: sequence of separate up immunoglobulin variable domains, preventing transmission of signal Wnt3a:

[360]

VHH OF ID SEQ ID N0:FR1CDR1FR2CDR2FR3CDR3FR4
Wnt3a-and-093α01 SEQ N0:22EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAM OF GWFRQAPGKEREFV A OFAISWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASPIPYGSLLRRRNNYDYWGQGTLVTVSS
Wnt3a-and-367β10 SEQ N0:23EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAM OF GWFRQAPGKEREFV A OFAISWRSGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADPRGYGVAYVSAYY Ε-ΥWGQGTLVTVSS

[361]

Table cooler: separate up immunoglobulin sequence variable domains, binding with serum albumin (domain A1Sh:

[362]

VHH OF ID SEQ ID N0:FR1CDR1FR2CDR2FR3CDR3FR4
Alb 11 SEQ N0:24EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEW VS OFSISGSGSDTLYADSVKGL-RFTISRDNAKTTYLQMNSLRPEDTAVYYCTIGGSLS OF RSSQGTLVTVSS

[363]

As is installed above, (at least two) separate up immunoglobulin variable domain in polypeptide according to the invention can be connected with each other directly, without using linker, or same through linker. Linker is preferably peptide linker and, according to the invention, formulas so, to make possible binding of at least two separate up immunoglobulin variable domains each from their epitopnykh targets.

[364]

Suitable linkers the Inter at Alia depend on epitopes and in particular of the distance between epitopes, with which are separate immunoglobulin variable domains, on molecules - targets. Skilled this will understandable on the basis of said description, optionally - after a certain of limited amount of routine experiments.

[365]

So, suitable linkers may contain amino acid sequence, Eg ., length in 9 or more amino acids, preferably at least 17 amino acids, as - is approximately 20 - 40 amino acids.

[366]

Sequence linker can be found in nature or not found in nature. In case of use in therapeutic purposes linker preferably is not immunogenic for subject, which polypeptide is introduced according to the invention.

[367]

One of suitable groups of linker sequences are linkers, obtained from hinge section heavily chained antibodies, according to description in wo1996/34103 and wo1994/04678. Other example are polialaninovyelinkernye sequence, such, as-Ala-Ala-Ala-.

[368]

Further preferable examples of linker sequences are linkers-Gly/Ser-of different length, such, as linkers (-Glyof X ser-of Y )Z of , including, nair ., (-Gly4 ser-)3 , (-Gly4 ser-)5 , (-Gly4 ser-)7 , (-Gly3 ser-)3 , (-Gly3 ser-)5 , (-Gly3 ser-) of V, (-Gly3 ser-2 )3 , (-Gly3 ser-2 )5 And (-Gly3 ser-2 )7 .

[369]

Alternatively or additionally to polypeptide to the linker at least two separate up immunoglobulin variable domain in polypeptide according to the invention can be connected with each other by means other fragment, such as other polypeptide, which in preferable, but not single version can be additional separate immunoglobulin variable domain, according to already given above determination of. Such fragment can be in fact inactive or can and as a biological effect, such as improved desired properties of polypeptide, or same can be due to polypeptide one or more additional desired property. As already presented above, preferable additional polypeptide domain extends the half polypeptide, being such, as domain, binding with (human) serum albumin, such as domain a1y1.

[370]

Therefore according to the next version of the invention especially embraces polypeptides, containing any of below the following sequences, the accurate amino acid sequences can be take from table III below:

[371]

Yet SEQ of n0:25 (=sequence of polypeptide f013500575),

[372]

Yet SEQ of n0:26 (=sequence of polypeptide f013500571),

[373]

Yet SEQ of n0:27 (=sequence of polypeptide f013500720).

[374]

According to more specific version of polypeptides according to the invention are selected from the following group molecules:

[375]

Polypeptide f013500575 with sequence SEQ ID n0:25, polypeptide f013500571 with sequence SEQ ID n0:26, and polypeptide f013500720 with sequence SEQ of n0:27.

[376]

Table III: Sequence of three specific versions of realization of polypeptides according to the invention

[377]

OF ID SEQ ID N0:Amino acid sequence of (sequence CDR-underlined)
F013500575 SEQ N0:25AVOLVESGGGLVOPGGSLRLSCAASGRTFSTYTVGWFROAPGK EREFVAAIRRRGSSTYYADSVKGRFTISRDNSKNTVYLOMNSLR PEDTAVYYCAADTRTVALLOYRYDYWGOGTLVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSE OF V QLVESGGGL OF V OF Q PGNSLRLSCAASGFTFSSFGMSWVROAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLOMNSLRPEDTAVYYCTIGGS LSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG S-GGGGSE OF V OL THE VE S-GGGL V OF THE OP GGSLRL OF C S-A-A-S-S-S-GRTFΥAMGWFROAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDTAVYYCAASPIPYGSLLRRRNNYDYWGOGTLVTV THE SSA
F013500571 SEQ N0:26AVOLVESGGGLVOPGGSLRLSCAASGGTFSSYAMGWFROAPGKEREFVAAIRRSGRRTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDT A OF OF V ΥYC A OF A OF ARRVRS S-ECO RI ΥΝΤGTWWWE ROTATION GOGTL VT CIRCUITS OF V S-S-OF G GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES OF G GGL OF V OPGNSLRLSCAASGFTF S-OF FRONTAL CROSS GMS OF W VROAPGKGLEW OF V S-STEROL GSGSDTLYADSVKGRFTISRDNAKTTLYLOMNSLRPEDTAVYYC TIGGSLSRSSOGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFSSYA MGWFROAPGKEREFVAAISWRSGSTYYADSVKGRFTISRDNSK
NT-VYLOMNSLRPEDTAVYY OF C AADPRGYGVAYVSAYYEYW CO GTLVTVSSA

[378]

OF ID SEQ ID N0:Amino acid sequence of (sequence CDR-underlined)
F013500720 SEQ N0:27AVOLVESGGGLVOPGGSLRLSCAASGLTFSRYTMGWFROAPGK EREFVAAIVRSGGSTYYADSVKGRFTISRDNSKNTVYLOMNSLRPEDΤ A OF OF V ΥΥ OF C OF A ADRRGRGENYILLΥ S-S-THE GR YEΥ OF W CO THE GTL VT CIRCUITS OF V S-S-OF G GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES OF G GGL OF V OPGNSLRLSCAASGFTF S-OF FRONTAL CROSS GMS OF W VROAPGKGLEW OF V S-STEROL GSGSDTLYADSVKGRFTISRDNAKTTLYLOMNSLRPEDTAVYYC TIGGSLSRSSOGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFSSYA MGWFROAPGKEREFVAAISWRSGSTYYADSVKGRFTISRDNSK
NT-VYLOMNSLRPEDTAVYY OF C AADPRGYGVAYVSAYYEYW CO GTLVTVSSA

[379]

As explained earlier, if not as indicated or other, the polypeptides according to the invention may contain further fragments and/or additional polypeptide domains, if only such additional fragment or domain does not prevent their bonding with lrp5/lrp6.

[380]

Polypeptides according to the invention may additionally include modification, such as glikozilnye residues or modified amino acid side chain, and also can pegilirovatsya in order for extending half-time and other properties of similar molecules.

[381]

Methods and reagents for pegylation biparatopnykh structures separate up immunoglobulin variable domains can be find, nair ., in wo2011/107507.

[382]

Polypeptides according to the invention may have modified HaNkontse sequence, Eg ., with removal of one or more of n-end amino acids, or with the replacement of Eg ., the first of n-final amino acids (Eg ., glutamate on alanine), for the purpose of optimization of molecules for expression in certain systems expression (such, as specific vectors or cell - hosts), or for expression of as inclusion bodies or in soluble form, or for the secretion in medium or periplazmaticheskoe space or for holding in a cell, or for production of more homogeneous product. Polypeptides according to the invention may have modified on with - end of sequence, Eg ., with additional alanine (as shown for three versions of realisation in the table III above) and/or with additional amino acid substitutes in with - end part or in other installed positions of any frame sections, according to the purpose of further improving stability or WiFi client continuously ny immunogenicity of such polypeptides.

[383]

In addition, half polypeptides according to the invention can be prolongation addition of albumin domain, T. e ., having transformed them in albumin fusion proteins. Examples suitable albumin fragments and methods of their addition to binding molecules are described, nair ., in w02001/079271 and w02003/059934.

[384]

Preferably polypeptides according to the invention have values binding (copepod), analysis of measured binding FACS, described in example 7.1 below, in the range of 10"6 mole/liter or less, more preferably 10"9 mole/liter or less, still more preferably in the range from th "10 to 10"13 mole/liter, or have value ic50, measured combined analysis of gene - to the reporter and WntlWnt3a, described in example 7.3 below, 10"9 mole/liter or less, more preferably in the range from 5 X th "10 mole/liter to 10"12 mole/liter.

[385]

Polypeptides according to the invention is possible more effective treatment of number of types of cancer, such as tnrmzh, operation switch and nmkrl. In them improved in vitro of characteristics (T. e ., more is high efficiency of suppression track of WNT), cm ., Eg ., examples 7 and 8 below, and also considerable ability to suppress growth of tumor in vivo of, that leads to more high efficiency of in vivo of as compared with other coupling LRP6 molecules, it is shown that, Eg ., examples 9 and 10 below.

[386]

In particular, as shown in vivo of on model tumor, caused by of WNT, biparatopnyeperekrestnoreaktivnye by lrp5/lrp6 humanised VHH-and-constructs with prolonged period have been half-are able to suppress of WNT-and-signaling and growth of tumor in vivo of, providing even considerable reduction tumor (T. e ., suppression of tumor growth more than 100%), which in the same experimental conditions may not be was to achieve by means of known binding LRP6 substance. Reduced tumor (T. e ., regress tumor) is, outside deserves the doubts, desirable therapeutic effect (T. e ., effectiveness) in treatment of cancer patients patients. Moreover, regression of tumor, which to the full patomorfologicheskomu response (CSPs), is recognized clinical final point, indicates considerable improvement of survival rate without progressing and common survival rate.

[387]

In the same experiments in vivo of not observed significant changes of body mass (<10%), and results are histopathological analysis of gastro - intestinal tissues not have shown any - or toxic effect of above mentioned polypeptides according to the invention. This especially unexpectedly in light of above mentioned research expression DKK1in vivo of (τ. ℮ ., deadly to izyazvleniyu intestinal mucosa and loss of body weight).

[388]

So, polypeptides according to the invention actually provide new therapeutic possibility for treatment of cancer diseases, especially in case of large unsold medical demand, such, as, nair ., at (three times negative) breast cancer.

[389]

As neither strange, inventors came to such decision by not traditional track, T. e ., trying to develop inhibitors or binding molecules with high specificity/selectivity one a certain target, such as LRP6 (referred to as a target for treatment of "diseases, mediated by signal transmission of WNT", nair ., in w02009/056634). Vice versa, inventors have developed molecules, targeting simultaneously on two related protein - LRP6 and LRP5 - and so reached above described considerably improved effects in vitro of and in vivo of, which may not be was assumed by known from level of technology information.

[390]

Molecular mechanism, in the base such advantages, not completely clear, however can be assume - not having intentions limited specific theory - that such perekrestnoreaktivnye molecules can possess additional, and therefore more strong effect on very muddled signal cascade of signal path of WNT.

[391]

Described above keep advantage effects additionally illustrated examples below and available in them comparative data.

[392]

Moreover, polypeptides according to the invention are simple in manufacturing and better are dissolved, that means, that their can be stored and/or introduce in more high in comparison with common antibodies concentrations. They are stable at room temperature and longer remain stable even at extreme values of pH of, so that may be made of, stored and/or be transported without using refrigerating equipment, thereby saving finance, time and preventing consequences for the environment.

[393]

Due to above set out, and also its low immunogenicity, they additionally opened a number of capabilities relative to ways of introduction, apart from injections and infusions, and also relative to introduction of protocols and use of specific devices.

Nucleic acids, vectors, cells - hosts

[394]

According to further aspects the invention relates to molecules nucleic acids and expression vectors, encoding polypeptides according to the invention, and also to expressing their cells - host. These nucleic acids, vectors and cells - hosts are useful in manufacture of polypeptides according to the invention, and their additional aspects and versions will be learn more are described below in correlation with outlining methods of production of polypeptides according to the invention.

Therapeutic application

[395]

Owing to their biological properties polypeptides according to the invention are useful for treatment of diseases, characterized by excessive or abnormal cell proliferation, such as cancer and idiopathic pulmonary fibrosis (ilf).

[396]

For example, polypeptides according to the invention can be to treat the following cancers, tumor and other proliferative diseases, not limited by them:

[397]

Cancer of head and neck; lung cancer, such as, nair ., nemelko cell lung cancer (nmkrl) and small cell lung cancer (mkrl); neoplasm mediastinum, such as, nair ., neurogenic tumor and mesenchymal tumor;

[398]

Cancer gastro - intestinal tract (gastrointestinal tract), such as, nair ., cancer esophagus, stomach, pancreas, liver and bile ducts (including, nair ., pechenochnokletochnuyu carcinoma (Framework)), and also of thin and thick intestines (including, nair ., colorectal cancer); prostate cancer; testicular cancer;

[399]

Gynecological types of cancer, such as, nair ., ovarian cancer; cancer, such as, nair ., mammary gland carcinoma, hormone - retseptorpolozhitelnyi cancer, [neg 2 - positive breast and three times negative cancer; cancer endocrine system;

[400]

Sarcoma of soft tissues, such as, nair ., fibrosarcoma, rhabdomyosarcoma, angiosarcoma, sarcoma sarcoma; sarcoma bones, such as, nair ., myeloma, osteosarcoma, tumor sarcoma, fibrosarcoma, the osteochondroma, osteoblastoma and khondroblastoma; mesothelioma;

[401]

Skin cancer, such as, nair ., carcinoma basal cells, flat cell carcinoma, carcinoma cells merkelya and melanoma; neoplasm of central nervous system and brain, such as, Eg ., astrocytoma, glioblastoma, glioma, the neuroblastoma and retinoblastoma; lymphoma and leukemia, such, as, nair ., in - cell nekhodzhkinskie lymphoma (NHL), T - cell nekhodzhkinskie lymphoma, chronic in - cell lymphocytic leukosis (in - khll), chronic T - cell lymphocytic leukosis (T - khll), disease s lymphoma (bkh), leukosis with large granular lymphocytes (bgl), chronic myelogenous leukosis (khml), acute myelogenous/myeloid leucosis (UML), acute lymphatic/lymphoblastic leucosis (oll), multiple tier myeloma (mm), plasmacytoma and myelodysplastic syndromes (mmf); and also tumor unknown primary localization.

[402]

Implied, that all above said cancer disease, tumor, neoplasm and T. d ., lithology specific localization of/point of origin in body of, include as primary tumor, and taking place from them metastatic tumor.

[403]

More specifically polypeptides according to the invention useful in treatment of such diseases, in particular cancer diseases, at which abnormal proliferation cells induced anomalous (activated) of WNT-and-signalingom, or it receives in it participation.

[404]

Therefore polypeptides according to the invention are particularly useful for the treatment of solid tumors, more specifically for treatment of lung cancer, liver, colon, brain, thyroid gland, pancreas, breast, ovarian and prostate, more and more specifically for treatment of nemelko cellular lung cancer (nmkrl), three times negative breast cancer (tnrmzh) and colorectal cancer (operation switch). In particular, polypeptides according to the invention can be used for treatment of patients with locally or of metastatic tnrmzh, patients with metastatic nmkrl or locally or of metastatic operation switch, as single agent or in combination, to prolongation survival rate without progressing (vbp) and common survival rate (s). In addition, polypeptides according to the invention can be used as neoadjuvant therapy for patients with breast cancer for the purpose of achieving complete pathomorphological response (CSPs; is determined as absence of residual of invasive cancer and cancer the in situ at gistopatologicheskoi evaluation of completely dissected sample breast and samples from all regional lymph nodes after neoadjuvant systemic therapy).

[405]

Polypeptides according to the invention can be used in therapeutic protocols in a context of the first line, second line or any further line treatment.

[406]

Polypeptides according to the invention can be used for prophylaxis, short-term or long-term treatment of above mentioned diseases, optionally in combination with radiation therapy and/or surgery.

[407]

Also polypeptides according to the invention especially are useful for treatment of other diseases, caused by abnormal cell proliferation, in which receives participation of signal path of WNT, such as idiopathic pulmonary fibrosis (ilf). (Konigshoffand DP. "Functional of WNT signaling Li increased in vitro idiopathicpulmonaryfibrosis". The One PLoS 2008 ; 3 (5): ℮2142;Lamand DP. "Of WNT coreceptorLrp5 Li a of Driver and care idiopathicpulmonaryfibrosis". AW JRespirCrit Care is the MED. 2014; 190 (2): 185 - 95).

[408]

In addition, polypeptides according to the invention especially are useful for treatment of retinopathies, especially for treatment of diabetic retinopathy from - the abnormal activation of WNT in cells inner retina, which causes intensified is anomalous formation of blood vessels of retina, that leads to the development and progressing diabetic retinopathy (-chen of Y. and others.

[409]

"The activation care of The of WNT pathwayplays a of pathogenic the Role in diabeticretinopathy in vitro humans and sort animalmodels" Of The AW j-Pathol. 2009; 175 (6): 2676 - 85 ., Gaoand DP.

[410]

"ElevatedLRP6 the Levels correlate Hotel With vascularendothelial growth options factor pluggable in vitro of The vitreous care proliferativediabeticretinopathy" Mol lando. 2015 ; 21:665 - 72).

[411]

And finally, since it is shown, that signal suppression of tract Wntl/Wnt3a may also influence on dendritic cells (DC) and function of dendritic cells, polypeptides according to the invention can be useful for treatment of immune and infectious diseases, as well as for influence on the micro-environment tumor at various cancer diseases from number of listed above. Tumor actively inhibit antitumor immunity, and DC stabilize the important role in mechanism deviation from cancer immunity. In particular, investigating have shown, that of WNT-and-ligands in the microenvironment of the tumor are able also initiate parakrinnyisignaling in immune cells and to regulate antitumor immunity host (the Hong and DP. "of BETA-and-cateninpromotesregulatory t the Cell responses in vitro tumors By the inducingvitamin a of metabolism in vitro dendriticcells". Cancer resolution RES.

[412]

2015 ; 75 (4): 656 - 65).

[413]

Of course, above the described covers usage of polypeptides according to the invention in different methods of treatment above of said diseases by administration of therapeutically effective dose to the patient, requiring that, as is equal to and application of these polypeptides for preparing drugs for treatment of such diseases, as is equal to and pharmaceutical compositions, containing such polypeptides according to the invention, as is equal to and preparation of and/or manufacture of medicinal agents, including such polypeptides according to the invention, and T. d. and T. U.

Combination with other active substances

[414]

Polypeptides according to the invention can be used in alone or in combination with other pharmacologically active substances, such as substance best practices level or standard treatment, such as, nair ., cytostatic or the cytotoxic substance, inhibitors of cell proliferation, antiangiogenic substance, steroids, immunomodulators/inhibitors of control points and T. U.

[415]

Cytostatic and/or the cytotoxic active substances, which may be introduced in combination with compounds according to the invention, include, not limited by them, hormones, analogs of hormones and the antihormones, inhibitors of aromatase, agonists and lhrh antagonists, inhibitors of growth factors (such growth factors, as, for example, growth factor platelet (PDGE), fibroblast growth factor (of FGF), factor vascular endothelial growth (of VEGF), epidermal growth factor (EGFR), -like growth factors (of IGF), epidermal growth factor human (of HER, nair ., the HER2, HER3, HER4) and growth factor hepatocytes (of HGF)), where inhibitors are, for example, antibodies against growth factor, antibodies against growth factor receptor and inhibitors of tyrosine kinase, such as, for example, cetuximab, gefitinib, afatinib, nintedanib, obtain imatinib, lapatinib, bosutinib and trastuzumab; antimetabolites (nair ., antifolaty, such as methotrexate, raltitreksed, pyrimidine analogs, such as 5 - fluorouracil (5 - fu), capecitabine and gemcitabine, purine and adenosine analogs, such as azathioprine, thioguanine, kladribin and pentostatin, cytarabine (Agha with), fludarabine); antitumor antibiotics (nair ., anthracyclines); derivatives of platinum (nair ., cisplatin, oxaliplatin, Mytomicin); alkylating agents (nair ., estramustine, mechlorethamine, melfalan, chlorambucil, busulfan, dakarbazin, cyclophosphamide, ifosfamid, controlled release, nitrosourea, such as, for example, carmustine and lomustin, tiotepa); antimitotic agents (nair ., alkaloids Vinca, such as, for example, vinblastine, vindesine, vinorelbin and vincristine; and also taxanes, such as paclitaxel, docetaxel); inhibitors of angiogenesis, inhibitors of tubulin; inhibitors of DNA synthesis, parp inhibitors, inhibitors of topoisomerase (for example, epipodofillotoksiny, such as, for example, etopozide and etopofos, teniposide, amsacrine, topotecan, irinotekan, cytostatic mitoxantrone), serine/threoninekinase (nair ., inhibitors of PDK1, inhibitors of staining RAF, inhibitors of a-- staining RAF, inhibitors of b of - staining RAF, inhibitors of c - staining RAF, inhibitors of mTOR, inhibitors of mTORCl/2, inhibitors of pi3k, inhibitors of ρ13κ of α, double inhibitors of mTOR/pi3k, inhibitors of STK33, inhibitors act, inhibitors of PLK1 (such as volasertib), inhibitors of CDK, the Aurora kinase inhibitors), tyrosine kinase inhibitors (nair ., inhibitors of PTK2/FAK), inhibitors of protein - protein interaction, inhibitors of MEK, inhibitors of ERK, inhibitors of FLT3, inhibitors of BRD4, inhibitors of IGF - 1r, agonists TRAILR2, inhibitors of showing Bcl - the XL, inhibitors of the Bel - 2, inhibitors of showing Bcl - 2/showing Bcl - the XL, inhibitors of receptor egv, inhibitors of bcr - abl kinase, inhibitors of abl kinase, inhibitors of Src of, rapamycin analogs (for example, everolimus, temzirolimus, ridaforolimus, sirolimus), inhibitors of synthesis of androgen, inhibitors of receptors androgen, inhibitors of DNMT, inhibitors of HDAC, inhibitors of ang1/2, inhibitors of Sur 17, radiopharmaceutical preparations, immunotherapeutic agents, such as inhibitors of immune of control points (nair ., molecules/immunoglobulins, binding CTLA4, pd1, of Pd - L1 of, LAG3 and TIM3, such as ipilimumab, nivolumab, pembrolizumab), anticancer vaccine, such as traditional tumor vaccine (cell vaccine, Eg ., Sipuleucel - T for of prostate cancer treatment), personalized neoantigennye vaccine and onkoliticheskie viruses, as well as various chemotheraputic agents, such as amiphostin, anagrelide, clodronate, filgrastin, interferon, interferon - alpha, leucovorin, rituximab, prokarbazin, levamisole, Meena, mitotan, pamidronate and porfimer.

[416]

Especially preferable are methods for treatment, including use of polypeptides according to the invention in combination with medicinal agent, selected from a group, consisting of:

[417]

(I) antibodies against of VEGF (bevacizumab and other antiangiogenic substance), with or without combination with chemotherapy (including combination of doxorubicin/cyclophosphamide and/or combination of capecitabine/docetaxel by neoadjuvant circuit; protocol taxane/platinum for the first and pozdneishikh treatment of lines) for patients with breast cancer;

[418]

(Ii) ITC EGFR is for nmkrl, mutant by EGFR is, or krizotiniba for nmkrl with adjustment of alk, with or without combination with chemotherapy (the cytotoxic combined therapy platinum medications, including gemcitabine/cisplatin therapy as the first line; docetaxel or pemetreksed as second line therapy for patients with with lung cancer;

[419]

(Iii) antibodies against EGFR is (cetuximab and panitumumab for tumors with KRAS of wild type), with or without combination with chemotherapy (including irinotekan), combination with antibodies against of VEGF (bevacizumab and other antiangiogenic substance) or combination with regorafenibom, nair ., for treatment of patients with operation switch.

[420]

(Iv) immunotherapeutic agents, including agents against of Pd-and-1, such as pembrolizumab and nivolumab, agents against of Pd-and-l1, agents against CTLA4, agents against BTLA, agents against LAG3 and agents against TIM3, such as antibodies against PDL1 and T. d ., nair ., for treatment of patients with breast cancer, lung and operation switch.

[421]

(V) chemotherapeutic agents, such as anti-neoplastic preparations based on platinum, or same in combination with chemotherapeutic protocol FOLFOX, including folinovuyu acid, 5 '- fluorouracil and oxaliplatin, or same in combination with chemotherapeutic protocol FOLFOXIRI, including folinovuyu acid, 5' - fluorouracil, oxaliplatin and irinotekan, nair ., for treatment of patients with breast cancer and operation switch.

[422]

If two or more substance or principle of action is used as part of the combination chemotherapy protocol, then their may be introduced by the same or different tracks, in fact one and the same time (T. e ., simultaneously, in parallel) or in different times (nair ., in series, running, alternately, according to order of or in any other form of alternating mode).

[423]

If substance or on principles of is introduced simultaneously one and the same by, then can be introduced in the form of different pharmaceutical preparations or compositions or as part of combined pharmaceutical preparation or composition. In addition, if two or more substance or principle is used as part of the combination chemotherapy protocol, then each substance or principle may be introduced in the same amount and along the same protocol, which are used for single use of the compound of or principle of, and such combined use may have synergistic effect or not have its. However if combined application of two or more active substances leads to synergistic effect, then can be reduced amount of one, several or all introduced substances or principles, preserving the desired therapeutic effect. This may be useful for, for example, to avoid, limiting or reducing any - or undesired by-effects, associated with application of one or more substances or principles in ordinary amounts, preserving the desired pharmacological or therapeutic effect.

[424]

Of course, above described are embraced by manufacture of and methods of making polypeptides according to the invention for combined application with above said combination partners. Are embraced by also manufacture and methods for manufacturing said above of combination partners for combined application with polypeptides according to the invention. So, invention are provided, nair ., methods of using or manufacturing for use immunomodulator/inhibitor of control points, such as antibodies against pd1, such as pembrolizumab or nivolumab, for introduction in combination with polypeptide according to the invention, and more specifically for introduction in as component of the combined therapeutic protocol with polypeptide according to the invention.

[425]

Moreover, invention also are embraced by sets of, included in at least one polypeptide according to the invention and one or more other components, selected from the group, consisting of other medicinal agents, used for treatment of diseases and disorders according to the description above, and also devices according to description below.

[426]

Pharmaceutical compositions, methods for administration, dosage Skilled will be clearly testify, that above described methods for treatment of disease imply manufacture of medicinal agent for treatment of said disease. So, the invention also relates to pharmaceutical compositions for treatment of above mentioned diseases, the similar compositions contain at least one polypeptide according to the invention.

[427]

Polypeptides according to the invention and/or compositions containing them may be introduced to the patient, requiring in this, by any method, depending on particular used pharmaceutical preparation or composition. So, polypeptides according to the invention and/or compositions containing them may be introduced, for example, intravenously (in. in.), subcutaneously (U. to.), intramuscularly (in. m.), intraperitoneally (in. b.), through skin, orally, sublingually (Eg ., in the form of sublingual tablets, spray or drops, placed under tongue and adsorb through mucous membrane in sternohyoid capillary network), (inside -) nasally (Eg ., in the form of nasal spray and/or aerosol), surface, by means of suppository, inhalation or any other suitable method in the effective amount or dose.

[428]

Polypeptides according to the invention and/or compositions containing them is introduced according to protocol of treatment of, suitable for treatment and/or relief of disease, disorder or condition, to be treatment or relief. Doctor, as a rule, can determine suitable protocol treatment of depending on such factors, as disease, disorder or state, subject to treatment or relief, disease severity, the severity degree of its symptoms, a specific used polypeptide according to the invention, a specific path administration and used pharmaceutical preparation or composition, age, field, weight, power supply mode, patient s general state and the like factors, well known a doctor. As a rule, protocol includes introduction of one or more polypeptides according to the invention or one or more containing their compositions in therapeutically effective amounts or doses.

[429]

As a rule, for treatment and/or relief of mentioned here diseases, disorders and conditions, depending on particular subject to treatment of diseases, disorder or condition, a specific force used polypeptide according to the invention, used a specific track administration and a specific pharmaceutical preparation or composition polypeptides according to the invention in common is introduced in an amount from 0.005 to 20.0 mg per kg of body weight and dose, preferably from 0.05 to 10.0 mg/kg/dose, more preferably from 0.5 to 10 mg/kg/dose, or continuously (Eg ., infusion), or, that more preferably, in the form of separate doses (such as, Eg ., received twice per week, a week, time per month; cm. below), but all this may significantly differ, especially depending on the above mentioned parameters. Therefore in some cases sufficient may be dose is less than said here minimum dose, while in other cases it may exceed upper boundary. At introduction of large amounts of their split is recommended on a number of smaller doses, received during the day.

[430]

Depending on particular polypeptide according to the invention, and its specific pharmacokinetic and other properties of, its may be introduced once a day, every second, third, fourth, fifth or sixth day, a week, time per month and T. U. Introduction of protocol may imply term weekly treatment. Under "term" there is in type of duration of at least two weeks, preferably months or years.

[431]

The validity of polypeptides according to the invention, and also containing their compositions can be verified by any suitable known of per of Se analysis of in vitro of, analysis on cells, analysis of in vivo of and/or on animal model, same or any of their combinations, depending on specific disease under consideration. Suitable analysis and animal model will be clear skilled and for coded include analysis and animal model, used in examples below.

[432]

Preferably polypeptides according to the invention have better characteristics, than common known antibodies (such, as binding LRP6, described in partition "level of equipment" above), in at least one of these analysis or models, and preferably in one or more models in vivo of.

Preparations

[433]

For pharmaceutical use polypeptides according to the invention may be introduced into composition of pharmaceutical preparation, including (1) at least one polypeptide according to the invention and (of II) at least one pharmaceutically acceptable carrier, diluent, auxiliary substance, adjuvant and/or stabilizer, and also (of III) optionally one or more additional pharmacologically active polypeptides or compounds. Under "pharmaceutically acceptable" should be understand material, not showing any - or biological or other undesirable effects at introduction to an individual and not interacting detrimentally with any from other components pharmaceutical composition (such as, nair ., pharmaceutically active ingredient), which contains corresponding material. Specific examples can be find in standard Benefits, such as, nair ., Remington's the PharmaceuticalSciences, 18 - E of 2 yr ., Mack Publishing by the COMPANY, USA (1990). For example, polypeptides according to the invention can be retseptirovat and introduce by any method, known of per of Se for normal antibodies and antibody fragments, and other pharmaceutically active proteins. So, according to the next version of invention relates to pharmaceutical composition or preparation, containing at least one polypeptide according to the invention and at least one pharmaceutically acceptable carrier, diluent, auxiliary substance, adjuvant and/or stabilizer, and also optionally one or more additional pharmacologically active substances.

[434]

Pharmaceutical preparations for parenteral administration, such as intravenous, intramuscular, hypodermic injection or intravenous infusion, can, for example, be sterile solutions, suspensions, variances, emulsions or powders, which contain active substance and can be used, optionally after additional dissolution step or dilution, for injection or infusion. Suitable carriers or diluents for similar preparations include, for coded and not for limiting, sterile water and pharmaceutically acceptable aqueous buffers and solutions, such as physiological phosphate - salt solution, Ringer solution, dextrose solution and solution khenka; oil with water; glycerol; ethanol;

[435]

glycols, such as propylene glycol, and mineral oil, animal oil and vegetable oils, for example, peanut oil, soya oil and suitable their mixture.

[436]

Solutions of polypeptides according to the invention may also contain preservative for preventing growth of microorganisms, such, as antibacterial and antifungal substance, for example, U - hydro ksibenzoaty, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, ethylenediaminetetraacetic acid (and its salts with alkaline metals) and T. U. In many cases preferably include isotonic agents, for example, sugar, buffers or sodium chloride. Optionally can be used emulsifiers and/or dispersers. Adequate fluidity may be maintained, for example, formation of liposomes, maintenance of the required particle size in case of dispersions and use of surface - active substances. May also be added other substances, delay absorption, for example, aluminum monostearate and gelatin. Solutions can be of teeming in injection vials, ampoule, bottle for infusions and T. U.

[437]

In any case final medicinal form should be sterile, liquid and stable during production and in storage. Sterile injection solutions is made connection of an active substance in required amount of appropriate solvent, when required with different other components from mentioned above of, with sterilizing filtration following. In case of sterile powders for preparing sterile injection solutions preferable methods of manufacturing methods are vacuum drying and freezing-out, making it possible to produce the powder of active substance and any desired additional ingredient from their solution, preliminarily passed sterilizing filtration.

[438]

Usually prefer aqueous solutions or suspension. As a rule, suitable preparations for therapeutic proteins, such as polypeptides according to the invention, are buffered solutions of proteins, such as solutions, containing protein in a suitable concentration (such, as from 0.001 to 400 mg/ml, preferably from 0.005 to 200 mg/ml, more preferably from 0.01 to 200 mg/ml, more preferably 1.0 - 100 mg/ml, such, as 1.0 mg/ml (in. in. introduction) or 100 mg/ml (and. to. introduction) and aqueous buffer, such as:

[439]

- phosphate - salt solution, pH of 7.4,

[440]

- other phosphate buffers, pH of 6.2 - 8.2,

[441]

- acetate buffers, pH of 3.2 - 7.5, preferably pH of 4.8 - 5.5

[442]

- gistidinovye buffers, pH of 5.5 - 7.0,

[443]

- succinate buffers, pH of 3.2 - 6.6, and

[444]

- citrate buffers, pH of 2.1 - 6.2,

[445]

and, optionally, salt (Eg ., of NaCl) and/or sugar (Eg ., sucrose and trehalose) and/or other polyols (Eg ., mannitol and glycerol) for providing isotonic solution.

[446]

Preferable buffered solutions of proteins are solutions, containing approximately 0.05 mg/ml polypeptide according to the invention, dissolved in 25 mm phosphate buffer, pH of 6.5, adjusted with addition of 220 mm trehalose byby.

[447]

Additionally similar solutions may contain other agents, such as detergents, Eg ., 0.02% Nonidet-and-20 or Nonidet-and-80. Preparations for subcutaneous application may contain considerably large concentration of polypeptide according to the invention, such, as to 100 mg/ml or even above 100 mg/ml.

[448]

However skilled in this area will be clearly testify, that above the indicated components and amount of are only one, preferable from capabilities. Skilled will be immediately evident, or same easily vyvodimy from above contained, their alternative to and versions.

[449]

Also as compared to ordinary antibodies or fragments of antibodies one from large advantages application polypeptides according to the invention is, that their can be easily introduced tracks, different from parenteral administration, and easily developing their product for such introduction. For example, as described in international patent application w02004/041867, like polypeptides can be retseptirovat as preparations for oral, intranasal, RR and transdermal administration.

[450]

According to the next aspect of the polypeptide according to the invention can be used in combination with device, lumps for introduction of polypeptide, such as syringe, handle - injector, micro or other device.

Methods of production and purification

[451]

Invention also presented methods for the production of polypeptide according to the invention, the such methods as a whole include stages:

[452]

- cultivation of cells - host, containing nucleic acid, encoding polypeptide according to the invention (hereinafter: "nucleic acid according to the invention") in conditions, expression of polypeptide according to the invention; and

[453]

- separation or isolation of polypeptide, of expressed byby, from culture; and

[454]

- optionally further cleaning and/or modification and/or retseptirovaniya polypeptide according to the invention.

[455]

Nucleic acid according to the invention can, nair ., be DNA molecule, containing coding sequence, and also regulatory sequence and optionally natural or artificial the nitrons, or can be molecule cDNA. It may have their initial codons or optimized codons, specially adapted for expression in supposed cell - or host organism - host. According to one of Embodiment of the Invention nucleic acid according to the invention is in in fact separated form according to determination of above.

[456]

Nucleic acid according to the invention may also be in the form of, be present in and/or be part of vector, such as, for example, plasmid, or kosmidaYAC, which again - still may be in in fact separated form. Vector may be in fact expression vector, T. e ., vector, capable to provide expression of polypeptide in vitro of and/or in vivo of (nair ., in a suitable cell - host, organism - host and/or expression system). Like expression vector, as a rule, comprises at least one nucleic acid according to the invention, functionally connected with one or more suitable regulator elements, such as promoter (- s), enhancer (- s), terminator (s -) and T. U. Specific examples similar regulatory elements and other elements, such as factors insertion, selection markers, signal or lidernye sequence, reporternye genes and T. U ., useful or required for expression of polypeptides according to the invention, invention describes, nair ., on temperature control system. 131 - 133 W02006/040153.

[457]

Nucleic acids according to the invention can be made or allows known of per of Se by (nair ., automated synthesis of DNA and/or recombinant DNA technology) on the basis of given here information about amino acid sequences of polypeptides according to the invention.

[458]

According to another version of the invention relates to host or cell - host, expressing or that is to express polypeptide according to the invention; and/or which contains nucleic acid, encoding polypeptide according to the invention.

[459]

According to especially preferable alternative of the mandate cells - hosts are bacterial cells, yeast cells, fungal cells or cells of mammals.

[460]

For production of in industrial scale preferred heterologous hosts for (industrial) production of separate up immunoglobulin variable domains - containing polypeptides and their protein drugs include strains E coli-., Of Pichia pastoris and S saccharomyces cerevisiae, which are suitable for large scale expression, production and fermentation, and in particular, for large scale (bio -) pharmaceutical expression, production and fermentation.

[461]

Polypeptides according to the invention, unusable in a cell according to the description above, can developed or vnutrikletochno (nair ., in cytosol, periplasm or the harvest) followed by isolation from cells - host and optional further cleaning; or they can be produced extracellularly (secreted in medium, where cultivated cells - hosts) followed by isolation from cultural medium and optional further cleaning.

[462]

Additional methods and reagents, used for recombinant production of polypeptides, such, as suitable expression vectors, methods of transformation or transfection, selection markers, methods of induction of expression of protein, culturing conditions and T. U. are known from level of technology.

[463]

The same way skilled are known equipment of isolation and purification of protein, suitable for method for production of polypeptide according to the invention.

[464]

Production of polypeptides according to the invention fermentation in suitable recombinant organisms - host, such as E. coli and yeast, is finance economy as compared to ordinary antibodies, which, as a rule, should be expensive equipment for cultivation of mammalian cells. Moreover, achievable expression, and output of polypeptides according to the invention is in the range of 1 - 10 g/l (E. coli-) and up to 10 g/l (yeast) and more.

EXAMPLES

Example 1: immunization Lamas LRP5 and LRP6 to induction of humoral immune response

[465]

For identification of perekrestnoreaktivnykh by lrp5/lrp6 VHH-and-domains binding it is necessary was to develop and translate the row of protocols immunization Lamas: first Lamas immunized the recombinant extracellular domains proteins LRP6 and LRP5 (from human and mouse). However functional evaluation of above of said recombinant protein LRP5 shows, that only epitope binding class Wntl was is laid appropriately. And vice versa, nothing indicated on adequate laying of domain binding class lrp5 - wnt3a. Therefore for development of suitable for immunization antigens has been practiced additional operation. As bypass track Lamas immunized cells [nek 293, stable transfected human LRP5 or human LRP6. However and then can be to achieve only very weak expression of human LRP5, as at time, and at stable transfection, and also with use of different cell lines (cells [nek 293, cho and NIH-and-3t3). Therefore for achievement of sufficient expression LRP5 has been practiced more additional operation. At the end of ends, after a certain amount of unsuccessful samples and errors, this makes it developed to achieve protocol, implying stable to - transfection cells η ε-κ293MesDC-and-2, shaperonom, which should increase exogenous expression of LRP5. But even in this case, T. e ., after koekspressiiMesDC-and-2 during creation of stable transfectant LRP5 cell line, repeatedly observed instability expression of protein. This drug problem: expression LRP5 during immunization and selection could be lost. To solve this additional problem, number of passages cells, expressing LRP5, restricted as little as possible and fulfil additional sorting cells to enrichment cells, expressing LRP5.

[466]

Lamas additionally immunized DNA, encoding LRP5, and DNA, encoding LRP6, with and without chaperone hMesDC-and-2 on opposite ends.

[467]

Several lamam introduced additional stimuli in an attempt to enhance perekrestnoreaktivnyi immune response, to to increase chances identification perekrestnoreaktivnykh by lrp5/lrp6 VHH-and-domains.

[468]

Through regular time intervals took samples immunized blood (CRC), determine serological response and prepared from isolated CRC common RNA. After immunization recombinant protein observed average serological response to LRP6, as distinct from weak serological response to LRP5. Average immune response to LRP5 observed in Lamas, immunized DNA. And vice versa, for immunizations cells observed very weak immune response. Additionally investigated synthetic library. The not to the grain, at the end of ends makes it to achieve sufficient variety of repertoire for the advancement of the next stage, as set out in example 2.

[469]

Example 2: isolation of monovalent VHH-and-domains gunn), binding LRP5 and LRP6

Creation of library:

[470]

Immediately after selection immune tissues allocate common RNA, affirmed integrity and concentration of RNA. Of these preparations RNA did samples cDNA. Nucleotide sequences, encoding VHH, amplifitsirovali from samples of cDNA a single-WS - PCR. Amplicons by 700 U. about ., specially amplified from cDNA present in sample IgG2 and of IgG3, allocate from agarose gel and subsequently used as template for "socket" PCR. Then products PCR split Sfil and BstEll and vshivali in corresponding restriction sites fagmidnogo vector p α-χ50. Cross-linked mixture BK 1.20 in Of Escherichia coli TG OF-AND-1. Pool obtained transformants reach genetic variety of library phage - display.

[471]

p α-χ50 - this expression vector, obtained from pUC119, containing a gene of resistance to ampicillin and promoter/Ac, which is followed by sequence encoding signal peptide rsh - protein in one reading frame with further site insertion VHH-and-domain. In reading frame with sequence, encoding VHH-and-domain, vector encodes with - final ICC, geksagistidinovuyu mark and rsh - protein kolifaga. After infection library clones E coli-. Tg of-and-1 phage of - presence of khelperom p α-χ50 makes possible production of particles in these phage clones, bringing individual VHH-and-domains in the form of protein, fused with rsh - protein.

[472]

Withdrawal of:

[473]

For taking created and used library VHH-and-blast furnace fagmid. Taking into account very high interspecies homology proteins (LRP5 and LRP6 human and Lama), was unclear, provides caused by whether in Lam immune response sufficient variety of VHH-and-domains. Therefore during taking in parallel with immune libraries used two synthetic library.

[474]

During taking used the following different strategy:

[475]

- Change obtained from LRP5 and LRP6 tools, so to chans to detect perekrestnoreaktivnye by lrp5/lrp6 VHH-and-domains, nair ., in selecting libraries from immunized LRP5 Lamas by means of obtained from LRP6 proteins, or field and LRP5, and LRP6 during taking by synthetic libraries.

[476]

- Type of change - source for taking human/murine perekrestnoreaktivnykh by lrp5/lrp6 VHH-and-domains (cross with mice reactivity such antagonists LRP5 and LRP6 allows to evaluate efficiency of, T. e ., suppression of tumor growth, and profile safety, necessary for evaluating therapeutic window in the same pre-clinical models (T. e ., in ksenotransplantatnykh models tumors mice)).

[477]

- Withdrawal of "in solution" by means of recombinant proteins, to hold epitopes in natural conformation: additional obstacle was in, that recombinant proteins LRP5 and LRP6 at direct application on boards is preferable for binding lose adequate laying. Therefore recombinant proteins biotinilirovali and, confirming adequate laying of functional analyses, used for taking "in solution".

[478]

- Withdrawal by means of cells, overexpressing LRP5 or LRP6, to to provide natural conformation receptors. Unexpectedly, but this present important intake, especially required for improvement of selection of domains, binding with domain LRP5 class Wnt3a, since functional data on recombinant protein have shown is insufficient correct laying of the \ up13a - binding epitope.

[479]

Example 3: screening monovalent VHH

[480]

After taking clones in density of 96 - them having agglutination with deep holes (volume 1 ml) and induced expression of VHH addition of iptg.

[481]

According to standard procedure, produced, nair ., in wo2011/107507, prepared periplazmaticheskie extracts separate clones was carried out and their screening on binding with human LRP6 and LRP5. Initially screening periplazmaticheskikh extracts was binding analysis is preferable with the recombinant LRP6 and LRP5, which is sensitive, by interference-resistive and high-producing analysis as compared with analysis of binding based on FACS. After cleaning VHH, identified analyses is preferable, gave additional characteristic by means of analysis of binding FACS, to confirm binding of purified VHH with receptors LRP6 and LRP5 in their natural conformation.

[482]

Usually expected good correlation analyses of binding by methods is preferable and FACS. However in this case VHH, preferably binding with LRP6 and LRP5 in analysis is preferable (τ. ℮ ., with high affinity to recombinant extracellular domain LRP6 or LRP5), expressed not necessarily any or very weak binding with human LRP6 and LRP5 in analysis of binding FACS, that shown on figure 2 on panel binding substances "2". Field different buffers coating (phosphate - salt buffer dulbekko and bicarbonate buffer) and blocking solutions at setting is preferable (the Marvel and BSA) avert not observed divergence. Instead was, that very weak according to is preferable svyazyvateli have shown high affinity to LRP5 and LRP6 in binding FACS using cells, expressing LRP5 and LRP6, as shown on figure 2 on panel binding substances "2". These additional data and experiments done possible withdrawal of high affine binding substances, of recognising natural conformation of two receptors. The above, has been confirmed by, that these high affine binding substance recognized in proteins LRP5 and LRP6 depending on conformation epitope, and not linear epitope. These additional irregular data and experiments done possible withdrawal of therapeutically important substances, binding LRP5 and LRP6, which should possess high affinity to LRP5 and LRP6, expressible on plasma membrane in its natural conformation.

[483]

Therefore, notwithstanding on (1) low productivity analyses of binding FACS, (of II) less than noise-immune setting analysis and (of III) complexity described above, during loading from - losses of the expression of the recombinant protein at passivating cells, overexpressing LRP5, these analysis all same used for subsequent selection and characteristics of high affine VHH-and-svyazyvatelei. If briefly, cells incubated with by the breedings purified VHH (serial dilution 1:5 from 1 μμ to 1 PM in final concentration) during 1.5 hours at 4 theoretically on tablet shaker. After 5 - multiple cells washing buffer FACS, consisting of ix phosphate - salt buffer (PRF)+ 10% embryonic bull serum (ebs) + 0.05% sodium azide, their incubated on length from 30 minutes to 1 hour at 4 theoretically with polyclonal murine antibody, binding with frame section VHH, and it, binding with all Check svyazyvatelyamiLRP5 and/or LRP6. After of triple washing cells buffer FACS their incubated on length from 30 minutes to 1 hour at 4 theoretically with labelled secondary antibody (protivomyshinoe, FE) followed by threefold washing buffer FACS. Fluorescence measured by means of FACS the Array (of bd).

[484]

On the basis of data about binding FACS and analysis of sequences in immune libraries and libraries synthetic origin identified in common hundred perekrestnoreaktivnykh by lrp5/lrp6 families/clusters VHH. Examples their representatives shown and are determined its sequence below. VHH expressed in E.coli and cleaned.

[485]

If expression in E.coli has been insufficient, VHH they produced in Of Pichia pastoris. Brief description of expression and purification of VHH is described below.

[486]

Total expression of VHH inE.coli:

[487]

Encoding sequence inserted in expression vector rakhyuo and expressed in E.coli in the form of with - ICC proteins with geksagistidinovoi mark.

[488]

Cells E.coli Tg of-and-1, containing picks needed VHH-and-constructs, density of (37 theoretically, about 250/min) in shaken flask of TV on medium with addition of kanamycin and induced addition of 1 mm iptg for expression. After centrifugation of cell cultures prepared periplazmaticheskie extracts by freezing method - thawing sediment and resuspension in fsbd.

[489]

Total expression of VHH in Of Pichia (p.) yastoris:

[490]

Encoding sequence inserted in expression vector p α-χ159 and expressed in P pastoris. in the form of with - ICC proteins with geksagistidinovoi mark. Cells P pastoris. -x-33, containing picks needed VHH-and-constructs, density of (30 theoretically, about 250/min) on medium BGCM (logging buffered Glycerol-and-for Complex Medium Monegal, complex medium with bufferized glycerol; Invitrogen). On third day medium changed on vmsm (logging buffered Methanol-and-for Complex Medium Monegal, complex medium with bufferized with methanol; Invitrogen) and density of culture further, regularly inducing its addition of 0.5 about.% methanol (100%). After centrifugation of cell cultures were singled supernatant (containing sekretiruemyeVHH).

[491]

Purification of VHH:

[492]

With VHHgeksagistidinovoi mark cleaned on the Tecan EVO150 affinity chromatography with an metal (RoboColumns 100ul NickelSepharose™ 6 FF of, Atoll), eluted with column 250 mm - imidazole and then desalted to fsbd. Integrity and purity of VHH affirmed SDS-polyacrylamide gel-and--PAGE and/immunoenzymometric or western blotting - with by the detection antibodies against the ICC and VHH.

[493]

Example 4: In vitro in /? - properties of purified monovalent VHH After screening purified VHH showing high affinity to cells, expressing LRP5 and LRP6, gave characteristic by means of a number of functional and biophysical analysis, described below:

[494]

4.1 Force binding with LRP5 and LRP6. cross-reactivity: analysis competition of with DKK1 based on FACS

[495]

During characteristics perekrestnoreaktivnykh by lrp5/lrp6 monovalent VHH observed, that obtained in analysis of binding FACS data are not always correlated with force, observed in analysis gene - to the reporter and WntlWnt3a, peer the whole, from - the high speed dissociation of some VHH. Therefore there necessity in development of this additional analysis (τ. ℮ ., FACS for competition of with DKK1), which proved a more reliable with respect to selectivity and of determining force of binding, and also comparison binding with LRP5 and LRP6. Was to selection of functional VHH, binding with LRP5 and LRP6 with the same force, to one and the same concentration to achieve blockade of both receptors.

[496]

Therefore identified functional VHH to Wntl and Wnt3a characterized FACS for competition of with DKK1 as follows:

[497]

For analysis of competition of with DKK1 based on FACS used cells [nek 293 with stable caused human LRP5 or human LRP6. Human recombinant DKK1 (rhDKKl - of R&d of the Systems, Part Number 5439-and-Dc/the CF) added to the to cells in constant final concentration 1 nm.

[498]

Cells incubated with rhDKKl and LRP5i/or 1ler6 - svyazyvatelyami (serial dilution of purified VHH 1:5) during 1.5 hour at 4 theoretically on tablet shaker. After of triple washing cells buffer FACS their incubated with biotinylated kozlinym antibody against human DKK1 (of R&d of the Systems, art. BAF1096) during 30 minutes at 4 theoretically on tablet shaker. After of triple washing cells buffer FACS their incubated with streptavidin FE (of bd Biosciences, art. 554061) on length from 30 minutes to 1 hour at 4 theoretically on tablet shaker in darkness.

[499]

Cells twice washed buffer FACS, measured fluorescence by means of FACS the Array (of bd) and marked value MCF.

[500]

Expected, that perekrestnoreaktivnye by lrp5/lrp6 VHH will be compete with human DKK1 behind binding with [nek 293, supering-express human LRP5, as is equal to and behind binding with [nek 293, supering-express human LRP6. Opposite to, by specific LRP5VHH would compete with human DKK1 behind binding with [nek 293, supering-express human LRP5, but not the binding with [nek 293, supering-express human LRP6 (or compete would, but very slightly (>200 [nm200) (and vice versa, the same relates to specific by LRP6VHH). As a result of this experiment dapsone displayed a, that present perekrestnoreaktivnye by lrp5/lrp6 VHH compete with human DKK1 behind binding with [nek 293, supering-express human LRP5, as is equal to and with [nek 293, supering-express human LRP6 (T. e ., WiFi client continuously tracks the value MCF at to increase the NII concentration svyazyvatelya, where complete suppression DKK1-and-binding at highest proven complies with concentration value MCF < 60).

[501]

4.2 Interspecies cross-reactivity: of a mouse and a javanese macaque to determine, whether is capable extracted panel perekrestnoreaktivnykh by lrp5/lrp6 VHH to bind with LRP5 and LRP6 from mouse and Javanese macaque, fulfil the following FACS for competition of with DKK1:

[502]

Serial dilution VHH incubated with cells of [nek 293, stably expressing LRP5 mouse, LRP5 macaque, LRP6 mouse or LRP6 macaque in the presence of 1 and 0.3 nm hDKKl (concentration below copepod for mouse and macaque respectively). Binding DKK1 with cells of identify by means of biotinylated antibodies against DKK1 with streptavidin - FE as secondary detector, according to the description above. As a result have been able to demonstrate such cross reactivity.

[503]

4.3 Epitope - specific sorting

[504]

Experiments by sorting fulfil for of the most strong blocking Wntl-and-signalingperekrestnoreaktivnykh by lrp5/lrp6 VHH, to determine different epitopic group. In particular, separate VHH tested ability to compete with other biotinilirovannymiVHH (T. consumed.

[505]

standard VHH) the binding with receptors LRP5 and LRP6 by means of analysis based on FACS. Serial dilution separate VHH incubated on the cells [nek 293, stably expressing human LRP5 or LRP6, together with 200 PM or 500 PM biotinylated reference VHH (concentration value below value of copepod). Binding of biotinylated reference VHH with cells of determined by means of streptavidine - FE.

[506]

Competition VHH with standard VHH behind binding with LRP5 and LRP6 WiFi client continuously effect was that fluorescence, measured FACS the Array.

[507]

As a result of these experiments blockers Wntlpodrazdelili on three groups.

[508]

More low affinity VHH not dimension owing to carry out experiments by epitopspetsificheskoi sorting for blockers of Wnt3a.

[509]

4.4 Analysis gene - to the reporter and WntlWnt3a

[510]

Capacity perekrestnoreaktivnykh by lrp5/lrp6 VHH suppress of WNT-and-signaling tested functional analysis and WntlWnt3a. In this is impossible with respect to was to use installed protocol, had to make several attempts of, to be set biochemical functional analysis, such as analysis blocking Wntl/wnt3a - lrp5/lrp6: to all complexity with the recombinant proteins LRP5 and LRP6 (cm. an example 1), no functional recombinant Wntl ligand (including available in sale). Proteins of WNT contain much conserved cysteine and modified by monounsaturated fatty acid (palmitinoleinovoi acid), connected to conservative to the serine. These posttranslational modification necessary for effective signalinga and the secretion of WNT. Structural analysis have shown, that one of domains, containing lipid palmitinoleinovuyu acid, is required for binding with receptor Frizzled, results to change of conformation, routine possible interaction of WNT-and-ligands with LRP5 and LRP6 on cellular surface. Present, such that posttranslational modification necessary for functional investigations with this protein, but at the same time similar lipid posttranslational modification of very highly complicated expression and purification of these proteins (from - due to bad solubility). This therefore to become large barrier for biochemical analysis.

[511]

Therefore for characteristics of purified VHH was developed functional analysis on cellular based: analysis of WNT gene - to the reporter beta - lactamase. In particular, for suppressing track Wntl cells CellSensorLEF/TCF-and-bla bar FreeStyle 293f (Invitrogen, art. κ1677) transfected human Wntl and selected clones with stable caused human Wntl. For checking suppression of track Wnt3a created cells CellSensorLEF/TCF-and-bla bar FreeStyle 293f with stable caused human Wnt3a. Cell line CellSensor® LEF/TCF - bla bar FreeStyle™ 293 has reporter gene beta - lactamase under control of induced of WNT promoter LEF/TCF, which stable is built in cells FreeStyle™ 293 (Invitrogen). So, expression of Wntl or Wnt3a in these cells leads to constitutive expression, and it, and to enzymatic activity of beta - lactamase. Therefore treatment of functional perekrestnoreaktivnymi by lrp5/lrp6 VHH should cause suppression track Wntl and Wnt3a, which will lead to suppression of enzymatic activity of beta - lactamase.

[512]

For analysis of 1e06/ml cells with caused Wntl or Wnt3a seeded on 384 - alveolar plotting board for tissue cultures and incubated during night at 37 theoretically. Next day prepared serial dilution of various perekrestnoreaktivnykh by lrp5/lrp6 VHH and added to the to cells in presence of LiCl in final concentration of 10 nm. As positive control to cells added to the DKK1 in final concentration of 200 nm.

[513]

Treatment DKK1 led to the full suppression track Wntl and Wnt3a and therefore to the full suppression of enzymatic activity of beta - lactamase.

[514]

Cells incubated during night at 37 theoretically. Next day enzymatic activity beta - lactamase measured according to instructions of a manufacturer of (Invitrogen, art. k1085). For fluorescent emission obtained value at 460 nm and 530 nm by means of standard fluorescent tablet Rieder, and ratio of emission at 460/530 nm applied on graph relative to said treatment. Efficiency expected relative to positive control (DKK1 in final concentration of 200 nm).

[515]

The whole selected twelve perekrestnoreaktivnykh by lrp5/lrp6 blockers of Wntl, complete efficiency and force of which was larger, than 50 nm. Identified fourteen perekrestnoreaktivnykh blockers of Wnt3a, mainly weak. Only one blocker Wnt3a proved good force (below 5 nm).

[516]

4.5 Analysis of phosphorylation of Wntl and Wnt3a

[517]

Construction of the powerful and effective samples from each group of blockers of Wntl, and also construction of the powerful and effective blockers Wnt3a further tested in analysis of phosphorylation of LRP5 and LRP6, dependent from Wntl and Wnt3a. In analysis of phosphorylation of used cells CellsensorLEF/TCF 293f from Invitrogen (art. k1677), to - transfected expression vectors, coding items or Wntl, or Wnt3a. As formation of complex of WNT-and-Frizzled-and-LRP5 or - LRP6 leads to phosphorylation LRP5 or LRP6 and subsequent descending transmission of signal, for measuring such transmission can be used quantitative determination of phosphorylation of. To produce specific by LRP5 and LRP6 readings, cells subjected lysis and fulfil the immunoprecipitation by means of antibodies, selective by LRP5 or LRP6 (directed against intracellular domain of two receptors).

[518]

Phosphorylated LRP5 or LRP6 identify Western - blotting using polyclonal antibodies against phospho - 1ltr6 (serl490) (the Cell Signaling the Technology), vyyavlyayushchego phosphorylated proteins and LRP5, and LRP6. Sampled panel purified VHH, blocking Wntl and Wnt3a, containing at least one representative VHH from each group, tested in final concentrations from 10 to 100 nm. In particular, cells incubated during night in the presence of blocking VHH before lysis cells and by the immunoprecipitation LRP5 and LRP6. Efficiency VHH, blocking Wntl and Wnt3a, at freezing of phosphorylation of LRP5 and LRP6 expected quantitative evaluation strips Western - blotting as compared with positive control (DKK1, final concentration 1 mcm).

4.6 Biophysical properties

[519]

Perekrestnoreaktivnym by lrp5/lrp6 VHH-and-domains gave further evaluation of expression and cleaning in E coli-. and Of Pichia pastoris, as described in example 3. in particular, output of expression monovalent specimens panel VHH believed acceptable, if it is higher than 0.1 mg/l. Selected perekrestnoreaktivnye by lrp5/lrp6 VHH have shown expression of in the range of 0.1 - 8.2 mg/l in E.coli and above Of Pichia pastoris (>1 mg/l). Expression of evaluation analysis of SDS-polyacrylamide gel-and-PPAGE.

[520]

Thermal stability monovalent perekrestnoreaktivnykh by lrp5/lrp6 VHH determined fluorescent analysis of thermal shift (exchange) by means of Lightcycler (Roche Airport). VHH incubated at different values of pH in the presence of the Orange Sypro, of a temperature gradient. After deployment the laying, caused by heat, strip down hydro fobnye sections proteins, with which binds Sypro the Orange, that leads to agreed to to increase the intensity of fluorescence (vozb/EM=465/580 nm). Bending point on the first derivative curve of fluorescence intensity serves as measure of melting point (Tf). In all VHH Tf at enhanced to increase the NII pH and it was equalized at pH 6, which is the typical for VHH circuit Tf. For perekrestnoreaktivnykh by lrp5/lrp6 VHH-and-blockers of Wntl and VHH-and-blockers of Wnt3a received average value of 82 theoretically at pH 7.

[521]

By method of analytical exclusion chromatography of investigated potential possibility of aggregation and multimerization of VHH to LRP5 and LRP6. For this purpose 8 mcg of purified sample VHH in concentration of 0.5 mg/ml injected with the aid of equipment Dionex the Ultimate 3000 in column Agilent the SEC-and-3. The mobile phase applied of L-argininovyi buffer (10 mm phosphate, 300 mm-Arg of HCl, pH of 6.0) on flow rate 1 ml/min. Neither of VHH to LRP5 and LRP6 not that large problems with thrombicytes during ekhanaliza: profiles pointed to more than 95% monomers in most specimens.

Example 5: creation and characteristic biparatopnykh constructs with prolonged half-period

[522]

Perekrestnoreaktivnye by lrp5/lrp6 VHH for Wntl and Wnt3a used as building blocks for creating biparatopnogo construct, pictured on figure 1. as method for extending half-time used genetic fusion with VHH, binding with serum albumin. Three building unit (Wntl-blocking agent-, Wnt3ablokator and albuminsvyazyvayushchii) unite the flexible linker. VHH formulated in P pastoris. and cleaned by description in example 3. obtained constructs, T. E. biparatopnyeperekrestnoreaktivnye by lrp5/lrp6 VHH-and-constructs inserted in expression vector p α-χ159 for P pastoris. in the form of VHH-and-constructs with smus - geksagistidinovoi mark on with - end, according to standard procedures, described, Eg ., in wo2012/131078. Investigated different orientation building blocks and different linkers, especially GSlinkery. On the basis of data simulating, reflecting increased area surface between potential sites binding Wntl and Wnt3a in LRP6 (T. e ., then, that beta - the propellers 1 and 2 not are located in close proximity from the beta - propeller 3), chosen relative to long the GS-linker.

[523]

The best results tangent force by combined analysis gene - to the reporter and WntlWnt3a received at insertion into VHH, binding human serum albumin/HAS, in the middle of. Used 35-and-GSlinker, and VHH-blockers and WntlWnt3a placing the in preferable order.

[524]

With the aim of selecting optimal VHH-and-svyazyvatelei and combinations of svyazyvatelei created library, in which VHH, binding human serum albumin/HAS, placed between lrp5/lrp6 Wntl-and-Wnt3ablokatorami. In particular, in library used panel high affine svyazyvatelei with large force and effectiveness according to analysis or WntlWnt3a (analysis gene - to the reporter and phosphorylation), to create biparatopnye constructs with prolonged period half-, designed, as shown on figure 1. after expression in Pichiapastoris (as described in example 3) and subsequent cleaning biparatopnye constructs with prolonged period half-subjected screening by means of analysis gene - to the reporter and WntlWnt3a (described in example 4) in the presence of 30 mcm HAS in three dilutions (1/100, 1/1000, 1/7000), to evaluate efficiency and relative force. As a whole watched good correlation of data reporter analysis Wntl and Wnt3a, and for multiple formats measured the high efficiency. For further evaluating selected the whole 11 biparatopnykh by LRP5 and LRP6 constructs with prolonged half-period, taking into account efficiency in both reporter analysis and variety of blockers of Wntl and Wnt3a. These further estimated analysis are described below.

[525]

Analysis gene - to the reporter and WntlWnt3a:

[526]

Analysis gene - to the reporter and WntlWnt3a fulfil according to the description in example 4.4, in the presence of final concentration HAS 30 mcm. Purified biparatopnye by lrp5/lrp6 constructs tested 12 dilutions, starting from 2.5 mcm.

[527]

Most constructs shown high force - from 1.7 nm to 0.16 nm and complete efficiency in both reporter analysis, that reflected in the table IV below. Sequence installed there individual VHH-and-domains, inhibiting Wntl and Wnt3a, are given in table V below:

[528]

Table IV: Force and essektivnost selected biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 unn - constructs with prolonged period in half-analysis gene - to the reporter in the presence of HAS

[529]

Reporter analysis WntlReporter analysis Wnt3a
ConstructOf n-end ofWith - end ofTHE IC50 (M)% ING.THE IC50 (M)% ING.
F013500053F0129093 [A 01F0130333G062, 3 Ε-- 101024, 1 Ε-- 1097
F013500039F0129093 [A 03F0130333E061, 7 Ε-- 091021, 6 Ε-- 0982
F013500046F0130332D03F0129093 [A 015, 1 Ε-- 101026, 0 Ε-- 1096
F013 500016F0130332D03F0130367B103, 1 Ε-- 101022, 7 Ε-- 1099
F013 500018F0130332D03F0130378B057, 4 Ε-- 101021, 3 Ε-- 0998
F013500047F0130333E06F0129093 [A 018, 8 Ε-- 101029, 1 Ε-- 1094
F013500026F0130333E06F0130367B103, 3 Ε-- 101022, 6 Ε-- 10100
F013500021F0130333G06F0130367B101, 6 Ε-- 101021, 8 Ε-- 10102
F013500032F0130378A04F0130333G062, 4 Ε-- 101024, 3 Ε-- 1094
F013500033F0130378B05F0130333G061, 8 Ε-- 101022, 5 Ε-- 1088
F013500030F0130378B05F0130372C081, 4 Ε-- 091021, 3 Ε-- 0996

[530]

Table of V: Sequence VHH-and-domains. given in table IV

[531]

VHH specificity of IDAmino acid sequence ofSEQ ID N0:
F0129093 [a 01 Wnt3aΕ-OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-S-S-GRTFΥ A OF MGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASPIPΥGSLLRRRNNYD ROTATION GOGTL VT CIRCUITS OF V S-S-Α-Α-Α-Ε-ΟKLISEEDLN OF G A OF Α-ΗΗΗΗΗΗ28
F0130333g06 WntlEVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAIRRSGRRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAARRVRSSTRYNTGTWWWEYWGQGTL VT CIRCUITS OF V S-S-A-A-Α-Ε-OKLISEEDLN OF G A OF Α-ΗΗΗΗΗΗ29
F0129093 α 03 Wnt3aΕ-OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-S-GRTFΤΥ OF V MGWFRQAPGKEREFVAAINWSGSRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA OF SRS S-YAGRTYYELYDYWGOGTL VT CIRCUITS VS OF S-Α-Α-Α-Ε-ΟKLISEEDLNG A OF Α-ΗΗΗΗΗΗ30
F0130333e06 WntlEVQEVESGGGEVQPGGSERESCAASGRTFSTYTVGWFRQAPGKEREFVAAIRRRGSSTYYSDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYY OF C PAAD TRTVALLOYRYDYWGOGTLVTVSSAAAEOKLISEEDLN OF G A OF Α-ΗΗΗΗΗΗ31
F0130332d03 WntlEVQLVESGGGLVQPGGSLRLSCAASGLTFSRYTMGWFRQ Α-P GKEREF OF V OF A AINRSGGSTYYSDS VK OF GRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGRGENΥSLLΥ S-SNRYE ROTATION GQGTLVTV S-S-A-A-Α-Ε-OKL LI EEDLNG A OF Α-ΗΗΗΗΗΗ32
F0130367b10 Wnt3aΕ-VQL THE VE S-GGGL ANY VQP GGSLRL OF C S-A-A-S-S-S-GGTFΥ A OF MGWFRQ Α-P GKEREFVAAISWRSGSTYY AD OF S-VK OF GRFTISRDNSKNTVYLQMNSLRPEGTAVYYCAADPRGY GV CHIPSETS A OF Υ VS OF A OF ΥYE ROTATION GOGTLVTV S-S-GA AND Α-Ε-OKLISEEDLNGAAHHHHHH33
F0130378b05 Wnt3aEVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAMGWFRQAPGKEREFVAAISRSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRVYSTLPPTTSRYNYWGOGTLVTVSSAAAEOKLISEEDLNG A OF Α-ΗΗΗΗΗΗ34
F0130378a04 Wnt3aΕ-OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-S-S-GRTFΥ A OF MGWFRQAPGKEREFVAAITRTGRRTΥΥ AD OF S-VK OF GRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGYYYYDSSFYDYWGOGTL VT CIRCUITS VS OF SAAAEOKLISEEDLNGAAHHHHHH35

[532]

VHH specificity of IDAmino acid sequence ofSEQ ID N0:
F0130372c08 WntlΕ-OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-GRTF OF SD SUBJECT MGWFRQAPGKEREFVAAISWSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAKRRGRGSVSPNS S-SRYNYWGQGTL VT CIRCUITS VS OF S-THE AAA EOKLISEEDLNGAAHHHHHH36
F013500016EVQLVESGGGLVQPGGSLRLSCAASGLTFSRYTMGWFRQ Α-P GKEREF OF V OF A AINRSGGSTYYSDS VK OF GRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGRGENYSLLΥ S-SNRYEYWGQGTL VT CIRCUITS OF V S-THE SG GGGSGGGGSGGGGSGGGGSGGGGSGGGGS GG IS GGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYY OF C TIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAISWRSGSTYYADSVKGRFTISRDNSK NT-OF V YLQMNSLRPEGTAVYY OF C OF A ADPRGY GV CHIPSETS HBSAG AY V-S-A-ΥYE ROTATION GOGTL VT CIRCUITS OF V S-S-GA AND AEOKLISEEDLN OF G37
Α-Α-ΗΗΗΗΗΗ
F013500018EVQLVESGGGLVQPGGSLRLSCAASGLTFSRYTMGWFRQ Α-P GKEREF OF V OF A AINRSGGSTYYSDS VK OF GRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGRGENYSLLYSSNRYE ROTATION GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GG IS GGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAMGWFRQAPGKEREFVAAISRSGGRTYYADSVKGRFTISRDNSKNΤVYLQMNSLRPEDTAVYY OF C AADRRVYSTLPPTTSRYNYWGOGTLVTVSSGAAEOKLISEEDLNGA Α-ΗΗΗΗΗΗ38

[533]

VHH specificity of IDAmino acid sequence ofSEQ ID N0:
F013500021EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAIRRSGRRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAARRVRSSTRYNTGTWWWEYWGQGTL VT CIRCUITS OF V S-THE SG GGGSGGGGSGGGGSGGGGSGGGGSGGGGS GG IS GGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYY OF C TIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAISWRSGSTYYADSVKGRFTISRDNSK NT-OF V YLQMNSLRPEGTAVYY OF C OF A ADPRGY GV CHIPSETS HBSAG AY V-S-A-ΥYE ROTATION GOGTL VT CIRCUITS OF V S-S-GA AND AEOKLISEEDLN OF G Α-Α-ΗΗΗΗΗΗ39
F013500026EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTVGWFRQAPGKEREFVAAIRRRGSSTYYSDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYY OF C PAAD TRTVALLQYRYD ROTATION GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQL OF V ESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS S-QGTL VT CIRCUITS VS OF S-GGGGSGGGGSGGGGSGGGGS GG IS GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAISWRSGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEGTAVYY OF C OF A ADPRGY OF G OF V OF A Υ V-S-A-ΥYE ROTATION GOGTL VT CIRCUITS VS OF SGAAEOKLISEEDLNGAAHHHHHΗ40
F013500030EVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAMGWFRQAPGKEREFVAAISRSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRVYSTLPPTTSRYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE OF V QL THE VE S-GGGL OF V QPGNSLRL S-OF C OF A GF AS OF THE TF S-OF FRONTAL CROSS OF GM S-WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS S-QGTL VT CIRCUITS VS OF S-GGGGSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSEVQL THE VE S-GGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVAAIS OF W SGGRTΥΥ AD OF S-VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAKRRGRGSVSPN S-S-SRYNYWGOGTLVTV S-S-GAAEOKLISEEDLNGAAHHΗΗΗΗ41

[534]

VHH specificity of IDAmino acid sequence ofSEQ ID N0:
F013500032EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQ AP GKEREF OF V OF A AITRTGRRTΥΥ AD OF S-VK OF GRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGYΥΥYD S-OF FRONTAL CROSS YD ROTATION GQGTL VT CIRCUITS OF V S-S-GGGGS OF G GGGSGGGGSGGGGSGGGGSGGGGSGGGGSE OF V OF Q LVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS RS-S-QGTL VT CIRCUITS VS OF S-GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAIRRSGRRTYYADSVKGRFTISRDNSKNTVYLQΜΝSLRPEDΤ A OF OF V ΥΥ OF C A OF A OF ARRVRS S-ECO RI ΥΝΤ GF OF W WWEYWGQGTLVTV S-S-GAAEOKLISEEDLNGAA42
ΗΗΗΗΗΗ
F013500033EVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAMGWFRQAPGKEREFVAAISRSGGRTYYADS VK OF GRFTISRDNSKNTVYLQMNSLRPEDTAVYY WITH Α Α-DRRVYSTLPPTTSRYNYWGQGTLVTV S-SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE OF V QL THE VE S-GGGL OF V QPGNSLRL S-OF C OF A GF AS OF THE TF S-OF FRONTAL CROSS OF GM S-WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS S-QGTL VT CIRCUITS VS OF S-GGGGSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSEVQLVE S-GGGLVQPGGSLRL S-OF C OF A AS OF GGTF S-S-ΥAMGWFRQAPGKEREF OF V OF A AIRRSGRRTYYADSVKGRFTISRDNSKNTVYLQΜΝSLRPEDΤAVYY OF C A OF A OF ARRVRS S-ECO RI ΥΝΤ GF OF W WWEYWGQGTLVTVSSGAAEOKLISEEDLNGAA43
ΗΗΗΗΗΗ
F013500039EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYVMGWFRQAPGKEREFVAAINWSGSRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA OF SRS S-YAGRTYYELYD ROTATION GQGTLVTV S-SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE OF V QL THE VE S-GGGL OF V QPGNSLRL S-OF C OF A GF AS OF THE TF S-OF FRONTAL CROSS OF GM S-WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS S-QGTL VT CIRCUITS VS OF S-GGGGSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSEVQLVE S-GGGLVQPGGSLRLSCAASGRTFSTYTVGWFRQAPGKEREF OF VA AIRRRGS S-ΤΥΥ OF SD SVKGRFTISRDNSKNT OF V YLQMNSLRPEDTAVYYCAADTRTVALLQYRYDYWGOGTLVTVSSGAAEOKLISEEDLNGAAHHHHHΗ44

[535]

VHH specificity of IDAmino acid sequence ofSEQ ID N0:
F013500046EVQLVESGGGLVQPGGSLRLSCAASGLTFSRYTMGWFRQ Α-P GKEREF OF V OF A AINRSGGSTYYSDS VK OF GRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGRGENΥSLLΥ S-SNRYE ROTATION GQGTL VT CIRCUITS OF V S-S-OF G GGGSGGGGSGGGGSGGGGSGGGGSGGGGS GG IS GGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYY OF C TIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASPIPYGSLLRRRNNYD ROTATION GOGTL VT CIRCUITS OF V S-S-GA AND AEOKLISEEDLN OF G Α-Α-ΗΗΗΗΗΗ45
F013500047EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTVGWFRQAPGKEREFVAAIRRRGSSTYYSDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYY OF C PAAD TRTVALLQYRYD ROTATION GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQL OF V ESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS S-QGTL VT CIRCUITS VS OF S-GGGGSGGGGSGGGGSGGGGS GG IS GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTΥΥADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASPIPYGSLLRRRNNYDYWGOGTLVTVSSGAAEOKLISEEDLNGAAHHHHHH46
F013500053Ε-OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-S-S-GRTFΥ A OF MGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASPIPΥGSLLRRRNNYD ROTATION GQGTLVTV S-SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE OF V QL THE VE S-GGGL OF V QPGNSLRL S-OF C OF A GF AS OF THE TF S-OF FRONTAL CROSS OF GM S-WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS S-QGTL VT CIRCUITS VS OF S-GGGGSGGGGSGGGGSGGGG S-GGGGSGGGGSGGGGSEVQL THE VE S-GGGLVQPGGSLRL S-OF C OF A AS OF GGTF S-S-ΥAMGWFRQAPGKEREF OF V OF A AIRRSGRRTYYADSVKGRFTISRDNSKNTVYLQΜΝSLRPEDΤAVYY OF C A OF A OF ARRVRS S-ECO RI ΥΝΤ GF OF W WWEYWGOGTLVTV S-S-GAAEOKLISEEDLN GA AND Α-ΗΗΗΗΗΗ47

[536]

(.Note: Described in the table of V molecules contain shusgeksagistidinovuyu mark (stressed), to facilitate cleaning it is recombinant expressed polypeptides; this mark not practiced for - and, as a rule, does not take in - binding molecules with their targets.

[537]

Example 6: optimization of sequences VHH and VHH -constructs

[538]

Optimization of sequences - this process, in which the parent sequence is subjected to mutations, to make its more similar with human embryonic consensus sequence IGHV3-and-IGHJ. For example, specific amino acids frame sections (except for so called distinguishing residues) are replaced with their human analogs so, to preserve structure, activity and stability of the protein.

[539]

These mutations may be divided into the following category :

[540]

1. Standard: optimization of sequences in these positions should not substantially change stability, activity or affinity VHH, therefore their all is changed simultaneously, to produce base version.

[541]

2. Unique: unknown, as optimization of sequences in these positions affect on stability, activity or affinity VHH, therefore their examined individually on the basis of base versions.

[542]

Known, that are the critical residues important for stability, activity and affinity VHH, therefore their is not changed.

[543]

In addition, amino acids CDR-, for which there is experimental confirmation the, that they sensitive to posttranslational modifications (PEJ), discoloration so, to inactivate the site PEJ, it before ever touching the structure, activity and stability of the protein. Most frequently posttranslational modification, described for antibodies and VHH, listed in the table VI below. Sensitivity VHH to posttranslational modifications analyzed by research with forced load, of a row of standard conditions, including treatment of H2 Exhaust gases for evaluating oxidation of methionine, high temperature, high pH and for long-term storage for evaluating dezamidirovaniya asparagine and isomerization of aspartate. Percentage share of oxidation, dezamidirovaniya and isomerization of measured according to standard procedures and compared with reference samples (VHH, stored at -20 theoretically). For determination of potentially sensitive residues fulfil analysis of integral proteins by reversed - phase chromatography (ofkh) and mapping peptides by mass - spectrometry (ms). If after stress - dough VHH postoperative period posttranslational modification, corresponding to (- MR) amino acid (s -) subjected mutations.

[544]

Table VI of: Potential posttranslational modification and potentially launching their motives

[545]

MotifModification of
MOxidation of met polypeptide
OF N-S-/ G/H/N/ADeamidization Asn-
-D-S-/ G/HIsomerization of Asp-
OF N-X S-/ T OF-X-Asn-glycosylation
(X ≠ Λ2 P)
Q/Epiroglutamat

[546]

As a result described above constructs introduced several mutations, the report among other things three construct, shown in the table III above, selected for further characteristics in vitro of and in vivo of, as shown in further examples.

[547]

Example 7: In vitrovtTro characteristic three biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged period half-; comparison with other coupling LRP6molecules

[548]

After optimization of sequences VHH three biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-construct with prolonged period half-it is recombinant expressed and cleaned, after which characterized by means of row functional and biophysical analysis, described below.

7.1 Binding analysis FACS

[549]

Binding with human LRP5 and LRP6 determined on the cells FACS analysis, as shown on shapes behind and of members. In particular, binding with human LRP5 tested on cells [nek 293 with stable caused human LRP5. For binding with human LRP6 used cells [nek 293 with stable caused human LRP6. Cells incubated with by the breedings LRP5ievrb - svyazyvatelei (serial dilution svyazyvatelei 1:5, corresponding final concentration of, indicated on shapes behind and of members) during 1.5 hour at 4 theoretically on tablet shaker. After 5 - multiple cells washing buffer FACS, consisting of ix PRF (Invitrogen, art. 141190 - 094) + 10% ebs (the Sigma, art.

[550]

F7524) + 0.05% sodium azide, their incubated during 1 hour at 4 theoretically with polyclonal murine antibody, binding with frame section VHH. After of triple washing cells buffer FACS their incubated during 1 hour at 4 theoretically with labelled secondary antibody (protivomyshinoe, FE (115 - 116 - 071)) followed by threefold washing buffer FACS. Fluorescence measured by means of FACS the Array (of bd). Bonding with human LRP5 and LRP6 at highest concentration of proven value complies MCF > 600. Negative control consisted of nenatselennogosvyazyvatelya (VHH-construct, binding with bacterial protein, which in cells [nek 293 not is expressed). As shown respectively on figure behind and figure of members, bonding with human LRP5 and LRP6 value complies MCF > 600 and > 1600, at highest concentration of proven three biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged half-period. These data is confirmed, that formatted biparatopnye binding molecules with optimized sequence in its natural conformation in system analysis on cells are as with human LRP5, and with human LRP6.

[551]

Value copepod binding with hLRP5 and hLRP6 are given in table VII below.

[552]

Table of VII: Value eszo binding with human LRP5 and LRP6.

certain binding FACS analysis

[553]

Binding analysis based on FACSF013500571F013500575F013500720
hLRP5, eszo (nm)0.010.0010.02
hLRP6, Eszo (nm)0.020.010.02

7.2 Analysis of competition of with DKK1 based on FACS

[554]

Force and efficiency three biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs additionally analyzed analysis competition of with DKK1 based on FACS, described in example 4.1. [Nek 293 cells with stable caused human LRP5 or human LRP6 incubated with serial by the breedings perekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs (serial dilution 1:5, corresponding final concentration of, indicated on shapes 4a and 4B).

[555]

Perekrestnoreaktivnye by lrp5/lrp6 VHH compete with human DKK1 behind binding with cells [nek 293, supering-express human LRP5, as is equal to and behind binding with cells [nek 293, supering-express human LRP6, as shown respectively on shapes 4a and 4B. Complete suppression of binding DKK1 reached at the highest checked concentrations (>10 nm), that corresponds to values of MCF < 60. Opposite to, by specific LRP5VHH would compete with human DKK1 behind binding with [nek 293, supering-express human LRP5, but not the binding with [nek 293, supering-express human LRP6 (or compete would, but very slightly (>200 nm) (and vice versa, the same relates to specific by LRP6VHH). As a result of this experiment dapsone displayed a, that present perekrestnoreaktivnye by lrp5/lrp6 VHH compete with human DKK1 behind binding with [nek 293, supering-express human LRP5, as is equal to and with [nek 293, supering-express human LRP6 (T. e ., WiFi client continuously tracks the value MCF at to increase the NII concentration svyazyvatelya, where complete suppression DKK1-and-binding at highest proven complies with concentration value MCF < 60). Value ic50 for competition of DKK1 behind binding with hLRP5 and hLRP6 with three perekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs are given in the table VIII below. These data is confirmed binding three perekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with human LRP5 and with human LRP6, and shown very similar affinity (here as determined by values of 1c 50 in the analysis of competition of with DKK1) of two receptors. Moreover, data are strengthened by position, that formatted biparatopnye binding molecules with optimized sequence in its natural conformation are as with human LRP5, and with human LRP6.

[556]

Table of VIII: Value IC^nDKK1 with competition of the binding with human LRP5 and LRP6. certain analysis of binding FACS analysis of competition of with DKK1 f013500571 f013500575 f013500720

[557]

Analysis of competition of with DKK1F013500571F013500575F013500720
hLRP5, 1c50 (nm)0.030.030.3
hLRP6, 1c50 (nm)0.01OD0.2

[558]

7.3 Combined analysis of gene - to the reporter and WntlWnt3a

[559]

Force and efficiency formatted biparatopnykh binding molecules with optimized sequence analyzed by combined analysis gene - to the reporter and WntlWnt3a, to provide functional check of blockers of Wntl and Wnt3a in one analysis.

[560]

Combined analysis of gene - to the reporter and WntlWnt3a is based on described in example 4.4 analysis, changes with the following protocol.

[561]

1E06/ml cells with caused Wntl seeded on 384 - alveolar plotting board for tissue cultures and treated recombinant human Wnt3a (rivers. cel. Wnt3a: of R&d of #5036-and-Wn/the CF), after that cells are incubated during night at 37 theoretically. Next day prepared serial dilution of various biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH and added to the to cells in presence of LiCl in final concentration of 10 nm. As positive control to cells added to the DKK1 in final concentration of 200 nm. Treatment DKK1 led to the full suppression combined track Wntl and Wnt3a and therefore to the full suppression of enzymatic activity of beta - lactamase. Cells incubated during night at 37 theoretically. Next day measured enzymatic activity beta - lactamase according to the instructions of a manufacturer. As specified in example 4.4, for fluorescent emission obtained value at 460 nm and 530 nm by means of standard fluorescent tablet Rieder, and ratio of emission at 460/530 nm applied on graph relative to said treatment. The ratio value fluorescence [460/535nm], designated on figure 5a as "base line", corresponds to complete inhibition of track Wntl and Wnt3a, a certain treatment with positive control (DKK1 in final concentration of 200 nm).

[562]

The ratio value fluorescence [460/535nm], designated as "Wntl", corresponds to activation of only track Wntl, defined by cells, sverkhekspressiruyushchimWntl (τ. ℮ ., not treated recombinant human Wnt3a). The ratio value fluorescence [460/535nm], designated as "Wntl + Wnt3a", corresponds to combined activation of tracts and WntlWnt3a, certain by treatment of cells, overexpressing Wntl, recombinant human Wnt3a. As shown on figure 5a, complete suppression of (T. e ., ratio of fluorescence [460/535nm], corresponding to base line) achieved by treatment of three perekrestnoreaktivnymi by lrp5/lrp6 formatted biparatopnymi coupling binding molecules with optimized sequence. In addition, was also considerable force, as shown in table IX below at values ic50.

[563]

Table of IX: Value MPP suppression of tracts and WntlWnt3a in combined analysis and WntlWnt3a gene - to the reporter

[564]

Combined analysis of Wntl and Wnt3a gene - to the reporterF013500571F013500575F013500720
1C50 (nm)0.050.20.06

[565]

Then force and efficiency perekrestnoreaktivnykh by lrp5/lrp6 formatted biparatopnykh binding molecules with optimized sequence compared with known first of molecules, coupling LRP6, mentioned in wo2011/138391 and wo2011/119661:

[566]

In wo2011/138391 have been described are multivalent antibodies, binding with LRP6 and inhibiting interaction with ligand as propeller 1 (nair ., Wntl), and propeller 3 (nair ., Wnt3). These multivalent antibodies, binding with LRP6, are biparatopnymi molecules, coupling LRP6, consisting of antibodies of IgG as the first binding receptor domain and fragment scFv antibody as the second binding receptor domain, the antibody IgG and scFv antibody fragment are connected linker. In wo2011/138391 communicates, that all binding LRP6 molecules have approximately equal force through reporternomu analysis and WntlWnt3a (Figure 18 wo2011/138391). Therefore for comparative experiments can be to select any of these multivalent molecules, binding with LRP6.

[567]

As therefore first connection for comparing was classified to use construct "901" (denoted MOR08168IgGlLALA 6475 scFv antibody; also shown on Figure 27 wo2011/138391).

[568]

In wo2013/067355 shown derivatives of this construct "901". More specifically invention describes compounds 801t and 802t (cm. information on temperature control system. 132 description), having two yarb - binding domain scFv antibody fragment plus, drives enhanced half. As, along the whole visibility, 801t and 802t have the same force and biophysical characteristics in vitro of, for the experiments described below took only one of them - version 802t.

[569]

In wo2011/119661 invention describes antibodies bispecific, binding with LRP6 and suppress transmission signal by many to the isoform of WNT. These bispecific antibodies against LRP6 are with two different sections LRP6 and inhibit signal transmission, inducible isoforms of WNT, among them Wntl and Wnt3a. For producing said bispecific antibodies against LRP6 used construction according to principle of "projections - vovpadiny" (Atwelland DP. "To stable heterodimers the From remodeling of The the Domain interface interface care of a homodimer the Using of a phage the Display the Library". JMolBiol. 1997; 270 (1): 26 - 35). In example 11 wo2011/119661 described is a hybrid structure of IgG byby heavy chain yw211.31.62 and yw2 10.09. Therefore for the purpose of comparison sgenerirovali two bispecific of hybrid antibodies of IgG byby heavy chain yw211.31.62 and yw2 10.09, use equipment "projections - in - cavity", T. e ., with amino acid changes, designed for creation of projection on snz heavy chain yw2 10.09 and recesses on snz heavy chain yw211.31.62 or vice versa, and their designated as Knob ns yw2 10.09 and Knob ns yw211.31.62, respectively. These two construct described by means of reporter analysis Wntl and Wnt3a for example according to 4.4. As expected, two bispecific hybrid of IgG byby heavy chain yw211.31.62 and yw2 10.09 in analysis Wntl and Wnt3a have shown equal force, that indicated in the table X below. Therefore shown for further comparative examples as the second comparative compounds chosen Knob ns yw2 10.09.

[570]

Table Of X: value IC^n suppression of tracts and WntlWnt3a in analysis Wntland Wnt3a gene - to the reporter

[571]

Reporter analysis of WNTKnob ns yw2 10.09Knob ns yw211.31.62
Wntl, the IC50 (nm)4.84.6
Wnt3a, the IC50 (nm)0.70.9

[572]

Force and efficiency biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged period half-compared with biparatopnoi molecule MOR08168IgGlLALA 6475 scFv antibody, binding LRP6, with of bispecific molecule Knob ns yw2 10.09 against LRP6 and with of bispecific molecule 802t against LRP6, using combined analysis of gene - to the reporter and WntlWnt3a. As shown on figure V, complete suppression of (T. e ., ratio of fluorescence [460/535nm], corresponding to base line on figure V) achieved by treatment of Knob ns yw2 10.09 (bispetsificheskim hybrid antibody of IgG byby heavy chain yw211.31.62 and yw2 10.09) also, as perekrestnoreaktivnoi by lrp5/lrp6 formatted biparatopnoi binding molecule with optimized sequence f013500571. However f013500571 proved greater force, as indicated in the table of the XI below. At the same time biparatopnye molecules MOR08168IgGlLALA 6475 scFv and 802t, binding LRP6, have shown is insufficient complete suppression of Wntl and Wnt3a (T. e ., ratio of fluorescence [460/535ημ] considerably above base line on figure V and on figure 5c, respectively). These data indicate then, that both biparatopnye molecules MOR08168IgGlLALA 6475 scFv and 802t, binding LRP6, have considerably smaller efficiency at suppression Wntl and Wnt3a as compared with f013500571. Therefore described for further experiments in vivo of as comparative compounds was selected Knob ns yw2 10.09 (An example 9; efficiency in vivo of).

[573]

Table of the XI: Value ICsn suppression of tracts and WntlWnt3a in combined analysis and WntlWnt3a gene - to the reporter

[574]

Combined analysis of Wntl and Wnt3a gene - to the reporterF013500571Knob ns yw2 10.09MOR08168IgGlLALA 6475 scFv antibody802Τ
1C50 (nm)0.051.150.190.01

Example 8: influence of three biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 unn - constructs with prolonged period half-transmission signal of WNT and viability of cancer cell lines

[575]

Capacity biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 unn - constructs with prolonged period half-suppress active of WNT-and-signaling additionally described by means of cell lines with active of WNT-and-signalingom, described earlier (Baficoand DP. "The AN autocrinemechanism for Laptops constitutive of WNT pathway the activation of in vitro human being cancercells". The Cell Cancer 2004 ; 6 (5): 497 - 506 ;DeAlmeidaand DP. "Of The soluble of WNT receptorFrizzled8CRD-and-the HFC inhibits of The growth options care teratocarcinomas in vivo of". Cancer resolution RES. 2007 ; 67 (11): 5371 - 9); Akiriand DP. "Of WNT pathwayaberrationsincludingautocrine of WNT the activation occur client terminal the High the Frequency of in vitro human being the non-the Small-and-the Cell lung-carcinoma". Oncogene. 2009;

[576]

28 (21): 2163 - 72). If briefly, line of cancer cells with active of WNT-and-signalingom, Pa - 1 and PA-TU-and-8988s, seeded on 12 - lunkovye boards and treated by perekrestnoreaktivnymi lrp5/lrp6 VHH-and-by the constructs in final concentration of 1 mcm on throughout two days. Capacity suppress of WNT-and-signaling identify by inhibition of mRNA expression Axin2, endogenic gene - target of WNT. Analysis of expression kptsr fulfil using standard techniques RNA: RNA recovery was by means of QIAGENRNeasy the Mini KIT is according to protocol of QIAGEN; synthesis of cDNA by means of SuperscriptVILOcDNASynthesis KIT is (Invitrogen, art. 11754050) and kptsr - by means of TaqManGene expression command Assay with primers/probes Axin2TaqMan (Hs00610344_mlAxin2 for FAM, the Life Technologies for) and also of c eucaryotic 18s endogenic control the VIC-and-MGB (4319413 ε-- 1307061, AutoMARK Biosystems).

[577]

As shown on figure 6a, and cancer cells the PA-te18988s, and Pa - 1 in treatment of three biparatopnymiperekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs with prolonged period half-have shown considerable WiFi client continuously tracks the relative levels of mRNA Axin2 (τ. ℮ ., of normalized by endogenous control) as compared with untreated (control) cells. These data show ability of biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged period half-suppress of WNT-and-signaling in cell lines with active of WNT-and-signalingom. In addition, investigated the effect of blockade of WNT-and-signalinga on viability of cells of cancer cell lines the PA-tu8988s and YAPC, about which earlier reported, that their proliferation depends on active of WNT-and-signalinga (Jiang offer and DP. "Inactivatingmutations care RNF43confer of WNT dependency of in vitro pancreaticductaladenocarcinoma". The proc NatlAcad SCI to the USA. 2013; 110 (31): 12649 - 54). Viability of cells measured analysis of Alamar the Blue (Invitrogen, art. DALI 100) through ten days after treatment of biparatopnymiperekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs with prolonged period half-(final concentration of 1 mcm) or comparison compound 802t (final concentration of 1 mcm). As shown on figure 6B, cancer cells the PA-tu8988s in treatment of three biparatopnymiperekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs with prolonged period half-have shown considerable WiFi client continuously tracks the percentage of viable cells (WiFi client continuously tracks on > 75%) as compared with untreated (control) cells. After treatment 802t influence on viability of cells not discovered (Figure 6 in, diagram the right side). These data show ability of biparatopnykhperekrestnoreaktivnykh by lrp5/lrp6 VHH-and-constructs with prolonged period half-suppress proliferation of cell lines, dependent from the active of WNT-and-signalinga, and also superiority of these constructs above 802t.

[578]

In addition, for f013500571 as compared with 802t on lines of cancer cells the PA-tu8988s (Figure 6 with) and YAPC (Figure 6 d of) appreciated dependence of viability of cells from dose. In treatment of f013500571 identified dose-dependent WiFi client continuously tracks the cell viability. Unlike it, in treatment of comparison compound 802t influence on viability of cells not discovered in one of lines of cancer cells the PA-tu8988s and YAPC. These data demonstrate, that biparatopnyeperekrestnoreaktivnye of NO lrp5/lrp6 VHH-and-constructs with prolonged period half-have best effect as compared with 802t.

Example 9: efficiency in vivo of

[579]

Perekrestnoreaktivnye by lrp5/lrp6 biparatopnyeVHH-and-constructs/binding molecules with prolonged period half-additionally described in vivo of on model tumor, caused by of WNT.

[580]

Implemented conducts experiments, designed to determine, whether suppress these binding molecules growth of tumor in vivo of. On the same model tumor, caused by of WNT, determine efficiency of comparative compounds Knob ns yw2 10.09.

[581]

Transgenic expression of WNT-and-ligands by means of LTR--and-enhancer of virus of breast tumor mice (promoter MMTV) in mice leads to extensive hyperplasia ducts with subsequent by the adenocarcinoma of mammary gland in transgenic (Tg) mice to 6 - month age. These mammary gland tumor called induced glucocorticoids caused of WNT-and-ligands and by their indices similar tnrmzhopukholyam, including expression of epithelial and mesenchymal markers (like basal phenotype) and active of WNT-and-signaling, assessed by intracellular localization of beta - katenina. In particular, breast tumor in transgenic mice MMTV-and-of WNT-and-1 depend on Wntl. In the same art, reported, that blocking activity of WNT by means of soluble receptor of WNT, including cysteine-rich domain (btsd) Frizzled8, fused with human fn-and-domain (f8crdhfc) (DeAlmeidaand DP. "Of The soluble of WNT receptorFrizzled8CRD-and-the HFC inhibits of The growth options care teratocarcinomas in vivo of". Cancer resolution RES. 2007 ; 67 (11): 5371 - 9), suppresses growth of tumor in vivo of. Therefore isolated from transgenic mice MMTV-and-Wntl tumor subcutaneously passaged in the form of bits tumor thymus-free mice in an amount of from 2 to 5 passages before the beginning of experience by efficiency of. Between 14 and 21 days after implantation, when the tumor reached average volume of approximately 150 - 250 mm3 , mice randomized by groups of by 7 mice and 1.20 to them in. in. compounds.

[582]

Perekrestnoreaktivnye by lrp5/lrp6 biparatopnyeVHH-and-constructs with prolonged period half-1.20 mice in. in. twice per week, in doses, shown on the figure 7a for f013500571 and on figure 7B for f013500720. Comparative compound Knob ns yw2 10.09 also 1.20 in. in.

[583]

twice per week, but in higher doses, T. e ., 30 and 45 mg/kg (Figure 7 with), from - the data, obtained for this compound in experiments described above in vitro of. During experience by efficiency of monitored tumour volume and body weight, and medians volumes of tumors shown on shapes 7a - 7c. At the end of experience by efficiency of determined suppression of tumor growth (pro). In particular, pro determined for each group treatment in the control group (treatment of mice gistidinovym buffer - buffer with 20 mm histidine and pH 6.5 - in experiment shapes 7a and 7B, or same buffer in experiment figure 7c). In addition, at the end of experience by efficiency of fulfil gastro - intestinal (liquid crystal) histopathological analysis (g&E - staining sections of liquid crystal - path from duodenal to rectum) to to estimate potential toxicity antagonists LRP5 and LRP6. Suppression of tumor growth (pro), result of liquid crystal - histopathological analysis in end of investigating effectiveness of in vivo of, lethality, corresponding to number of mice, which had death from - the considerable body mass loss (>18% losses of body mass as compared with beginning of experience by efficiency of), and the number of regressov tumors (when volume of tumor at the end of experience is less than, than the volume of the tumor at the beginning of treatment), for each group of treatment are given in the tables khpa, khpb and khpv, data and experiments shown also on shapes 7 of α, 7B and 7c, respectively.

[584]

Table khpa:Essektivnost f013500571 in vivo of at in. in. introduction of twice per week. Results of this experiment shown also on figure 7a,

[585]

Dose [mg/kg]PRO [%]Regressov [X/7]Lethality [X/7]G istopatologicheskaya evaluation of gastrointestinal tract
ControlGistidinovyi buffer----
F0135005711012870Without features of
411370Without features of
26100not ODA.

[586]

Table khpb:Essektivnost f013500720 in vivo of at in. in. introduction of twice per week. Results of this experiment shown also on figure 7B.

[587]

Dose [mg/kg]PRO [%]Regressov [X/7]Lethality [X/7]G istopatologicheskaya evaluation of gastrointestinal tract
ControlGistidinovyi buffer----
F013500720112870Without features of
0.46000Without features of
0.21700not ODA.

[588]

Table khpv:EssektivnostKnob ns yw2 10.09 in vivo of at in. in.

[589]

introduction of twice per week. Results of this experiment shown also on Figure 7c.

[590]

in. in.Dose [mg/kg]PRO [%]Regressov [X/7]Lethality [X/7]G istopatologicheskaya evaluation of gastrointestinal tract
ControlCitrate buffer----
Knob ns yw2 10.09308000-
458500Without features of

[591]

As can be understand by figures 7a - 7c and tables khpa - khpv, treatment biparatopnymiperekrestnoreaktivnymi by lrp5/lrp6 VHH-and-by the constructs with prolonged period half-(f013500571 in a dose of 4 and 10 mg/kg and f013500720 in a dose of 1 mg/kg by the curve 2p/week) actually resulted tumor regression (T. e ., suppression of growth tumor (pro) > 100%, which corresponds to reduction of tumor; decreased volume of the tumor at the end of experience by efficiency of as compared with tumor volume at the beginning of experience), the are not found significant changes of body mass (<10%) and on histopathological analysis of gastrointestinal tract are not found features. Is important, that unlike it, in treatment of ytrb - specific svyazyvatelemKnob ns yw2 10.09 not observed any regress tumors, even in maximum introduced mice by plot in. in. dose, which corresponds to 90 mg/kg by the curve zr/week.

[592]

To additionally it possible to study observed in efficiency of difference experiment described above in vivo of, put one more experience, allowing more frequent introduction of mice (three times per week) still increased dose comparative compounds. Briefly, to achieve such amplified effect of, comparative compound 1.20 in B.., as specified in below the following table XIID:

[593]

Table XIID:EssektivnostKnob ns yw2 10.09 in vivo of at in. b.

[594]

introduction of twice or three times a week, soglazno said.

[595]

in. b.Dose [mg/kg]GraphPRO [%]Regressov [X/5]Lethality [X/5]
ControlTsitratn th bufferThree times a week
Knob ns yw2 10.0945Twice per week5510
90Twice per week8500
90Three times a week8700

[596]

As seen by data, given in the table XIID, in this setting also not reached considerable amplification effect in plan pro. Other words, these experiments, data and results clearly indicate more high efficiency biparatopnykhVHH-and-constructs with prolonged half-period as compared with Knob ns yw2 10.09, and also unprecedented ability of polypeptides according to the invention not only release growth of tumor, but even cause its reduced. Reduced tumor (T. e ., regress tumor) is, outside deserves the doubts, desirable therapeutic effect (T. e ., effectiveness) in treatment of cancer patients patients. In the same art, in clinical investigations drug, causing regression of tumor, which to the full patomorfologicheskomu response (CSPs), positively lead to considerable improvement of survival rate without progressing and common survival rate in case of large unsold medical demand, such, as at breast cancer.

[597]

Described above comparative examples also demonstrate, that perekrestnoreaktivnye by lrp5/lrp6 biparatopnyeVHH-and-constructs with prolonged period half-not only are better by their indices binding, such as affinity or value of to, but they also have large advantages and better characteristics in conditions of in vivo of.

[598]

Then studied, can whether soedineniemszh081681§shaa 6475 scFv antibody to provide the same effect. For this purpose fulfilled the following examination tolerance in vivo in mice: compound MOR08168IgGlLALA 6475 scFv antibody 1.20 in. in. in a dose of 3 mg/kg twice per week (2qw); this the same construction of the dose and protocol, for which in wo2011/138391 on ksenotransplantatnoi model tumor has been detected efficiency in vivo of, according to description on fig.. 22 of this document. The first treatment MOR08168IgGlLALA 6475 scFv antibody implemented in day 1, and starting from the days 6, in mice marked considerable loss of body weight. On 10 - day some mouse, treated compound MOR08168IgGlLALA 6475 scFv antibody, have shown considerable loss of body mass (>10%). On 11 - day mice put to death, and histopathological analysis of gastrointestinal tract proved inflammation with erosion of colon and blind gut mice. These data indicate then, that MOR08168IgGlLALA 6475 scFv antibody in effective dose/protocol neperenosim.

[599]

So, biparatopnyeperekrestnoreaktivnye by lrp5/lrp6 VHH-and-constructs with prolonged period half-have the advantage with respect to therapeutic window; T. e ., they cause regression of tumor without significant changes of body mass (<10%) and without features, which would identified histopathological analysis of gastrointestinal tract.

Example 10: suppression of track of WNT in vivo of

[600]

To additionally evaluate influence of perekrestnoreaktivnykh by lrp5/lrp6 biparatopnykhVHH-and-constructs/binding molecules with prolonged period of half-on of WNT-and-signaling, at the end of as described example 9 experience by efficiency of allocate tumor. In particular, tumor allocate through 16 hours after the last injection of compounds or control treatment. Suppression of WNT-and-signalinga determined by WiFi client continuously agreed to mRNA expression Axin2 in tumors, analyzed according to the description in example 8. ratio change mRNA expression Axin2 relative to control group is indicated on figure 8a for efficiency of f013500571 in vivo of and on figure 8B for efficiency of f013500720 in vivo of. Quantitative determination of WiFi client continuously ny mRNA expression Axin2 in each group of treatment is presented in the tables khsha and khshb below.

[601]

Table XIIIA: WiFi client continuously tracks the mRNA expression Axin2 in tumors in treatment of F013500571, data field figures 7a and 8a,

[602]

Dose [mg/kg]PRO [%]WiFi client continuously tracks Axin2 (%)
ControlGistidinovyi buffer-
F0135005711012883
411377
26135

[603]

Table khshb: WiFi client continuously tracks the mRNA expression Axin2 in tumors in treatment of F013500720. Data field figures 7B and 8B,

[604]

Dose [mg/kg]PRO [%]WiFi client continuously tracks Axin2 (%)
ControlGistidinovyi buffer-
F013500720112892
0.460-21
0.21735

[605]

As seen by figures 8a and 8B, and also tables khsha and khshb, in tumors, treated molecules, perekrestnoreaktivno coupling lrp5/lrp6, the postoperative period considerable WiFi client continuously tracks and dose-dependent reduction of (in particular, for treatment of f013500571) mRNA expression Axin2 in the control group. These results are make assume, that molecules, perekrestnoreaktivno binding lrp5/lrp6, actually are able to suppress growth of tumor, suppressing of WNT-and-signaling in tumor cells.

Example 11: method of industrial production of

[606]

11.1 Fermentation: Any of polypeptides, listed in the tables III and V of above, can expressed in cytoplasm of various strains of E. coli-, such as w3110, TGi specification, bl21, bl21 (de3), HMS174, HMS174 (de3), mm294, under control of inducible promoter. This promoter may be selected from lacUV5, the TAC, τ7, teterapeptide Trp, T5, agav. Cultivation medium preferably completely determined by Wilmsand other ., 2001 (Wilms in ., Hauck a. The, Reuss μ is ., Syldatk s., Mattes R, Siemann μ is. and Altenbuchner J: the High-the Cell-and-DensityFermentation in production for Laptops care of L-of n-Carbamoylase the Using the AN expression command the System based hotel booking on line of The of Escherichia coli rhaBADPromoter. Biotechnology and sort Bioengineering, 73:95 - 103 (2001)), DeLisaand ∂ρ ., 1999 (DeLisa μ is. p ., lithium J s., Rao g, Weigand of W.

[607]

And. and of Bentley w ε-.: to Monitoring GFP--and-operon the Fusion protein expression command during the High the cell densitycultivation care of Escherichia coli the Using the AN of an line the Optical the Sensor.

[608]

Biotechnology and sort Bioengineering, 65:54 - 64. (1999)) or are equivalent to them. Nevertheless, addition medium such amino acids, as isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, or complex components, such as soya peptone or yeast extract, can be useful. Enzymatic process fulfil semicontinuous method. Conditions: temperature 30 - 40 theoretically, pH of 6 - 7, 5, dissolved oxygen is more than 20%. After consumption of initial carbon source culture fuel above this (or equivalent) nutrient medium. At accumulation in fermenter 40 - 90 g/l of dry weight of cells culture induced adequately inductor, corresponding used promotory system (nair ., iptg, lactose, arabinozoi).

[609]

Induction can be performed complete pulse induction or as partial induction, by delivering corresponding inductor in fermenter for prolonged time. Phase production should last not less than 4 hours. Cells separated by centrifugation in rotor centrifuges, tubular centrifuges or plate separators, culture supernatant is poured off.

[610]

11.2 Cleaning: Cell mass E. coli-resuspended in 6 - 8 - fold amount lizisnogo buffer (phosphate or tris - buffer, pH of 7 - 8, 5). Lysis of cells preferably is homogenization under high pressure with subsequent removal of fragments cells by centrifugation in rotor, tubular centrifuges or plate separators. Supernatant, containing target protein, optionally filtered through filter on 0.22 - 10 mcm and separated cation-exchange chromatography (nair ., ToyopearlMegaCap® of II Ultra SP-550ec, ToyopearlGigaCap S-S 650m, Sp-Sepharose β-β-, Sp-Sepharose FF of or S HyperCel™) at pH 7 - 8, 5. Elution is linearly the aid of gradient of NaCl at pH 7 - 8, 5. Fraction, containing target protein, are combined and then incubated with 5 - 10 mm dtt to to prevent dimerization or aggregation, mediated free reactive cysteine residues. After further addition of 0.8 - 1 m ammonium sulfate or 2 - 3 M of NaCl solution is separated hydro philic chromatography (nair ., on PhenylSepharose HCl1, PhenylSepharose FF of, ButylSepharose HCl1, ButylSephrose FF of, ButylToyopearl 650 (s-, of m, of c), PhenylToyopearl 650 (s-, of m, of c)) at pH 7 - 8, 5. Elution is linearly reducing gradient of ammonium sulfate or NaCl at pH 7 - 8, 5 in the presence of 5 mm dtt. Fraction, containing target protein with minimum degree of purity 90%, are combined and diafiltration is desalted in the presence of 5 mm dtt followed by concentration to approximately 5 mg/ml. Is then refolding, separating protein solution 1:5 - 1:20 50 mm tris, 150 mm of NaCl, 4 mm cystamine, 10 mm CHAPS at pH 8.5 to final protein concentration 0.25 - 1 mg/ml. Refoldingovyi solution incubated, stirring, during 12 - 36 hours at room temperature and then separated cation-exchange chromatography (nair ., Sp-Sepharose FF of, Sp-Sepharose HCl1, Toyopearl Ultra SP-650 (s-, μ is, with)) at pH 7 - 8, 5. Elution is linearly the aid of gradient of NaCl at pH 7 - 8, 5. Fraction, containing monomeric target protein, are combined and introduced in the composition of 25 mm sodium phosphate - and 220 mm purified from endotoxins of trehalose at pH 7.5 by diafiltration. Solution is sterilized by filtration and stored at 2 - 8 theoretically.

Example 12: pharmaceutical preparation for U. to. introduction

[611]

Any of above described biparatopnykh polypeptide constructs according to the invention can be selected for production of pharmaceutical preparation for subcutaneous administration following composition of:

[612]

Medicinal substance: 100 mg/ml (from 1 to 3 nmol/ml) acetate buffer: 25 mm

[613]

Trehalose: 220 mm

[614]

Ammonium-and-20: 0.02%

[615]

Medicinal substance is introduced in solution composition above said composition, sterilized and stored at 2 - 8 theoretically.

[616]

Example 13: pharmaceutical application article on Manufactured in example 11.2 solution is introduced to the patient, requiring in this, such as a man, patient cancer, sensitive to inhibitors of WNT-and-signalinga, by intravenous infusion (in a dose of from 100 to 200 mg) with interval of from two to four weeks.

[617]

Example 14: effect of suppression of the \ ¥ U! 3a - signalinga on release of pro-inflammatory cytokines dendritic cells in analysis for ex-vivo of In healthy donors with their informed agreement took mkpk.

[618]

Human dendritic cells of origin (Mo - DC) created as follows: mkpk cultured on medium-x-vivo of with addition of 50 ng/ml of GM-CSF and 50 ng/ml of IL-and-4. After 24 h of culturing supernatant carefully selected and substituted medium-x-vivo of with addition of the same of GM-CSF and IL-and-4. On the fourth day supernatant carefully selected and substituted medium-x-vivo of only in presence of lps or in combination with human Wnt3a or with human Wnt3a and molecules, perekrestnoreaktivno coupling lrp5/lrp6. Next day supernatants collected and subjected analysis TNF-alpha - by method is preferable according to the instructions of a manufacturer. As earlier reported (Oderupand other " The Canonical and sort noncanonical of WNT proteins the Program dendritic the Cell responses for Laptops tolerance ". JImmunol. 2013; 190 (12): 6126 - 34), and as shown on figure 9a, Wnt3a directly suppresses secretion of pro-inflammatory cytokines (T. E. release of TNF-alpha -) differentsiirovannymi dendritic cells (DC). Caused by suppression of Wnt3a release TNF-alpha - from DC resumptions of the meeting at addition of molecules, binding perekrestnoreaktivno lrp5/lrp6.

[619]

These data demonstrate, that formatted biparatopnye binding molecules with optimized sequence are able to restore secretion of TNF-alpha - treated in Wnt3a dendritic cells, thereby suppressing inhibitory effect of WNT on dendritic cells.

[620]

It should be mentioned, that blocking track of WNT in dendritic cells of the microenvironment of the tumor may be potential therapeutic approach to disturbance opukholeoposredovannogo suppression of immunity and of addition antitumor immunity.

[621]

That it possible to study the effect of DC on T - cells (effector T - cells), pretreated Wnt3a DC, with or without molecules, binding perekrestnoreaktivno lrp5/lrp6, cultured together with T - cells, isolated from mkpk, as described earlier (Oderup and others.

[622]

"The Canonical and sort noncanonical of WNT proteins the Program dendritic the Cell responses for Laptops tolerance". Of j Immunol. 2013; 190 (12): 6126 - 34). Through 3 days joint cultivation DC/T - cells collected supernatants and subjected their analysis on IFN - gamma by method is preferable according to the instructions of a manufacturer.

[623]

Secretion IFN - gamma is a marker activation of T - cells. As shown on figure 0.9B, the \ up13a - mediated suppression of DC leads to WiFi client continuously nnoi secretion IFN - gamma T - cells (suppression of function of T - cells), which fully is reduced by treatment of molecules, perekrestnoreaktivno coupling lrp5/lrp6.

[624]

In this these data show, molecules that, perekrestnoreaktivno binding lrp5/lrp6, inhibit inhibitory effect of WNT on dendritic cells, results restoration of function of T - cells.

[625]

Known, that constant activation/stimulation of T - cells causes final differentsiirovanie, leading to istoshchennomu phenotype T - cells, progressive loss of function of T - cells. Therefore is provided, that influence of molecules, binding perekrestnoreaktivno lrp5/lrp6, on T - cells, mediated activation of DC, can be limited by depletion T - cells.

[626]

Expected therefore, that combined treatment, in which introduction of molecules, binding perekrestnoreaktivno lrp5/lrp6, merged with by administration of inhibitor of immune of control points, blocking exhausted T - cells, should help to activate and maintain function T - cells, thereby changing the micro-environment tumour and maintaining therapeutic effect molecules according to the invention.

LIST OF SEQUENCES

[627]

<110> BERINGER INGELHEIM GOLDEN SANDS RESORT GMBH

[628]

<120> BIPARATOPNYEPOLIPEPTTHE IDES - ANTAGONISTS OF SIGNAL TRANSMISSION OF WNT IN TUMOR CELLS

[629]

<130> P12-AND-0401/WO/1

[630]

<160> 47

[631]

<170> BiSSAP 1.2

[632]

<210> 1

[633]

<211> 5

[634]

<212> Protein

[635]

<213> Artificial sequence

[636]

<22 0>

[637]

<223> CDRlWntl-and-333E06mod

[638]

<400> 1

[639]

Of Thr tug of Thr-Val-Gly

[640]

1 5

[641]

<210> 2

[642]

<211> 17

[643]

<212> Protein

[644]

<213> Artificial sequence

[645]

<22 0>

[646]

<223> CDR2Wntl-and-333E06mod

[647]

<400> 2

[648]

-Ala lie of Arg Arg-Gly Ser-Ser-of Thr-Tyr-Tyr-Ala-Asp-Val-Gly Ser-Lys-15 10 15

[649]

<210> 3

[650]

<211> 14

[651]

<212> Protein

[652]

<213> Artificial sequence

[653]

<22 0>

[654]

<223> CDR3Wntl-and-333E06mod

[655]

<400> 3

[656]

Asp-of Thr Arg of of Thr-Val-Ala-Leu-Leu-Tyr-Tyr-Tyr-Asp Arg of Gin

[657]

15 10

[658]

<210> 4

[659]

<211> 5

[660]

<212> Protein

[661]

<213> Artificial sequence

[662]

<22 0>

[663]

<223> CDRlWntl-and-333g06

[664]

<400> 4

[665]

Ser-met polypeptide-Gly-Tyr-Ala

[666]

1 5

[667]

<210> 5

[668]

<212> Protein

[669]

<213> Artificial sequence

[670]

<22 0>

[671]

<223> CDR2Wntl-and-333g06

[672]

<400> 5

[673]

lie-Ala Arg Ser--Gly Arg of Thr-Tyr-Tyr-Ala-Asp-Val-Gly Ser-Lys-15 10 15

[674]

<210> 6

[675]

<211> 19

[676]

<212> Protein

[677]

<213> Artificial sequence

[678]

<22 0>

[679]

<223> CDR3Wntl-and-333g06

[680]

<400> 6

[681]

-Ala Arg-Val Arg of Ser-Ser-of Thr Arg of Tyr-Asn-of Thr-Gly of Thr teterapeptide Trp teterapeptide Trp 15 10 15 teterapeptide Trp Glu-Tyr-

[682]

<210> 7

[683]

<211> 5

[684]

<212> Protein

[685]

<213> Artificial sequence

[686]

<22 0>

[687]

<223> CDRlWntl-and-332D03mod

[688]

<400> 7

[689]

Of Arg-Tyr met polypeptide of Thr-Gly

[690]

1 5

[691]

<210> 8

[692]

<211> 17

[693]

<212> Protein

[694]

<213> Artificial sequence

[695]

<22 0>

[696]

<223> CDR2Wntl-and-332D03mod

[697]

<400> 8

[698]

lie-Ala-Val Arg of Ser--Gly-Gly Ser-of Thr-Tyr-Tyr-Ala-Asp-Val-Gly Ser-Lys-15 10 15

[699]

<210> 9

[700]

<211> 20

[701]

<212> Protein

[702]

<213> Artificial sequence

[703]

<22 0>

[704]

<223> CDR3Wntl-and-332D03mod

[705]

<400> 9

[706]

Asp-Arg-Gly of Arg-Gly-Glu-Asn-Tyr lie-Leu-Leu-Tyr Ser-Ser--Gly 15 10 15

[707]

Of Arg tug Glu-tug

[708]

20

[709]

<210> <211> <212> <213>10 5 Protein artificial sequence
<22 0> <223>CDRl wnt3a - 093a01
<400>10
Ser-tug-Ala-Gly met polypeptide
<210> <211> <212> <213>11 17 Artificial protein sequence
<22 0> <223>CDR2 wnt3a - 093a01
1-Gly5 10 15
<210> <211> <212> <213>12 17 Artificial protein sequence
<22 0> <223>CDR3 wnt3a - 093a01
<400>12
1 Tug5 10 15
<210> <211> <212> <213>13 5 Protein artificial sequence
<22 0> <223>CDRlWnt3a-and-3 67 β-10
<400>13
<210> <211> <212> <213>14 17 Artificial protein sequence
<22 0> <223>CDR2Wnt3a-and-3 67 β-10

[710]

1 5

[711]

<400> 11

[712]

-Ala lie Ser-teterapeptide Trp Ser--Gly-Gly Ser-of Thr-Tyr-Tyr-Ala-Asp-Val Ser-Lys-1 5 10 15

[713]

Ser-of Pro lie of Pro-Tyr-Gly-Leu-Leu Ser-Arg Arg of Asn-Asn-Tyr-Asp-1 5 10 15

[714]

Ser-met polypeptide-Gly-Tyr-Ala

[715]

1 5

[716]

<400> 14

[717]

-Ala lie Ser-teterapeptide Trp Arg of Ser-Ser-of Thr-Tyr-Tyr-Gly-Ala-Asp-Val-Gly Ser-Lys-15 10 15

[718]

<210> 15

[719]

<211> 16

[720]

<212> Protein

[721]

<213> Artificial sequence

[722]

<22 0>

[723]

<223> CDR3Wnt3a-and-3 67 β-10

[724]

<400> 15

[725]

Of Pro-Asp of Arg-Tyr-Gly-Gly-Val-Ala-Tyr-Val-Ala-Tyr-Tyr Ser-Glu-Tyr-15 10 15

[726]

<210> 16

[727]

<211> 5

[728]

<212> Protein

[729]

<213> Artificial sequence

[730]

<22 0>

[731]

<223> CDRlAlbll

[732]

<400> 16

[733]

Ser-Phe-met polypeptide of Ser-Gly

[734]

1 5

[735]

<210> 17

[736]

<211> 17

[737]

<212> Protein

[738]

<213> Artificial sequence

[739]

<22 0>

[740]

<223> CDR2Albll

[741]

<400> 17

[742]

Ser-lie of Ser-Gly-Gly Ser-Ser-Asp-of Thr-Leu-Tyr-Ala-Asp-Val-Gly Ser-Lys-15 10 15

[743]

<210> 18

[744]

<211> 6

[745]

<212> Protein

[746]

<213> Artificial sequence

[747]

<22 0>

[748]

<223> CDR3Albll

[749]

<400> 18

[750]

-Gly-Gly-Leu Ser-Ser-of Arg

[751]

1 5

[752]

<210> 19

[753]

<211> 123

[754]

<212> Protein

[755]

<213> Artificial sequence

[756]

<22 0>

[757]

<223> VHH-and-domain Wntl-and-333E06mod

[758]

-Ala-ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Of ThrTyr-
202530
Of Thr-Val-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieOf ArgOf ArgOf Arg-GlySer-Ser-Of ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ThrOf ArgOf Thr-Val-Ala-Leu-LeuGinTyr-Of ArgTyr-Asp-Tyr-
100105110
Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser-
115120
<400> 20
-Ala 1-ValGin-Leu-Val 5Glu-Ser--Gly-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Ala-AlaSer-25-Gly-GlyOf ThrPhe-Ser-30Ser-Tyr-
-AlaMet polypeptide-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
-Ala-Ala 50lieOf ArgOf ArgSer--Gly 55Of ArgOf ArgOf ThrTyr-Tyr-60-AlaAsp-Ser--Val
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of ArgAsp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf ProGlu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
-Ala-Ala-AlaArg of 100Of Arg-ValOf ArgSer-Ser-105Of ThrOf ArgTyr-Asn-Of Thr 110-GlyOf Thr
Teterapeptide TrpTeterapeptide TrpTeterapeptide Trp 115Glu-Tyr-Teterapeptide Trp-GlyGin 120-GlyOf Thr-Leu-ValOf Thr 125-ValSer-Ser-

[759]

<210> 20

[760]

<211> 128

[761]

<212> Protein

[762]

<213> Artificial sequence

[763]

<22 0>

[764]

<223> VHH-and-domain Wntl-and-333g06

[765]

<210> 21

[766]

<211> 129

[767]

<212> Protein

[768]

<213> Artificial sequence

[769]

<22 0>

[770]

<223> VHH-and-domain Wntl-and-332D03mod

[771]

<400> 21

[772]

Gin-Ala-Val-Leu-Val-Gly-Gly-Gly-Leu Glu-Ser-Gin-Val Gly-Gly

[773]

1 5 10 15

[774]

Ser-of Arg-Leu-Leu Ser-is Cys-Ala-Ala-Gly-Leu Ser-of Thr Phe-Ser-of Arg-Tyr

[775]

20 25 30

[776]

Of Thr met polypeptide-Gly teterapeptide Trp Phe-Arg of Gin-Ala Gly Lys-Arg-Glu-Phe--Val

[777]

35 40 45

[778]

lie-Ala-Ala-Val Arg of Ser--Gly-Gly Ser-of Thr-Tyr-Tyr-Ala-Asp-Val Ser-

[779]

50 55 60

[780]

Of Arg-Gly Lys-Phe-of Thr lie Ser-of Arg Asp-Asn-Ser-Lys-Asn-of Thr-Val-Tyr

[781]

65 70 75 80

[782]

-Leu Gin met polypeptide Ser-Asn-Leu is of Arg of Pro Glu-Asp-of Thr-Ala-Val-Tyr-Tyr is Cys

[783]

85 90 95

[784]

-Ala-Ala-Asp Arg-Gly of Arg-Gly-Glu-Asn-Tyr-Leu-Leu-Tyr Ser-lie

[785]

100,105,110

[786]

Of Ser-Gly Arg of Tyr-Glu-Tyr-teterapeptide Trp-Gly Gin of Thr-Gly-Leu-Val-Val Ser-

[787]

115,120,125

[788]

Ser-

[789]

Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieSer-Teterapeptide TrpSer--Gly-GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaSer-Of ProlieOf ProTyr--GlySer--Leu-LeuOf ArgOf ArgOf ArgAsn-Asn-
100105110
Tyr-Asp-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser-
115120125
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieSer-Teterapeptide TrpOf ArgSer--GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ProOf Arg-GlyTyr--Gly-Val-AlaTyr--ValSer--AlaTyr-Tyr-
100105110
Glu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser-
115120125

[790]

<210> 22

[791]

<211> 126

[792]

<212> Protein

[793]

<213> Artificial sequence

[794]

<22 0>

[795]

<223> VHH-and-domain wnt3a - 093a01

[796]

<400> 22

[797]

<210> 23

[798]

<211> 125

[799]

<212> Protein

[800]

<213> Artificial sequence

[801]

<22 0>

[802]

<223> VHH-and-domain wnt3a - 367b10

[803]

<400> 23

[804]

<210> 24

[805]

<22 0>
Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15Asn-
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Ala-AlaSer-25-GlyPhe-Of ThrPhe-Ser-30Ser-Phe-
-GlyMet polypeptideSer-35Teterapeptide Trp-ValOf ArgGin-Ala 40Of Pro-GlyLys--GlyLeu is 45Glu-Teterapeptide Trp-Val
Ser-Ser-50lieSer--GlySer--Gly 55Ser-Asp-Of Thr-LeuTyr-60-AlaAsp-Ser--Val
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of ArgAsp-Asn--Ala 75Lys-Of ThrOf Thr-LeuTyr-80
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf ProGlu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
Of Thrlie-Gly-Gly 100Ser--LeuSer-Of ArgSer-105Ser-Gin-GlyOf ThrLeu is 110-ValOf Thr
-Ala-ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Of ThrTyr-
202530
Of Thr-Val-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieOf ArgOf ArgOf Arg-GlySer-Ser-Of ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ThrOf ArgOf Thr-Val-Ala-Leu-LeuGinTyr-Of ArgTyr-Asp-Tyr-
100105110
Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Gly-Gly-GlySer-
115120125
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
130135140
-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-Glu--Val
145150155160
Gin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-GlyAsn-Ser--Leu
165170175
Of Arg-LeuSer-Is Cys-Ala-AlaSer--GlyPhe-Of ThrPhe-Ser-Ser-Phe--GlyMet polypeptide
180185190
Ser-Teterapeptide Trp-ValOf ArgGin-AlaOf Pro-GlyLys--Gly-LeuGlu-Teterapeptide Trp-ValSer-Ser-
195200205
lieSer--GlySer--GlySer-Asp-Of Thr-LeuTyr--AlaAsp-Ser--ValLys--Gly
210215220
Of ArgPhe-Of ThrlieSer-Of ArgAsp-Asn--AlaLys-Of ThrOf Thr-LeuTyr--LeuGin
225230235240

[806]

<212> Protein

[807]

<213> Artificial sequence

[808]

<223> VHH-and-domain Albll

[809]

<400> 24

[810]

-Val Ser-Ser-

[811]

115

[812]

<210> 25

[813]

<211> 435

[814]

<212> Protein

[815]

<213> Artificial sequence

[816]

<22 0>

[817]

<223> VHH-construct f013500575

[818]

<400> 25

[819]

Met polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is CysOf Thrlie
245250255
-Gly-GlySer--LeuSer-Of ArgSer-Ser-Gin-GlyOf Thr-Leu-ValOf Thr-ValSer-
260265270
Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
275280285
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
290295300
-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-Val
305310315320
GinOf Pro-Gly-GlySer--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf Thr
325330335
Phe-Ser-Ser-Tyr--AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-
340345350
Of ArgGlu-Phe--Val-Ala-AlalieSer-Teterapeptide TrpSer--Gly-GlySer-Of ThrTyr-Tyr-
355360365
-AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-
370375380
Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala
385390395400
-ValTyr-Tyr-Is Cys-Ala-AlaSer-Of ProlieOf ProTyr--GlySer--Leu-LeuOf Arg
405410415
Of ArgOf ArgAsn-Asn-Tyr-Asp-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-Val
420425430
Ser-Ser--Ala
435
<210> 26
<211> 439
<212> Protein
<213> Artificialsequence
<22 0>
<223> VHH-constructF013500571
<400> 26
-Ala-ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieOf ArgOf ArgSer--GlyOf ArgOf ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-Ala-AlaOf ArgOf Arg-ValOf ArgSer-Ser-Of ThrOf ArgTyr-Asn-Of Thr-GlyOf Thr
100105110
Teterapeptide TrpTeterapeptide TrpTeterapeptide TrpGlu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser-
115120125
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
130135140
-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly
145150155160
-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGin
165170175
Of Pro-GlyAsn-Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyPhe-Of ThrPhe-
180185190
Ser-Ser-Phe--GlyMet polypeptideSer-Teterapeptide Trp-ValOf ArgGin-AlaOf Pro-GlyLys--Gly-Leu
195200205
Glu-Teterapeptide Trp-ValSer-Ser-lieSer--GlySer--GlySer-Asp-Of Thr-LeuTyr--Ala
210215220

[820]

Asp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn--AlaLys-Of Thr
225230235240
Of Thr-LeuTyr--LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-Val
245250255
Tyr-Tyr-Is CysOf Thrlie-Gly-GlySer--LeuSer-Of ArgSer-Ser-Gin-GlyOf Thr
260265270
-Leu-ValOf Thr-ValSer-Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
275280285
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
290295300
-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser-
305310315320
-Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-GlySer--LeuOf Arg-LeuSer-Is Cys-Ala
325330335
-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr--AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin
340345350
-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val-Ala-AlalieSer-Teterapeptide TrpOf ArgSer-
355360365
-GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-
370375380
Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--LeuOf Arg
385390395400
Of ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys-Ala-AlaAsp-Of ProOf Arg-GlyTyr-
405410415
-Gly-Val-AlaTyr--ValSer--AlaTyr-Tyr-Glu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr
420425430
-Leu-ValOf Thr-ValSer-Ser--Ala
435
<400> 27
-Ala-ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--Gly-LeuOf ThrPhe-Ser-Of ArgTyr-
202530
Of ThrMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-Alalie-ValOf ArgSer--Gly-GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ArgOf Arg-GlyOf Arg-GlyGlu-Asn-Tyr-lie-Leu-LeuTyr-Ser-
100105110
Ser--GlyOf ArgTyr-Glu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-
115120125
Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
130135140
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
145150155160
-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-Val
165170175
GinOf Pro-GlyAsn-Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyPhe-Of Thr
180185190
Phe-Ser-Ser-Phe--GlyMet polypeptideSer-Teterapeptide Trp-ValOf ArgGin-AlaOf Pro-GlyLys--Gly
195200205

[821]

<210> 27

[822]

<211> 440

[823]

<212> Protein

[824]

<213> Artificial sequence

[825]

<22 0>

[826]

<223> VHH-construct f013500720

[827]

-LeuGlu-Teterapeptide Trp-ValSer-Ser-lieSer--GlySer--GlySer-Asp-Of Thr-LeuTyr-
210215220
-AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn--AlaLys-
225230235240
Of ThrOf Thr-LeuTyr--LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala
245250255
-ValTyr-Tyr-Is CysOf Thrlie-Gly-GlySer--LeuSer-Of ArgSer-Ser-Gin-Gly
260265270
Of Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-Gly
275280285
Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
290295300
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-
305310315320
Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-GlySer--LeuOf Arg-LeuSer-Is Cys
325330335
-Ala-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr--AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of Arg
340345350
Gin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val-Ala-AlalieSer-Teterapeptide TrpOf Arg
355360365
Ser--GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of Thrlie
370375380
Ser-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--Leu
385390395400
Of ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys-Ala-AlaAsp-Of ProOf Arg-Gly
405410415
Tyr--Gly-Val-AlaTyr--ValSer--AlaTyr-Tyr-Glu-Tyr-Teterapeptide Trp-GlyGin-Gly
420425430
Of Thr-Leu-ValOf Thr-ValSer-Ser--Ala
435440
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieSer-Teterapeptide TrpSer--Gly-GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaSer-Of ProlieOf ProTyr--GlySer--Leu-LeuOf ArgOf ArgOf ArgAsn-Asn-
100105110
Tyr-Asp-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Ala-Ala
115120125
-AlaGlu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-
130135140
His-His-His-His-His-

[828]

<210> 28

[829]

<211> 149

[830]

<212> Protein

[831]

<213> Artificial sequence

[832]

<22 0>

[833]

<223> VHH-and-domain f0129093a01

[834]

<400> 28

[835]

<210> 29

[836]

<211> 151

[837]

<212> Protein

[838]

<213> Artificial sequence

[839]

<22 0>
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieOf ArgOf ArgSer--GlyOf ArgOf ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-Ala-AlaOf ArgOf Arg-ValOf ArgSer-Ser-Of ThrOf ArgTyr-Asn-Of Thr-GlyOf Thr
100105110
Teterapeptide TrpTeterapeptide TrpTeterapeptide TrpGlu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser-
115120125
-Ala-Ala-AlaGlu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala
130135140
-AlaHis-His-His-His-His-His-
145150
<210> 30 <211> 148 <212> Protein
<213> Artificialsequence
<22 0>
<223> VHH-and-domainF0129093A03
<400> 30
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Of ThrTyr-
202530
-ValMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieAsn-Teterapeptide TrpSer--GlySer-Of ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaSer-Of ArgSer-Ser-Tyr--Ala-GlyOf ArgOf ThrTyr-Tyr-Glu--LeuTyr-
100105110
Asp-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Ala-Ala-Ala
115120125
Glu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-His-
130135140
His-His-His-His-
145
<210> 31 <211> 146 <212> Protein

[840]

<223> VHH-and-domain f0130333g06

[841]

<400> 29

[842]

<213> Artificial sequence

[843]

<22 0>

[844]

<223> VHH-and-domain f0130333e06

[845]

Glu-Gin-Val-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Of Ser-Leu Arg of-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Of ThrTyr-
202530
Of Thr-Val-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-Ala lieOf ArgOf ArgOf Arg-GlySer-Ser-Of ThrTyr-Tyr-Ser-Asp-Ser--Val
505560
Lys-of Arg-GlyPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-Leu Gin met polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-Ala-AspOf ThrOf ArgOf Thr-Val-Ala-Leu-LeuGinTyr-Of ArgTyr-Asp-Tyr-
100105110
Teterapeptide Trp-Gly Gin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Ala-Ala-AlaGlu-Gin
115120125
Lys-Leu is lieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-His-His-His-
130135140
His-His-145
<210> 32 <211> 152 <212> Protein
<213> Artificialsequence
<22 0>
<223> VHH-and-domainF0130332D03
<400> 32 Glu-Gin-Val-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Of Ser-Leu Arg of-LeuSer-Is Cys-Ala-AlaSer--Gly-LeuOf ThrPhe-Ser-Of ArgTyr-
202530
Of Thr met polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-Ala lieAsn-Of ArgSer--Gly-GlySer-Of ThrTyr-Tyr-Ser-Asp-Ser--Val
505560
Lys-of Arg-GlyPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-Leu Gin met polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-Ala-AspOf ArgOf Arg-GlyOf Arg-GlyGlu-Asn-Tyr-Ser--Leu-LeuTyr-Ser-
100105110
Ser-Asn-Arg ofTyr-Glu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-
115120125
Of Ser-Ala-Ala-AlaGlu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly
130135140
-Ala-Ala-HisHis-His-His-His-His-
145150
<210> 33 <211> 148 <212> Protein
<213> Artificialsequence
<22 0>
<223> VHH-and-domainF0130367B10
<400> 33 Glu-Gin-Val-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015

[846]

Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieSer-Teterapeptide TrpOf ArgSer--GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu--GlyOf Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ProOf Arg-GlyTyr--Gly-Val-AlaTyr--ValSer--AlaTyr-Tyr-
100105110
Glu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Ala-Ala
115120125
Glu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-His-
130135140
His-His-His-His-
145
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Val-AlaSer--GlyOf ArgOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieSer-Of ArgSer--Gly-GlyOf ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ArgOf Arg-ValTyr-Ser-Of Thr-LeuOf ProOf ProOf ThrOf ThrSer-Of Arg
100105110
Tyr-Asn-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Ala-Ala
115120125
-AlaGlu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-
130135140
His-His-His-His-His-

[847]

<210> 34

[848]

<211> 149

[849]

<212> Protein

[850]

<213> Artificial sequence

[851]

<22 0>

[852]

<223> VHH-and-domain f0130378b05

[853]

<400> 34

[854]

145

[855]

<210> 35

[856]

<211> 147

[857]

<212> Protein

[858]

<213> Artificial sequence

[859]

<22 0>

[860]

<223> VHH-and-domain f0130378a04

[861]

<400> 35

[862]

Glu-Gin-Val-Leu-Val-Gly-Gly-Gly-Leu Glu-Ser-Gin-Val Gly-Gly

[863]

1 5 10 15

[864]

Ser-of Arg-Leu-Leu Ser-is Cys-Ala-Ala-Gly Ser-of Arg of Thr Phe-Ser-Ser-Tyr-

[865]

20 25 30

[866]

Met polypeptide-Ala-Gly teterapeptide Trp Phe-Arg of Gin-Ala Gly Lys-Arg-Glu-Phe--Val

[867]

35 40 45

[868]

-Ala-AlalieOf ThrOf ArgOf Thr-GlyOf ArgOf ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ArgOf Arg-GlyTyr-Tyr-Tyr-Tyr-Asp-Ser-Ser-Phe-Tyr-Asp-
100105110
Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Ala-Ala-AlaGlu-
115120125
GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-His-His-
130135140
His-His-His-
145
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Asp-Tyr-
202530
-GlyMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieSer-Teterapeptide TrpSer--Gly-GlyOf ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaLys-Of ArgOf Arg-GlyOf Arg-GlySer--ValSer-Of ProAsn-Ser-Ser-Ser-
100105110
Of ArgTyr-Asn-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Ala
115120125
-Ala-AlaGlu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly-Ala-Ala
130135140
His-His-His-His-His-His-
145150
Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Ala-AlaSer-25-Gly-LeuOf ThrPhe-Ser-30Of ArgTyr-
Of ThrMet polypeptide-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
-Ala-Ala 50lieAsn-Of ArgSer--Gly 55-GlySer-Of ThrTyr-Tyr-60Ser-Asp-Ser--Val
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of ArgAsp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80

[869]

<210> 36

[870]

<211> 150

[871]

<212> Protein

[872]

<213> Artificial sequence

[873]

<22 0>

[874]

<223> VHH-and-domain f013 0372c08

[875]

<400> 36

[876]

<210> 37

[877]

<211> 462

[878]

<212> Protein

[879]

<213> Artificial sequence

[880]

<22 0>

[881]

<223> VHH-construct f013500016

[882]

<400> 37

[883]

-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ArgOf Arg-GlyOf Arg-GlyGlu-Asn-Tyr-Ser--Leu-LeuTyr-Ser-
100105110
Ser-Asn-Of ArgTyr-Glu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-
115120125
Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
130135140
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
145150155160
-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-Val
165170175
GinOf Pro-GlyAsn-Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyPhe-Of Thr
180185190
Phe-Ser-Ser-Phe--GlyMet polypeptideSer-Teterapeptide Trp-ValOf ArgGin-AlaOf Pro-GlyLys--Gly
195200205
-LeuGlu-Teterapeptide Trp-ValSer-Ser-lieSer--GlySer--GlySer-Asp-Of Thr-LeuTyr-
210215220
-AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn--AlaLys-
225230235240
Of ThrOf Thr-LeuTyr--LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala
245250255
-ValTyr-Tyr-Is CysOf Thrlie-Gly-GlySer--LeuSer-Of ArgSer-Ser-Gin-Gly
260265270
Of Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-Gly
275280285
Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
290295300
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-
305310315320
Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-GlySer--LeuOf Arg-LeuSer-Is Cys
325330335
-Ala-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr--AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of Arg
340345350
Gin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val-Ala-AlalieSer-Teterapeptide TrpOf Arg
355360365
Ser--GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of Thrlie
370375380
Ser-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--Leu
385390395400
Of ArgOf ProGlu--GlyOf Thr-Ala-ValTyr-Tyr-Is Cys-Ala-AlaAsp-Of ProOf Arg-Gly
405410415
Tyr--Gly-Val-AlaTyr--ValSer--AlaTyr-Tyr-Glu-Tyr-Teterapeptide Trp-GlyGin-Gly
420425430
Of Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Ala-AlaGlu-GinLys--LeulieSer-
435440445
Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-His-His-His-His-His-
450455460

[884]

<210> 38

[885]

<211> 463

[886]

<212> Protein

[887]

<213> Artificial sequence

[888]

<22 0>

[889]

<223> VHH-construct f013500018

[890]

<400> 38

[891]

Glu-Gin-Val-Leu-Val-Gly-Gly-Gly-Leu Glu-Ser-Gin-Val Gly-Gly

[892]

1 5 10 15

[893]

Ser-of Arg-Leu-Leu Ser-is Cys-Ala-Ala-Gly-Leu Ser-of Thr Phe-Ser-of Arg-Tyr

[894]

20 25 30

[895]

Of Thr met polypeptide-Gly teterapeptide Trp Phe-Arg of Gin-Ala Gly Lys-Arg-Glu-Phe--Val

[896]

35 40 45

[897]

-Ala-Ala 50lieAsn-
Lys-65-GlyOf ArgPhe-
-LeuGinMet polypeptideAsn-
-Ala-AlaAsp-Arg of 100
Ser-Asn-Arg of 115Tyr-
Ser--Gly 130-Gly-Gly
-Gly 145-Gly-Gly-Gly
-Gly-Gly-GlySer-
GinOf Pro-GlyAsn-180
Phe-Ser-Ser-195Phe-
-LeuGlu-210Teterapeptide Trp-Val
-Ala 225Asp-Ser--Val
Of ThrOf Thr-LeuTyr-
-ValTyr-Tyr-Is Cys 260
Of Thr-Leu-Val 275Of Thr
Ser--Gly 290-Gly-Gly
-Gly 305-Gly-Gly-Gly
Ser--Gly-Gly-Gly
-Val-AlaSer--Gly 340
Gin-AlaOf Pro 355-Gly
-Gly-Gly 370Of ArgOf Thr
Ser-385Of ArgAsp-Asn-
Of ArgOf ProGlu-Asp-
Tyr-Ser-Of ThrLeu is 420
-GlyOf ThrLeu is 435-Val
Ser-Glu-450Glu-Asp-
Of ArgSer--Gly 55-Gly
Of Thrlie 70Ser-Of Arg
Ser-85-LeuOf ArgOf Pro
Of Arg-GlyOf Arg-Gly
Glu-Tyr-Teterapeptide Trp-Gly 120
-GlySer--Gly 135-Gly
Ser--Gly 150-Gly-Gly
Glu-165-ValGin-Leu
Ser--LeuOf Arg-Leu
-GlyMet polypeptideSer-Teterapeptide Trp 200
Ser-Ser-lie 215Ser-
Lys--Gly 230Of ArgPhe-
Leu is 245GinMet polypeptideAsn-
Of Thrlie-Gly-Gly
-ValSer-Ser--Gly 280
-GlySer--Gly 295-Gly
Ser--Gly 310-Gly-Gly
Leu is 325-ValGinOf Pro
Of ArgOf ThrPhe-Ser-
Lys-Glu-Of ArgGlu-360
Tyr-Tyr--Ala 375Asp-
Ser-Lys-390Asn-Of Thr
Of Thr 405-Ala-ValTyr-
Of ProOf ProOf ThrOf Thr
Of Thr-ValSer-Ser-440
-LeuAsn--Gly 455-Ala
Ser-Of ThrTyr-Tyr-60
Asp-Asn-Ser-75Lys-
Glu-Asp-90Of Thr-Ala
Glu-105Asn-Tyr-Ser-
Gin-GlyOf Thr-Leu
-Gly-GlySer--Gly 140
-GlySer--Gly 155-Gly
-ValGlu-170Ser--Gly
Ser-185Is Cys-Ala-Ala
-ValOf ArgGin-Ala
-GlySer--GlySer-220
Of ThrlieSer-235Of Arg
Ser-Leu is 250Of ArgOf Pro
Ser-265-LeuSer-Of Arg
-Gly-Gly-GlySer-
-Gly-GlySer--Gly 300
-GlySer-Glu-315-Val
-Gly-Gly 330Ser--Leu
Ser-345Tyr--AlaMet polypeptide
Phe--Val-Ala-Ala
Ser--ValLys--Gly 380
-ValTyr-Leu is 395Gin
Tyr-Is Cys 410-Ala-Ala
Ser-425Of ArgTyr-Asn-
-Gly-Ala-AlaGlu-
-AlaHis-His-His-460
Ser-Asp-Ser--Val
Asn-Of Thr-ValTyr-80
-ValTyr-Tyr-95Is Cys
-LeuLeu is 110Tyr-Ser-
-Val 125Of Thr-ValSer-
-Gly-Gly-GlySer-
-Gly-GlySer--Gly 160
-Gly-Gly-Leu 175-Val
Ser--Gly 190Phe-Of Thr
Of Pro 205-GlyLys--Gly
Asp-Of Thr-LeuTyr-
Asp-Asn--AlaLys-240
Glu-Asp-Of Thr 255-Ala
Ser-Ser-270Gin-Gly
-Gly 285-Gly-Gly-Gly
-Gly-Gly-GlySer-
Gin-Leu-ValGlu-320
Of Arg-LeuSer-335Is Cys
-GlyTeterapeptide Trp 350Phe-Of Arg
lie 365Ser-Of ArgSer-
Of ArgPhe-Of Thrlie
Met polypeptideAsn-Ser-Leu is 400
Asp-Of ArgArg of 415-Val
Tyr-Teterapeptide Trp 430-GlyGin
Gin 445Lys--Leulie
His-His-His-

[898]

<210> 39 <211> 461 <212> Protein <213> Artificial sequence

[899]

<22 0>

[900]

<223> VHH-construct f013500021

[901]

<400> 39 Glu-Gin-Val-Leu-Val-Gly-Gly-Gly-Leu Glu-Ser-Gin-Val Gly-Gly 15 10 15

[902]

Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr-
202530
-AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieOf ArgOf ArgSer--GlyOf ArgOf ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-Ala-AlaOf ArgOf Arg-ValOf ArgSer-Ser-Of ThrOf ArgTyr-Asn-Of Thr-GlyOf Thr
100105110
Teterapeptide TrpTeterapeptide TrpTeterapeptide TrpGlu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser-
115120125
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
130135140
-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly
145150155160
-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGin
165170175
Of Pro-GlyAsn-Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyPhe-Of ThrPhe-
180185190
Ser-Ser-Phe--GlyMet polypeptideSer-Teterapeptide Trp-ValOf ArgGin-AlaOf Pro-GlyLys--Gly-Leu
195200205
Glu-Teterapeptide Trp-ValSer-Ser-lieSer--GlySer--GlySer-Asp-Of Thr-LeuTyr--Ala
210215220
Asp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn--AlaLys-Of Thr
225230235240
Of Thr-LeuTyr--LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-Val
245250255
Tyr-Tyr-Is CysOf Thrlie-Gly-GlySer--LeuSer-Of ArgSer-Ser-Gin-GlyOf Thr
260265270
-Leu-ValOf Thr-ValSer-Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
275280285
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
290295300
-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser-
305310315320
-Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-GlySer--LeuOf Arg-LeuSer-Is Cys-Ala
325330335
-AlaSer--Gly-GlyOf ThrPhe-Ser-Ser-Tyr--AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin
340345350
-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val-Ala-AlalieSer-Teterapeptide TrpOf ArgSer-
355360365
-GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-
370375380
Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--LeuOf Arg
385390395400
Of ProGlu--GlyOf Thr-Ala-ValTyr-Tyr-Is Cys-Ala-AlaAsp-Of ProOf Arg-GlyTyr-
405410415
-Gly-Val-AlaTyr--ValSer--AlaTyr-Tyr-Glu-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr
420425430
-Leu-ValOf Thr-ValSer-Ser--Gly-Ala-AlaGlu-GinLys--LeulieSer-Glu-
435440445
Glu-Asp--LeuAsn--Gly-Ala-AlaHis-His-His-His-His-His-
450455460

[903]

<210> 40

[904]

<211> 456

[905]

<212> Protein

[906]

<213> Artificial sequence

[907]

<22 0>

[908]

<223> VHH-construct f013500026

[909]

Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Ala-Ala
Of Thr-Val-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40
-Ala-Ala 50lieOf ArgOf ArgOf Arg-Gly 55Ser-
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of Arg
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf Pro
-Ala-AlaAsp-Of Thr 100Of ArgOf Thr-Val-Ala
Teterapeptide Trp-GlyGin 115-GlyOf Thr-Leu-ValOf Thr 120
-Gly-Gly 130-Gly-GlySer--Gly-Gly 135-Gly
-Gly 145-Gly-GlySer--Gly-Gly 150-Gly-Gly
Gin-Leu-ValGlu-Ser-165-Gly-Gly-Gly
Of Arg-LeuSer-Is Cys 180-Ala-AlaSer--Gly
Ser-Teterapeptide Trp-Val 195Of ArgGin-AlaOf Pro-Gly 200
lieSer-210-GlySer--GlySer--Asp 215Of Thr
Arg of 225Phe-Of ThrlieSer-Arg of 230Asp-Asn-
Met polypeptideAsn-Ser--LeuArg of 245Of ProGlu-Asp-
-Gly-GlySer-Leu is 260Ser-Of ArgSer-Ser-
Ser--Gly-Gly 275-Gly-GlySer--Gly-Gly 280
-Gly-Gly 290-Gly-GlySer--Gly-Gly 295-Gly
-Gly 305-Gly-GlySer-Glu--Val 310Gin-Leu
GinOf Pro-Gly-GlySer-325-LeuOf Arg-Leu
Phe-Ser-Ser-Tyr-340-AlaMet polypeptide-GlyTeterapeptide Trp
Of ArgGlu-Phe-355-Val-Ala-AlalieSer-360
-AlaAsp-370Ser--ValLys--GlyArg of 375Phe-
Asn-385Of Thr-ValTyr--LeuGin 390Met polypeptideAsn-
-ValTyr-Tyr-Is Cys-Ala 405-AlaAsp-Of Pro
Ser--AlaTyr-Tyr-420Glu-Tyr-Teterapeptide Trp-Gly
Ser--Gly-Ala 435-AlaGlu-GinLys-Leu is 440
-Ala-Ala 450His-His-His-His-His-455His-
-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser-25-GlyOf ArgOf ThrPhe-Ser-30Of ThrTyr-
Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
Ser-Of ThrTyr-Tyr-60Ser-Asp-Ser--Val
Asp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80
Glu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
Leu is 105-LeuGinTyr-Of ArgTyr-110Asp-Tyr-
-ValSer-Ser--Gly-Gly 125-Gly-GlySer-
-GlySer--Gly-Gly 140-Gly-GlySer--Gly
Ser--Gly-Gly 155-Gly-GlySer-Glu--Val 160
-Leu-Val 170GinOf Pro-GlyAsn-Ser-175-Leu
Phe-185Of ThrPhe-Ser-Ser-Phe-190-GlyMet polypeptide
Lys--Gly-LeuGlu-Teterapeptide Trp 205-ValSer-Ser-
-LeuTyr--AlaAsp-220Ser--ValLys--Gly
-AlaLys-Of Thr 235Of Thr-LeuTyr--LeuGin 240
Of Thr-Ala 250-ValTyr-Tyr-Is CysOf Thr 255lie
Gin 265-GlyOf Thr-Leu-ValOf Thr 270-ValSer-
-Gly-GlySer--Gly-Gly 285-Gly-GlySer-
-GlySer--Gly-Gly 300-Gly-GlySer--Gly
-ValGlu-Ser-315-Gly-Gly-Gly-Leu-Val 320
Ser-Is Cys 330-Ala-AlaSer--Gly-Gly 335Of Thr
Phe-345Of ArgGin-AlaOf Pro-Gly 350Lys-Glu-
Teterapeptide TrpOf ArgSer--GlySer-365Of ThrTyr-Tyr-
Of ThrlieSer-Arg of 380Asp-Asn-Ser-Lys-
Ser--LeuArg of 395Of ProGlu--GlyOf Thr-Ala 400
Of Arg-Gly 410Tyr--Gly-Val-AlaTyr-415-Val
Gin 425-GlyOf Thr-Leu-ValOf Thr 430-ValSer-
lieSer-Glu-Glu-Asp-445-LeuAsn--Gly

[910]

<210> 41

[911]

<211> 461

[912]

<212> Protein

[913]

<213> Artificial sequence

[914]

Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Val-Ala
-AlaMet polypeptide-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40
-Ala-Ala 50lieSer-Of ArgSer--Gly 55-Gly
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of Arg
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf Pro
-Ala-AlaAsp-Arg of 100Of Arg-ValTyr-Ser-
Tyr-Asn-Tyr-115Teterapeptide Trp-GlyGin-GlyOf Thr 120
-Gly-Gly 130Ser--Gly-Gly-Gly-Gly 135Ser-
-Gly 145Ser--Gly-Gly-Gly-Gly 150Ser--Gly
Ser-Glu--ValGinLeu is 165-ValGlu-Ser-
Asn-Ser--LeuArg of 180-LeuSer-Is Cys-Ala
Phe--GlyMet polypeptide 195Ser-Teterapeptide Trp-ValOf ArgGin 200
-ValSer-210Ser-lieSer--GlySer-215-Gly
-Val 225Lys--GlyOf ArgPhe-Of Thr 230lieSer-
Tyr--LeuGinMet polypeptideAsn-245Ser--LeuOf Arg
Is CysOf Thrlie-Gly 260-GlySer--LeuSer-
Of Thr-ValSer-275Ser--Gly-Gly-Gly-Gly 280
-Gly-Gly 290Ser--Gly-Gly-Gly-Gly 295Ser-
-Gly 305Ser--Gly-Gly-Gly-Gly 310Ser-Glu-
-Gly-Leu-ValGinOf Pro 325-Gly-GlySer-
-GlyOf ArgOf ThrPhe-340Ser-Asp-Tyr--Gly
-GlyLys-Glu-355Of ArgGlu-Phe--Val-Ala 360
Of ThrTyr-370Tyr--AlaAsp-Ser--Val 375Lys-
Asn-385Ser-Lys-Asn-Of Thr-Val 390Tyr--Leu
Asp-Of Thr-Ala-ValTyr-405Tyr-Is Cys-Ala
-ValSer-Of ProAsn-420Ser-Ser-Ser-Of Arg
-Leu-ValOf Thr 435-ValSer-Ser--Gly-Ala 440
Glu-Asp-450-LeuAsn--Gly-Ala-Ala 455His-

[915]

<22 0>

[916]

<223> VHH-construct f013500030

[917]

<400> 41

[918]

-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser-25-GlyOf ArgOf ThrPhe-Ser-30Ser-Tyr-
Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
Of ArgOf ThrTyr-Tyr-60-AlaAsp-Ser--Val
Asp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80
Glu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
Of Thr 105-LeuOf ProOf ProOf ThrOf Thr 110Ser-Of Arg
-Leu-ValOf Thr-ValSer-125Ser--Gly-Gly
-Gly-Gly-Gly-Gly 140Ser--Gly-Gly-Gly
-Gly-Gly-Gly 155Ser--Gly-Gly-Gly-Gly 160
-Gly-Gly 170-Gly-Leu-ValGinOf Pro 175-Gly
-Ala 185Ser--GlyPhe-Of ThrPhe-190Ser-Ser-
-AlaOf Pro-GlyLys--Gly 205-LeuGlu-Teterapeptide Trp
Ser-Asp-Of ThrLeu is 220Tyr--AlaAsp-Ser-
Of ArgAsp-Asn-235-AlaLys-Of ThrOf ThrLeu is 240
Of ProGlu-250Asp-Of Thr-Ala-ValTyr-255Tyr-
Arg of 265Ser-Ser-Gin-GlyOf Thr 270-Leu-Val
Ser--Gly-Gly-Gly-Gly 285Ser--Gly-Gly
-Gly-Gly-Gly-Gly 300Ser--Gly-Gly-Gly
-ValGinLeu is 315-ValGlu-Ser--Gly-Gly 320
-LeuArg of 330-LeuSer-Is Cys-Ala-Ala 335Ser-
Met polypeptide 345-GlyTeterapeptide TrpPhe-Of ArgGin 350-AlaOf Pro
-AlalieSer-Teterapeptide TrpSer-365-Gly-GlyOf Arg
-GlyOf ArgPhe-Of Thr 380lieSer-Of ArgAsp-
GinMet polypeptideAsn-395Ser--LeuOf ArgOf ProGlu-400
-AlaLys-410Of ArgOf Arg-GlyOf Arg-Gly 415Ser-
Tyr-425Asn-Tyr-Teterapeptide Trp-GlyGin 430-GlyOf Thr
-Ala-His-Glu-HisGin-HisLys-His-460445-His-LeulieSer-Glu-

[919]

Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Ala-Ala
-AlaMet polypeptide-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40
-Ala-Ala 50lieOf ThrOf ArgOf Thr-Gly 55Of Arg
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of Arg
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf Pro
-Ala-AlaAsp-Arg of 100Of Arg-GlyTyr-Tyr-
Tyr-Teterapeptide Trp-Gly 115Gin-GlyOf Thr-Leu-Val 120
Ser--Gly 130-Gly-Gly-GlySer--Gly 135-Gly
-Gly 145-Gly-Gly-GlySer--Gly 150-Gly-Gly
-ValGin-Leu-ValGlu-165Ser--Gly-Gly
-LeuOf Arg-LeuSer-180Is Cys-Ala-AlaSer-
Met polypeptideSer-Teterapeptide Trp 195-ValOf ArgGin-AlaOf Pro 200
Ser-lie 210Ser--GlySer--GlySer-215Asp-
-Gly 225Of ArgPhe-Of ThrlieSer-230Of ArgAsp-
GinMet polypeptideAsn-Ser-Leu is 245Of ArgOf ProGlu-
lie-Gly-GlySer-260-LeuSer-Of ArgSer-
Ser-Ser--Gly 275-Gly-Gly-GlySer--Gly 280
Ser--Gly 290-Gly-Gly-GlySer--Gly 295-Gly
-Gly 305-Gly-Gly-GlySer-Glu-310-ValGin
-ValGinOf Pro-Gly-Gly 325Ser--LeuOf Arg
Of ThrPhe-Ser-Ser-340Tyr--AlaMet polypeptide-Gly
Glu-Of ArgGlu-355Phe--Val-Ala-Alalie 360
Tyr--Ala 370Asp-Ser--ValLys--Gly 375Of Arg
Lys-385Asn-Of Thr-ValTyr--Leu 390GinMet polypeptide
-Ala-ValTyr-Tyr-Is Cys 405-Ala-Ala-Ala
Tyr-Asn-Of Thr-Gly 420Of ThrTeterapeptide TrpTeterapeptide TrpTeterapeptide Trp

[920]

<212> Protein

[921]

<213> Artificial sequence

[922]

<22 0>

[923]

<223> VHH-construct f013500032

[924]

<400> 42

[925]

-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser-25-GlyOf ArgOf ThrPhe-Ser-30Ser-Tyr-
Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
Of ArgOf ThrTyr-Tyr-60-AlaAsp-Ser--Val
Asp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80
Glu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
Tyr-105Tyr-Asp-Ser-Ser-Phe-110Tyr-Asp-
Of Thr-ValSer-Ser--Gly 125-Gly-Gly-Gly
-Gly-GlySer--Gly 140-Gly-Gly-GlySer-
-GlySer--Gly 155-Gly-Gly-GlySer-Glu-160
-GlyLeu is 170-ValGinOf Pro-GlyAsn-175Ser-
-Gly 185Phe-Of ThrPhe-Ser-Ser-190Phe--Gly
-GlyLys--Gly-LeuGlu-205Teterapeptide Trp-ValSer-
Of Thr-LeuTyr--Ala 220Asp-Ser--ValLys-
Asn--AlaLys-235Of ThrOf Thr-LeuTyr-Leu is 240
Asp-Of Thr 250-Ala-ValTyr-Tyr-Is Cys 255Of Thr
Ser-265Gin-GlyOf Thr-Leu-Val 270Of Thr-Val
-Gly-Gly-GlySer--Gly 285-Gly-Gly-Gly
-Gly-GlySer--Gly 300-Gly-Gly-GlySer-
-Leu-ValGlu-315Ser--Gly-Gly-GlyLeu is 320
-LeuSer-330Is Cys-Ala-AlaSer--Gly 335-Gly
Teterapeptide Trp 345Phe-Of ArgGin-AlaOf Pro 350-GlyLys-
Of ArgOf ArgSer--GlyArg of 365Of ArgOf ThrTyr-
Phe-Of ThrlieSer-380Of ArgAsp-Asn-Ser-
Asn-Ser-Leu is 395Of ArgOf ProGlu-Asp-Of Thr 400
Of ArgArg of 410-ValOf ArgSer-Ser-Of Thr 415Of Arg
Glu-425Tyr-Teterapeptide Trp-GlyGin-Gly 430Of Thr-Leu

[926]

-Val-Val Ser-Ser--Gly-Ala-Ala Glu-Gin Lys-Leu is lie Ser-Glu-Glu-

[927]

435,440,445

[928]

Asp-Asn--Gly-Ala-Leu-Ala-His-His-His-His-His-His

[929]

450,455,460

[930]

<400> 43
Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Val-Ala
-AlaMet polypeptide-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40
-Ala-Ala 50lieSer-Of ArgSer--Gly 55-Gly
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of Arg
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf Pro
-Ala-AlaAsp-Arg of 100Of Arg-ValTyr-Ser-
Tyr-Asn-Tyr-115Teterapeptide Trp-GlyGin-GlyOf Thr 120
-Gly-Gly 130Ser--Gly-Gly-Gly-Gly 135Ser-
-Gly 145Ser--Gly-Gly-Gly-Gly 150Ser--Gly
Ser-Glu--ValGinLeu is 165-ValGlu-Ser-
Asn-Ser--LeuArg of 180-LeuSer-Is Cys-Ala
Phe--GlyMet polypeptide 195Ser-Teterapeptide Trp-ValOf ArgGin 200
-ValSer-210Ser-lieSer--GlySer-215-Gly
-Val 225Lys--GlyOf ArgPhe-Of Thr 230lieSer-
Tyr--LeuGinMet polypeptideAsn-245Ser--LeuOf Arg
Is CysOf Thrlie-Gly 260-GlySer--LeuSer-
Of Thr-ValSer-275Ser--Gly-Gly-Gly-Gly 280
-Gly-Gly 290Ser--Gly-Gly-Gly-Gly 295Ser-
-Gly 305Ser--Gly-Gly-Gly-Gly 310Ser-Glu-
-Gly-Leu-ValGinOf Pro 325-Gly-GlySer-
-Gly-GlyOf ThrPhe-340Ser-Ser-Tyr--Ala
-GlyLys-Glu-355Of ArgGlu-Phe--Val-Ala 360
Of ThrTyr-370Tyr--AlaAsp-Ser--Val 375Lys-
Asn-385Ser-Lys-Asn-Of Thr-Val 390Tyr--Leu

[931]

<210> 43

[932]

<211> 462

[933]

<212> Protein

[934]

<213> Artificial sequence

[935]

<22 0>

[936]

<223> VHH-construct f013500033

[937]

-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser-25-GlyOf ArgOf ThrPhe-Ser-30Ser-Tyr-
Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
Of ArgOf ThrTyr-Tyr-60-AlaAsp-Ser--Val
Asp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80
Glu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
Of Thr 105-LeuOf ProOf ProOf ThrOf Thr 110Ser-Of Arg
-Leu-ValOf Thr-ValSer-125Ser--Gly-Gly
-Gly-Gly-Gly-Gly 140Ser--Gly-Gly-Gly
-Gly-Gly-Gly 155Ser--Gly-Gly-Gly-Gly 160
-Gly-Gly 170-Gly-Leu-ValGinOf Pro 175-Gly
-Ala 185Ser--GlyPhe-Of ThrPhe-190Ser-Ser-
-AlaOf Pro-GlyLys--Gly 205-LeuGlu-Teterapeptide Trp
Ser-Asp-Of ThrLeu is 220Tyr--AlaAsp-Ser-
Of ArgAsp-Asn-235-AlaLys-Of ThrOf ThrLeu is 240
Of ProGlu-250Asp-Of Thr-Ala-ValTyr-255Tyr-
Arg of 265Ser-Ser-Gin-GlyOf Thr 270-Leu-Val
Ser--Gly-Gly-Gly-Gly 285Ser--Gly-Gly
-Gly-Gly-Gly-Gly 300Ser--Gly-Gly-Gly
-ValGinLeu is 315-ValGlu-Ser--Gly-Gly 320
-LeuArg of 330-LeuSer-Is Cys-Ala-Ala 335Ser-
Met polypeptide 345-GlyTeterapeptide TrpPhe-Of ArgGin 350-AlaOf Pro
-AlalieOf ArgOf ArgSer-365-GlyOf ArgOf Arg
-GlyOf ArgPhe-Of Thr 380lieSer-Of ArgAsp-
GinMet polypeptideAsn-395Ser--LeuOf ArgOf ProGlu-400

[938]

Of Thr-Asp-Ala-Val-Tyr-Tyr is Cys-Ala-Ala-Ala-Arg-Val Arg of Ser-Ser-

[939]

405,410,415

[940]

Of Thr Arg of Tyr-Asn-of Thr-Gly of Thr teterapeptide Trp teterapeptide Trp teterapeptide Trp Glu-Tyr-teterapeptide Trp-Gly-Gly Gin

[941]

420,425,430

[942]

Of Thr-Leu-Val-Val Ser-Ser--Gly-Ala-Ala Glu-Lys-Leu is lie Ser-Gin

[943]

435,440,445

[944]

Glu-Glu-Asp-Asn--Gly-Leu-Ala-Ala-His-His-His-His-His-His

[945]

450,455,460

[946]

<400> 44
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Of ThrTyr-
202530
-ValMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieAsn-Teterapeptide TrpSer--GlySer-Of ArgOf ThrTyr-Tyr--AlaAsp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaSer-Of ArgSer-Ser-Tyr--Ala-GlyOf ArgOf ThrTyr-Tyr-Glu--LeuTyr-
100105110
Asp-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Gly-Gly
115120125
-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-Gly
130135140
Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
145150155160
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-GlyAsn-
165170175
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyPhe-Of ThrPhe-Ser-Ser-Phe-
180185190
-GlyMet polypeptideSer-Teterapeptide Trp-ValOf ArgGin-AlaOf Pro-GlyLys--Gly-LeuGlu-Teterapeptide Trp-Val
195200205
Ser-Ser-lieSer--GlySer--GlySer-Asp-Of Thr-LeuTyr--AlaAsp-Ser--Val
210215220
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn--AlaLys-Of ThrOf Thr-LeuTyr-
225230235240
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
245250255
Of Thrlie-Gly-GlySer--LeuSer-Of ArgSer-Ser-Gin-GlyOf Thr-Leu-ValOf Thr
260265270
-ValSer-Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly
275280285
-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-Gly
290295300
Ser--Gly-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly
305310315320
-Leu-ValGinOf Pro-Gly-GlySer--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--Gly
325330335
Of ArgOf ThrPhe-Ser-Of ThrTyr-Of Thr-Val-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-Gly
340345350
Lys-Glu-Of ArgGlu-Phe--Val-Ala-AlalieOf ArgOf ArgOf Arg-GlySer-Ser-Of Thr
355360365

[947]

<210> 44

[948]

<211> 456

[949]

<212> Protein

[950]

<213> Artificial sequence

[951]

<22 0>

[952]

<223> VHH-construct f013500039

[953]

Tyr-Tyr-Ser-Asp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-
370375380
Ser-Lys-Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-
385390395400
Of Thr-Ala-ValTyr-Tyr-Is Cys-Ala-AlaAsp-Of ThrOf ArgOf Thr-Val-Ala-Leu-Leu
405410415
GinTyr-Of ArgTyr-Asp-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-
420425430
Ser--Gly-Ala-AlaGlu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn--Gly
435440445
-Ala-AlaHis-His-His-His-His-His-
450455
<400> 45
Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Ala-Ala
Of ThrMet polypeptide-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40
-Ala-Ala 50lieAsn-Of ArgSer--Gly 55-Gly
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of Arg
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf Pro
-Ala-AlaAsp-Arg of 100Of Arg-GlyOf Arg-Gly
Ser-Asn-Arg of 115Tyr-Glu-Tyr-Teterapeptide Trp-Gly 120
Ser--Gly 130-Gly-Gly-GlySer--Gly 135-Gly
-Gly 145-Gly-Gly-GlySer--Gly 150-Gly-Gly
-Gly-Gly-GlySer-Glu-165-ValGin-Leu
GinOf Pro-GlyAsn-180Ser--LeuOf Arg-Leu
Phe-Ser-Ser-195Phe--GlyMet polypeptideSer-Teterapeptide Trp 200
-LeuGlu-210Teterapeptide Trp-ValSer-Ser-lie 215Ser-
-Ala 225Asp-Ser--ValLys--Gly 230Of ArgPhe-
Of ThrOf Thr-LeuTyr-Leu is 245GinMet polypeptideAsn-
-ValTyr-Tyr-Is Cys 260Of Thrlie-Gly-Gly
Of Thr-Leu-Val 275Of Thr-ValSer-Ser--Gly 280
Ser--Gly 290-Gly-Gly-GlySer--Gly 295-Gly
-Gly 305-Gly-Gly-GlySer--Gly 310-Gly-Gly
Ser--Gly-Gly-GlyLeu is 325-ValGinOf Pro

[954]

<210> 45

[955]

<211> 463

[956]

<212> Protein

[957]

<213> Artificial sequence

[958]

<22 0>

[959]

<223> VHH-construct f013500046

[960]

-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser-25-Gly-LeuOf ThrPhe-Ser-30Of ArgTyr-
Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
Ser-Of ThrTyr-Tyr-60Ser-Asp-Ser--Val
Asp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80
Glu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
Glu-105Asn-Tyr-Ser--LeuLeu is 110Tyr-Ser-
Gin-GlyOf Thr-Leu-Val 125Of Thr-ValSer-
-Gly-GlySer--Gly 140-Gly-Gly-GlySer-
-GlySer--Gly 155-Gly-Gly-GlySer--Gly 160
-ValGlu-170Ser--Gly-Gly-Gly-Leu 175-Val
Ser-185Is Cys-Ala-AlaSer--Gly 190Phe-Of Thr
-ValOf ArgGin-AlaOf Pro 205-GlyLys--Gly
-GlySer--GlySer-220Asp-Of Thr-LeuTyr-
Of ThrlieSer-235Of ArgAsp-Asn--AlaLys-240
Ser-Leu is 250Of ArgOf ProGlu-Asp-Of Thr 255-Ala
Ser-265-LeuSer-Of ArgSer-Ser-270Gin-Gly
-Gly-Gly-GlySer--Gly 285-Gly-Gly-Gly
-Gly-GlySer--Gly 300-Gly-Gly-GlySer-
-GlySer-Glu-315-ValGin-Leu-ValGlu-320
-Gly-Gly 330Ser--LeuOf Arg-LeuSer-335Is Cys

[961]

-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Ser-Tyr--AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of Arg
340345350
Gin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val-Ala-AlalieSer-Teterapeptide TrpSer-
355360365
-Gly-GlySer-Of ThrTyr-Tyr--AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of Thrlie
370375380
Ser-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--Leu
385390395400
Of ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys-Ala-AlaSer-Of ProlieOf Pro
405410415
Tyr--GlySer--Leu-LeuOf ArgOf ArgOf ArgAsn-Asn-Tyr-Asp-Tyr-Teterapeptide Trp-GlyGin
420425430
-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Ala-AlaGlu-GinLys--Leulie
435440445
Ser-Glu-Glu-Asp--LeuAsn--Gly-Ala-AlaHis-His-His-His-His-His-
450455460
<400> 46
Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-Gly-Gly
151015
Ser--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf ThrPhe-Ser-Of ThrTyr-
202530
Of Thr-Val-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-Of ArgGlu-Phe--Val
354045
-Ala-AlalieOf ArgOf ArgOf Arg-GlySer-Ser-Of ThrTyr-Tyr-Ser-Asp-Ser--Val
505560
Lys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-Asn-Of Thr-ValTyr-
65707580
-LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is Cys
859095
-Ala-AlaAsp-Of ThrOf ArgOf Thr-Val-Ala-Leu-LeuGinTyr-Of ArgTyr-Asp-Tyr-
100105110
Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-ValSer-Ser--Gly-Gly-Gly-GlySer-
115120125
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
130135140
-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-Glu--Val
145150155160
Gin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-ValGinOf Pro-GlyAsn-Ser--Leu
165170175
Of Arg-LeuSer-Is Cys-Ala-AlaSer--GlyPhe-Of ThrPhe-Ser-Ser-Phe--GlyMet polypeptide
180185190
Ser-Teterapeptide Trp-ValOf ArgGin-AlaOf Pro-GlyLys--Gly-LeuGlu-Teterapeptide Trp-ValSer-Ser-
195200205
lieSer--GlySer--GlySer-Asp-Of Thr-LeuTyr--AlaAsp-Ser--ValLys--Gly
210215220
Of ArgPhe-Of ThrlieSer-Of ArgAsp-Asn--AlaLys-Of ThrOf Thr-LeuTyr--LeuGin
225230235240
Met polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala-ValTyr-Tyr-Is CysOf Thrlie
245250255
-Gly-GlySer--LeuSer-Of ArgSer-Ser-Gin-GlyOf Thr-Leu-ValOf Thr-ValSer-
260265270
Ser--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer-
275280285
-Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly-Gly-Gly-GlySer--Gly
290295300

[962]

<210> 46

[963]

<211> 457

[964]

<212> Protein

[965]

<213> Artificial sequence

[966]

<22 0>

[967]

<223> VHH-construct f013500047

[968]

-Gly-Gly-GlySer-Glu--ValGin-Leu-ValGlu-Ser--Gly-Gly-Gly-Leu-Val
305310315320
GinOf Pro-Gly-GlySer--LeuOf Arg-LeuSer-Is Cys-Ala-AlaSer--GlyOf ArgOf Thr
325330335
Phe-Ser-Ser-Tyr--AlaMet polypeptide-GlyTeterapeptide TrpPhe-Of ArgGin-AlaOf Pro-GlyLys-Glu-
340345350
Of ArgGlu-Phe--Val-Ala-AlalieSer-Teterapeptide TrpSer--Gly-GlySer-Of ThrTyr-Tyr-
355360365
-AlaAsp-Ser--ValLys--GlyOf ArgPhe-Of ThrlieSer-Of ArgAsp-Asn-Ser-Lys-
370375380
Asn-Of Thr-ValTyr--LeuGinMet polypeptideAsn-Ser--LeuOf ArgOf ProGlu-Asp-Of Thr-Ala
385390395400
-ValTyr-Tyr-Is Cys-Ala-AlaSer-Of ProlieOf ProTyr--GlySer--Leu-LeuOf Arg
405410415
Of ArgOf ArgAsn-Asn-Tyr-Asp-Tyr-Teterapeptide Trp-GlyGin-GlyOf Thr-Leu-ValOf Thr-Val
420425430
Ser-Ser--Gly-Ala-AlaGlu-GinLys--LeulieSer-Glu-Glu-Asp--LeuAsn-
435440445
-Gly-Ala-AlaHis-His-His-His-His-His-
450455
<400> 47
Glu-1-ValGin-Leu-Val 5Glu-Ser--Gly
Ser--LeuOf ArgLeu is 20Ser-Is Cys-Ala-Ala
-AlaMet polypeptide-Gly 35Teterapeptide TrpPhe-Of ArgGin-Ala 40
-Ala-Ala 50lieSer-Teterapeptide TrpSer--Gly 55-Gly
Lys-65-GlyOf ArgPhe-Of Thrlie 70Ser-Of Arg
-LeuGinMet polypeptideAsn-Ser-85-LeuOf ArgOf Pro
-Ala-AlaSer-Of Pro 100lieOf ProTyr--Gly
Tyr-Asp-Tyr-115Teterapeptide Trp-GlyGin-GlyOf Thr 120
-Gly-Gly 130Ser--Gly-Gly-Gly-Gly 135Ser-
-Gly 145Ser--Gly-Gly-Gly-Gly 150Ser--Gly
Ser-Glu--ValGinLeu is 165-ValGlu-Ser-
Asn-Ser--LeuArg of 180-LeuSer-Is Cys-Ala
Phe--GlyMet polypeptide 195Ser-Teterapeptide Trp-ValOf ArgGin 200
-ValSer-210Ser-lieSer--GlySer-215-Gly
-Val 225Lys--GlyOf ArgPhe-Of Thr 230lieSer-
Tyr--LeuGinMet polypeptideAsn-245Ser--LeuOf Arg
Is CysOf Thrlie-Gly 260-GlySer--LeuSer-
-Gly-Gly 10-Leu-ValGinOf Pro-Gly 15-Gly
Ser-25-GlyOf ArgOf ThrPhe-Ser-30Ser-Tyr-
Of Pro-GlyLys-Glu-Arg of 45Glu-Phe--Val
Ser-Of ThrTyr-Tyr-60-AlaAsp-Ser--Val
Asp-Asn-Ser-75Lys-Asn-Of Thr-ValTyr-80
Glu-Asp-90Of Thr-Ala-ValTyr-Tyr-95Is Cys
Ser-105-Leu-LeuOf ArgOf ArgArg of 110Asn-Asn-
-Leu-ValOf Thr-ValSer-125Ser--Gly-Gly
-Gly-Gly-Gly-Gly 140Ser--Gly-Gly-Gly
-Gly-Gly-Gly 155Ser--Gly-Gly-Gly-Gly 160
-Gly-Gly 170-Gly-Leu-ValGinOf Pro 175-Gly
-Ala 185Ser--GlyPhe-Of ThrPhe-190Ser-Ser-
-AlaOf Pro-GlyLys--Gly 205-LeuGlu-Teterapeptide Trp
Ser-Asp-Of ThrLeu is 220Tyr--AlaAsp-Ser-
Of ArgAsp-Asn-235-AlaLys-Of ThrOf ThrLeu is 240
Of ProGlu-250Asp-Of Thr-Ala-ValTyr-255Tyr-
Arg of 265Ser-Ser-Gin-GlyOf Thr 270-Leu-Val

[969]

<210> 47

[970]

<211> 462

[971]

<212> Protein

[972]

<213> Artificial sequence

[973]

<22 0>

[974]

<223> VHH-construct f013500053

[975]

Of Thr-ValSer-275Ser--Gly-Gly-Gly-Gly 280
-Gly-Gly 290Ser--Gly-Gly-Gly-Gly 295Ser-
-Gly 305Ser--Gly-Gly-Gly-Gly 310Ser-Glu-
-Gly-Leu-ValGinOf Pro 325-Gly-GlySer-
-Gly-GlyOf ThrPhe-340Ser-Ser-Tyr--Ala
-GlyLys-Glu-355Of ArgGlu-Phe--Val-Ala 360
Of ThrTyr-370Tyr--AlaAsp-Ser--Val 375Lys-
Asn-385Ser-Lys-Asn-Of Thr-Val 390Tyr--Leu
Asp-Of Thr-Ala-ValTyr-405Tyr-Is Cys-Ala
Of ThrOf ArgTyr-Asn-420Of Thr-GlyOf ThrTeterapeptide Trp
Of Thr-Leu-Val 435Of Thr-ValSer-Ser--Gly 440
Glu-Glu-450Asp--LeuAsn--Gly-Ala 455-Ala
Ser--Gly-Gly-Gly-Gly 285Ser--Gly-Gly
-Gly-Gly-Gly-Gly 300Ser--Gly-Gly-Gly
-ValGinLeu is 315-ValGlu-Ser--Gly-Gly 320
-LeuArg of 330-LeuSer-Is Cys-Ala-Ala 335Ser-
Met polypeptide 345-GlyTeterapeptide TrpPhe-Of ArgGin 350-AlaOf Pro
-AlalieOf ArgOf ArgSer-365-GlyOf ArgOf Arg
-GlyOf ArgPhe-Of Thr 380lieSer-Of ArgAsp-
GinMet polypeptideAsn-395Ser--LeuOf ArgOf ProGlu-400
-Ala-Ala 410Of ArgOf Arg-ValOf ArgSer-415Ser-
Teterapeptide Trp 425Teterapeptide TrpGlu-Tyr-Teterapeptide Trp-Gly 430Gin-Gly
-Ala-His-Ala-His-Glu-HisGin-His 460Lys-445-His-Leu-HislieSer-



[976]

The invention provides novel biparatopic LRP5/LRP6 cross-reactive binding polypeptides, and more specifically novel biparatopic LRP5/LRP6 cross-reactive immunoglobulin single variable domain constructs which can inhibit Wnt signaling pathways. The invention also relates to specific sequences of such polypeptides, methods of their production, and methods of using them, including methods of treatment of diseases such as cancer.



E polypeptide, specifically binding with LRP5 or LRP6, containing

- the first immunoglobulin separate variable domain, selected from the group of separate up immunoglobulin variable domains

(I)- (of III), determined the presence of the following CDR--sequences:

(I):

CDR1: TYTVG (=SEQ N0:1)

CDR2: AIRRRGSSTYYADSVKG (=SEQ N0:2)

CDR3: DTRTVALLQYRYDY (=SEQ N0:3)

(II):

CDR1: SYAMG (=SEQ N0:4)

CDR2: AIRRSGRRTYYADSVKG (=SEQ N0:5)

CDR3: ARRVRSSTRYNTGTWWWEY (=SEQ N0:6)

(III):

CDR1: RYTMG (=SEQ N0:7)

CDR2: AIVRSGGSTYYADSVKG (=SEQ N0:8)

CDR3: DRRGRGENYILLΥSSGRYEΥ (=SEQ N0:9),

and

- the second immunoglobulin separate variable domain, selected from the group of separate up immunoglobulin variable domains (of IV) and (V-), determined the presence of the following CDR--sequences:

(IV):

CDR1: SYAMG (=SEQ N0:10)

CDR2: AISWSGGSTYYADSVKG (=SEQ N0:11)

CDR3: SPIPΥGSLLRRRNNYDΥ (=SEQ N0:12)

(V):

CDR1: SYAMG (=SEQ N0:13)

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15).

2. Polypeptide by and. 1, differs, that

- said first separate immunoglobulin variable domain contains the following sequence CDR-:

CDR1: TYTVG (=SEQ N0:1)

CDR2: AIRRRGSSTYYADSVKG (=SEQ N0:2)

CDR3: DTRTVALLQYRYDY (=SEQ N0:3)

and where

- specified second separate immunoglobulin variable domain contains the following sequence CDR-:

CDR1: SYAMG (=SEQ N0:10)

CDR2: AISWSGGSTYYADSVKG (=SEQ N0:11)

CDR3: SPIPΥGSLLRRRNNYDΥ (=SEQ N0:12).

3. Polypeptide of NO of π. 1, differs, that

- said first separate immunoglobulin variable domain contains the following sequence CDR-:

CDR1: SYAMG (=SEQ N0:4)

CDR2: AIRRSGRRTYYADSVKG (=SEQ N0:5)

CDR3: ARRVRSSTRYNTGTWWWEY (=SEQ N0:6)

and where

- specified second separate immunoglobulin variable domain contains the following sequence CDR-:

CDR1: SYAMG (=SEQ N0:13)

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15).

4. Polypeptide of NO of π. 1, differs, that

- said first separate immunoglobulin variable domain contains the following sequence CDR-:

CDR1: RYTMG (=SEQ N0:7)

CDR2: AIVRSGGSTYYADSVKG (=SEQ N0:8)

CDR3: DRRGRGENYILLΥSSGRYEΥ (=SEQ N0:9)

and where

- specified second separate immunoglobulin variable domain contains the following sequence CDR-:

CDR1: SYAMG (=SEQ N0:13)

CDR2: AISWRSGSTYYADSVKG (=SEQ N0:14)

CDR3: DPRGYGVAYVSAYYEY (=SEQ N0:15).

5. Polypeptide according to any of the and. and. 1 - 4, characterized in that said immunoglobulin separate variable domains are domains VHH, preferably humanized domains VHH.

6. Polypeptide by and. 1, differs, that

- said first separate immunoglobulin variable domain is selected from group of separate up immunoglobulin variable domains (1) - (of III) with the following sequences:

(I):

AVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTVGWFRQAPGKEREFV AAIRRRGS ST-ΥΥ AD OF S-VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTRTVALLQYRYDYWGQGTLVTVSS

(=SEQ N0:19),

(II):

AVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAIRRSGRRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAARRVRS ST-RYNTGTWWWEYWGQGTL VT CIRCUITS OF V S-S-

(=SEQ N0:20),

(III):

AVQLVESGGGLVQPGGSLRLSCAASGLTFSRYTMGWFRQAPGKEREFV AAIVRSGGSTΥΥ THE ADS VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRGRGENYILLΥ S-S-THE GR YE ROTATION GQGTLVTV S-S-

(=SEQ N0:21),

and where

- specified second separate immunoglobulin variable domain is selected from the group, consisting of separate up immunoglobulin variable domains (of IV) and (V-) with the following sequences:

EVQLVE S-GGGLVQPGGSLRLS OF C OF A AS OF GRTF S-S-ΥAMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASPI P YGSLLRRRNNYDYWGQGTLVT VS OF S-

(=SEQ N0:22),

(V):

Ε-OF V QL THE VE S-GGGL OF V QUANTIZATION GGSLRL OF C S-A-A-S-S-S-GGTFΥAMGWFRQ Α-P GKEREF OF V A OF THE AIS OF W RSGSTΥΥ AD OF S-VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADPRGYGVAYVSAYYEYWGQGTLVTVSS

(=SEQ N0:23).

7. Polypeptide of NO of π. 1 or of π. 2, differs, that

- said first separate immunoglobulin variable domain has amino acid sequence according to SEQ n0:19, and

- specified second separate immunoglobulin variable domain has amino acid sequence according to SEQ n0:22.

8. Polypeptide according to Claim 1 or U. 3, differs, that

- said first separate immunoglobulin variable domain has amino acid sequence according to SEQ n0:20, and

- specified second separate immunoglobulin variable domain has amino acid sequence according to SEQ n0:23.

9. Polypeptide according to Claim 1 or U. 4, differs, that

- said first separate immunoglobulin variable domain has amino acid sequence according to SEQ n0:21, and

- specified second separate immunoglobulin variable domain has amino acid sequence according to SEQ n0:23.

10. Polypeptide according to any of claims U. 1 - 9, characterized in that said first and second separate immunoglobulin variable domains covalently are connected peptide - linker, where indicated peptide - linker optionally contains third immunoglobulin separate variable domain or consists of it.

11. Polypeptide according to any of claims U. 1 - 9, characterized in that additionally contains polypeptide fragment, drives enhanced half, said fragment, drives enhanced half, is covalently bound with said polypeptide and optionally is selected from the group, consisting of albuminsvyazyvayushchego fragment, such as albuminsvyazyvayushchii peptide or albuminsvyazyvayushchii immunoglobulin domain, transferrinsvyazyvayushchego fragment, such as antitransferrinovyi immunoglobulin domain, molecules of polyethylene glycol, human serum albumin, and also fragment of human serum albumin.

12. Polypeptide by and. 11, characterized in that said fragment, drives enhanced half, is albuminsvyazyvayushchim separate immunoglobulin variable domain, preferably domain Alb 11, determined by SEQ n0:24.

13. Polypeptide, selected from the group of polypeptides, containing or consisting of SEQ n0:25, SEQ n0:26 and SEQ n0:27.

14. Nucleic acid molecule, preferably in isolated form, encoding polypeptide according to any of claims U. 1 - 13.

15. Expression vector, which contains nucleic acid by and. 14.

16. Cell - host, carrying expression vector by and. 15.

17. Method of production of polypeptide according to any of claims U. 1 - 13, including stages

- cultivation of cells - by host and. 16 in conditions, expression of polypeptide according to any of claims U. 1 - 13; and

- isolation of said polypeptide.

18. The method and. 17, additionally including stage

- purification of the said polypeptide.

19. Pharmaceutical composition, containing (1) as an active substance polypeptide according to any of the and. and. 1 - 13 and (of II) a pharmaceutically acceptable carrier, and also optionally (of III) diluent, auxiliary substance, adjuvant and/or stabilizer.

20. Polypeptide according to any of the and. and. 1 - 13 for use as a medicinal agent in method for treatment, prophylaxis or relief of disease, disorder or condition in human or animal.

21. Polypeptide according to any of the and. and. 1 - 13 for using in cancer treatment, preferably breast cancer, lung cancer, pancreas cancer, colorectal cancer, sarcoma, ovarian cancer or pechenochnokletochnoi carcinoma, or for use in the treatment of idiopathic pulmonary disease, or for use in the treatment of retinopathy, caused by abnormal signal transmission of WNT.

22. Polypeptide according to any of the and. and. 1 - 13 for use in the treatment three times negative breast cancer (tnrmzh).

23. Polypeptide according to any of the and. and. 1 - 13 for application in combination with chemotherapeutic agents, therapeutically active compound, suppresses angiogenesis, track signal transduction inhibitor, inhibitor of EGFR is, immunomodulator, immune inhibitor of control points or means for hormonal therapy.

24. Therapeutic agent, selected from the group, consisting of chemotherapeutic agents, therapeutically active compounds, suppress angiogenesis, inhibitors of track signal transduction, inhibitors of EGFR is, immunomodulators, inhibitors of immune of control points and the means for hormonal therapy, for application in combination with polypeptide according to any of the and. and. 1 - 13.

25. Method for treating cancer, idiopathic pulmonary disease or retinopathy in patient, in need, including introduction of to the indicated patient the effective amount of polypeptide according to any of claims U.

1 - 13 or pharmaceutical composition according to Claim 19.

26. Polypeptide, containing

- the first immunoglobulin binding protein, which is capable of specifically to bind and with LRP5, and with LRP6 ("perekrestnoreaktivnyi by lrp5/lrp6") and suppresses caused Wntl transcription gene - target, and -

- the second immunoglobulin binding protein, which is capable of specifically to bind and with LRP5, and with LRP6 ("perekrestnoreaktivnyi by lrp5/lrp6") and suppresses caused Wnt3a transcription gene - target.

27. Polypeptide according to Claim 26, characterized in that said first and second immunoglobulin binding proteins are separate immunoglobulin variable domains.

28. Use of polypeptide according to any of claims U. 1 - 13, 26 or 27 for modification of the microenvironment of the tumor by suppression caused by Wntl and Wnt3a gene transcription - target in dendritic cells.

29. Polypeptide according to any of claims U. 1 - 13, 26 or 27dlya application in treatment of cancer.

3 0. Polypeptide according to any of claims U. 1 - 13, 26 or 27dlya application in treatment of cancer in combination with immune inhibitor of control points, selected from a group, consisting of antibodies against of Pd-and-1, antibodies against of Pd-and-l1, antibodies against CTLA4, antibodies against BTLA, antibodies against LAG3 and antibodies raised against TIM3, or same in combination with anticancer vaccine.