Specific and high affinity binding proteins comprising modified sh3 domains of fyn kinase

[0001] The present invention relates to a recombinant binding protein as defined in the claims, wherein said recombinant binding protein comprises at least one 3 Src (SH3) homology domain from FYN kinase, characterized in that at least one amino acid within or located at a distance of two amino acids adjacent to the src loop and at least one amino acid within or located up to two amino acids adjacent to the RT loop, subject to substitution, deletion or addition. In addition, the invention is devoted to fusion proteins containing a binding protein as defined in the claims, fused to a pharmaceutically and / or diagnostically active ingredient. In addition, the invention relates to nucleotides encoding for these binding and / or fusion proteins as well as corresponding vectors and host cells. Finally, the present invention relates to the use of binding and / or fusion proteins as defined in the claims for the preparation of a medicament or diagnostic method as well as pharmaceutical or diagnostic compositions containing said binding and / or fusion proteins.

Background of the invention [0002] Specific high affinity binding agents are irreplaceable tools in biological and medical research as well as being useful in medical diagnostics, prevention and treatment. Currently, monoclonal antibodies are a major class of binding molecules with high affinity and specificity for virtually any target that can be quickly isolated. However, immunoglobulins have some limitations that are primarily associated with their general biophysical properties and their rather complex molecular structure. Thus, as early as the 1990s, several research groups discovered small globular proteins that were substitutes for antibodies. The idea underlying this approach is to transfer the universal binding site from the antibody structure to alternative protein structures, so-called scaffolds. To date, more than 40 scaffolds have been described, including two SH3 domains from Abl and Src kinases (see Binz et al., Nature Biotechnology, Vol. 23, No. 10, 1257-1268, 2005).

[0003] SH3 domains can be found in a wide variety of proteins involved in intracellular signal transduction and cytoskeleton organization (Cohen et al., "Modular binding domains in signal transduction proteins." Cell 80 (2): 237-48, 1995). These SH3 domains, despite the variation in their primary structures, show a very similar overall structure and binding method for proteins containing the minimal PxxP consensus sequence, which is a critical factor determining natural SH3 binding. An important function of SH3 domains is participation in highly selective protein-protein interactions.

[0004] EP 1541694 A1 describes a method for identifying, selecting and / or characterizing a compound that regulates

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The activity of at least one kinase in the Src family. In addition, it relates to compounds identified by the method, pharmaceutical compositions and the use of these compounds and pharmaceutical compositions in the treatment of diseases that are at least partially caused by Src family kinase. [0005] US 6,326,469 B1 relates to cytoplasmic tyrosine kinases isolated from megakaryocytes (megakaryocyte kinase or MKK) which are involved in cellular signaling pathways and the use of these proteins in the diagnosis and treatment of the disease. The US patent additionally relates to specific megakaryocyte kinases referred to as MKK1, MKK2 and MKK3 and their use as diagnostic and therapeutic agents.

[0006] Erpel et al. ("Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions." Embo J. 14 (5): 963-75,1995) studied the effect of mutations in RT and n loops -Src of Src SH3 domains and has shown that mutations in both loops that are adjacent to the hydrophobic surface can affect the ability of these domains to participate in inter- and intramolecular interactions.

[0007] Hiipakka et al. ("SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef." J. Mol. Biol. 293 (5): 1097-106, 1999) studied the ability of the RT loop from the Hck SH3 domain to act as a comprehensive specificity factor and affinity. The authors created a phage library of Hck domains in which 6 amino acids of the RT loop were random (referred to as RRT-SH3). Using this strategy, they identified individual RRT-SH3 domains that can bind to Nef protein with HIV up to 40 times better than Hck-Sh3. The authors point to the essence of the RT loop in the selection of SH3 ligand as a general strategy when creating SH3 domains with desired binding properties.

[0008] Lee et al. ("A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein." Embo. J. 14 (20): 5006-15, 1995) studied the structural basis of the binding affinity of various SH3 and Hck specificity for HIV-1 Nef protein and were able to transfer Nef Hck SH3 binding properties to the Fyn SH3 domain by a single mutation in the RT loop of the Fyn SH3 domain (R96I).

[0009] Hosse et al. ("A new generation of protein display scaffolds for molecular recognition", Protein Science, 15: 14-27, 2006) specifically refers to the requirements for binding proteins suitable for therapeutic applications. The authors emphasize the essence of certain features of therapeutically useful binding proteins, such as serum stability, tissue penetration, blood clearance, retention in target tissue, and immune response. In the latter respect, it is emphasized that therapeutic non-human therapeutic proteins should be produced in a form as similar to their human counterparts as possible, and human scaffolds may already be less immunogenic as a starting point. The authors conclude:

"However, even a fully human scaffold does not guarantee that the protein will not trigger an immune response in humans, especially if it is an intracellular protein. Randomization of amino acids during library creation can potentially introduce new T-cell epitopes. Even single mutations can make a human protein immunogenic In addition, most human scaffolds elicit some autoimmune response. "

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[0010] Currently, protein scaffolds for the production of protein binding agents with assigned specificity are considered SH3 domains from Abl and Hck kinases, even though only binding agents have been identified to date with known ZnPyc2h0li5g4an4d3óww, such as Nef proteins or synthetic peptides ( Look ^B"^andH^nzthat^ee^t v^ande^l.r^pe^aboutvⁱⁿen^YZand^en^j).entirely human scaffold is no guarantee for a protein that does not elicit a human immune [0re0s1p1o] nsDeo, mesepneac iazl lykiinfaitziys aFnynin t (raFcyenll uSlaHr 3p) rootebien.jmRuajnedo6m3i zraetsiozntyof (a1^pe^aboutm^thesebⁿand^thiae^llyt ^andandⁿl^t.^ro(^d"^ats^ce^es -ⁿr^aboute^vla^et^le^Td^-CEp^lr^labout^et^pabout^andabout^thisn^pc^eabout^sg^. e^En^veeⁿ, ^ss^andsⁿn^g,^leb^pe^aboutl^andaboutⁿn^t g^ms^utt^andabout^{ti o}tⁿh^se^capⁿro^rt^eeⁿin^de-t^rs^andro^hs^atin^me^ank^pin^roand^ts^eeⁱⁿ_f^andand^mm^mand^atlyⁿ.^about" ^gP^enro^cc^.. Natl. Acad. Sci. USA 83 (15): 5459-63, 1986) and Kawakami et al. ("Isolation and oncogenic potential of a novel⁰h¹at⁰m^] and^Tn^abouts^dr^andc^s-^,l^tk^he g^Se^Hn³e^d."^ohmM^andabout^andl^nsC^aboute^fll^ANDB^band^labout^andlⁿ. ^d6(^H1^c2^k):^ki4ⁿ1^ace9^e5^s-2^and0^re1,^ac1^k9ⁿ8^about6ⁱⁿ)^l.^eF^dgs^en^dc^andfrom^sBovine^pn^roki^teim^nsr^cabout^andd^fffrom^aboutand^ln^ds^sfS^orc^gekiⁿn^eand^rafrom^titⁿs^gro^pfrom^ros^tn^eaboutⁱⁿwych o pmtidaessieha5v8e bkeDean.idWentiwfieydniskoufasrp (slieceinBgiunzaeltearl.nabtyowven) e.go Fyn protein occurs in two different isoforms ⁰r¹about¹from^]it^Tc^hs^ech^SHs³ut^downd^mabout^andmⁱⁿe^aboutn^fand^thm^eand ^Fk^sandⁿand^kfromⁱⁿs^and;^sethem⁽d^Fn^sand^{n S.}p^Habout³s⁾this^cÆ^ohmin^ps^rist^eę^sp⁶at³them^rein^siduyou^em^s about^(andc^andyt⁸and³c^-1h⁴, ⁵s^aboutp^fle^thn^eabout^sc^es^qthis^eucⁿh^ceand ^rp^ee^pin^orn^theses^dch^wouldlines ^kAND^about8^m3^about(1^r5^e)^k: 5^h4^e5^m9-^and6^t3^about,^l1^m9^f8^about6^id) an^lnd^sK^chand^,inⁿak^andand^thism^me^iat ^sand^tl.^d("^rAND^ats^gabout^andyears^pand^aboutn^sand^thisn^Æd ^andhe^kc^atabout^mge^atn^luc^themafter^st^ande^ęn^gthia^LOl ⁱⁿofⁿand^enⁱⁿove^ml ^abouthu^fromm^overan^(Csr^aboutc^about-l^kike ^andge^Pn^ee^r.^l"^mutter,"^lE^Cx^ep^llre^Bs^ands^land^.about⁶n^{( 1}about²f^):and⁴n¹about⁹⁵v^-e²l⁰f¹about^,rm¹⁹⁸about⁶f ⁾t^.h^Fe^millF y^isn^andpr⁵about⁹this^k-^Dabout^andnc^mabout^eg^me^bn^er and^aboutn^f h^the^em^Sand^rt^cop^faabout^me^loamt^sc^ofce^youl^rls^about.^s"ⁱⁿN^ee^kin^inaB^sand^eabout^sl.^. 1^AND(^s1)^and: ^r6^e6^s-^at7^lt4^about, ^f1989). ^Fm^ataboutⁿc^kyt^ce^js^e, ^bsp^andle^lon^gabout^andc^cs^fromtheseⁿs^e and^Fn^sdⁿso^sm^and e^zrh^aboute^frommⁿand^ctolⁱⁿwhite^andpⁿh^eoid^aboutc^be^ell^IUtench^ate^js^and, ^pin^rh^fromhow much^kand ^froms^seⁱⁿWhat^andnⁿd^efo^srm^ygandⁿc^andc^Lum^pat^aboutla^pt^re^froms^ep^fromri^rn^ec^cip^eand^pll^ts^orin^kb^aboutr^main^{T daughters,}o.o.

^-"c⁷ft⁴y_about^,nn¹,⁹and⁸are⁹in^).Se^Tlr_l^hca^es^bfaa^anddm^lh^aboute^glys^cand^andtn^ls^fr^atcanⁿse^cid^tn^andand^aboutaeⁿthese^skd^aboutins^f ia^Fgs^snⁿe_and._l^andl '_and^rn^egin^d(t^andR^v. ^eEJ^rs^s.h^e, B^andMⁿio.^dcD^andhⁿ. ^ce_"F^lm^ats^d.n^e, C.^sand^geSⁿLLR^andc^lB^lf^andandⁿiom^gl.^vil y^ia3t^ty0^hr (^eat 1_s^T1in)^ce^Elk1^land^rn1^ea5^cs9^ee^p-.6^t"^about2_AND^rn^,t^r.^e1J^g9.^at9B^la8io^t)^andc.^abouthⁿEJ^aboutme^fp.^btC^raebⁱⁿl_lowl e3w0 (n1ą1t) r: z1k1o5m9-ó6r2k, o1w9y9m8). ItSisEQan iInDtr acNeOllu: la1r prportzeeind.sStaEwQi aID sNeOk: all fthyen FSynHS3H (3asaeq8u3e-n1c4e5 (aak i8n3a-z1y45Foyfn et al., See in Kawba et al.

GVTLFVALYDYEARTEDDLSFHKGEKFQ1LNSSĘGDWWEARSLTTGETGYIPSNY VAPVDSIQ (SEQ ID NO: 1) ⁰[¹0²0^]12^T] ^hS^ee^sk^ein^qe^atn^enc^cj ^e p^aboutę^{f th}l^eR^RT^T-S^-Sr^rc^c and^an-^dS^tR^heCⁿs^-Sand^rcfrom^lop^aboutabout^pin^andand^re^ed^atnⁿand^dep^rabout^tenchd^ek^dre^andśⁿl^dhe^de^ouand^bp^leabout^-d^atin^ndabout^ejn^RLIe^ADp^dabout^,d^rek^sr^pe^eś^clo^tivn^El.^y n^[0S⁰H¹3 ^]di^Sffer^ksⁱⁿin^enabout^cn^je^and, vol^andh^me ^andaboutⁿn^aboute^kaboutⁱⁿf^andX^se^aboutnⁱⁿabout^andpu ^Fs ^slaⁿe ^Svi^Hs ³in ^jt^ein^sabout^t andⁱⁿm^pin^eabout^LNand^andc^kid^aboutpⁿabout^ss^eand^rtiⁱⁿhe^ands^youfⁱⁿroⁿm^andth^ate^cc^fromabout^Bovinerrⁱⁿes^ep^kabout^andn^,di^mng^ysh^fromat^sm^,and^sn^fromd^cabout^fromm^urand^andin^and. ^monkeys (gibbon). Chicken Fyn SH3 differs from the corresponding human domain one from Xenopus laevis two

0^p1^about4^from]^moonAND^jn^amsu^andmm^amar^andsⁿ,^aboutt^khⁱⁿe^andp^sri^aboutaboutⁱⁿra^sr^mtt^ande^. ac^Ph^aboute^ds^aboutp^broⁿt^ande^ein ^jf^andra^kmeⁱⁿinⁿabout^erks^d, ^aboutth^me^esⁿabout^sca^Slle^Hd³s^,ca^Fff^sabout ⁿlds,^Sand^Hs³alte^fromr^bn^atand^dti^aboutveⁱⁿs^andtⁿabout ^andes^jt^eab^sl^tand sh^fromed^two przeciwrówno^jβ-sheets, which include two elastic loops (referred to as RT-Src and n-Src loops) that allow interaction with other proteins.

In summary, the knowledge to date in the art includes protein skeletons, so-called scaffolds, as alternatives to established antibody structures. The 3 Src (SH3) homology domain is one of these approximately 40 or more scaffolds). Among the many different SH3 domains (about 300 in the human genome and several thousand described for this poay in nature), Fyn SH3 is the one that was previously used to explain binding specificity and total affinity. One skilled in the art is also aware that intracellular proteins are particularly prone to elicit an immune response, and thus, are usually less useful or even useless in in vivo applications such as terrace and diagnostics.

[0015] The object of the present invention is to provide improved proteins ^kin^andble^vfrom^tand^dLc^ksⁱⁿc^dh^{and ng}about^{l kr}s^andp^ktc^ts^itfi^kc^lfrom^jiWell^lrSC^sand^ond^ltyoc^gtl^tabout^kin^. high affinity binding that is suitable for use as research agents, and in particular as diagnostic and medical factors. In addition, these binding proteins should be stable and soluble under physiological conditions^/they have little or no immunological effect on bored ye recipients and should promote a binding structure that

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EP 2 054 432 B1 is also available for large target structures, i.e. it is not masked by steric obstacles.

Description of the invention [0016] Surprisingly, it has been found that the SH3 domain of the Fyn kinase of the Src family provides excellent properties when designing recombinant binding domains with specificity and high affinity for selected targets. In particular, it has been found that target specificity can be designed by mutating the RT loop and / or src loop, resulting in higher variability and improved binding properties for multiple purposes.

[0017] Furthermore, it was surprisingly found that not only the native Fyn SH3 binding protein, but also the binding proteins obtained from the mutated Fyn SH3 were not immunogenic in vivo. Thus, recombinant mutant Fyn SH3 binding proteins are particularly useful in developing non-immunogenic therapeutic and / or diagnostic proteins.

[0018] The present invention is defined in the claims. Accordingly, the first aspect of the present invention relates to a recombinant binding protein with a specific binding affinity for a protein or peptide, characterized in that said protein or peptide is not a natural SH3 binding ligand containing at least one derivative of the 3 Src (SH3) kinase homology domain Fyn wherein (a) at least one amino acid within or located at a distance of two amino acids adjacent to the src loop and (b) at least one amino acid within or located at a distance of two amino acids adjacent to the RT loop is subject to substitution, deletion or addition, wherein the derivative of SH3 domain comprises an amino acid sequence having at least 70, preferably at least 80, more preferably at least 90 and most preferably at least 95% identity s2e0k5w4e4nc3j2i with amino acid sequence of SEQ ID NO: 1 and wherein the derivative of domain SH3 has at least 85% identity with the amino acids of SEQ ID NO: 1, % ętsleaqmuienscrce idRenTti tiyztoztahestarzmeinżoenaiceidm that _b^eand^qand^atL^ekⁿabout^cr^ee^aboutk^fabout^Sm^Eb^Qin^ANDabout^Din^Nand^ABOUTn^:e^1,n^ande^ndthemⁱⁿs^ht ^ein^res^andsⁿthis^arep^andat^dI^Sc^Hs³m^downin^mn^andandⁱⁿhere^dr^efrom^re^{iv a}n^tand^ivt^eur^hal^sn^ands^tm^leab^sia^t L⁸k⁵e^%m^idfrom^eandⁿin^titand^se^tr^aboutoh^tand^hc^es^{am and}d^Wellm^ace^idnę^{of S}H^E3^Q. ^[r⁰about⁰vi¹s⁹about^]th^Rand^ett^kh^aboute^mre^bcⁱⁿohm^thatbi^andnⁿand^ent ^bp^andr^andabout^Lt^ke^aboutin ⁱⁿis^blen^fromabout^andthis^ceonⁱⁿhere^er^dal^ŁuS^gH3ⁱⁿd^saboutⁿm^alai^fromn^kc^atont^andand^ein^aboutin^bg^ep^IUro^atthese^themin ^se^exi^ksⁱⁿtin^egⁿand^cn^jion^maurochsⁱⁿe.^{acid SEQ ID}⁰N⁰ABOUT¹⁹: ^]2. ^TS^hek^rin^ece^aboutn^mc^bPH^inanm^tbinⁱⁿabout^dk^andin^ngace^pabout^roin^theseand^{in of}S^thE^eQ^invAND^eD^ntN^andABOUT^nd: ^about2^es(in^Welland^tr^cia^aboutn^mt^pR^ris9^e6^tAND^hedL^amFⁱⁿs^aboutn^acS^idH^s3^eqin^eudⁿL^cat^eg^ofL^Se^Ee^Qe^ANDt^Dand^Nl.^ABOUT, ^:see above) are shown below.

GVTLFVALYDYEAITEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNYV APVDSIQ (SEQ ID NO: 2)

0 [00202] 0] InWthekoconntetekxśtcoifethtiseginovenwtiyonatlhaezRkuT, l optholtolaf thReTF ynkikninaazsye (Fsyonme (tcimzaeseamlso doeksreigśnlatnead RrTó-wSnrta)^feb_l^te^he_t^eejm_d^and at^moh_n^andeⁿd^about/and_about^andmr^cai^andn^dd^sOdek^Ed^AND,and^Ris_e^Ts.^Ethese^DoA_b^thRe^andtm^tE^andat^rD_t^eand,_t^le^aboutkd^cthis^andand^tr_n^ee^doz_rⁱⁿlaod^pkj^aboutaa^sLCI^andez^tiWell^abouttⁿt^soa¹nth²ee^ts^aboutra¹T⁷in_loⁱⁿop_p^Soz^Eay_r^Qec j^ANDaa^Dmch^Nin^ABOUTat 1^:2a¹c^.did^Taxis^h11^e07^pt^aboutthat^s1^itS^and9^aboutEⁿp^sQre^thisfI_eD^bra^enb^sLO_s^ub:₁^s11^ti.t^tabout^atP^t1^eat 8^dfrom,^,positions, reprmefaejraabplyod12letgoa1ć7s. of constitution, deletion and / or addition, i.e. they are to be mutated within or adjacent to⁰p⁰ę²tl¹and ^]RTⁱⁿabout^tb^he^eIU^Whatatⁿj^tand^exand^tm^ofin^thabout^iskwⁱⁿand^vesⁿs^ti1^about0^ntd^habout^es1^r9^c,^lok^aboutabout^pgovernment^abouts^fs^thtn^ee^FY1^N1 ^kdⁱⁿabout^and1^s8^e,^(pk^aboutabout^mgovernment^es^tis^mtn^eand^se^andj ^l1^s2^fromd^eabout^si1^g7ⁿ.^{ated n-Src-loop) consists}

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[0021] In the context of this invention, the Fyn kinase src loop (sometimes also referred to as the n-Src loop) includes NSSE amino acids that are located at positions 31 to 34 in SEQ ID NO: 1. The positions to be subject to substitution, deletion and / or addition, i.e. they are intended to mutate within or adjacent to the RT loop, include amino acids 29 to 36, preferably 30 to 35, more preferably 31 to 34.

[0022] The recombinant protein of the invention is not a natural protein containing an SH3 domain found in or isolated from nature. In other words, the scope of the invention excludes proteins containing the wild type SH3 domain. There are many proteins in the nature that contain the SH3 domain. These natural SH3 proteins have binding affinity for their natural ligands. Most, if not all, of these natural SH3 ligands contain the PxxP motif. However, the recombinant proteins of the invention are genetically modified proteins designed to have affinity for non-natural targets, i.e. unnatural targets are any targets e.g. in nature, preferably in mammals, more preferably in humans, excluding natural (wild-type) SH3 ligands . The recombinant proteins of the invention generally show no binding affinity for any natural SH3 binding ligands, most preferably no natural SH3 binding ligands containing the PxxP motif.

[0023] Preferably, the number of amino acids to be added to one and / or both loops is 1 to 20, more preferably 1 to 10 or 1 to 5 amino acids, and most preferably no amino acids are added to the loop. [0024] In another preferred embodiment, fragments of the SH3 derivative domain that lie outside the RT and src loops are conservative to the maximum extent possible, so as to avoid the introduction of immunogenic motifs. [0025] It is preferred that the recombinant proteins of the invention do not substantially induce an immunogenic reaction in mammals, preferably in mice, rats and / or humans, most preferably in humans. Of course, the immunogenicity of the full recombinant protein of the invention will depend not only on the fragment of the derivative of the SH3 domain, but may be influenced by other fragments of the whole protein.

[0026] In a preferred embodiment of the invention, at least a fragment of the recombinant SH3 domain derivative is substantially non-immunogenic in mammals, preferably in mice, rats and / or humans, most preferably in humans.

[0027] For example, one skilled in the art can determine immunogenic reactions of a recombinant protein or a fragment thereof derived from the SH3 domain using standard and routine techniques, e.g. administration (e.g. iv injection) of the recombinant protein of interest, or a derivative of its SH3 domain to a mammal, such as a mouse and analysis of blood cell immune responses and / or factors (e.g. interleukin) after an adequate period of time to allow the immune reaction to occur.

[0028] In a more preferred embodiment of the invention, the binding protein according to the invention is one in which said derivative of the SH3 domain has at least 90, preferably at least 95, most preferably at least 98 to 100% identity with the Src (SH3) kinase homology domain in addition to the src and RT loops FYN.

[0029] In a preferred embodiment of the invention, the mutations are introduced in both RT and src loops.

[0030] In a further more preferred embodiment of the invention, the binding protein of the invention comprises one or preferably two altered residues at positions 37 and / or 50 of the derivative domain SH3, preferably two hydrophobic altered residues, more preferably Trp37 and / or Tyr50, Trp37 and Tyr50 are most preferred. How

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Figure 3b below shows their randomization can increase affinity.

[0031] The term "derivative of 3 Src (SH3) homology domain from FYN kinase" as used herein, means that it includes an amino acid sequence having at least 70, preferably at least 80, more preferably at least 90 and most preferably at least 95% sequence identity with the amino acid sequence with SEQ ID NO: 1. The same meaning remains true for a derivative of SH3 domain exhibiting at least 85, preferably at least 90, more preferably at least 95, most preferably at least 98% sequence identity with 3 Src (SH3) homology domain from FYN kinase outside the src and RT loops, except that when sequence identity is determined, the amino acids forming said loops are omitted.

[0032] To determine the degree of sequence identity of the derived SH3 domain from Fyn with the amino acid sequence of SEQ ID NO: 1, for example, SIM Local similarity software (Xiaoquin Huang and Webb Miller, "A Time-Efficient, Linear-Space Local Similarity Algorithm. "Advances in Applied Mathematics, vol. 12: 337-357, 1991), available free of charge from authors and their institutions (see also the website http: //www.ex-pasy.org/tools/sim-prot.html); ClustalW (Thompson et al., "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice." can be used to analyze multiple matches. ", Nucleic Acids Res., 22 (22) : 4673-4680, 1994.). Preferably, the extent of derivative sequence identity to SEQ ID NO: 1 is determined relative to the full sequence of SEQ ID NO: 1.

[0033] In a preferred embodiment of the invention, the binding protein of the invention comprises at least two derivatives of the Fyn SH3 domain. More preferably, it is a bivalent binding protein. The at least two derivatives of the SH3 domain may be the same or different. Preferably, they are the same.

[0034] The binding protein of the invention may be designed to have any specific binding affinity for a given peptide or protein, such as that containing the PxxP motif, provided that said protein or peptide is not a natural SH3 binding ligand. Of course, only a small proportion of the natural and physiologically relevant target proteins contain the PxxP motif. The examples below show that binding proteins of the invention are available for purposes (e.g. ED-B fibronectin domain) with motifs other than PxxP. Thus, the binding protein of the invention is in no way limited to the PxxP motif and may exhibit specific binding affinity for any given peptide or protein characterized in that said protein or peptide is not a natural SH3 binding ligand.

[0035] More preferably, the binding protein of the invention has specific binding affinity for a target of 10^-7 up to 10^-12 M, preferably 10^-8 up to 10^-12 M, preferably a therapeutically and / or diagnostically relevant target, more preferably an amino acid based target comprising the PxxP motif.

[0036] In a most preferred aspect, the binding protein of the invention has a specific binding affinity (in vivo and / or in vitro) of 10^-7 up to 10^-12 M, more preferably 10^-8 up to 10^-12 M to the extracellular tumor-fetal fibronectin domain (ED-B).

[0037] Also described herein is a recombinant binding protein comprising at least one derivative of a 3 Src (SH3) homology domain from FYN kinase, characterized in that (a) at least one amino acid within or located within a distance of two amino acids

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Adjacent to the src loop and / or (b) at least one amino acid within or located at a distance of two amino acids adjacent to the RT loop is subject to substitution, deletion or addition, wherein the derivative of the SH3 domain comprises an amino acid sequence of at least 70, preferably at least 80, more preferably at least 90 and most preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, preferably with the proviso that the recombinant binding protein does not comprise the amino acid sequence of SEQ ID NO: 2, and preferably with the proviso that the recombinant protein is not a naturally occurring protein containing the SH3 domain, wherein said binding protein exhibits (in vivo and / or in vitro) a specific binding affinity of preferably 10^-7 up to 10^-12 M, more preferably 10^-8 up to 10^-12 M to the extracellular tumor-fetal fibronectin domain (ED-B).

[0038] The derivative of the SH3 domain, in addition to the src and RT loops, has at least 85, preferably at least 90, more preferably at least 95, most preferably at least 98 to 100% identity with the Src (SH3) homology domain 3 of FYN kinase.

[0039] The above binding protein specific for ED-B may contain at least two derivatives of the SH3 domain, preferably it is a bivalent binding protein.

Also, said ED-B specific binding protein may contain one or more, preferably two, altered, preferably hydrophobic, residues at positions 37 and / or 50 of the derivative SH3 domain, in particular Trp37 and / or Tyr50, most preferred are Trp37 and Tyr50.

[0041] For use in, e.g., arthritis or cancer therapy, many antibody-cytokine fusion proteins have already been studied, often with impressive results. For example, to deliver proinflammatory cytokines (such as IL-2, IL-12 or TNF) to solid tumors, human L19 antibody specific for the ED-B domain of fibronectin (an angiogenesis marker) has sometimes been used with huge therapeutic benefits [to For a review and relevant literature references, see Neri & Bicknell, Nat. Rev. Cancer (2005) 5: 436-446 and also WO 01/62298].

[0042] The binding protein of the present invention now allows the replacement of antibodies known from prior art in the field of fusion proteins and also the design of new and less immunogenic fusion proteins for in vivo and in vitro pharmaceutical and diagnostic applications.

[0043] In a second aspect, the invention relates to a fusion protein comprising the binding protein of the invention fused to a pharmaceutically and / or diagnostically active ingredient.

[0044] The fusion protein of the invention may contain non-polypeptide components, e.g. non-peptide linkers, non-peptide ligands, e.g. for therapeutically or diagnostically suitable radionuclides.

[0045] Preferably, said active ingredient is a cytokine, preferably a cytokine selected from the group consisting of IL-2, IL-12, TNF-alpha, IFN alpha, IFN beta, IFN gamma, IL-10, IL-15, IL- 24, GM-CSF, IL-3, IL-4, IL-5, IL-6,

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EP 2 054 432 B1

IL-7, IL-9, IL-11, IL-13, LIF, CD80, B70, TNF beta, LT-beta, CD-40 ligand, Fas ligand, TGF-beta, IL-1alpha and IL-1beta.

[0046] More preferably, said active ingredient is a toxic compound, preferably a small organic compound or polypeptide, preferably a toxic compound selected from the group consisting of calicheamycin, neocarzinostatin, esperamycin, dynemycin, kedarcidin, maduropeptin, doxorubicin, daunorubicin, ruristin truncated Pseudomonas exotoxin A, modecin, diphteria toxin and recombinant gelonin.

[0047] In another preferred embodiment, the fusion protein of the invention is a protein in which the active ingredient is a chemokine, preferably a chemokine selected from the group consisting of IL-8, GRO alpha, GRO beta, GRO gamma, ENA-78, LDGF- PBP, GCP-2, PF4, Mig, IP-10, SDF-1alfa / beta, BUNZO / STRC33, ITAC, BLC / BCA-1, MIP-1alfa, MIP-1 beta, MDC, TECK, TARC, RANTES, HCC -1, HCC-4, DC-CK1, MIP-3 alpha, MIP-3 beta, MCP-1-5, Eotaxin, Eotaxin-2, I-309, MPIF-1, 6Ciny, CTACK, MEC, Lymphactactin and Fractalkins.

[0048] In a further preferred embodiment of the invention, the binding protein according to the invention comprises synthetic amino acids.

[0049] In further preferred embodiments of the fusion protein of the present invention, the active ingredient is a fluorescent dye, preferably a component selected from the Alexa Fluor groups or Cy dyes (Berlier et al., "Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes:

Fluorescence of the Dyes and Their Bioconjugates ", J Histochem Cytochem. 51 (12): 1699-1712, 2003);

a photosensitizing agent, preferably bis (triethanolamine) Sn (IV) chlorin e6 (SnChe6); a procoagulant factor, preferably a tissue factor; a prodrug activating enzyme, preferably an enzyme selected from the group consisting of carboxypeptidase, glucuronidase and glucosidase; a radionuclide or from a group of 99m 123 111 isotopes emitting gamma rays, preferably Tc, I, In, or from a group of positron emitting agents, preferably¹⁸F ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y ¹²⁴And, or from the beta-emitter group, preferably ¹³¹AND, ⁹⁰Y, 177 67 213 211

Lu, Cu, or from the group of alpha-emitters, preferably Bi, At; and / or a functional Fc domain, preferably a human functional Fc domain.

[0050] The above-mentioned functional Fc domain will allow directing the mammalian immune response to the target specific binding site of the binding protein component in the fusion protein, e.g. for therapeutic, prophylactic and / or diagnostic applications.

[0051] A further preferred embodiment of the invention relates to the fusion proteins of the present invention, as mentioned above, further comprising a serum half life modification component, preferably a component selected from the group consisting of polyethylene glycol (PEG), immunoglobulin and albumin binding peptides.

[0052] In a most preferred embodiment of the invention, the fusion protein of the invention as mentioned above comprises a binding protein of the invention having specific binding affinity (in vivo and / or in vitro) of 10^-7 up to 10^-12 M, more preferably 10^-8 up to 10^-12 M to the extracellular tumor domain of fibronectin (ED-B). Preferably, said ED-B specific binding protein contains one or more, preferably two hydrophobic residues at positions 37 and / or 50 of the derivative domain SH3, in particular Trp37 and / or Tyr50, most preferably Trp37 and Tyr50.

VP / 3260 / RW

[0053] The binding and fusion proteins of the invention can be prepared by any of a number of conventional and well-known techniques, such as simple organic synthesis strategies, solid phase assisted synthesis techniques, or through commercially available automated synthesizers. On the other hand, they can also be prepared by conventional recombinant techniques used alone or in combination with conventional synthetic techniques.

[0054] Further aspects of the present invention are devoted to (i) a polynucleotide encoding said binding protein or fusion protein of the invention, (ii) a vector comprising said polynucleotide, (iii) a host cell comprising said polynucleotide and / or said vector.

[0055] The polynucleotides may be DNA, RNA, PNA and any other analogues thereof. The vectors and host cells may be of any conventional type which corresponds to the intended purpose, e.g. production of binding and fusion proteins of the invention, therapeutically useful vectors and host cells e.g. for gene therapy. The skilled artisan, based on extensive prior knowledge in the art, will be able to select these polynucleotides, vectors and host cells and confirm their particular suitability for the desired purposes by routine methods and without undue burden.

[0056] The binding and fusion proteins of the present invention do not induce a strong and preferably do not substantially induce any immune response in mammals, in particular humans and mice, as has been shown for mice and analogously this is also true for humans because Fyn SH3 is identical in both species of mammals. It has been surprisingly found that neither native Fyn SH3 nor mutated Fyn SH3 elicits an immune response in mice injected with iv each of them was given. This was unexpected because Fyn kinase is an intracellular protein and does not participate in neonatal B cell selection. Thus, binding and fusion proteins derived from Fyn SH3 with designed specificity and target affinity are particularly well suited for in vivo therapeutic, prophylactic and / or diagnostic applications .

[0057] Consequently, a highly important aspect of the present invention relates to the use of a binding or fusion protein of the invention in the manufacture of a medicament.

[0058] In a further aspect, the binding or fusion protein of the invention is used to produce diagnostic agents, in particular for in vivo applications.

[0059] Preferably, the ED-B specific binding or fusion protein as described above is used in the preparation of a medicament or diagnostic agents for the treatment or diagnosis of cancer.

[0060] Another aspect of the present invention relates to a pharmaceutical composition comprising the binding or fusion protein of the invention and optionally a pharmaceutically acceptable excipient.

[0061] Another aspect of the present invention relates to a diagnostic composition, preferably for in vivo applications, comprising the binding or fusion protein of the invention and an optional pharmaceutically acceptable excipient.

[0062] Preferably, the pharmaceutical or diagnostic composition comprises an ED-B specific binding or fusion protein of the invention and an optional pharmaceutically acceptable excipient.

[0063] Pharmaceutical compositions and diagnostic agents for in vivo uses according to the present

VP / 3260 / RW

The inventive compositions usually contain a therapeutically or diagnostically effective amount of the binding and / or fusion protein of the invention and optionally excipients such as pharmaceutically acceptable excipients. Said pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art. The carrier or excipient may be a liquid material that can serve as a carrier or medium for the active substance. Suitable carriers or excipients are well known in the art and include, for example, stabilizers, antioxidants, pH regulators, controlled release excipients. The pharmaceutical preparation of the invention may be adapted for, for example, parenteral use and may be administered to a patient in the form of solutions and the like.

[0064] Also described herein is a method of treatment or diagnosis characterized in that an effective amount of the above pharmaceutical or diagnostic composition is administered to a patient in need thereof, preferably a patient suffering from or suspected of cancer and / or inflammatory diseases.

[0065] When performing treatment or diagnosis of a diseased subject, the binding or fusion protein of the present invention may be administered in any form or manner that makes the therapeutic or diagnostic compound bioavailable in an effective amount, including oral or parenteral. For example, the compositions of the present invention may be administered subcutaneously, intramuscularly, intravenously and the like. A specialist in formulation preparation can easily choose the appropriate form and route of administration, depending on the specific characteristics of the selected product, the disease or condition being treated or diagnosed, the stage of development of the disease or condition and other relevant conditions (see e.g. Remington's Pharmaceutical Sciences, Mack Publishing Co (1990)). The compositions of the present invention may be administered alone or in the form of a pharmaceutical or diagnostic preparation in combination with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the solubility and chemical properties of the selected product, the chosen route of administration and standard pharmaceutical and diagnostic practice. The products of the present invention, while effective in themselves, can be formulated and administered in the form of their pharmaceutically acceptable salts, such as acid addition salts or basic addition salts, for stability, easy crystallization, increased solubility and the like.

Figures [0066]

Fig. 1 shows a dot blot analysis. The percentage of clones expressing a detectable amount of soluble Fyn SH3 mutants was determined by dot blot analysis of bacterial lysates using anti-HIS-HRP antibody conjugate (Sigma) as detection reagent. Peroxidase activity was detected using the ECL plus Western blotting detection system (Amersham).

VP / 3260 / RW

EP 2 054 432 B1

A) FynSH3 mutants with a randomized RT-Src loop.

B) FynSH3 mutants with extended (4-> 6) and randomized n-Src loop.

C) FynSH3 with randomized RT- and n-Src loops.

Fig. 2 shows the monoclonal phage ELISA. After the third round of panning against MSA, monoclonal bacterial supernatants containing phages displaying Fyn SH3 mutants were tested by ELISA using MaxiSorp (Nunc) coated plates with MSA (100 μg / ml overnight, 100 μl per well). Bound phages were detected using M-13-HRP antibody conjugates.

Figure 3 shows the monoclonal phage ELISA (versus MSA) after one round of affinity maturation selection using MSA coated MaxiSorp (Nunc) plates (100 μg / ml overnight, 100 μl per well).

A) phage ELISA of the first G4 sub-library (randomized loop of n-Src and Trp37 and Tyr50). The parent G4 clone is indicated by an arrow.

B) phage ELISA of the second G4 sub-library (randomized and extended n-Src loop). The parent G4 clone is indicated by an arrow.

C) phage ELISA of the first and second sub-library after one round of panning, carried out under conditions favorable for long koff binding agents. The parent G4 clone is indicated by an arrow.

Fig. 4 shows a soluble ELISA (using MaxiSorp (Nunc) plates coated with MSA (100 μg / ml overnight, 100 μl per well) of several MSA binding clones, after cloning (pQE-12 vector), expression and purification of soluble protein , according to the manufacturer's instructions (Qiagen, native conditions) Anti-HIS-HRP antibody conjugates were used as detection factors. As control, the same binding proteins were added to the wells blocked only with 4% MPBS.

Fig. 5 ELISA specificity for soluble protein. Selected MSA Fyn SH3 mutants were tested for binding to human serum albumin (HSA), rat serum albumin (RSA), bovine serum albumin (BSA) and ovalbumin using MaxiSorp (Nunc) plates coated with different albumin (each 100 μg / ml overnight, 100 μl per well).

Fig. 6 BIACore analysis for D3. Concentrations used: 4, 2, 1, and 0.5 μM (from above).

Fig. 7 ELISA analysis of blood samples for the presence of mouse antibodies.

A) MaxiSorp (Nunc) plates were coated with Fyn SH3 (20 μg / ml overnight, 100 μl per well). Blood samples (in the range 75-200 μθ for each of 5 mice were used in serial dilutions (from 1: 4 to 1: 100). Antibody detection was performed using

VP / 3260 / RW

Anti-mouse-IgG-HRP conjugate (Sigma). Anti-HIS-HRP conjugates (Sigma) were used as control performance for coating.

B) MaxiSorp (Nunc) plates were coated with Fyn SH3 D3 (20 μg / ml overnight, 100 μΐ per well). Blood samples (in the range 75-200 μθ for each of the 5 mice were used in serial dilutions (from 1: 4 to 1: 100). Antibody detection was performed using anti-mouse-IgG-HRP antibody conjugate (Sigma). As a performance control Anti-HIS-HRP (Sigma) conjugates were used for coating.

C) MaxiSorp (Nunc) plates were coated with FscFv (60 μg / ml overnight, 100 μΐ per well). Blood samples (in the range 75-200 μΐ) for each of the 4 mice were used in serial dilutions (from 1: 4 to 1: 100). Antibody detection was performed using anti-mouse-IgG-HRP antibody conjugate (Sigma). Anti-myc-HRP conjugates (Roche) were used as control for coating efficiency.

Figure 8 shows D3 immunoflouorescence (Figure 8a), corresponding to negative control (8.b), anti-CD31 staining (Figure 8.c) and corresponding negative control (8.d) on histological sections of murine F9 teratocarcinoma.

Fig. 9 shows Fyn SH3-D3 tumor retention (Fig. 9.a), while no accumulation of Fyn SH3wt was observed (Fig. 9.b). Targeting results are expressed as% given by injection

125 dose of retained I-labeled protein per g tissue (% ID / g).

[0067] The object of the invention is described in more detail below with reference to certain preferred embodiments which were not intended to limit the invention.

Examples

Example 1: Expression of Fyn SH3 mutants [0068] To assess the expression of Fyn SH3 mutants, a dot blot analysis was carried out for three different Fyn SH3 sub-libraries (Fig. 1): in the first library only the RT loop was randomized, in the second the Src loop was randomized and extended to 6 residues, and in the third library the RT- and Src loops were randomized simultaneously, the second loop was extended from 4 to 6 residues. The percentage of expressed Fyn SH3 mutants ranged from 59-90%.

Table 1

<td>Library</td><td>Mutants expressed (%)</td><td>Number of clones tested</td>

<td>RT-Src</td><td> 59</td><td> 29</td>

<td>n-Src</td><td> 90</td><td> 29</td>

<td>RT-Src and n-Src</td><td> 62</td><td> 58</td>

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EP 2 054 432 B1

Example 2: Selections by phage display relative to mouse serum albumin [0069] Library 10 was created⁷ various Fyn SH3 (only the RT loop was randomized), which was cloned into the pHEN1 phagemid vector (Hoogenboom et al. "Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains", Nucleic Acids Res , 19 (15): 4133-7, 1991). The library was presented on phages and three rounds of panning were performed relative to murine serum albumin (MSA). After the third round, screening was performed using a monoclonal phage ELISA; 13 positive colonies were detected (Fig. 2). Sequencing of 13 clones showed that two different sequences designated G4 and C4 were enriched.

[0070] However, after subcloning and expression of G4 in the pQE-12 vector (Qiagen, expression and purification were carried out according to the manufacturer's manual, under native conditions) protein binding to MSA could not be detected by ELISA analysis (Fig. 4) due to low affinity (phage ELISA is more sensitive than soluble protein ELISA). Thus, the G4 sequence was used in two affinity maturation libraries (size: 10⁷ clones for each library). In the first, 4 residues of the n-Src loop and residues Trp37 (SEQ ID NO: 1) and Tyr50 (SEQ ID NO: 1) were random, in the second, the n-Src loop was extended from 4 to 6 random residues. After one round of panning, several clones from both sub-libraries showed stronger signals in the phage ELISA compared to the G4 parent clone (Fig. 3). After subcloning and expression of several clones, the binding of soluble protein was confirmed by ELISA analysis (Fig. 4). Visible dissociation constants were in the range of 100 nM (as determined by BIAcore). Some clones showed cross-reactivity with other serum albumin (human serum albumin (HSA), rat serum albumin (RSA), bovine serum albumin (BSA) and ovalbumin) were tested, while other clones were highly specific for MSA, indicating that Isolation of high specificity binding proteins is possible (Fig. 5).

Example 3: Selections by phage display relative to the additional b domain of fibronectin (ED-B) [0071] ED-B was selected as the target protein to demonstrate the possibility of selecting Fyn SH3-derived agents that bind pharmaceutically significant protein. ED-B is a type III homology domain of 91 amino acids that is inserted into a fibronectin molecule by an alternative splicing mechanism at the level of the primary transcript when tissue remodeling occurs (Zardi et al., "Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon. "Embo J. 6 (8): 2337-42, 1987). It is a good qualitative marker of angiogenesis, which is overexpressed in various solid tumors (e.g. kidney cancer, colon cancer, liver cancer, high grade astrocytoma, head and neck cancer, bladder cancer, etc.) but is virtually undetectable in normal adult tissues (for except for the endometrium in the proliferative phase and certain vessels in the ovaries). For details of ED-B as a target, see Menrad and Menssen, "ED-B fibronectin as a target for antibody-based cancer treatments." Expert Opin. Ther. Targets 9 (3): 491500, 2005).

PnoZm / 3a2,6h0ig / Rh WGR

1la4d

EctPab2le0in5 4no4r3m2alB1 ^[about⁰n⁰E⁷²D^]-B^Pand^rs^zyand^gt^aboutand^tr^aboutgeⁱⁿt^andsⁿe^aboute M^be^bn^lr^andad^theseand^kn^ęd ^pM^abouteⁿn^ands^ddream¹, "E^mD^loam-^iaB^rdfib^andro^mAD^atc^tt^andin^ntand^aboutsⁱⁿat^Far^sgⁿet ^Sfo^Hr ³and^,nti^kb^tabout^aboutd^rs^and- b^pace^ree^fromd^ecⁿand^tn^aboutcⁱⁿe^andrⁿtr^abouteatⁿm^anden^ft^ands^g."^ah(EjexdpnerotcOzpeisnn. AThrear.nTdaormgeiztsac9j (a3): p4ę9tl1i -5R0T0-, S2r0c05i) n.-Src). After three rounds of panning relative to ED-B using f^[oa⁰mg⁰and⁷from²wa^]these_andg_n^ANDoo^ltf^ande^bR^rs^andTTU^r-^sSE^aboutrc^fL^mIaSn^aboutad^renz^t-and^hsd^andreⁿcn_l¹toyo^bfip^andk^lls^andabout^about)in_.ⁿaa^Ffn_t^seoⁿr ^St3_h^Hrk_e³leo^mnroy^atat^thiswnⁿdi^tace^sżoⁱⁿaf^andcp^se_and.^pnS^rn^ee^pnk^andg^reae^dgn_and^andcinⁿjos^dtn^deo^isDw^p-and^lB^andn^s3i^ee^dbw_and^aboutnyⁿdk_in^pag^hfrom^andac^gLł_about^ehe^sde⁽s^siwi^mee^atrr^lEO^thisfrom_andⁿdn^eee^aboutn^atts_and^sfe_e^rk^anddⁿbe^ds^-functions (pkhloangey EoLzInSaAc.zSoenqeuejanckiongBr1e1veialDed3) t.wSo tdaiłfąferdeynst oscejqaucejincDe3s (all-known information about oBp1r1zeazndaDn3he)^aboutfrom^fAD_d^Dc³s_hsⁱⁿthat^andwi^ssetd^ds ^eIt has^thesev^rand^mr_le_atⁱⁿez^eoo^dnf a₈^bn.^s5s^sxu^ur1^f0^andp^cl8a^eMz^pm^l(^andFo^sin_g^mUO^aboutrw_eⁿ 6^re) p_.^sabout^aboutin^oneⁿr^cfrom^ech^ren^andand^ltin^thems^ech^intefrom^{r ac}from^tand^andsⁿthis^andsⁿabout^andin^lyand^sin^se^atm^singat^andgovernment^Band^ANDd^ANDfrom^ce^aboutn^reia³⁰⁰B⁰IAⁱⁿc^sabout^trr^ate^m3^e0ⁿ0^t0 and a value of 8.5 X 10 was shown^-8 (Figure 6).

D3 (SEQ ID NO: 3)

GVTLFVALYDYHAQSGADLSFHKGEKFQILKFGRGKGDWWEARSLTTGETGYIPSNYV

APVDSIQ

Example 4: Immunogenicity [_andⁱⁿm0^v0m^about7^lat^v3_n^and]ⁿabout^gGim_e^renm^pc^eat_p^tin^tabout^andabout^vthese^egne^andtin^dan_l^moo^andfⁿfA^andHs^se^trb^andF^tand_s^andand^aboutNLⁿeS^skH^about3j^few^andsilt^dd^rie^atye^gpd^.en^DPa^atro^ezTE^thising^tI (^hof^ewyn^cn^aboutSyⁿcH^sh3^erin^vt^and)and^tand^andd_nⁿdt^aboutae^f r_F^tand^hyp_n^eand iS^FH^sfromⁿin₃^SMia^Huz_t³aann^ss^et (c^qfh^ats^enⁿfrom S^cehb₃andⁱⁿaDłk^m3a_,^cam^ebi,^andinⁿdz^dew_r^mL_andand^egsⁿuntil_andc^tn^hzs^eat w tEraDk-tBo) wwaansi aincvhe sotibgeajtmeduijnej cvyivcohbpyoinwjetactriznagn5empicoedraenpieaaltekdluy.wZithutwhaegtwi onap rkooteninsse.rMwiacteywenroeśićn jescetkewj eHntc^te^hxy^irace^dm^dy_n^andand^sin⁾gcⁱⁿfrom_th^andL^tabout^hew_p²ir⁰eek^mare_e^g,_n^aboutcp^feo^pt_about^re^aboutrn^tand^ec_b^andjⁿare^.eł^ABOUTit_cⁿe^ecan^duf^andnm^sou^andgr^fand^ten^eAD^r n^tand^hs_n^etb_and⁴-Fia_sL^andknⁿand^jS^eH^cF^t3y^andwnⁿt S^manh^cd3^eandⁱⁿt_ns^etp_and^r-^eFu_s^sdn^andfrom^csi^rkH^fand^ande3^cgD^eabout^d3 ^andand(ⁿF_n^dyou_bn^babout^loS_d^aboutH_e^ds3_.^sin^andAND^mts) ^pai^lebp^sohm_sⁱⁿat_it^eit_v^rabout^eew^tc^andao^knn^ee_tⁿrg_about^fol^about4^rFyn SmHic3e (wFeyrne iSnjHec3tDed3, (ecqzuyanlntimkae in conjunction with this code) - sbaagdeasn (o = 6in0 vmivgo) Mice were irradiated 4 times (gco on the third day) with 20 pg of protein. The day after the 4th injection, mice were sacrificed and blood samples were taken to test for the presence or absence of murine anti-Fyn SH3wt and anti-Fyn SH3D3 antibodies. The positive control was 4 mice injected with human antibody in the form of a single Fv chain (scFv) by injection (same injection time points and equal doses (= 60 pg)). However, one mouse from the scFv group died 20 minutes after the third injection, and the other three were close to death, so blood samples were taken after the third injection. Figures 7 a and b show that it was impossible to detect antibodies to Fyn SH3wt and Fyn SH3D3, while strong signals were observed in the control group (Fig. 7c).

Example 5: Immunohistofluorescence [0074] To examine whether Fyn SH3-D3 (D3, binding agent for ED-B) recognizes its target in native tissue conformation, immunofluorescence staining of F9 teratocarcinoma sections was performed. Figure 8 shows that D3 binds to the tumor stroma around the blood vessels (Fig. 8.a). Detection was carried out using anti-His-Alexa488 antibody conjugate. No D3 protein was added in the negative control (Fig. 8b). To image blood vessels, the same sections were simultaneously stained with rat anti-mouse-CD31 antibody, and donkey anti-rat Alexa594 antibody conjugate was used (Fig. 8.c). As a negative control, a secondary antibody without a primary antibody was used (Fig. 8.d).

Example 6: Quantitative in vivo biodistribution [0075] Fyn SH3-D3 targeting behavior (ED-B binding agent) and wild-type Fyn SH3

VP / 3260 / RW

EP 2 054 432 B1 (non-binding ED-B) in vivo was evaluated in biodistribution experiments in mice loaded subcutaneously transplanted with F9 mouse teratocarcinoma. Because ED-B is identical in mice and humans, results of tumor targeting studies should predict D3 behavior in humans. 125 injected mice were dosed with D3 I and SH3wt labeled mice and 24 h later the mice were sacrificed, organs_in^ths^ec^tand^atę^mthis^about,^rfrom^sin^trand^aboutfrom^mabout^andn^andabout^roand^atfromⁿm^dand^be^lr^aboutfrom^aboutabout^dn^vabout^esra^sed^land^soa^(Fk^andt^gs^.in^8.n^andabout^).ś^TÆ^h. ^eN^dand^eF^theseand^cg^tat^andrⁿthat^wa9^s.and^fullp^rabout^fok^rand^mfrom^eand^dnⁱⁿabout,^iththat^anD^ti3^-Hs^ise^-l^ANDe^lk^et^xs^andin⁴⁸n⁸e^andand^ntk^bat^aboutm^dat^sI am in love^gs^atin^zie^ere ⁽c^sabout^t-^abouts^sthis^atine^kd^and in^gand^atth^from:arⁿand^andt ^rand^fromn^andti^dMO-^mat^ands^ee^ś-^cC^andD^BRIEFINGS31^sand^utntibⁱⁿA young^from and^kn^rd^eand^ss^eace³e^:1cond^aboutary¹and⁰n^:t¹b^),from^ps^aboutd^dabout^cn^fromk^ande^sya^gnt^dand-^sinstallmentsⁿAND^elex^about5^b9^s4^erwowano watzebwoagsaucseendia (Fdigla. 8b.cia) .ł kTahetynpeugadtizviekiceognotroFlywnaSs dHo3ne (Fuisgi.ng9.tbh) e. secondary antibody without the primary antibody pLleIS6T: AQ uSaEntKitWatiEveNbCioJdIistribution in vivo

B [0) 0w7a6s] evaluated by biodistribution experiments in mice bearing a sc grafted F9 murine teratocarcinoma. Since ed a &lt;n1d1r0a&gt; di oEaicdtgiveitnyoweasssicsocuhneteTde. cFhignuirsech9.eaHshoocwhscthautl eDZ3 userlei chtively accumulated in the tumor (tumor: organ

ENC &lt;E12L0IS&gt; TsINpG Specific high affinity binding proteins containing modified SH3 domains from FYN kinase

110&gt;_&lt;E₁and₃d₀g_&gt;en₅about₀e₀ss₂and₇sc_Ph_Ce_TTechnische Hochschule Zuerich &lt;160&gt; 3 ^160&gt;&lt;³170&gt; PatentIn version 3.3

170&gt;^&lt;P²and¹t⁰e^&gt;nt¹In version 3.3 &lt;211&gt; 63 ₂²¹1⁰1^&gt;&gt;&lt;₆¹2₃12&gt; PRT

212&gt; &lt;P21R3T&gt; Homo sapiens

400&gt;&lt;1400&gt; 1

<td>Gly</td><td>val</td><td>Thr</td><td>Leu</td><td>phe</td><td>val</td><td>ala</td><td>Leu</td><td rowspan="2">Tyr</td><td>Asp</td><td>Tyr</td><td>Glu</td><td>ala</td><td>Arg</td><td>Thr</td><td>Glu</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td rowspan="2">Asp</td><td rowspan="2">Asp</td><td>Leu</td><td>Cheese</td><td>phe</td><td>His</td><td rowspan="2">lys</td><td rowspan="2">Gly</td><td>Glu</td><td rowspan="2">lys</td><td>phe</td><td>Gin</td><td>how much</td><td>Leu</td><td>own</td><td>Cheese</td>

<td></td><td> 20</td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>cheese</td><td>Glu</td><td>Gly</td><td rowspan="2">Asp</td><td rowspan="2">Trp</td><td rowspan="2">Trp</td><td>Glu</td><td>ala</td><td rowspan="2">Arg</td><td>cheese</td><td>Leu</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Glu</td><td>Thr</td>

<td></td><td></td><td> 35</td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Gly</td><td>Tyr</td><td>how much</td><td>Pro</td><td>cheese</td><td>own</td><td>Tyr</td><td>val</td><td>ala</td><td>pro</td><td>val</td><td>Asp</td><td>cheese</td><td>how much</td><td>Gin</td><td></td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

^&gt;&lt;⁶2³10&gt; 2

VP / 3260 / RW

EP 2 054 432 B1 &lt;211&gt; 63 &lt;212&gt; PRT &lt;213&gt; Synthetic

<td>Gly</td><td>val</td><td>Thr</td><td>Leu</td><td>phe</td><td>val</td><td>ala</td><td>Leu</td><td rowspan="2">Tyr</td><td>Asp</td><td rowspan="2">Tyr</td><td>Glu</td><td>ala</td><td>how much</td><td>Thr</td><td>Glu</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>

<td rowspan="2">Asp</td><td rowspan="2">Asp</td><td>Leu</td><td>Cheese</td><td>phe</td><td>ΗΪ E.</td><td rowspan="2">L2S</td><td rowspan="2">Gly</td><td>2LU</td><td rowspan="2">lys</td><td>phe</td><td>Gln</td><td>how much</td><td>Leu</td><td>own</td><td>Cheese</td>

<td></td><td> 20</td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Cheese</td><td>Glu</td><td>Gly</td><td rowspan="2">Asp</td><td rowspan="2">Trp</td><td rowspan="2">Trp</td><td>Glu</td><td>ala</td><td rowspan="2">Arg</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Glu</td><td>Thr</td>

<td></td><td></td><td> 35</td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Gly</td><td>Tyr</td><td>how much</td><td>Pro</td><td>Cheese</td><td>own</td><td>Tyr</td><td>val</td><td>ala</td><td>Pro</td><td>val</td><td>Asp</td><td>Cheese</td><td>how much</td><td>Gln</td><td></td>

55 60 &lt;400&gt; 2 &lt;21 0&gt; 3 &lt;211&gt; 65 ²2^&lt;&gt;3⁰²³&gt;^&gt;1v²and^&gt;ri^Pan^Rt ^Tof SH3 domain of Fyn kinase with high affinity to ED-B domain of fibronectin &lt;^&gt;2⁶1⁵3&gt; Synthetic &lt;220&gt;

&lt;223&gt; variant SH3 domain from Fyn kinase with high affinity for the fibronectin ED-B domain

<td colspan="7"> &lt;400&gt; 3</td><td colspan="4" rowspan="2">Leu Tyr Asp Tyr 10</td><td rowspan="2">His</td><td rowspan="2">ala</td><td rowspan="2">Gln</td><td rowspan="2">Cheese 15</td><td rowspan="2">Gly</td>

<td colspan="3">Gly val Thr 1</td><td>Leu</td><td colspan="3">Phe Val Ala 5</td>

<td>ala</td><td rowspan="2">Asp</td><td>Leu</td><td>cheese</td><td>phe</td><td>His</td><td rowspan="2">lys</td><td rowspan="2">Gly</td><td>Glu</td><td rowspan="2">lys</td><td>phe</td><td>Gln</td><td>How much</td><td>Leu</td><td>lys</td><td>phe</td>

<td></td><td></td><td> 20</td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Arg</td><td>Gly</td><td>lys</td><td>Gly</td><td>Asp</td><td>Trp</td><td>Trp</td><td>Glu</td><td>ala</td><td rowspan="2">Arg</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Thr</td><td>Gly</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Glu</td><td>Thr</td><td rowspan="2">Gly</td><td rowspan="2">Tyr</td><td>how much</td><td>pro</td><td>Cheese</td><td>own</td><td rowspan="2">Tyr</td><td>val</td><td>ala</td><td>Pro</td><td>val</td><td rowspan="2">Asp</td><td>cheese</td><td>how much</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

blah

VP / 3260 / RW

EP 2 054 432 B1