PL236281B1 - Protein sequence, preferably constituting a fragment of antibody, in a specific way interacting with human or mouse vascular endothelial growth factor C, and its application - Google Patents

Abstract

The application relates to novel protein sequences that interact specifically with human and murine vascular endothelial growth factor C (VEGF-C). In particular, protein sequences that are single chain human antibody (scFv) fragments. The application includes a method for isolating VEGF-C from a solution, especially an aqueous solution, and the use of protein sequences to prepare VEGF-C protein preparations.

Description

Description of the invention

The invention relates to an antibody of the scFv format which is an inhibitor of the vascular endothelial growth factor C protein (VEGF-C). In particular, the protein sequences of the invention are single-chain human antibody fragments (scFv). An object of the invention is to provide molecules that can efficiently and specifically bind human and murine VEGF-C proteins. An additional object of the invention is to provide a method for detecting human and murine VEGF-C protein and determining its amount and / or concentration.

Cancer is one of the leading causes of death in the world. Effective therapy related to the surgical removal or irradiation of primary tumors is often impossible due to the migration of cancer cells from the primary environment to other organs and tissues in the body. Malignant neoplasms use both blood vessels and lymph vessels to form metastases (Bzowska M et al., Acta Biochim Pol 2013, 60 (3): 263-75). Until now, researchers focused on the phenomenon of angiogenesis, i.e. the formation of new blood vessels. It has become the main target of cancer metastasis inhibiting therapies, although their efficacy has been lower than expected. One of the many possible reasons for the failure of this type of therapy is the neglect of the role of lymphatic vessels as an alternative route for cancer cells to escape from the primary tumor (Potente M et al. Cell 2011; 146 (6): 873-87). The most promising target for inhibiting lymphangiogenesis is the blockade of the signal axis between the VEGF-C protein (vascular endothelial growth factor C) and its receptors VEGFR-2 and VEGFR-3 (vascular endothelial growth factor receptor; vascular endothelial growth factor receptor) (Stacker SA et al. Nat Rev Cancer 2014; 14 (3): 159-72). VEGF-C belongs to the family of vascular endothelial growth factors, consisting of seven proteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF (placental growth factor). growth factor). Discovered in 1996, the VEGF-C protein was described as a non-covalent homodimer ligand for VEGFR-2 and VEGFR-3 (Joukov V et al. EMBO J. 1996; 15 (7): 290-298), although some reports also report on the presence of covalently linked VEGF-C dimers (Leppanen VM et al. Proc. Natl Acad Sci USA 2009; 107 (6): 2425-2430). Its affinity for receptors is regulated by a multistage proteolytic processing, during which the strength of the interaction of successively emerging forms of VEGF-C with VEGFR-3 increases. In turn, VEGFR-2 can only recognize the mature form of VEGF-C (Joukov V et al. EMBO J 1997; 16 (13): 3898-3911). The sequences of the mature forms of human and murine VEGF-C proteins differ by only two amino acid residues (UniProtKB / Swiss-Prot database records P97953 and P49767 for murine and human VEGF-C, respectively). VEGFR-2 and VEGFR-3 are receptors with tyrosine kinase activity, which, as a result of ligand binding, become activated - they undergo dimerization and mutual phosphorylation of cytoplasmic domains (Joukov 1996, loc. Cit.). Under the influence of VEGF-C interaction with its receptors, only VEGFR-3 homodimers or VEGFR-2/3 heterodimers may be formed (Nilsson I et al. EMBO J 2010; 29 (8): 1377-88). Probably an important role in the signaling axis between VEGF-C and its receptors is played by neuropilins that do not have enzymatic activity, which act as co-receptors for VEGFR-2 and VEGFR-3 (Karpanen T et al. FASEB J 2006; 20 (9): 1462-72 ).

VEGF-C plays an important role in lymphangiogenesis, and therefore takes part in many different physiological and pathological processes, such as: development of the lymphatic system during embryogenesis, wound healing, chronic inflammation, autoimmune diseases or tumor metastasis (Chachaj A and Szuba A [in:] Angiogenesis and Vascularisation Dulak J et al. (eds.); Springer Vienna 2013, pp. 27-66). VEGF-C, by activating VEGFR-3, stimulates the proliferation and migration of lymphatic endothelial cells and provides them with a survival signal (Makinen T et al. EMBO J 2001; 20 (17): 4762-4773). Moreover, while VEGF-A remains the major pro-angiogenic factor, VEGF-C also can stimulate angiogenesis by interacting with VEGFR-2/3 heterodimers (Nilsson 2010, loc. Cit.).

The main pro-lymphangiogenic factors of key importance in tumor metastasis are the VEGF-C and VEGF-D proteins, which induce the formation of new lymphatic vessels around the tumor by activating VEGFR-3 and the neuropilin-2 coreceptor (Karpanen 2006; op. Cit .; Mandriota) SJ et al. EMBO J 2001; 20 (4): 672-82; Chen Z et al. Cancer Res 2005; 65 (19): 9004-9011; Xu Y et al. J Cell Biol 2010; 188 (1): 115-30). In this way, cancer cells, secreting VEGF-C, provide an escape route to a nearby lymph node, from which they can spread further both through the lymphatic and bloodstream (Stacker SA et al. Nat Rev Cancer 2002; 2 (8): 573-583 ). Essential

The role of VEGF-C in tumor-associated lymphangiogenesis was confirmed by in vivo experiments in which blocking this factor inhibited metastasis of neoplastic cells (Chen 2005; op. Cit .; Wang F et al. Exp Ther Med 2010; 1 (5) : 899-904).

Thus, the assessment of the presence and / or level of the VEGF-C protein in biological material may be of diagnostic importance in medicine. Patent description US20100317533 discloses a panel of biomarkers for the diagnosis and monitoring of the course of neoplastic disease; one of the panel components is VEGF-C, the presence of which is associated with the metastatic potential of the tumor. The patent specification US2012231480 further discloses a method of diagnostic evaluation of pancreatic lesions by measuring the level of VEGF-A and VEGF-C proteins and prostaglandin E2.

The VEGF-C protein in biological material can be determined, among others, by antibodies, especially monoclonal antibodies. A VEGF-C specific IgG antibody is disclosed in US2011143428. Antibodies are molecules of relatively large size, which may limit their analytical and diagnostic use. Moreover, the production of monoclonal antibodies is expensive (e.g. requires the use of eukaryotic cell culture). Single-chain variable fragment (scFv) antibodies, which are about 6 times smaller than IgG class antibodies and can be produced with the use of prokaryotic expression systems, do not have these drawbacks.

ScFv antibodies that specifically interact with the VEGF-C protein are known in the art (Rinderknecht M et al. PLoS One 2010; 5 (8): e11941). However, the antibodies described have a number of limitations. The protein used to select the scFv antibodies came from the yeast organism, as it was produced in a system using Pichia pastoris cells. Therefore, this protein was characterized, among others, by a glycosylation pattern different from that of the mammalian VEGF-C protein. Moreover, the biological activity of the protein used to select scFv antibodies has not been confirmed.

The object of the invention is to provide molecules that can efficiently and specifically interact with the biologically active VEGF-C protein. An additional object of the invention is to provide a method for purifying, detecting and / or quantifying VEGF-C protein.

The present invention relates to an antibody of the scFv format which is an inhibitor of the VEGF-C protein comprising an amino acid sequence selected from the following group: Seq. No.14, Seq. No.15, Seq. No.16, Seq. No. 17, Seq. No.18, Seq. No.19, Seq. No. 20, Seq. No.21, Seq. No.22, Seq. No. 23, Seq. No. 24, Seq. No.25, Seq. no 26.

The invention also relates to a method for isolating VEGF-C from a solution, in particular an aqueous solution, characterized in that:

(a) contacting a protein sequence of the invention with a solution containing a VEGF-C protein to obtain a protein complex comprising a protein sequence of the invention and a non-covalently associated VEGF-C protein;

(b) separating the resulting protein complex from the solution;

(c) the VEGF-C protein is dissociated from the resulting protein complex;

(d) the VEGF-C protein is isolated from the dissociated solution.

Preferably, in step (b) of the above-described process, the isolation of VEGF-C from the solution is carried out by affinity chromatography, using a chromatographic medium containing an immobilized protein sequence according to the invention as the medium.

In another aspect, the invention also relates to a method for detecting the presence of VEGF-C in a biological material, characterized in that:

(a) contacting a protein sequence of the invention with biological material to obtain a protein complex comprising a protein sequence of the invention and a non-covalently associated VEGF-C protein;

(b) detecting the obtained protein complex by means of indirect or direct biochemical detection methods.

The invention also relates to a method for determining the concentration of VEGF-C in an aqueous solution, in particular in physiological fluid, characterized in that:

(a) contacting a protein sequence of the invention with an aqueous solution containing VEGF-C to obtain a protein complex comprising a protein sequence of the invention and a non-covalently associated VEGF-C protein;

(b) the concentration of the resulting protein complex is determined by indirect or direct biochemical detection methods.

PL 236 281 B1

In another aspect, the invention also relates to the use of a protein sequence according to the invention for the preparation of VEGF-C protein preparations.

The invention also includes the use of a protein sequence according to the invention for the detection of a VEGF-C protein in biological material.

The invention also includes the use of a protein sequence according to the invention for determining the concentration of VEGF-C protein in an aqueous solution.

The subject of the invention is presented in non-limiting examples of implementation and in the drawing, in which:

Figure 1 illustrates that the selected monoclonal antibodies are specific for both murine VEGF-C (A) and human VEGF-C (B);

Figure 2 illustrates antigen binding by selected soluble anti-scFv

VEGF-C;

Figure 3 illustrates that the 1F2, 1F5 and 1F6 antibodies are specific for VEGF-C only;

Fig. 4 illustrates the amino acid sequences of the antibodies isolated from the phagemid library, converged to MSA (Multi-Sequence Alignment).

W episode of performance

Isolation of phages showing the expression of recombinant monoclonal antibodies of the scFv format specific for the murine and human VEGF-C protein

A phage display method based on the use of a phagemid library of human recombinant antibodies of the Tomlinson I + J scFv format was chosen for the production of antibodies. Using polyclonal phages, three selection rounds were carried out on the immobilized murine VEGF-C protein produced by mammalian cells (the antigen was therefore the mature recombinant VEGF-Canac protein). The biological activity of the protein used for selection had previously been confirmed by biological methods using in vitro cell culture. For this purpose, the Immortalized Human Lymphatic Endothelial Cells (ihLEC) assay under the influence of the biologically active VEGF-C protein was used. As a result of selection on the antigen, phages were obtained which expressed on their surface antibodies capable of binding the murine recombinant VEGF-C protein (Fig. 1A). Although the phage selection of the library was performed using the murine VEGF-C protein as the antigen, it was confirmed that antibodies expressed on the surface of the isolated phages also bind human VEGF-C (Fig. 1B). Thus, the selected antibodies are specific for both murine and human VEGF-C proteins.

Obtaining soluble antibodies of the scFv format binding murine and human VEGF-C

An antibody expressed on the surface of a filamentous phage is not a soluble protein - it is produced as a fusion protein together with the pIII protein, a phage's structural coat protein. To express the soluble scFv antibodies, the coding sequence of the selected antibody was cloned into the pETscFv expression vector. The expression of soluble anti-VEGF-C antibodies was obtained in E. coli Rosetta Blue bacteria. The antibodies were isolated from the periplasmic region of the bacteria and purified using protein A affinity chromatography. Figure 2 shows the binding of selected scFv monoclonal antibodies to the antigen.

Confirmation of the specificity for the VEGF-C protein

Since the proteins belonging to the VEGF family show high similarity in the amino acid sequence, it was confirmed using an ELISA test that the obtained antibodies 1F5, 1F6 and 1F2 recognize and bind VEGF-C while showing no affinity for VEGF-A and VEGF-D (fig. 3).

Obtaining the sequence of antibodies of the scFv format binding murine and human VEGF-C

The pIT2 phagemid vectors encoding the antibodies displayed on the surface of the extracted phages were sequenced to obtain the amino acid sequence of the encoded antibodies. The sequences obtained were then aligned in the form of MSA (Multi-Sequence Alignment) and presented in Fig. 4. Within them, CDR regions can be distinguished that are responsible for the binding of the protein sequence to the antigen.

The individual CDRs in the antibody sequence can be thought of as interchangeable fragments since, as shown in Figure 4 for the 1F5 and 1F10 antibody sequences, the CDRH2 sequences are

PL 236 281 B1 and CDRH3 are identical, although the corresponding CDRL2 and CDRL3 sequences are different; analogously, the 1F5 and 1G4 sequences have identical CDRL2 fragment but different CDRH2, CDRH3 and CDRL3 fragments.

In addition, the above sequences containing heavy chain fragments and light chain fragments can be subjected to further optimization; in particular, they can be converted into a single camelid heavy chain antibodies which characterize naturally occurring immunoproteins (Hamers-Casterman et al. Nature 1993; 363: 446-448, Muyldermans S and Lauwereys M. J Mol Recognit 1999; 12: 131-140). In this case, the entire protein sequence constituting the scFv antibody is not required for antigen binding, only a fragment (single chain) thereof. Thus, the use of such a fragment will be sufficient to preserve the antigen binding ability of the protein sequence. The use of such an optimization strategy for antibodies of the scFv format is known from the literature (Rinderknecht M et al., Loc. Cit.).

The selected monoclonal antibodies are specific for both murine and human VEGF-C as shown in Figures 1A and 1B, respectively. The analysis was performed using an ELISA test. Plates coated with antigens (mouse or human VEGF-C) were incubated with the phage extracted and the non-binding VEGF-C phage, kn negative control. The obtained result confirms that 1) the antibodies are capable of recognizing and specifically binding the murine VEGF-C protein and the human VEGF-C protein, and 2) the antibodies can be used to detect VEGF-C in both murine samples and human material.

Antigen binding by selected soluble anti-VEGF-C scFv antibodies is illustrated in Figure 2. The analysis was performed using an ELISA assay. Expression of anti-VEGF-C antibodies was obtained in E. coli Rosetta blue bacteria. The antibodies were isolated from the periplasmic area of the bacteria. The antibodies were bound to the antigen immobilized on the ELISA plate. Detection of antigen-bound scFvs was made using a solution of L-HRP (horseradish peroxidase-labeled L protein) as it binds to the scFv light chain. kn denotes an antibody not specific for VEGF-C. The obtained result confirms that not only the antibodies expressed on the surface of the selected phages are able to bind the VEGF-C protein, but also the corresponding soluble antibodies that were produced using the bacterial expression system based on the pETscFv vector.

The selected 1F2, 1F5 and 1F6 antibodies were tested for the specificity of their interaction with VEGF-C (Figure 3). The analysis was performed using an ELISA test. The plate was coated with VEGF-C, VEGF-A and VEGF-D proteins at a concentration of 2 pg / ml. All antibodies were diluted to a concentration of 5 µg / ml in PBS buffer containing 3% bovine albumin. Detection of antigen bound scFvs was accomplished with the L-HRP protein. K - control scFv antibodies specific for a protein other than VEGF-C. The obtained result confirms that the obtained scFv antibodies are specific only for the VEGF-C protein. The VEGF-C protein is very similar to other proteins of the VEGF family (for example, VEGF-A or VEGF-D), so these antibodies are a very valuable tool for the specific detection and selective binding of VEGF-C.

Antibody sequences (Figure 4) were determined by phagemid vector sequencing. As sequencing templates, pIT2 phagemids isolated from TOP10 bacteria, containing the sequences of individual selected antibodies, were used. The sequencing process used LMB3 (5 'CAG GAA ACA GCT ATG AC 3') and pHEN (5 'CTA TGC GGC CCC ATT CA 3') primers. The obtained sequences were analyzed with the use of programs available on the Mobyle @ Pasteur internet platform (revseq, transeq, needle), ClustalX and Jalview.

PL 236 281 Β1

Sequence e

Sequence No. 1a: 1D2_CDRH2 AIDSKGSFTV Sequence No. Ib:> 1D2_CDRH3 EGLRF

Sequence No. 1c:> 1D2_CDRL2 HASH

Sequence no. Ld:> 1D2_CDRL3 GPIDPQ

Sequence No. 2a:> 1E7_CDRH2 AISAQGTSTL

Sequence No. 2b:> 1E7_CDRH3 KYHMF

Sequence # 2c:> 1E7_CDRL2 HAST

Sequence # 2d:> 1E7_CDRL3 RRAMPT

Sequence # 3a:> 1F2_CDRH2 SIQAAGPTTD

Sequence # 3b:> 1F2_CDRH3 ADWEF

Sequence # 3c:> 1F2_CDRL2 GASV

Sequence # 3d:> 1F2_CDRL3 ALRVPS

PL 236 281 Β1

Sequence # 4a:> 1F5_CDRH2 SILDHGQGTQ

Sequence No. 4b:

> 1F5_CDRH3

EGTML

Sequence No. 4c:

> 1F5_CDRL2

RASR

Sequence # 4d:> 1F5_CDRL3

LRASPR

Sequence No. 5a:> 1F6_CDRH2

SITGHGSTTE

Sequence # 5b:> 1F6_CDRH3

EGAVF

Sequence # 5c:> 1F6_CDRL2

QASS

Sequence # 5d:> 1F6_CDRL3

TARYFD

Sequence number 6a:> 1F1O_CDRH2 SILDHGQGTQ Sequence number 6b:> 1F1O_CDRH3 EGTML

Sequence # 6c:> 1F1O_CDRL2 YASS

Sequence # 6d:> 1F1O_CDRL3 PSVGPK

PL 236 281 Β1

Sequence # 7a:> 1G2_CDRH2 DIQPKGTATN

Sequence # 7b:> 1G2_CDRH3 STNQF

Sequence # 7c:> 1G2_CDRL2 PASQ

Sequence # 7d:> 1G2_CDRL3 QFRNPV

Sequence No. 8a:

> 1G4_CDRH2

SIAAQGSPTA

Sequence No. 8b:

> 1G4_CDRH3

EGTNF

Sequence No. 8c:

> 1G4_CDRL2

RASR

Sequence No. 8d:

> 1G4_CDRL3

PASWPP

Sequence No. 9a:> 1G5_CDRH2 SITQGGTLTQ Sequence No. 9b:> 1G5_CDRH3 PGLAF

Sequence No. 9c:> 1G5_CDRL2 GASA

Sequence # 9d:> 1G5_CDRL3 TSRRPK

PL 236 281 Β1

Sequence No. 10a:> 1G6_CDRH2 TITDSGYQTL

Sequence No. 10b:

> 1G6_CDRE3

EALHF

Sequence No. 10c:> 1G6_CDRL2

HASR

Sequence No. 10 from:> 1G6_CDRL3 LPPSPK

Sequence No. 11a:> 1G8_CDRH2 SITHKGGLTQ

Sequence no. Llb:

> 1G8_CDRE3

EGAPF

Sequence no. Lic:> 1G8_CDRL2

AASS

Sequence no. Lid:> 1G8_CDRL3

SY3TPN

Sequence No. 12a:> 1G1O_CDRH2 TIALDGQRTN

Sequence No. 12b:

> 1G1O_CDRH3

HWAPF

Sequence No.12c:> 1G1O_CDRL2

TASS

Sequence No. 12d:

> 1G1O CDRL3

LAQFPP

PL 236 281 Β1

Sequence No. 13a:

> 2F10_CDRH2

HIMQSGYTTV

Sequence No. 13b:

> 2F10_CDRH3

YSPKF

Sequence No. 13c:

> 2F10_CDRL2

VASQ

Sequence No. 13d:

> 2F10_CDRL3

AQEPPS

Sequence No. 14:

> 1D2

MKYLLPTAAAGLLLIAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFS3YAMSWVR QAPGKGLEWVS AlDS KGS FTYYAD SVKGRFTIS DAM S KNT LYLQMNS L RAEDTAYYYCAK EGLRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYHASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQGPIDPQTFGQGTKVEIKRAAAHHHHHH

Sequence No. 15:

> 1E7

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAP GKGLEWYSAlS AQGT Ξ T L YAD SVKGRFT13 DAM SKNT LY LQMN3 L RAEDT AVYYCAK KYHMFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS QSIS3YLNWYQQKPGKAPKLLIYHASTLQSGVPSRFSGSG3GTDFTLTISSLQPEDFATY YCQQRRAMPTTFGQGTKVEIKRAAAHHHHHH

Sequence No. 16:

> 1F2

MKYLLPTAAAGLLLLAAOPAMAEYGLLESGGGLYOPGGSLRLSCAASGFTFSSYAMSWYR QAPGKGLEWVSSIQAAGPTTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK ADWEFDYWGQGTLWVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYGASVLQSGVPTRFSGSGSGTDFTLTISSLQPEDFATY YCQQALRVPSTFGQGTKVEIKRAAAHHHHHH

Sequence No. 17:

> 1F5

PL 236 281 Β1

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSSILDHGQGTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK EGTMLDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYRASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQLRASPRTFGQGTKVEIKRAAAHHHHHH

Sequence No. 18:

> 1F6

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLΞCAASGFTFSSYAMSWVR QAPGKGLEWVSSITGHGSTTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK EGAVFDYWGQGTLVTVSSGGGG3GGGGSGGGGSTDIQMTQSP33LSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYQASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQTARYPDTFGQGTKVETKRAAAHHHHHH

Sequence No. 19:

> 1F1O

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRL3CAASGFTFS3YAMSWYR QAPGKGLEWVSSILDHGQGTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK EGTMLDYWGQGTLVTVS3GGGG3GGGGSGGGGSTDIQMTQSP33LSASVGDRVTITCRAS 03Ι33ΥΕΝΜΥΰ2ΚΡ6ΚΑΡΚΕΕΙΥΥΑ33Ε036νΡ3ΕΡ36303ΘΤ0ΡΤΕΤΙ33Ε0ΡΕ0ΡΑΤΥ YCQQPSVGPKTFGQGTKVEIKRAAAHHHHHH

Sequence No. 20:

> 1G2

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSDIQPKGTATNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STNQFDYWGQGTLVTVSSGGGG3GGGGSGGGG3TDIQITQSP33L3ASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYPASQLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQQFRNPVTFGQGTKVEIKRAAAHHHHHH

Sequence No. 21:

> 1G4

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSSIAAQGSPTAYADSVKGRFTISRDNSKNTLYLQMNΞLRAEDTAVYYCAK EGTNFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS 03Ι33ΥΕΝΜΥ0ΩΚΡΟΚΑΡΚΕΕΙΥΡΑ3ΚΕ03θνΡ3ΕΚ3Ο303 & ΤΟΡΤΕΤΙ33Ε0ΡΕϋΕΑΤΥ YCQQPASWPPTFGQGTKVEIKRAAAHHHHHH

PL 236 281 Β1

Sequence No. 22:

> 1G5

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWYR QAPGKGLEWVS ΞITQGGT LTQYADSVKGRFTIS DAM S KNT LYLQMN 3 LRAEDTAVYYCAK PGLAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS ς3Ι33ΥΕΝΉΥ2ςΚΡ6ΚΑΡΚΕΕΙΥΰΑ3ΑΕς36νΡ3ΗΡ36363 & ΤΏΡΤΕΤΙ33Ε2ΡΕΏΕΑΤΥ YCQQTSRRPKTFGQGTKVEIKRAAAHHHHHH

Sequence No. 23:

> 1G6

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVS TIT FOR GYQT LYADSVKGRFTIS DAM S KNT LYLQMN S LRAEDTAVYYCAK EALHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRWITCRAS QSISSYLNWYQQKPGKAPKLLIYHASRLQSGVP3RFSGSGSGTDFTLTISSLQPEDFATY YCQQLPPSPKTFGQGTKVEIKRAAAHHHHHH

Sequence No. 24:

> 1G8

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSSITHKGGLTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK EGAPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS GSISSYLNWYGeKPGKAPKLLIYAASSLGSGYPSRFSGSGSGTDFTLTISSLGPEDFATY YCQQSYSTPNTFGQGTKVEIKRAAAHHHHHH

Sequence No. 25:

> 1G1O

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYANSWVR QAPGKGLEWVS TIALDGQRTNYADSVKGRFTIS DAM S S KNTLYLQMN LRAEDTAVYYCAK HWAPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTTSSLQPEDFATY YCQQLAQFPPTFGQGTKVEIKRAAAHHHHHH

Sequence No. 26:

> 2F10

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSHTMQSGYTTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK YSPKFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRWITCRAS QSISSYLNWYQQKPGKAPKLLIYVASQLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQAQEPPSTFGQGTKVEIKRAAAHHHHHH

Claims (8)

Patent claims 1. An antibody of the scFv format which is an inhibitor of a VEGF-C protein comprising an amino acid sequence selected from the following group: Seq. No.14, Seq. No.15, Seq. No.16, Seq. No. 17, Seq. No.18, Seq. No.19, Seq. No. 20, Seq. No.21, Seq. No.22, Seq. No. 23, Seq. No. 24, Seq. No.25, Seq. no 26. 2. A method for isolating VEGF-C from a solution, especially an aqueous solution, characterized by: (a) contacting an antibody according to claim 1; 1 with a solution containing the VEGF-C protein to obtain a protein complex comprising the antibody of claim 1, 1 and a non-covalently associated VEGF-C protein; (b) separating the resulting protein complex from the solution; (c) the VEGF-C protein is dissociated from the resulting protein complex; (d) the VEGF-C protein is isolated from the dissociated solution. 3. The method according to p. 2. A method according to claim 2, characterized in that in step (b) the isolation is performed by affinity chromatography, wherein the medium is a chromatographic medium containing an immobilized antibody according to claim 1. 1. 4. A method of detecting the presence of VEGF-C in biological material, characterized by the fact that: (a) contacting an antibody according to claim 1; 1 with biological material to obtain a protein complex containing the antibody according to claim 1, 1 and a non-covalently associated VEGF-C protein; (b) detecting the obtained protein complex by means of indirect or direct biochemical detection methods. 5. A method for determining the concentration of VEGF-C in an aqueous solution, in particular in physiological fluid, characterized by: (a) contacting an antibody according to claim 1; 1 with an aqueous solution containing VEGF-C to obtain a protein complex containing the antibody of claim 1, 1 and a non-covalently associated VEGF-C protein associated therewith; (b) the concentration of the resulting protein complex is determined by indirect or direct biochemical detection methods. 6. Use of an antibody according to claim 1 1 for the preparation of VEGF-C protein preparations. 7. The in vitro use of an antibody according to claim 1 1 for the detection of VEGF-C protein in biological material. 8. Use of an antibody according to claim 1 1 for determining the concentration of the VEGF-C protein in an aqueous solution.