DE102004050101A1 - Recombined polypeptide antibody or antibody fragment binds the ED-B domain of Fibronectin - Google Patents

Abstract

Novel recombined polypeptide which is an antibody or an antibody fragment, which binds the ED-B domain of Fibronectin, and inhibits the interaction between the ED-B domain and its receptors. Independent claims are also included for the diagnostic and pharmaceutical applications of the recombinant polypeptide. The polypeptide has an affinity for the ED-B domain of K(D value equal to or less than 1 nM. ACTIVITY : Cytostatic. No biological data given. MECHANISM OF ACTION : Antibody therapy.

Description

The The invention relates to recombinant polypeptides, in particular antibodies or Antibody fragments that bind to the ED-B isoform of fibronectin and block its function can. Furthermore, the diagnostic and pharmaceutical application of polypeptides of the invention disclosed.

During the Tumorigenesis and progression becomes the extracellular matrix (ECM), in which the tumor grows, rebuilt by proteolytic degradation of existing ECM. This process generates a tumor-induced matrix differs from that occurring in normal tissue ECM. The tumor-induced ECM seems to be the optimal environment for tumor growth and growth Tumor angiogenesis (1-4).

at Tumor angiogenesis forms new blood vessels from existing ones Vessels. This process implements the proteolysis of ECM, the targeted growth and differentiation of endothelial cells into new vascular structures which is essential for the further tumor growth are (5).

fibronectins are an important class of matrix glycoproteins. Your main role It is the attachment of cells to a variety of different extracellular To allow matrices. The presence of fibronectins on the surface of non-transformed cells in culture as well as their absence transformed cells for the identification of fibronectins as important adhesion proteins. she interact with many different other molecules, e.g. Collagen, heparan sulfate proteoglycans and fibrin, and regulate hence the cell shape and the structure of the cytoskeleton. Furthermore are she is responsible for Cell migration and cell differentiation during embryogenesis. she are as well important for Wound healing, during which they migrate from macrophages and others Enable immune cells in the affected area, and in the formation clots by attaching platelets damaged Regions of the blood vessels allow.

fibronectins are dimers of two similar Peptides, each chain being approximately 60-70 nm is long. At least 20 different fibronectin chains are all of which have been identified by alternative splicing of the RNA transcripts of a single fibronectin gene are generated.

fibronectins are high molecular weight, adhesion promoting Glycoproteins, which play an important role in the development of the blood vessel system play. Furthermore, fibronectins act chemotactically on endothelial cells, modulate the effect of growth factors and support that linear growth of endothelial cells during angiogenesis (6-9). An analysis of proteolytic fibronectin cleavage products shows that the polypeptides consist of six strongly folded domains, each of which domain contains repetitions called "repeats" whose similarities in terms of their amino acid sequence allow classification into three types (Type I, II, III). The central region of both chains from the dimer consists of a section of so-called type III repeats, which average 90 amino acids in length are (10). Structural studies have shown that every type III repeat from seven beta strands which are folded into two antiparallel leaflets, with short ones Loop regions are exposed as potential protein-protein interaction sites (11).

The Enable type III repeats that fibronectins act as adhesion molecules, those with cell surface molecules, the so-called "integrins" interact The term integrin was first used in 1987 in (12) to to describe a related group of heterodimeric cell surface molecules, as mediators between the extracellular matrix and the intracellular cytoskeleton and thus cell adhesion and induce migration. These heterodimeric receptors "integrate" or mediate Signals from the extracellular Environment with specific cellular Functions. To date, 17 beta subunits are known that with more than 20 alpha subunits interact specifically and noncovalently can, um to form more than 20 different families (13). Especially mediates the sequence RGDS, which in the tenth repeat of the type III of fibronectin (III-10), the interaction of fibronectin with at least 8 different integrations. In addition, it was shown that at least four integrins in an RGDS-independent manner specific to Fibronectin can interact (13). The group of type III repeat sequences comprises in addition to the III7, III8, III9 and III10 sequences also the repeats EIIIB and EIIIA (ED-B and ED-A).

The ED-B fibronectin is present in normal adult tissue (only exception: proliferating Endometrium) is undetectable while being highly expressed in fetal tissue and tumor growth, in addition to stromal local expression of ED-B. In addition, ED-B fibronectin localizes perivascularly to blood vessels that have newly formed during angiogenesis. For this reason, ED-B fibronectin is a specific marker protein for the process of (tumor) angiogenesis (14).

The ED-B domain is a highly conserved, complete type III homology building block consisting of 91 amino acids. Of the Homology between humans and rats is 100%, between humans and rats Chicken 96%. about the function of the ED-B domain is very little known in the literature. A few publications (15-17) speculate about a general adhesive agent Effect for different cells. A specific effect on endothelial cells has not been shown yet.

In WO 02/20563 has demonstrated that recombinant ED-B has a specific pro-angiogenic Effect in vitro shows: (i) the proliferation of bFGF-stimulated human dermal microvascular Endothelial cells (HDMVEc) are amplified by recombinant ED-B, (ii) the protein mediates the adhesion of HDMVEC and (iii) recombinant ED-B stimulates the invasion and differentiation (tube formation) of HDMVEc in collagen gels.

Furthermore, these ED-B-mediated effects could be specifically blocked by synthetic peptides derived from the ED-B domain. The peptide sequences thus represent the binding region for an ED-B specific receptor on the endothelial cell surface, which was identified by affinity chromatography as the α ₂ β ₁ integrin. The specific interaction between the α ₂ β ₁ integrin and the ED-B domain has not previously been described in the literature.

Further, WO 02/20563 discloses proteins which are specifically regulated by the ED-B fibronectin domain and which comprise the α ₂ β ₁ integrin, the focal adhesion kinase, the CD6 ligand (ALCAM), the alpha chain of the vitronectin receptor, comprises the integrated alpha 8 subunit or the precursor of the follistatin-related protein.

A Variety of integrin receptors with partially overlapping properties are expressed by endothelial cells (Lit .: DG Stupack and DA Cheresh, SciSTKE, 2002 Feb 12; 2002). These expression patterns show that various integrins (e.g., alphavbeta3 and alpha5beta1) are similar biological phenomena (Adhesion, Migration and survival) mediate and therefore represent redundant systems for the endothelial cell, their behavior and survival to secure. So far, it was known that alpha2beta1 with its natural Ligands, the collagens, interacts. The blockade of this interaction can lead to an anti-angiogenic effect (Y Funahashi et al., Cancer Res. 62: 6116-6123, 2002).

An object of the present invention was therefore to provide functionally-blocking binding molecules, such as antibodies, which specifically block receptor binding sites of the ED-B domain. These binding molecules have an anti-angiogenic effect on (tumor) endothelial cells. In contrast to the relatively broadly expressed α ₂ β ₁ integrin, the ED-B domain is an ideal and specific target molecule for such binding molecules.

The Structure of ED-B makes the development of monoclonal and polyclonal antibodies difficult as ED-B is expected to have low immunogenicity in vivo Has. This is how the antibody reacts BC-1 (J. Cell Biol. 108 (1989), 1139-1148) with a cryptic Epitope of fibronectin present only in the presence of ED-B and therefore not directly with ED-B. The antibody L19 (Tarli et al., Blood 94 (1999), 192-198 and WO 01/62800) obtained by using recombinant ED-B as Immunogen, again is biologically inactive, i. he can cell adhesion at ED-B does not know significantly.

Surprisingly it has now been found that functionally blocking ED-B binding molecules are produced can be such as the ED-B function blocking antibody MOR03255, which is the adhesion of cells at ED-B strongly inhibit and in vivo a significant reduction of tumor growth. The caused by the binding molecules according to the invention Blockade of ED-B obviously can not be compensatory Mechanisms of collagen-integrin interaction are balanced.

By means of the HuCAL ^® GOLD antibody library, a library of about 1.6 x 10E10 different antibodies in the Fab fragment format, starting from the HuCAL consensus sequences (WO 97/08320;, Knappik A, Ge L, Honegger A, pack P Fischer M, Wellnhofer G, Hoess A, Wolle J, Plunckthun A, Biol. 296: 57-86) was generated by diversification according to the diversity of human antibodies in all six CDR regions and by CysDisplay (WO 01/05950), a variant of the phage display method, was screened, in the experiments leading to the present invention functionally blocking and selectively binding to the ED-B domain Fab antibody fragments were identified. The efficacy of these antibody fragments could be demonstrated in an in vitro adhesion test, which reflects the specific interaction between recombinant ED-B and isolated HDMVEc. Through targeted modification of the binding molecules, the binding affinity to the ED-B domain could be significantly improved. Furthermore, it could be shown in vivo that the binding molecules in an animal model inhibit the growth of tumors.

• (i) spezifisch an die ED-B-Domäne von Fibronektin bindet und
• (ii) die Interaktion zwischen der ED-B-Domäne und ihrem Rezeptor inhibiert.

A first aspect of the invention is thus a polypeptide which

• (i) specifically binds to the ED-B domain of fibronectin and
• (ii) inhibits the interaction between the ED-B domain and its receptor.

The polypeptide of the invention is preferably an antibody or antibody fragment. The term "antibody" for the purposes of the present invention includes polyclonal, monoclonal, chimeric, humanized or human antibodies as well as recombinant antibodies, for example single-chain antibodies, or antigen-binding antibody fragments, for example monovalent antibody fragments such as Fab fragments or scFv fragments, or divalent ones Antibody fragments such as F (ab ') ₂ fragments.

essential to the invention It is in this context that the antibodies have one or more antigen-binding sites containing the o.g. Conditions, i. specific binding to the ED-B domain and Inhibition of the interaction between the ED-B domain and its receptor, in particular their receptor on endothelial cells. These antigen binding properties are preferably achieved by combining a VH and VL region, these regions consist of so-called framework regions (FR1, FR2, FR3 and FR4) as well as the antigen binding CDR regions (H-CDR1, H-CDR2, H-CDR3 for the VH region and L-CDR1, L-CDR2, L-CDR3 for the VL region) are constructed.

The polypeptide of the invention preferably has an affinity for the ED-B domain corresponding to a K _D value of ≦ 1 μM, preferably ≦ 100 nM, more preferably ≦ 10 nM, even more preferably ≦ 1 nM, and most preferably ≦ 0.1 nM wherein the affinity may be as indicated in the examples is determined by a test on the BIAcore ^® system.

The polypeptide of the invention indicates that it binds specifically to the ED-B domain of fibronectin, i.e. it binds with a significantly lower affinity to others fibronectin domains, in particular to the fibronectin domain 6 (FN6) or / and the fibronectin domains 7-8-9 (7-8-9). In a preferred embodiment, the polypeptide of the invention binds and the fibronectin ED-B domain with at least the factor 2, in particular by at least the factor 5 and more preferably by at least a factor of 10 higher affinity than FN6 or / and 7-8-9, such as by the binding assay given in the Examples can be determined.

In a further preferred embodiment shows the polypeptide of the invention in vitro an inhibition of adhesion of recombinant ED-B to HDMVEc cells, preferably one Inhibition of at least 50% and more preferably of at least 75% in a concentration of 10 μg / ml each.

Furthermore it is preferred that the polypeptide of the invention in vivo a Inhibition of growth by implantation of F9 teratocarcinoma cells (ATCC CRL-1720) in experimental animals such as nude mice Tumor shows.

• (a) eine VH-Region (i) kodiert von einer Nukleinsäuresequenz SEQ ID NO. 1 (MOR 02610), SEQ ID NO. 5 (MOR 02611), SEQ ID NO. 9 (MOR 02613), SEQ ID NO. 13 (MOR 02614), SEQ ID NO. 17 (MOR 02616), SEQ ID NO. 21 (MOR 02618), SEQ ID NO. 25 (MOR 02619), SEQ ID NO. 29 (MOR 02622), SEQ ID NO. 33 (MOR 02715), SEQ ID NO. 37 (MOR 02718), SEQ ID NO. 41 (MOR 02721), SEQ ID NO. 45 (MOR 02722) oder zumindest eine H-CDR1-, H-CDR-2- oder/und H-CDR3-Region einer der genannten VH-Regionen oder (ii) abgeleitet von einer VH-Region gemäß (i) durch Veränderung von zumindest einer H-CDR-Region oder/und
• (b) eine VL-Region (i) kodiert von einer Nukleinsäuresequenz SEQ ID NO. 3 (MOR 02610), SEQ ID NO. 7 (MOR 02611), SEQ ID NO. 11 (MOR 02613), SEQ ID NO. 15 (MOR 02614), SEQ ID NO. 19 (MOR 02616), SEQ ID NO. 23 (MOR 02618), SEQ ID NO. 27 (MOR 02619), SEQ ID NO. 31 (MOR 02622), SEQ ID NO. 35 (MOR 02715), SEQ ID NO. 39 (MOR 02718), SEQ ID NO. 43 (MOR 02721), SEQ ID NO. 47 (MOR 02722) oder zumindest eine L-CDR1-, L-CDR2- oder/und L-CDR3-Region einer der genannten VL-Regionen oder (ii) abgeleitet von einer VL-Region gemäß (i) durch Veränderung von zumindest einer L-CDR-Region.

The polypeptide of the invention is preferably selected from antibodies and antibody fragments comprising

• (a) a VH region (i) encodes a nucleic acid sequence SEQ ID NO. 1 (MOR 02610), SEQ ID NO. 5 (MOR 02611), SEQ ID NO. 9 (MOR 02613), SEQ ID NO. 13 (MOR 02614), SEQ ID NO. 17 (MOR 02616), SEQ ID NO. 21 (MOR 02618), SEQ ID NO. 25 (MOR 02619), SEQ ID NO. 29 (MOR 02622), SEQ ID NO. 33 (MOR 02715), SEQ ID NO. 37 (MOR 02718), SEQ ID NO. 41 (MOR 02721), SEQ ID NO. 45 (MOR 02722) or at least one H-CDR1, H-CDR-2 or / and H-CDR3 region of one of said VH regions or (ii) derived from a VH region according to (i) by altering at least one H-CDR region or / and
• (b) a VL region (i) encoded by a nucleic acid sequence SEQ ID NO. 3 (MOR 02610), SEQ ID NO. 7 (MOR 02611), SEQ ID NO. 11 (MOR 02613), SEQ ID NO. 15 (MOR 02614), SEQ ID NO. 19 (MOR 02616), SEQ ID NO. 23 (MOR 02618), SEQ ID NO. 27 (MOR 02619), SEQ ID NO. 31 (MOR 02622), SEQ ID NO. 35 (MOR 02715), SEQ ID NO. 39 (MOR 02718), SEQ ID NO. 43 (MOR 02721), SEQ ID NO. 47 (MOR 02722) or at least one L-CDR1, L-CDR2 and / or L-CDR3 region of one of said VL regions or (ii) derived from a VL region according to (i) by altering at least one L-CDR region.

Prefers are changes in the VL region the L-CDR3 region and / or in the VH region changes in the H-CDR2 region.

• (i) kodiert von der Nukleinsäuresequenz SEQ ID NO. 25 (MOR 02619) oder SEQ ID NO. 33 (MOR 02715) oder zumindest eine H-CDR1-, H-CDR-2- oder/und H-CDR3-Region einer der genannten VH-Regionen oder
• (ii) abgeleitet von einer VH-Region gemäß (i) durch Veränderung von zumindest einer H-CDR-Region.

For example, the polypeptide of the invention has a VL region derived from the VL region encoded by the nucleic acid sequence SEQ ID NO. 27 (MOR 02619) or SEQ ID NO. 35 (MOR 02715). Particularly preferred is a polypeptide comprising a VH region

• (i) encodes the nucleic acid sequence SEQ ID NO. 25 (MOR 02619) or SEQ ID NO. 33 (MOR 02715) or at least one H-CDR1, H-CDR-2 or / and H-CDR3 region of one of said VH regions or
• (ii) derived from a VH region according to (i) by altering at least one H-CDR region.

• (a) eine VH-Region (i) kodiert von der Nukleinsäuresequenz SEQ ID NO. 63 (MOR 03243), SEQ ID NO. 67 (MOR 03245), SEQ ID NO. 71 (MOR 03246), SEQ ID NO. 75 (MOR 03251), SEQ ID NO. 79 (MOR 03252), SEQ ID NO. 81 (MOR 03253), SEQ ID NO. 83 (MOR 03255), SEQ ID NO. 85 (MOR 03257), SEQ ID NO. 87 (MOR 03258) oder zumindest die H-CDR2-Region einer der genannten VH-Regionen oder (ii) abgeleitet von einer VH-Region gemäß (i) durch Veränderung von zumindest einer H-CDR-Region oder/und
• (b) eine VL-Region (i) kodiert von der Nukleinsäuresequenz SEQ ID NO. 65 (MOR 03243), SEQ ID NO. 69 (MOR 03245), SEQ ID NO. 73 (MOR 03246), SEQ ID NO. 77 (MOR 03251 sowie MOR03252, MOR03253, MOR03255 und MOR03257), SEQ ID NO. 89 (MOR 03258) oder zumindest die L-CDR1-, L-CDR2- oder L-CDR3- Region einer der genannten VL-Regionen oder (ii) abgeleitet von einer VL-Region gemäß (i) durch Veränderung von zumindest einer L-CDR-Region.

Preferably, the polypeptide has a VH region derived by altering the H-CDR2 region of a VH region encoded by the nucleic acid sequence of SEQ ID NO. 25 (MOR 02619) or SEQ ID NO. 33 (MOR 02715). Particularly preferred is a polypeptide comprising

• (a) a VH region (i) encoded by the nucleic acid sequence SEQ ID NO. 63 (MOR 03243), SEQ ID NO. 67 (MOR 03245), SEQ ID NO. 71 (MOR 03246), SEQ ID NO. 75 (MOR 03251), SEQ ID NO. 79 (MOR 03252), SEQ ID NO. 81 (MOR 03253), SEQ ID NO. 83 (MOR 03255), SEQ ID NO. 85 (MOR 03257), SEQ ID NO. 87 (MOR 03258) or at least the H-CDR2 region of one of said VH regions or (ii) derived from a VH region according to (i) by altering at least one H-CDR region and / or
• (b) a VL region (i) encoded by the nucleic acid sequence SEQ ID NO. 65 (MOR 03243), SEQ ID NO. 69 (MOR 03245), SEQ ID NO. 73 (MOR 03246), SEQ ID NO. 77 (MOR 03251 and MOR03252, MOR03253, MOR03255 and MOR03257), SEQ ID NO. 89 (MOR 03258) or at least the L-CDR1, L-CDR2 or L-CDR3 region of one of said VL regions or (ii) derived from a VL region according to (i) by altering at least one L-CDR3 region. CDR region.

Specific examples of polypeptide of the invention are as follows:
A polypeptide comprising the VH region encoded by SEQ ID NO. 1 and the VL region encoded by SEQ ID NO. 3 (MOR 02610) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 5 and the VL region encodes SEQ ID NO. 7 (MOR 02611) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 9 and the VL region encodes SEQ ID NO. 11 (MOR 02613) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 13 and the VL region encodes SEQ ID NO. 15 (MOR 02614) one of at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 17 and the VL region encodes SEQ ID NO. 19 (MOR 02616) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 21 and the VL region encodes SEQ ID NO. 23 (MOR 02618) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 27 (MOR 02619) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 29 and the VL region encodes SEQ ID NO. 31 (MOR 02622) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 33 and the VL region encoded by SEQ ID NO. 35 (MOR 02715) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 37 and the VL region encodes SEQ ID NO. 39 (MOR 02718) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 41 and the VL region encodes SEQ ID NO. 43 (MOR 02721) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 45 and the VL region encodes SEQ ID NO. 47 (MOR 02722) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 49 (MOR 03055) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 51 (MOR 03066) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 53 (MOR 03075) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 55 (MOR 03069) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 57 (MOR 03071) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 33 and the VL region encodes SEQ ID NO. 59 (MOR 03064) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 33 and the VL region encodes SEQ ID NO. 61 (MOR 03062) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 63 and the VL region encodes SEQ ID NO. 65 (MOR 03243) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 67 and the VL region encodes SEQ ID NO. 69 (MOR 03245) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 71 and the VL region encodes SEQ ID NO. 73 (MOR 03246) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 75 and the VL region encodes SEQ ID NO. 77 (MOR 03251) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 79 and the VL region encodes SEQ ID NO. 77 (MOR 03252) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 81 and the VL region encodes SEQ ID NO. 77 (MOR 03253) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 83 and the VR region encodes SEQ ID NO. 77 (MOR 03255) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 85 and the VR region encodes SEQ ID NO. 77 (MOR 03257) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

One A polypeptide comprising the VH region encoded by SEQ ID NO. 87 and the VR region encodes SEQ ID NO. 89 (MOR 03258) or at least an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof.

Of the Construction of the VH and VR chains of the polypeptides according to the invention is as follows:

VH chain:

• - The Framework 1 region ranges from nt 1-78 (ie 26 aa), the last codon (aa) of FR1 is always: TCC (Cys).
• - The "CDR1 region" can be two different lengths either 30 nt (10 aa) (possible in families VH1A, VH1B, 3, 4 and 5); or 36 nt (12 aa) (possible in families VH2, VH4 and VH6).
• - The Framework 2 region always has 33 nt (ie 11 aa), the first one Codon (aa) of FR2 is always: TGG (Trp), the last two codons are always CTC.GAG (LeuGlu).
• - The "CDR2 region" can be three different lengths either 57 nt (19 aa) (possible in families VH2 and VH4); 60 nt (20 aa) (possible in families VH1A, VH1B, 3 and 5) or 63 nt (21 aa) (possible in family VH6).
• - The Framework 3 region always has 96 nt (ie 32 aa), the third one Codon (aa) of FR3 is always: ACC (Thr), the last five codons are always TAT.TAT.TGC.GCG.CGT (Tyr Tyr Cys Ala Arg).
• - The "CDR3 region" can be several different lengths have, from 12-69 nt (4-23 aa) (in all families VH2 and VH4); 60 nt (20 aa) (possible in families VH1A, VH1B, 3 and 5) or 63 nt (21 aa) (possible in Family VH6).
• - The Framework 4 region always has 33 nt (ie 11 aa) and is in all 7 families identical: TGG.GGC.CAA.GGC.ACC.CTG.GTG.ACG.GTT.AGC.TCA

VL kappa chain:

• - The Framework 1 region ranges from nt 1-69 (ie 23 aa), the last one Codon (aa) of FR1 is always: TGC (Cys).
• - The "CDR1 region" can be four different lengths either 24 nt (8 aa) (possible in families Vk1 and Vk3); 27 nt (9 aa) (possible in family Vk3); 39 nt (13 aa) (possible in family Vk2); or 42 nt (14 aa) (possible in family Vk4); first codon (aa) always AGA (Arg), penultimate codon (aa) of CDR1 region always: CTG (Leu).
• - The Framework 2 region always has 33 nt (ie 11 aa), the first ones both codons (aa) of FR2 are always: TGG.TAC (Trp Tyr), the penultimate one Codon is always CCG (Pro).
• The "CDR2 region" always has 33 nt (ie 11 aa), with the first three codons (aa) always CTA.TTA.ATT (Leu Leu Ile) are.
• - The Framework 3 region always has 96 nt (ie 32 aa), the first ones three codons (aa) of FR3 are always: GGG.GTC.CCG (Gly Val Pro), the last three codons are always TAT.TAT.TGC (Tyr Tyr Cys).
• - The "CDR3 region" always has 24 nt (ie 8 aa), where the second codon (aa) is always CAG (Gln).
• - The Framework 4 region always has 39 nt (ie 13 aa) and is in all identical to four families: ACC.TTT.GGC.CAG.GGT.ACG.AAA.GTT.GAA.ATT.AAA.CGT.ACG.

VL lambda chain:

• - The Framework 1 region ranges from nt 1-66 (ie 22 aa), the last one Codon (aa) of FR1 is always: TGT (Cys).
• - The "CDR1 region" can be three different lengths either 33 nt (11 aa) (possible in family VI3); 39 nt (13aa) (possible in family VI1); or 42 nt (14 aa) (possible in families VI1 and VI2).
• - The Framework 2 region always has 33 nt (ie 11 aa), the first ones both codons (aa) of FR2 are always: TGG.TAC (Trp Tyr), the third last Codon is always GCG (Ala).
• - The "CDR2 region" always has 33 nt (ie 11 aa), where the third codon (aa) is always ATT (Ile), and the last three codons are always CGT.CCC.TCA (Arg Pro Ser).
• - The Framework 3 region always has 96 nt (ie 32 aa), where the first codon (aa) of FR3 is always: GGC (Gly) and the last four Codons are always GAT.TAT.TAT.TGC (Asp Tyr Tyr Cys).
• - The "CDR3 region" can be three different Have long, either 24 nt (8 aa); 27 nt (9 aa); or 30 nt (10aa).
• - The Framework 4 region always has 39 nt (ie 13 aa) and is in all 3 families identical: GTG.TTT.GGC.GGC.GGC.ACG.AAG.TTA.ACC.GTT.CTT.GGC.CAG.

The polypeptide of the invention can for therapeutic or diagnostic purposes, e.g. for in vitro or in vivo Diagnosis can be used.

For therapeutic Applications, the polypeptide of the invention in the form of a Conjugate with a therapeutic agent, for example selected from Radio or chemotherapeutic agents, e.g. low molecular weight or biological cytostatic or cytotoxic agents present. The conjugation of therapeutic agent to the polypeptide may be according to known Methods, preferably over a covalent coupling to reactive amino, carboxy, hydroxy or / and Thiol groups of the protein, optionally using homo- or heterobifunctional linkers, according to known methods.

In addition, the polypeptide may also be in the form of a fusion protein which, in addition to the antibody, eg in the form of an IgG molecule or a fragment thereof, contains a cytokine fused thereto, eg IL2, II12 or TNFα polypeptide. Furthermore, the fusion protein may be present in the form of a bispecific antibody, wherein, in addition to the binding to the ED-B domain, binding to a further antigen is preferred. Further antibody specificities of bispecific antibodies are binding domains against chelators for diagnostically or / and therapeutically relevant radionuclides, eg α, β or γ emitters such as ⁹⁰ Y, diagnostic NIR (near infrared) dyes, therapeutically active dyes, surface molecules on immunological effector cells (eg NK cells, cytotoxic T cells or NKT cells), angiogenesis-relevant integrins, in particular binding domains which block their function (eg α ₁ , β ₃ , α ₁ β ₃ , α ₂ β ₁ , α ₂ β ₂ ), inactivating anti-VEGF binding domains and inactivating binding domains against VEGF Receptors 1, 2 and 3.

For diagnostic Applications may include the polypeptide in the form of a conjugate of a diagnostically detectable Labeling group, e.g. a marker group for an in in vitro or in vivo diagnostics. Examples of marking groups are radioactive label groups, NMR, dye, enzyme and Fluorescence (e.g., near-infrared fluorescence) label groups.

For therapeutic applications, the polypeptide is preferably formulated as a pharmaceutical composition containing as active ingredient the polypeptide of the invention, optionally further active ingredients and pharmacologically conventional carriers, excipients and / or diluents. The pharmaceutical composition contains the active ingredient in a therapeutically effective dose which can be readily determined by a person skilled in the art by in vitro experiments, for example on suitable cell cultures, or in animal models. The administration of the composition is preferably by injection or infusion, but other modes of administration are contemplated. Preferably, an intravenous and / or subcutaneous administration takes place. The dose of the drug administered depends on the nature and severity of the disease, as well as the condition of the patient being treated. Preferably, the therapeutic composition is administered several times over a longer period of, for example, at least 2-4 weeks. It In this connection, reference is made to known methods for the administration of antibodies or antibody conjugates, such as in Ferarra, N. et al., Nature Reviews Drug Discovery, Volume 3, May 2004, 391-400 and Salgaller, ML, Current Opinion in Molecular Therapeutics 2003 5 (6): 657-667, or referred to existing pharmaceutical antibody administration protocols such as rituximab, CAMPATH, Remicade, etc.

One Another object of the invention is a diagnostic composition, comprising as diagnostic reagent a polypeptide according to the invention. In addition, the diagnostic composition may contain other diagnostically common reagents, Carrier, Excipients and / or diluents contain. The diagnostic composition contains the polypeptide in an amount sufficient to provide diagnostic evidence in the particular format, e.g. in an in vivo or in vitro diagnostic format to enable. It In this connection, reference is made to known methods for in vivo and in vitro diagnosis using labeled antibodies.

The Pharmaceutical and diagnostic composition can be used for Therapy and diagnosis of all diseases associated with ED-B expression, for example, a stromal and / or perivascular ED-B expression accompanied. examples for such diseases are hyperproliferative diseases, e.g. diseases associated with angiogenesis, especially cancer, ocular fundus diseases, hypertrophic scars, etc., disorders with a malfunction of myofibroblasts, e.g. Endometriosis, arteriosclerotic Plaques etc. or inflammatory Diseases, e.g. Psoriasis, Crohn's disease, rheumatoid arthritis or multiple sclerosis.

The Pharmaceutical according to the invention or diagnostic composition may be one or more effective Containing polypeptides, e.g. a combination of polypeptides that bind to different regions of the ED-B domain. The compositions are for use in human and veterinary medicine suitable.

One Another object of the invention is a nucleic acid, the for a polypeptide according to the invention or fusion polypeptide encoded. This nucleic acid can be single-stranded or double Be DNA or an RNA. The nucleic acid is preferably in operative shortcut with an expression control sequence expressing in a suitable host cell or a suitable host organism allows. The nucleic acid kan be present on a vector for introduction into a host cell or a host organism is suitable. The vector may, for example a prokaryotic vector suitable for introducing prokaryotic cells, e.g. a plasmid or bacteriophage. On the other hand, the Vector also a eukaryotic vector, suitable for introduction into eukaryotic host cells or host organisms, e.g. a plasmid, an artificial chromosome or a viral vector. suitable Vectors are known to those skilled in the art, for example, from Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, and Ausubel et al. (1989), Current Protocols in Molecular Biology, John Wiley and Sons, known.

Yet Another object of the invention is a cell, e.g. a prokaryotic cell or eukaryotic cells, such as a human cell treated with a nucleic acid according to the invention or a vector of the invention is transformed. Another object of the invention is a non-human organism, e.g. a transgenic animal with a nucleic acid according to the invention or a vector of the invention is transformed. The term "transformation" in the context of the present Invention includes all options for introducing foreign nucleic acids into a cell or a Including organism Transfection or infection.

The polypeptide of the invention can be obtained by culturing a cell according to the invention or a non-human cell according to the invention Organism is carried out under conditions involving expression of the polypeptide, and then the expressed polypeptide, e.g. from the cell, the culture medium, the organism or excretory products of the organism.

Farther the invention is intended by the following figures and examples explained become:

1 shows the schematic implementation of an inhibition test for the identification of functionally active antibodies.

Human endothelial cells derived from microvessels of the skin (human dermal microvessel endothelial cells, HDMVEc) were co-incubated with ED-B-specific Fab antibody fragments cubed. The number of bound cells is visualized by crystal violet staining and measured in the photometer. High color intensity means many adherent cells and no binding blocking antibody. Low color intensity means little adhesion and a binding blocking antibody.

2 Figure 14 shows a screening scheme for identifying ED-B fibronectin functional blocking of the antibody intermediate Fab fragment display.

In a first step is a recombinant panning procedure ED-B performed. The antibody fragments obtained in this way become a specificity test (ELISA), a functional adhesion test and subjected to an immunohistochemical examination. After that becomes the affinity who fulfills these tests antibody certainly. Subsequently In order to improve the affinity, changes in the CDR domains are performed Affinity maturation 1 a change the L-CDR3 domain and affinity maturation 2 a change the H-CDR2 domain means.

After this by affinity maturation obtained antibody The previously mentioned tests will be followed by the tested in vivo efficacy.

3 shows the results of a specificity assay in ELISA format with the antibody Fab fragments generated by panning with recombinant ED-B.

ED-B Binding to recombinant ED-B, 6-FN means binding to the recombinant fibronectin domain 6, domain 7-8-9 means binding to the recombinant fibronectin domains 7-8-9 (without ED-B) and domain 7-EDB-8-9 means binding to the recombinant fibronectin domains 7-8-9 with ED-B. The relationship of ED-B to 6-FN and 7-8-9, respectively, gives the specificity of the antibody tested. AP-39 means a covalent dimer of the biologically inactive anti-ED-B scFv antibody L 19th

4 shows the result of an adhesion test with the examined antibodies.

It became the inhibition of adhesion of HDMVEc cells to ED-B coated plates by the indicated Fab fragments at concentrations of 25 μg / ml and 0.4 μg / ml Fab, respectively examined. The values given are the result of a triple determination.

5 shows the immunohistochemical reaction pattern of function-blocking anti-ED-B-Fab fragments using the example of MOR 02616. In den 5A and 5C are the results with a negative control (negative HuCAL-Fab) and in the 5B and 5D the results with the antibody MOR 02616 on human neuroblastoma cell IRM xenografts ( 5A and 5B ) as well as murine F9 teratocarcinoma cells ( 5C and 5D ).

Cryo-sections of the cells (thickness: 10 μm) were air-dried and then fixed in ice-cold acetone for 10 min. The sections were then washed in PBS and incubated for 60 min with 2 μg / ml MOR 02616 or negative control at room temperature. As secondary antibody, a peroxidase-labeled polyclonal goat anti human F (ab) ₂ antiserum (diluted 1: 100 in PBS, Dianova) in combination with diaminobenzidine (Sigma Chemicals) was used as chromogenic substrate.

6 shows the results of a specificity assay in ELISA format with the maturation-optimized antibody Fab fragments.

The experimental procedure was as in 3 described.

7 shows the immunohistochemical reaction pattern of the maturation-optimized antibody Fab fragment MOR 03255 on cryostat sections of F9 teratocarcinoma cells ( 7A ) and SKMEL-28 human melanoma cell xenografts in the nude mouse ( 7B ).

The experimental procedure was as in 5 described.

8th shows the result of an adhesion test with the antibody Fab fragments according to the invention compared to the comparative antibodies L19 and Ly6 0.3.

The experiment was similar to in 4 described. The antibody concentrations were 1 μl / ml, 10 μl / ml and 20 μl / ml, respectively.

9 shows the stability of the antibody Fab fragments according to the invention after incubation for 4 h in human plasma or PBS at 37 ° C.

The immunoreactivity was in the ELISA test with concentrations ranging from 0.039 to 5 μg / ml certainly. As a 100% value, the signal was 0.62 μg / ml in human plasma defined without incubation.

10 shows the therapeutic efficacy of anti-ED-B-function blocking Fab antibody fragments using the example of MOR 03255.

11 shows the Nukleotidseqenzen coding for the VH and VL regions of the antibodies of the invention.

1. Material and methods

1.1 proteins and antibodies

Purified recombinant fibronectin domain ED-B (His) ₆ , fibronectin domain 6 (6FN), fibronectin domain 7-8-9 and domain 7-ED-B-8-9 were prepared according to standard procedures (eg Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press).

Mouse antibodies against Purified ED-B receptor were also purified by standard methods prepared by a mouse 50 micrograms purified receptor in Freund's Complete adjuvant was administered, 3 weeks later 25 μg purified Receptor in friend's Incomplete adjuvant were administered and another 3 weeks later all Applications in incomplete adjuvant administered intraperitoneally were. 14 days after the 3rd immunization was then blood on the Taken from retroorbital sinus.

plasma fibronectin was purchased from Sigma.

1.2 Selection of ED-B specific phages

Selection was for the HuCAL ^® Gold phage selection protocol performed (see HuCAL ^® Gold Manual Version 2). The HuCAL ^® Gold library has been divided into six framework-specific pools. The ED-B specific phages were enriched in recombinant protein immobilized in 96-well plates (Maxisorb, Nunc, Rochester, NY, USA) in three consecutive rounds of selection. For panning, A is the plate with 1 ug / well of ED-B (His ₆₎ (10 ug / ml in Ampuwa ^®) was coated for 2 h at 37 ° C. For panning B the plate with ED-B at the same concentration in PBS (pH 7.2 with 1 mM MgCl ₂ / 1mM CaCl ₂₎ was coated overnight at 4 ° C. Plates were blocked with 5% skim milk powder in PBS, 0.05% Tween 20 (Sigma, St. Louis, MO, USA) (blocking buffer) and incubating ^® with 1 x 10 ¹³ HuCAL GOLD phages were pre-incubated with one volume Blocking Buffer , For panning B, the phage were additionally pre-incubated with 0.5 μg / ml fibronectin domain 6 to reduce the selection of binding molecules specific for the conserved structure of a type 3 domain repeat of fibronectin. After incubation with the phage for 2 h at room temperature, the wells were washed 5x with PBS, 0.05% Tween20, and 5x with PBS. The remaining phages were eluted 20 mM dithiothreitol (DTT) in 10 mM Tris / HCl (pH 8.0) for 10 min at room temperature. This was followed by incubation with E. coli TG-1 (Stratagene, OD ₆₀₀ = 0.5) for infection. Thereafter, the wells were incubated with E. coli TG-1 as an additional elution step.

1.3 phagemid recovery, Phage amplification and purification

The HuCAL ^® Gold phage were amplified in 2 x TY medium with 34 ug / ml chloramphenicol and 1% glucose (2x TY-CG). After infection with a helper phage (VCSM13) at 37 ° C and an OD ₆₀₀ of about 0.5, centrifugation and resuspension in 2x TY-CG / 50 μg / ml kanamycin, the cells were induced with 0.25 mM IPTG and overnight 22 ° C cultivated. The phage were precipitated from the supernatant with polyethylene glycol (Ausubel et al., 1998, Current Protocols of Microbiology), resuspended in PBS, and used for subsequent rounds of selection.

1.4 Subcloning of selected Fab fragments and expression of soluble Fab fragments

The Fab encoding inserts of the selected HuCAL ^® Gold phages were in the Expressions subcloned vector pMORPHx9-FS. The plasmid DNA preparation of the selected HuCAL ^® Gold clones was digested with the restriction enzymes XbaII / EcoRI, the Fab-encoding insert was excised (ompA-VL and phoA-Fd). Fab molecules expressed in this vector contain two C-terminal labels (FLAG ^™ and Strep-tagII) for detection and purification.

1.5 Expression and purification of HuCAL ^® Gold antibodies in E. coli

The expression of Fab fragments encoded on pMORPHx9-FS in E. coli TG-1 F cells was carried out in shake flask cultures with 0.75 l 2xTY medium and 34 μg / ml chloramphenicol. After induction with 0.75 mM IPTG, the cells were cultured for 16 h at 30 ° C. Alternatively, Fab clones obtained from the second maturation pool 2 were induced with 0.1 mM IPTG and then cultured at 22 ° C. Periplasmic extracts of cell pellets were prepared by osmotic shock, and the Fab fragments were isolated by Strep-Tactin ^® chromatography (IBA, Göttingen, Germany). The apparent molecular weights were determined by size exclusion chromatography (SEC) with calibration standards. The concentrations were determined by UV spectrophotometry.

1.6 Identification of ED-B binding Fab fragments by ELISA

96-well Maxisorb ELISA plates were coated with 100 μl ED-B solution (10 μg / ml in coating buffer (PBS pH 7.4, 1 mM CaCl ₂ , 1 mM MgCl ₂ ) overnight at 4 ° C crude lysates or purified Fab molecules were added, non-binding Fab molecules were removed by washing five times with washing buffer (PBS pH 7.4, 0.05% Tween20) .The Fab fragments were removed by incubation with anti-human Fab. Antibody-peroxidase conjugates (Dianova), followed by development with a soluble peroxidase substrate (Roche) and measurement at 370 nm. The clones expressing for ED-B specific Fab molecules were compared by a positive ELISA signal on immobilized ED-B no or only a small signal at 6FN identified.

1.7 Determination of plasma stability under physiological temperature conditions by ELISA

The Coating with ED-B (2.5 μg / ml) was performed essentially as described above. The Fab fragments were used for 4 h at 37 ° C and a concentration of 50 μg / ml in human plasma (German Red Cross, lot 9985550). After incubation, the Fab molecules for the ELISA test on the concentrations of 5.0, 2.5, 1.25, 0.62, 0.31, 0.156, 0.078 and 0.039 μg / ml in PBS diluted with 1 bovine serum albumin to the linear range the signal intensity to determine. Functional Fab molecules were labeled with the anti-Flag M2 antibody (Sigma F3165) and a secondary one anti-mouse antibody-alkaline phosphatase conjugate, followed by development with Attophos (Roche) and by measurement determined at 535 nm.

1.8 HDMVEc cell culture and cell adhesion test

Human dermal microvascular endothelial cells (HDMVEc) isolated from juvenile foreskin were grown in EGM MV medium (Clonetics Inc.) with the addition of 10% fetal calf serum, 2 mM glutamine, 20 μg / ml heparin and 3 ng / ml bFGF in vials , coated with 0.1% gelatin (Sigma), cultured. For adhesion tests, cells that had no longer been cultured as passage 8 were used. ED-B (10 μg / ml) or plasma fibronectin (2.5 μg / ml) was incubated in PBS pH 7.4, 0.9 mM CaCl ₂ , 0.5 mM MgCl ₂ in 96-well ELISA plates overnight immobilized at 4 ° C and blocked with 1% BSA in PBS pH 7.4, 0.9 mM CaCl ₂ , 0.5 mM MgCl ₂ for 1 h at 37 ° C. The cells were labeled with 0.15 μg / ml calcein for 30 min at 37 ° C and washed once in adhesion medium (MCDB 131, 2 mM glutamine, 0.1% BSA, 20 μg / ml heparin). 1 × 10 ⁵ cells were incubated with the respectively indicated antibody concentration, diluted in adhesion medium (final volume 100 μl) for 20 min at 37 ° C. The cell suspension was added to the ED-B coated plates for 2 h at 37 ° C. After washing the cells twice in PBS pH 7.4, 0.9 mM CaCl ₂ , 0.5 mM MgCl ₂ , adherent cells were detected in a plate fluorimeter (excitation 485 nm, emission 530 nm).

to Control was (A) cell binding to ligand-coated plates without the addition of specific antibodies and (B) cell adhesion holes determined without ligand coating, the latter in the latter Control value obtained was defined as the minimum of cell adhesion in the test.

1.9 Affinity maturation of selected Fab molecules through gradual replacement of CDR cassettes

to increase the affinity and biological activity of selected ones Antibody Fragments CDR regions were detected by cassette mutagenesis using Trinucleotide-directed mutagenesis optimized.

For this purpose, Fab fragments from the expression vector pMORPHx9 were cloned into the phagemid vector pMORPH-23 using the EcoRI / XbaI restriction sites. To optimize the affinity of the selected Fab fragments, two consecutive maturation steps were performed for each maturation pool. In the first step, an antibody fragment phage library was generated in which the L-CDR3 region of the parent clones is varied by a repertoire of 7 x 10 ⁸ (pool 1) and 3.8 x 10 ⁸ (pool 2) single light chain CDR sequences has been. Affinity-enhanced derivatives from the first maturation step were then subjected to a second round of maturation by replacing the H-CDR-2 region with a library of diversified elements. In this way, two phage libraries were generated, each with 1 × 10 ⁸ individual clones.

The Affinitätsmaturierungsbibliotheken were generated by transformation into E. coli TOP10F. The phages were prepared as previously described. Selection for Fab fragments with improved affinity under stringent conditions for three rounds at the first maturity and for two rounds at the second Step performed.

1.10 Affinity Determination by Surface Plasmon Resonance (BIAcore ^® )

To determine the K _D values, monomer fractions (at least 80 monomer content as determined by analytical SEC; Superdex75, Amersham Pharmacia) of Fab fragments were used. F1 chips (Biacore, Sweden) were supplemented with approximately 200 RU ED-B (30 μg / ml, 10 mM acetate buffer, pH 4.0) and reference flow cells with an appropriate amount of human serum albumin (20 μg / ml, 10 mM acetate buffer , pH 4.5) using standard EDC-NHS amine coupling chemistry. The antigen density was reduced to about 100 RU for the Fab characterization of the second maturation. Regeneration was with 10 mM HCl. All kinetic measurements were performed in PBS buffer (136 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.76 mM KH ₂ PO ₄ pH 7.4) at a flow rate of 20 μl / min using a Fab concentration range of 1.5-500 nM. The injection time was 1 min each. All sensograms were evaluated using BIA evaluation software 3.1.

Abbreviations: EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, NHS: N-hydroxysuccinimide, RU: Resonance units

2 results

2.1 Preparation of antibodies against human ED-B

It Two different panning approaches were performed. The Coating with ED-B for The first approach (panning A) was made according to the biological Tests known conditions performed to ED-B in a conformation showcase, which the adhesion of HDMVEc cells allowed to the immobilized antigen. At the second panning (B) were the phages in addition with a surplus blocked at 6FN to avoid selection of binding molecules, which cross-react with the fibronectin domain 6.

A total of 23 different HuCAL Fab molecules were found from 1315 primary hits that meet the criteria of binding to ED-B and not to 6FN. Furthermore, the antibodies were tested for their ability to bind to a recombinantly expressed fibronectin comprising domains 7, 8 and 9 with and without the inserted ED-B domain in their natural position (see 3 ). All antibodies tested showed specific recognition for the 7-ED-B-8-9 construct, but did not react with the corresponding construct without the ED-B domain.

2.2 Functional characterization the ED-B specific binding molecules

The antibodies identified as specific in the ELISA were purified and tested for their ability to block the adhesion of HDMVEc cells to ED-B coated plates. It was the ad In the presence of ED-B specific Fab fragments, HDMVEc cells were tested in two concentrations as compared to a non-specific HuCAL control Fab molecule and mouse positive control sera.

Twelve of the 23 Fab molecules described in Section 2.1 showed inhibition of cell adhesion to immobilized ED-B. A representative selection of the six most potent Fab molecules is in 4 shown. At a concentration of 25 μg / ml, cell adhesion was inhibited by 50% to nearly 100%. At a concentration of 0.4 μg / ml only a few Fab molecules, eg MOR02610, MOR02616, MOR02715 and MOR02718 showed a reproducible inhibition of cell adhesion.

The functionally-blocking Fab antibody fragments were immunohistochemically characterized for their localization on F9 mouse teratocarcinoma cells and human IRM30 neuroblastoma xenograft cryosections. All antibodies tested showed vascular localization on both tissue sections but no staining of vessels for normal tissue (eg normal and sclerotic liver and skin). In FIG 5B and 5D a perivascular abluminal staining of vessels, proliferating endothelium and tumor stroma by MOR02616 is shown. An accumulation of ED-B in the vicinity of neovascular structures of exponentially growing tumors, but not on normal vessels, can be seen. No staining was visible with a negative control antibody to lysozyme at the same concentration. 5A and 5C ).

The properties of twelve selected binding molecules were determined by BIAcore analysis (see Table 1). The affinities (K _on ) ranged from 15 to 500 nM. The affinity of the bivalent L19 derivative AP39 (a biologically inactive reference antibody against ED-B) was determined to be 2.4 nM. Table 1 also shows the CDR sequences (L-CDR3 and H-CDR3 only) and the framework combinations. At least two independent measurements were made with Fab molecules from different approaches to protein expression and purification. The binding rate (K _on ) and the dissociation rate (K _off ) of the Fab molecules are given in separate columns.

Table 1

2.3 Affinity maturation of functionally blocking ED-B antibodies Optimization of the L-CDR3 and H-CDR2 regions

The Affinitätsmaturierungen the Fab molecules were carried out in two steps. First, the L-CDR3 sequence diversified and then H-CDR2 sequence of the obtained in the first step, improved Fab molecules exchanged.

Two affinity maturation libraries were generated for each step. The optimization was done in two separate pools. The L-CDR3 library 1 with a degree of diversity of 7 x 10 ⁸ elements contained the four starting Fab molecules MOR02610, MOR02616, MOR02619 and MOR02622. The L-CDR3 Bibiliothek 2 with a degree of diversity of 3.8 x 10 ⁸ elements contained only derivatives of MOR02715 and MOR02718. In each case, the Fab molecules were combined in groups according to their affinity and biological activity in the adhesion test.

Both Bibliotheken were during the selection procedure is kept separate. There were two panning strategies carried out. Panning I was performed with ED-B immobilized on Maxisorb plates for three rounds, as previously stated. Panning II was selected biotinylated ED-B in solution. The phage-antigen complex was streptavidin-coated Particles in the first round and Neutravidin plates in the two intercepted subsequent rounds. As part of the selection procedure stringent conditions were used.

The screening for optimized Fab molecules was carried out by determining the K _off values in the BIAcore system. A total of 11 derivatives of library 1 were characterized. The 5 highest affinity derivatives are given in Table 2. All improved clones were derivatives of MOR02619 with an up to 7-fold affinity enhancement. The clone MOR03075 found an affinity of 2.4 nM.

Out In the library 2, 2 derivatives of MOR02715 were analyzed in detail. For the Clone MOR03062 was found to be a 15-fold affinity enhancer, leaving a Monomeraffinität of 2.4 nM. The changes in the amino acid sequence the L-CDR3 region is shown in Tables 2a and 2b.

Table 2a

Table 2b

The specificity for ED-B (determined by ELISA, immunohistochemistry and cell adhesion test) All derivatives from the first maturity round were unchanged.

The Fab molecules shown in Tables 2a and 2b were selected for subsequent H-CDR2 maturation in two pools. In pool 1, five Fab molecules with a similar activity but different L-CDR3 regions were diversified into H-CDR2 (library size of 7x10 ⁷ elements). For pool 2 (bibliography size of 8.5 x 10 ⁷ elements), two Fab molecules were selected. The libraries were selected separately as described above. Increased affinity binding molecules were enriched in solution under stringent conditions by two rounds of panning.

Out Pool 1 became three Fab molecules selected. For the Derivatives MOR03243 and MOR03245 found affinities of about 0.7 nM. For the Derivative MOR03246 an affinity of 0.6 nM was found.

The H-CDR2 sequences of the corresponding clones and the affinity data are shown in Table 3a.

Table 3a

Also from pool 2, derivatives with improved properties could be selected become. Compared to the parent clones, the affinities of the Derivatives improved by a factor of 26 up to 250 times. The affinities of Clones MOR03255 and MOR03258 were determined to be 30 pM and 40 pM, respectively.

In Table 3b are H-CDR2 amino acid sequences of the selected clones from pool 2 and the corresponding affinity data shown.

Table 3b

2.4 Characterization of high-affinity function-blocking ED-B antibodies

To test whether the antibodies obtained by affinity maturation are still specific for ED-B, specificity tests were performed in ELISA format ( 6 ), immunohistochemical studies ( 7 ) and adhesion tests ( 8th ) carried out. In all three tests it was found that the specificity of the affinity-matured Fab molecules remained unchanged for ED-B. Out 8th It can also be seen that both the parent clones such as MOR 02715 and the clones produced by affinity maturation such as MOR 03062, MOR 03255, MOR 03064 and MOR 03259 have a significantly higher biological activity (adhesion inhibition) than the known antibody L19.

In addition, the thermal stability of Fab molecules in human plasma was tested. For this purpose, the Fab molecules were incubated with human plasma for 4 h at 37 ° C, without a significant loss of immunoreactivity determined by ELISA ( 9 ) was found.

All selected Fab molecules are able to detect the interaction of a receptor on human microvascular endothelial cells at ED-B on dose-dependent Way to block one property, the well-known ED-B antibody L19 does not have. Because the adhesion from HDMVEc to the extracellular Matrix one of the early Steps in neoangiogenesis is blocking this Operation by the binding molecules according to the invention a therapeutic Agent for inhibiting the formation of new vessels and inhibiting tumor growth represents.

2.5 In vivo efficacy

F9 teratocarcinoma cells (10 ⁶ / mouse in Matrigel with / without 100 μg Fab MOR 03255) were sc. Implanted into the flank of nude mice. After 3 days, the animals were treated with 100 μg MOR03255 / mouse iv for 7 consecutive days. Both treatment groups show growth inhibition of the tumor in the range of 42-64% compared to the solvent control. A summary of the test results is in 10 shown.

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It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (66)

Polypeptide, characterized in that it (i) specifically binds to the ED-B domain of fibronectin and (ii) inhibits the interaction between the ED-B domain and its receptor. Polypeptide according to claim 1, characterized in that that it is an antibody or antibody fragment. Polypeptide according to claim 1 or 2, characterized in that it has an affinity for the ED-B domain corresponding to a K _D value ≤ 100 nM. Polypeptide according to claim 3, characterized in that it has an affinity for the ED-B domain corresponding to a K _D value ≤ 10 nM. Polypeptide according to claim 3, characterized in that it has an affinity for the ED-B domain corresponding to a K _D value ≤ 1 nM. Polypeptide according to one of Claims 1 to 5, characterized that there is one with at least a factor of 5 higher specificity for the fibronectin ED-B domain as for the fibronectin domain 6 and / or the fibronectin domains 7-8-9. Polypeptide according to one of Claims 1 to 6, characterized that in vitro inhibits the adhesion of ED-B to HDMVEc cells, tumor cells and / or stromal cells shows. Polypeptide according to one of Claims 1 to 6, characterized that in vivo it inhibits the growth of one through implantation of tumor cells produced by F9 teratocarcinoma cells in experimental animals. Polypeptide according to one of claims 1 to 8, characterized in that it is selected from antibodies or antibody fragments comprising: (a) a VH region (i) encoded by a nucleic acid sequence SEQ ID NO. 1 (MOR 02610), SEQ ID NO. 5 (MOR 02611), SEQ ID NO. 9 (MOR 02613), SEQ ID NO. 13 (MOR 02614), SEQ ID NO. 17 (MOR 02616), SEQ ID NO. 21 (MOR 02618), SEQ ID NO. 25 (MOR 02619), SEQ ID NO. 29 (MOR 02622), SEQ ID NO. 33 (MOR 02715), SEQ ID NO. 37 (MOR 02718), SEQ ID NO. 41 (MOR 02721), SEQ ID NO. 45 (MOR 02722) or at least one H-CDR1, H-CDR-2 or / and H-CDR3 region from one of said VH regions or (ii) derived from a VH region according to (i) by alteration at least one H-CDR region and / or (b) one VL region (i) encoded by a nucleic acid sequence SEQ ID NO. 3 (MOR 02610), SEQ ID NO. 7 (MOR 02611), SEQ ID NO. 11 (MOR 02613), SEQ ID NO. 15 (MOR 02614), SEQ ID NO. 19 (MOR 02616), SEQ ID NO. 23 (MOR 02618), SEQ ID NO. 27 (MOR 02619), SEQ ID NO. 31 (MOR 02622), SEQ ID NO. 35 (MOR 02715), SEQ ID NO. 39 (MOR 02718), SEQ ID NO. 43 (MOR 02721), SEQ ID NO. 47 (MOR 02722) or at least one L-CDR1, L-CDR2 or / and L-CDR3 region of one of said VL regions or (ii) derived from a VL region according to (i) by altering at least one L-CDR region. Polypeptide according to Claim 9, characterized that has a VL region derived from a VL region according to (b) (i) through change the L-CDR3 region. Polypeptide according to claim 10, characterized that has a VL region derived from a VL region encodes the nucleic acid sequence SEQ ID NO. 27 (MOR 02619) or SEQ ID NO. 35 (MOR 02715). A polypeptide according to any one of claims 9 to 11 comprising (A) a VH region (i) encodes the nucleic acid sequence SEQ ID NO. 25 (MOR 02619) or SEQ ID NO. 33 (MOR 02715) or at least one H-CDR1, H-CDR-2 or / and H-CDR3 region of one of said VH regions or (ii) derived from a VH region according to (i) change of at least one H-CDR region and / or (b) a VL region (I) encodes the nucleic acid sequence SEQ ID NO. 49 (MOR 03055), SEQ ID NO. 51 (MOR 03066), SEQ ID NO. 53 (MOR 03075), SEQ ID NO. 55 (MOR 03069), SEQ ID NO. 57 (MOR 03071), SEQ ID NO. 59 (MOR 03064), SEQ ID NO. 61 (MOR 03062), or at least the L-CDR3 region of one of said VL regions or (Ii) derived from a VL region according to (i) by variation of at least one L-CDR region. Polypeptide according to one of Claims 9 to 12, characterized that it has a VH region derived from a VH region according to (a) (i) through change the H-CDR2 region. Polypeptide according to claim 13, characterized in that that it has a VH region derived from the VH region encodes the nucleic acid sequence SEQ ID NO. 25 (MOR 02619) or SEQ ID NO. 33 (MOR 02715). A polypeptide according to any one of claims 9 to 14 comprising (A) a VH region (i) encodes the nucleic acid sequence SEQ ID NO. 63 (MOR 03243), SEQ ID NO. 67 (MOR 03245), SEQ ID NO. 71 (MOR 03246), SEQ ID NO. 75 (MOR 03251), SEQ ID NO. 79 (MOR 03252), SEQ ID NO. 81 (MOR 03253), SEQ ID NO. 83 (MOR 03255), SEQ ID NO. 85 (MOR 03257), SEQ ID NO. 87 (MOR 03258) or at least the H-CDR2 region thereof or (ii) derived from a VH region according to (i) change of at least one H-CDR region and / or (b) a VL region (I) encodes the nucleic acid sequence SEQ ID NO. 65 (MOR 03243), SEQ ID NO. 69 (MOR 03245), SEQ ID NO. 73 (MOR 03246), SEQ ID NO. 77 (MOR 03251), SEQ ID NO. 89 (MOR 03258) or at least the L-CDR1, L-CDR2 or L-CDR3 region of one of said VL regions or (ii) derived from a VL region according to (i) change of at least one L-CDR region. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 1 and the VL region encoded by SEQ ID NO. 3 (MOR 02610) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 5 and the VL region encoded by SEQ ID NO. 7 (MOR 02611) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 9 and the VL region encoded by SEQ ID NO. 11 (MOR 02613) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2- or L-CDR3 region thereof. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 13 and the VL region encodes SEQ ID NO. 15 (MOR 02614) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14 comprising the VH region encoded by SEQ ID NO. 17 and the VL region encodes SEQ ID NO. 19 (MOR 02616) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 21 and the VL region encodes SEQ ID NO. 23 (MOR 02618) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 27 (MOR 02619) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 29 and the VL region encodes SEQ ID NO. 31 (MOR 02622) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 33 and the VL region encodes SEQ ID NO. 35 (MOR 02715) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 37 and the VL region encodes SEQ ID NO. 39 (MOR 02718) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 41 and the VL region encodes SEQ ID NO. 43 (MOR 02721) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 45 and the VL region encodes SEQ ID NO. 47 (MOR 02722) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 49 (MOR 03055) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 51 (MOR 03066) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 53 (MOR 03075) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 55 (MOR 03069) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 25 and the VL region encodes SEQ ID NO. 57 (MOR 03071) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 33 and the VL region encodes SEQ ID NO. 59 (MOR 03064) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 33 and the VL region encodes SEQ ID NO. 61 (MOR 03062) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14 comprising the VH region encoded by SEQ ID NO. 63 and the VR region encoded by SEQ ID NO. 65 (MOR 03243) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region thereof. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 67 and the VR region encoded by SEQ ID NO. 69 (MOR 03245) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 71 and the VL region encodes SEQ ID NO. 73 (MOR 03246) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 75 and the VL region encodes SEQ ID NO. 77 (MOR 03251) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 79 and the VL region encodes SEQ ID NO. 77 (MOR 03252) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 81 and the VL region encodes SEQ ID NO. 77 (MOR 03253) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 83 and the VL region encodes SEQ ID NO. 77 (MOR 03255) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 85 and the VL region encodes SEQ ID NO. 77 (MOR 03257) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. A polypeptide according to any one of claims 9 to 14, comprising VH region encoded by SEQ ID NO. 87 and the VL region encodes SEQ ID NO. 89 (MOR 03258) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 or L-CDR3 region from that. Polypeptide according to one of claims 1 to 43 in the form of a Conjugate with a therapeutic agent. Polypeptide according to Claim 44, characterized that the therapeutic agent is selected from radio and chemotherapeutic agents. Polypeptide according to one of claims 1 to 43 in the form of a Fusion protein. A polypeptide according to claim 46 as a fusion protein with a cytokine or as a bispecific antibody. Polypeptide according to one of claims 1 to 43 in the form of a Conjugate with a diagnostically detectable labeling group. Polypeptide according to Claim 46, characterized that the diagnostically detectable labeling group is selected from radio, NMR, dye, enzyme and fluorescent labeling groups. Pharmaceutical composition comprising as active ingredient a polypeptide according to any one of claims 1 to 47 and pharmacologically conventional carriers, excipients and / or diluents. A composition according to claim 50 for prevention or treatment of hyperproliferative disorders. A composition according to claim 50 or 61 for the prevention or treatment of cancer. Diagnostic composition comprising as a diagnostic Reagent a polypeptide according to any one of claims 1 to 43, 48 or 49. Composition according to claim 53 for the diagnosis of hyperproliferative diseases or a predisposition to it. Composition according to claim 53 or 54 for diagnosis Cancer of a predisposition from that. A composition according to any one of claims 50 to 55 for use in human medicine. Nucleic acid, the for a polypeptide according to any one of claims 1 to 43 or 46 to 47 coded. nucleic acid according to claim 57 in operative association with an expression control sequence. Vector, characterized in that it is a nucleic acid according to one of the claims 57 to 58 contains. Cell, characterized in that it is provided with a nucleic acid according to one of the claims 57 to 58 or a vector according to claim 59 is transformed. Non-human organism, characterized that he is using a nucleic acid according to one of the claims 57 to 58 or a vector according to claim 59 is transformed. A method for producing a polypeptide according to one of the claims 1 to 43, characterized in that one cell according to claim 60 or a non-human organism according to claim 61 under conditions cultured in which expression of the polypeptide takes place, and the expressed polypeptide wins. Use of a polypeptide according to any one of claims 1 to 47 for the manufacture of a medicament for the prevention or treatment of ED-B-associated diseases. Use according to claim 62 for the preparation of a Medicament for the prevention or treatment of cancer. Use of a polypeptide according to any one of claims 1 to 43, 48 or 49 for the preparation of a detection reagent for diagnosis of ED-B-associated diseases or a predisposition thereto. Use according to claim 65 for the preparation of a Detection reagent for the diagnosis of cancer or a predisposition to it.