CA2628552A1 - Identification and characterization of function-blocking anti-ed-b-fibronectin antibodies - Google Patents
Identification and characterization of function-blocking anti-ed-b-fibronectin antibodies Download PDFInfo
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Abstract
The invention relates to recombinant polypeptides, in particular antibodies or antibody fragments, which are able to bind the ED-B isoform of fibronectin and block the function thereof. Further diagnostic and pharmaceutical application of the polypeptides of the invention is also disclosed.
Description
Identification and Characterization of Function-Blocking Anti-ED-B-Fibronectin Antibodies Description This application claims the benefit of the filing date of U.S. Provisional Application Serial No. 60/697,565 filed July 11, 2005 which is incorporated by reference herein.
The invention relates to recombinant polypeptides, in particular antibodies or antibody fragments that bind to the ED-B-isoform of fibronectin and can block their function. In addition, the diagnostic and pharmaceutical application of the polypeptides according to the invention is disclosed.
During tumor development and progression, the extracellular matrix (ECM), in which the tumor grows, is modified by proteolytic degradation of already existing ECM.
This process generates a tumor-induced matrix, which is distinguished from that of the ECM
that occurs in normal tissue. The tumor-induced ECM appears to be the optimum environment for tumor growth and tumor angiogenesis (1-4).
In tumor angiogenesis, new blood vessels are formed from already existing vessels. This process requires the proteolysis of ECM, the targeted growth and differentiation of endothelial cells in new vascular structures, which are essential for further tumor growth (5).
Fibronectins are an important class of matrix-glycoproteins. Their main role consists in facilitating the adhesion of cells to a number of different extracellular matrices. The presence of fibronectins on the surface of non-transformed cells in culture as well as their absence in the case of transformed cells resulted in the identification of fibronectins as important adhesion proteins.
They interact with numerous other different molecules, e.g., collagen, heparan sulfate-proteoglycans and fibrin, and thus regulate the cell shape and the creation of the cytoskeleton. In addition, they are responsible for cell migration and cell differentiation during embryogenesis. In addition, they are important for wound healing, in which they make possible the migration of macrophages and other immune cells in the field in question and in the formation of blood clots by making possible the adhesion of blood platelets to damaged regions of the blood vessels.
Fibronectins are dimers of two similar peptides, whereby each chain is approximately 60-70 nm long. At least 20 different fibronectin chains have been identified, of which all are produced by alternative splicing of the RNA transcript of a single fibronectin gene.
Fibronectins are high-molecular, adhesion-mediating glycoproteins that play an important role in the development of the vascular system. In addition, fibronectins act chemotactically on endothelial cells, modulate the action of growth factors and support the linear growth of endothelial cells during angiogenesis (6-9). An analysis of proteolytic fibronectin-cleavage products shows that the polypeptides consist of six heavily folded domains of which each domain in turn contains so-called repetition sequences ("repeats") whose similarities with respect to their amino acid sequence allow a classification in three types (types I, II, and III). The central region of both chains of the dimer consists of a section of so-called type-III
repetitions, which on average are 90 amino acids long (10). Structural studies have revealed that each type-III
repetition consists of seven beta-strands, which are folded into two antiparallel folded sheets, whereby short loop regions are exposed as potential protein-protein-interaction sites (11).
These type-III repetitions make it possible for fibronectins to act as adhesion molecules that interact with cell surface molecules, the so-called "integrins." The term "integrin" was used for the first time in 1987 in (12) to describe a related group of heterodimeric cell surface molecules that act as mediators between the extracellular matrix and the intracellular cytoskeleton and thus induce cell adhesion and migration. These heterodimeric receptors "integrate" or mediate signals from the extracellular environment with specific cellular functions.
Up until now, 17 beta-subunits have been known that can interact specifically and non-covalently with more than 20 alpha-subunits, particularly to forrn as 20 different families (13).
The sequence RGDS, which is found in the tenth repetition of type III of the fibronectin (111-10), in particular mediates the interaction of fibronectin with at least 8 different integrins. Moreover, it was shown that at least four integrins can interact specifically with fibronectin in an RGDS-independent way (13). In addition to the 1117-, 1118-, 1119- and III10 sequences, the group of repetition sequences of type III also comprises the EIIIB and EIIIA (ED-B and ED-A) repetitions.
The ED-B-fibronectin cannot be detected in the normal tissue of adults (sole exception:
proliferating endometrium), while it is strongly expressed in fetal tissue and in tumor growth, in addition to a stromal local expression of ED-B. Moreover, ED-B-fibronectin is localized perivascularly around blood vessels that have newly formed during the 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 component that consists of 91 amino acids. The degree of homology between humans and rats is 100%, between humans and chickens 96%. In literature, very little is known on the function of the ED-B-domains. A few publications (15-17) speculate on a general adhesion-mediating action for various cells. A specific action on endothelial cells was still not shown to date.
In WO 02/20563, it was shown that recombinant ED-B shows a specific pro-angiogenic action in vitro: (i) the proliferation of bFGF-stimulated human dermal microvascular endothelial cells (HDMVEc) is enhanced 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.
In addition, these ED-B-mediated effects could be specifically blocked by synthetic peptides that were derived from the ED-B domains. The peptide sequences thus represent the binding region for an ED-B-specific receptor on the endothelial cell surface that was identified by means of affinity chromatography as the a2P I -integrin. The specific interaction between the aZ(31-integrin and the ED-B domain was not previously described in the literature.
Also disclosed in WO 02/20563 are proteins that are regulated specifically by the ED-B-fibronectin domain, which comprises the a2p I -integrin, the focal adhesion kinase, the CD6 ligand (ALCAM), the alpha chain of the vitronectin receptor, the integrated alpha-8 subunit or the precursor of the follistatin-related protein.
A number of integrin receptors with partially overlapping properties are expressed by endothelial cells (lit.: D. G. Stupack and D. A. Cheresh, SciSTKE, 2002 Feb 12; 2002). These expression patterns show that different integrins (e.g., alphavbeta3 and alpha5betal) mediate similar biological phenomena (adhesion, migration and survival) and therefore represent redundant systems for the endothelial cells that safeguard their behavior and survival. It was previously known that alpha2betal interacts with its natural ligands, the collagens. The blocking of this interaction can result in an anti-angiogenic action (Y. Funahashi et al. Cancer Res. 62:
6116-6123, 2002).
One object of this invention was therefore the preparation of function-blocking binding molecules, such as, e.g., antibodies that specifically block receptor binding sites of the ED-B-domains. These binding molecules have an anti-angiogenic action on (tumor) endothelial cells.
In contrast to relatively broadly expressed a2(3I -integrin, the ED-B domain represents an ideal and specific target molecule for such binding molecules.
The structure of ED-B makes the development of monoclonal and polyclonal antibodies difficult since it thus can be expected that ED-B has a low immunogenicity in vivo. The antibody BC-1 (J. Cell Biol. 108 (1989), 1139-1148) thus reacts with a cryptic epitope of fibronectin, which is present only in the presence of ED-B and therefore not directly with ED-B. Antibody L19 (Tarli et al. Blood 94 (1999), 192-198 and WO 01/62800), which was produced by use of recombinant ED-B as an immunogen, is in turn biologically inactive, i.e., it cannot recognize cell adhesion to ED-B to a significant extent.
Surprisingly enough, it was now found that function-blocking ED-B-binding molecules can be produced, such as, for example, the ED-B-function-blocking antibody MOR03255, which greatly inhibit the adhesion of cells to ED-B and bring about in vivo a significant reduction of the tumor growth. The blocking of ED-B produced by the binding molecules according to the invention obviously cannot be compensated for by compensatory mechanisms of collagen-integrin.
By means of the HuCAL -GOLD antibody library -- a library with, for example, 1.6 x 10E10 different antibodies in the Fab-fragment format, which was generated 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. and Virnekas, B. (2000) J.
Mol. Biol. 296:57-86) by diversification corresponding to the diversity of human antibodies in all six CDR areas and is closely examined by means of CysDisplay (WO 01/05950) of a variant of the phage display process -- function-blocking Fab-antibody fragments that bind selectively to the ED-B-domain were identified in the tests leading to this invention. The effectiveness of these antibody fragments could be shown in an in vitro adhesion test that reflects the specific interaction between recombinant ED-B and isolated HDMVEc. The binding affinity to the ED-B-domain could be considerably improved by a specific change in the binding molecules. It was also possible to show in vivo that the binding molecules in an animal model inhibit the growth of tumors.
A first aspect of the invention is thus a polypeptide that (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 according to the invention is preferably an antibody or antibody fragment. The term "antibody" in terms of this invention comprises polyclonal, monoclonal, chimera, humanized or human antibodies as well as recombinant antibodies, e.g., single-chain antibodies, or antigen-binding antibody fragments, e.g., monovalent antibody fragments, such as, for example, Fab fragments or scFv fragments, or divalent antibody fragments, such as, for example, F(ab')2 fragments.
In this connection, it is essential to the invention that the antibodies contain one or more antigen binding sites that meet the above-mentioned requirements, i.e., specific binding to the ED-B domain and inhibition of the interaction between the ED-B domain and its receptor, especially its receptor on endothelial cells. These antigen binding properties are preferably achieved by combination of a VH and VL region, whereby these regions are built up from the thus-mentioned skeleton regions (FR1, FR2, FR3 and FR4) as well as the CDR
regions that mediate the antigen bond (H-CDRI, H-CDR2, H-CDR3 for the VH region and L-CDR1, L-CDR2, L-CDR3 for the VL region).
The polypeptide according to the invention preferably has an affinity to the ED-B domain corresponding to a Kp value of 5 1 m, preferably <_ 100 nM, especially preferably < 10 nM, still more preferably <- 1 nM, and most preferably <- 0.1 nM, whereby the affinity can be determined as indicated in the examples by a test on the BIAcore system.
The polypeptide according to the invention is distinguished in that it specifically binds to the ED-B domain of fibronectin, e.g., it binds with a significantly lower affinity to other fibronectin domains, in particular to fibronectin domain 6 (FN6) and/or fibronectin domains 7-8-9 (7-8-9). In a preferred embodiment, the polypeptide according to the invention and the fibronectin ED-B domain bind with an affinity that is higher by at least a factor of 2-- in particular by at least a factor of 5, and especially preferably by at least a factor of 10 -- than that of FN6 and/or 7-8-9, as can be determined, for example, by the binding test that is indicated in the examples.
In another preferred embodiment, the polypeptide according to the invention shows in vitro an inhibition of the adhesion of recombinant ED-B to HDMVEc cells, preferably an inhibition of at least 50% and especially preferably of at least 75% in a concentration of 10 g/ml in each case.
Moreover, it is preferred that the polypeptide according to the invention shows in vivo an inhibition of the growth of a tumor that is produced by implantation of F9-teratocarcinoma cells (ATCC CRL- 1720) in test animals, for example hairless mice.
The polypeptide according to the invention is preferably selected from antibodies and antibody fragments that comprise (a) a VH region (i) Coded 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- and/or H-CDR3 region of one of the above-mentioned VH regions, or (ii) Derived from a VH region according to (i) by a change in at least one H-CDR region and/or (b) A VL region (i) Coded 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-CDR 1-, L-CDR2- and/or L-CDR3- region of one of the above-mentioned VL regions or (ii) Derived from a VL region according to (i) by a change iri at least one L-CDR region.
Changes in the L-CDR3 region in the VL region and/or changes of the H-CDR2 region in the VH region are preferred.
For example, the polypeptide according to the invention exhibits a VL region that is derived from the VL region and that is coded by the nucleic acid sequence SEQ
ID NO. 27 (MOR 02619) or SEQ ID NO. 35 (MOR 02715). Especially preferred is a polypeptide that comprises a VH region (i) Coded by 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 and/or H-CDR3 region of one of the above-mentioned VH regions, or (ii) Derived from a VH region according to (i) by a change in at least one H-CDR
region.
The polypeptide preferably exhibits a VH region that is derived by a change in the H-CDR2 region and is coded by a VH region from the nucleic acid sequence SEQ ID
NO. 25 (MOR 02619) or SEQ ID NO. 33 (MOR 02715). Especially preferred in this case is a polypeptide, comprising (a) A VH region (i) Coded 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 the above-mentioned VH regions or (ii) Derived from a VH region according to (i) by a change in at least one H-CDR region and/or (b) a VL region (i) Coded 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 as well as MOR 03252, MOR 03253, MOR 03255 and MOR 03257), SEQ ID NO. 89 (MOR 03258) or at least the L-CDRI, L-CDR2 or L-CDR3 region of one of the above-mentioned VL regions, or (ii) Derived from a VL region according to (i) by a change in at least one L-CDR region.
Specific examples of the polypeptide according to the invention are as follows:
A polypeptide that comprises the VH region that is coded by SEQ ID NO. I and the VL
region that is coded 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 that comprises the VH region that is coded by SEQ ID NO. 5 and the VL
region that is coded by SEQ ID NO. 7 (MOR 02611) or at least one H-CDR 1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 9 and the VL
region that is coded by SEQ ID NO. 11 (MOR 02613) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 13 and the VL
region that is coded by SEQ ID NO. 15 (MOR 02614) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 17 and the VL
region that is coded by SEQ ID NO. 19 (MOR 02616) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 21 and the VL
region that is coded by SEQ ID NO. 23 (MOR 02618) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 27 (MOR 02619) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 29 and the VL
region.that is coded by SEQ ID NO. 31 (MOR 02622) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 33 and the VL
region that is coded by SEQ ID NO. 35 (MOR 02715) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 37 and the VL
region that is coded by SEQ ID NO. 39 (MOR 02718) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 41 and the VL
region that is coded by SEQ ID NO. 43 (MOR 02721) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 45 and the VL
region that is coded by SEQ ID NO. 47 (MOR 02722) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof. .
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 49 (MOR 03055) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 51 (MOR 03066) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR 1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 53 (MOR 03075) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 55 (MOR 03069) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 57 (MOR 03071) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 33 and the VL
region that is coded by SEQ ID NO. 59 (MOR 03064) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 33 and the VL
region that is coded by SEQ ID NO. 61 (MOR 03062) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 63 and the VL
region that is coded by SEQ ID NO. 65 (MOR 03243) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 67 and the VL
region that is coded by SEQ ID NO. 69 (MOR 03245) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 71 and the VL
region that is coded by SEQ ID NO. 73 (MOR 03246) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 75 and the VL
region that is coded by SEQ ID NO. 77 (MOR 03251) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 79 and the VL
region that is coded by SEQ ID NO. 77 (MOR 03252) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 81 and the VL
region that is coded by SEQ ID NO. 77 (MOR 03253) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 83 and the VR
region that is coded by SEQ ID NO. 77 (MOR 03255) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 85 and the VR
region that is coded by SEQ ID NO. 77 (MOR 03257) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 87 and the VR
region that is coded by SEQ ID NO. 89 (MOR 03258) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
The creation of the VH and VR chains of the polypeptides according to the invention is as follows:
VH Chain:
- The 1-region framework extends from nt 1-78 (that is, 26 aa); the last codon (aa) of FR1 is always: TCC (Cys).
- The "CDRI region" can be of 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 V142, VH4 and VH6).
- The 2-region framework always exhibits 33 nt (that is, 11 aa); the first codon (aa) of FR2 is always: TGG (Trp); the last two codons are always CTC.GAG (LeuGlu).
- The "CDR2 region" can be of three different lengths; either 57 nt (19 aa) (possible in families VH2 and VH4), 60 nt (20 aa) (possible in families VH1A, VHI B, 3 and 5); or 63 nt (21 aa) (possible in family V146).
- The 3-region framework always exhibits 96 nt (that is, 32 aa); the third 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 of several different lengths: from 12-69 nt (4-23 aa) (in all families VH2 and VH4), 60 nt (20 aa) (possible in families VH 1 A, VH 1 B, 3 and 5), or 63 nt (21 aa) (possible in family VH6).
- The 4-region framework always exhibits 33 nt (that is, 11 aa) and is identical in all 7 families: TGG.GGC.CAA.GGC.ACC.CTG.GTG.ACG.GTT.AGC.TCA
VL kappa Chain:
- The 1-region framework extends from nt 1-69 (that is, 23 aa); the last codon (aa) of FRI
is always: TGC (Cys).
- The "CDRI region" can be of four different lengths: either 24 nt (8 aa) (possible in families Vkl and Vk3), 27 nt (9aa) (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 CDRI region always: CTG (Leu).
- The 2-region framework always exhibits 33 nt (that is, 1 I aa); the first two codons (aa) of FR2 are always: TGG.TAC (Trp Tyr); the penultimate codon is always CCG (Pro).
- The "CDR2 region" always exhibits 33 nt (that is, 1 l aa), whereby the first three codons (aa) are always CTA.TTA.ATT (Leu Leu Ile).
- The 3-region framework always exhibits 96 nt (that is, 32 aa); the first 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 exhibits 24 nt (that is, 8 aa), whereby the second codon (aa) is always CAG (Gin).
- The 4-region framework always exhibits 39 nt (that is, 13 aa) and is identical in all four families: ACC.TTT.GGC.CAG.GGT.ACG.AAA.GTT.GAA.ATT.AAA.CGT.ACG.
VL lambda Chain:
- The 1-region framework extends from nt 1-66 (that is, 22 aa); the last codon (aa) of FRI
is always: TGT (Cys).
- The "CDRI region" can exhibit three different lengths: either 33 nt (11 aa) (possible in family V13), 39 nt (13 aa) (possible in family VI1), or 42 nt (14 aa) (possible in families Vll and V12).
- The 2-region framework always exhibits 33 nt (that is, 11 aa); the first two codons (aa) of FR2 are always: TGG.TAC (Trp Tyr); the third last codon is always GCG (Ala).
- The "CDR2 region" always exhibits 33 nt (that is, 11 aa), whereby the third codon (aa) is always ATT (Ile), and the last three codons are always CGT.CCC.TCA (Arg Pro Ser).
- The 3-region framework always exhibits 96 nt (that is, 32 aa), whereby 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 exhibit three different lengths: either 24 nt (8 aa), 27 nt (9 aa), or 30 nt (10 aa).
- The 4-region framework always exhibits 39 nt (that is, 13 aa) and is identical in all 3 families: GTG.TTT.GGC.GGC.GGC.ACG.AAG.TTA.ACC. GTT.CTT. GGC.CAG.
For therapeutic or diagnostic purposes, e.g., for an in vitro or in vivo diagnosis, the polypeptide according to the invention can be used.
For therapeutic applications, the polypeptide according to the invention can be present in the form of a conjugate with a therapeutic active ingredient, for example selected from radiotherapy agents or chemotherapy agents, e.g., low-molecular or biological cytostatic or cytotoxic active ingredients. The conjugation of the therapeutic active ingredient on the polypeptide can be carried out according to known methods, preferably via a covalent coupling to reactive amino, carboxy, hydroxy and/or thiol groups of the protein, optionally with use of homo-or hetero-bifunctional linkers, according to known methods.
Moreover, the polypeptide can also be present in the form of a fusion protein, which, in addition to the antibody, e.g., in the form of an IgG molecule or a fragment thereof, contains a cytokine fused thereto, e.g., IL2,1112 or TNFa-polypeptide. In addition, the fusion protein can be present in the form of a bispecific antibody, whereby in addition to the binding to the ED-B
domain, a binding to another antigen is preferred. Other antibody specificities of bispecific antibodies are binding domains against chelating agents for diagnostically and therapeutically relevant radionuclides, e.g., a-,13- or r-emitters, such as, for example, 90Y, diagnostic NIR (near-infrared)-dyes, therapeutically effective dyes, surface molecules on immunological effector cells (e.g., NK cells, cytotoxic T cells, or NKT cells), angiogenesis-relevant integrins, in particular binding domains that block their function (e.g., (11,133, aI133, a2131, a2132), inactivating anti VEGF-binding domains and inactivating binding domains against VEGF receptors 1, 2 and 3.
For diagnostic applications, the polypeptide can be present in the form of a conjugate of a diagnostically detectable labeling group, e.g., a labeling group for an in vitro or an in vivo diagnosis. Examples of labeling groups are radioactive labeling groups, NMR-, dye-, enzyme-and fluorescence- (e.g., fluorescence in the near-infrared range) labeling groups.
For therapeutic applications, the polypeptide is preferably formulated as a pharmaceutical composition that as active ingredient contains the polypeptide according to the invention, optionally additional active ingredients as well as pharmacologically common vehicles, adjuvants and/or diluents. The pharmaceutical composition contains the active ingredient in a therapeutically effective dose that can be determined by one skilled in the art in a simple way by in vitro tests, for example on suitable cell cultures, or in animal models.
The administration of the composition is preferably carried out by injection or infusion, but also other types of administration are conceivable. An intravenous and/or subcutaneous administration is preferably carried out. The dose of the administered active ingredient depends on the type and the severity of the disease as well as the condition of the patient to be treated. The therapeutic composition is preferably administered several times over an extended period of, for example, at least 2-4 weeks. In this connection, reference is made to known processes for administering antibodies or antibody conjugates, as described in, for example, Ferarra, N. et al., Nature Reviews Drug Discovery, Volume 3, May 2004, 391-400 and Salgaller, M. L., Current Opinion in Molecular Therapeutics 2003 5(6): 657-667, or to existing administration procedures for pharmaceutical antibodies, such as Rituximab, CAMPATH, Remicade, etc.
Another subject of the invention is a diagnostic composition that comprises a polypeptide according to the invention as a diagnostic reagent. In addition, the diagnostic composition can still contain additional diagnostically common reagents, vehicles, adjuvants and/or diluents. The diagnostic composition contains the polypeptide in a sufficient amount to make possible diagnostic detection in the respective format, e.g., in an in vivo or in vitro diagnostic format. In this connection, reference is made to known processes for in vivo and in vitro diagnosis with use of labeled antibodies.
The pharmaceutical and diagnostic compositions can for therapy and diagnosis of all diseases that are accompanied by an ED-B expression, for example a stromal and/or perivascular ED-B expression. Examples of such diseases are hyperproliferative diseases, e.g., diseases that are associated with angiogenesis, in particular cancer, ocular fundus diseases, hypertrophic scars, etc., diseases that are associated with a myofibroblast malfunction, e.g., endometriosis, arteriosclerotic plaque, etc., or inflammatory diseases, e.g., psoriasis, Crohn's disease, rheumatoid arthritis or multiple sclerosis.
The pharmaceutical or diagnostic composition according to the invention can contain one or more active polypeptides, e.g., a combination of polypeptides, which bind to different areas of the ED-B domain. The compositions are suitable for use in human and veterinary medicine.
Another subject of the invention is a nucleic acid that codes for a peptide or fusion polypeptide according to the invention. This nucleic acid can be a single-strand or double-strand DNA or an RNA. The nucleic acid is preferably in operative linkage with an expression monitoring sequence, which makes possible an expression in a suitable host cell or a suitable host organism. The nucleic acid can be present on a vector that is suitable for introduction into a host cell or a host organism. The vector can be, for example, a prokaryotic vector, suitable for introducing prokaryotic cells, e.g., a plasmid or bacteriophage. In contrast, the vector can also be 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 one 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.
Still another subject of the invention is a cell, e.g., a prokaryotic cell or a eukaryotic cell, such as, for example, a human cell, which is transformed with a nucleic acid according to the invention or a vector according to the invention. Another subject of the invention is a non- -human organism, e.g., a transgenic animal, which is transformed with a nucleic acid according to the invention or a vector according to the invention. In this case, the term "transformation"
within the, scope of this invention contains all possibilities for introducing foreign nucleic acids into a cell or an organism including transfection or infection.
The polypeptide according to the invention can be made by cultivating a cell according to the invention or a non-human organism according to the invention under conditions in which an expression of the polypeptide is accomplished, and then the expressed polypeptide is obtained, e.g., from the cell, the culture medium, the organism or excretion products of the organism.
In addition, the invention is to be explained by the figures and examples below:
Figure 1 shows the diagrammatic implementation of an inhibition test for identifying functionally active antibodies.
Human endothelial cells, which were obtained from microvessels of the skin (human dermal microvessel endothelial cells, HDMVEc), were incubated together with ED-B-specific Fab-antibody fragments. The number of bonded cells is made visible by means of crystal violet staining and measured in a photometer. High color intensity means many adherent cells and no binding-blocking antibodies. Low color intensity means little adhesion and a binding-blocking antibody.
Figure 2 shows a screening diagram for identifying ED-B-fibronectin-function-blocking antibodies by means of a Fab-fragment display.
In a first step, a panning procedure with recombinant ED-B is performed. The antibody fragments that are obtained in this way are subjected to a specificity test (ELISA), a functional adhesion test as well as an immunohistochemical study. Then, the affinity of the antibodies that carry out these tests is determined. Then, changes in the CDR domains are performed to improve the affinity, whereby affinity maturation I means a change in the L-CDR3 domain and affinity maturation 2 means a change in the H-CDR2 domain.
After the antibodies, obtained by affinity maturation, have passed through the above-mentioned tests, then the in vivo effectiveness is tested.
Figure 3 shows the results of a specificity test in ELISA format with the antibody-Fab fragments that are produced by panning with recombinant ED-B.
ED-B means the binding to recombinant ED-B, 6-FN means the binding to the recombinant fibronectin-domain 6, domain 7-8-9 means the binding to recombinant fibronectin domains 7-8-9 (without ED-B), and domain 7-EDB-8-9 means the binding to the recombinant fibronectin domains 7-8-9 with ED-B. The ratio of ED-B to 6-FN or 7-8-9 produces the specificity of the antibody under study. AP-39 means a covalent dimer of the biologically inactive anti-ED-B scFv antibody L19.
Figure 4 shows the result of an adhesion test with the antibodies under study.
The inhibition of the adhesion of HDMVEc cells to ED-B-coated plates by the indicated Fab fragments at concentrations of 25 g/ml or 0.4 g/ml Fab was examined. The indicated values are the result of 3x determination.
Figure 5 shows the immunohistochemical reaction pattern of function-blocking anti-ED-B-Fab fragments in the example of MOR 02616. In Figures 5A and 5C, the results with a negative control (negative HuCAL-Fab), and in Figures 5B and 5D, the results with the antibody MOR 02616 on human neuroblastoma cell-IRM xenotransplants (Figures 5A and 513) as well as on murine F9-teratocarcinoma cells (Figures 5C and 5D) are shown.
Cryosections of cells (thickness: 10 m) were air-dried and then set in ice-cold acetone for 10 minutes. Then, the sections were washed in PBS and incubated for 60 minutes with 2 g/ml of MOR 02616 or negative controls at room temperature. As a secondary antibody, a peroxidase-labeled polyclonal goat anti-human F(ab)2 antiserum (1:100 diluted in PBS, Dianova) was used in combination with diaminobenzidine (Sigma Chemicals) as a chromogenic substrate.
Figure 6 shows the results of a specificity test in ELISA format with the antibody Fab fragments that are optimized by maturation.
The execution of the test was as described in Figure 3.
Figure 7 shows the immunohistochemical reaction pattern of the antibody Fab fragments MOR 03255, optimized by maturation, on cryostat sections of F9-teratocarcinoma cells (Figure 7A) and on SKMEL-28 human-melanoma cell xenotransplants in hairless mice (Figure 7B).
The execution of the test was as described in Figure 5.
Figure 8 shows the result of an adhesion test with the antibody Fab fragments according to the invention relative to the comparison antibodies L19 and Ly6 0.3.
The execution of the test was described very much like in Figure 4. The antibody concentrations were I l/ml, 10 l/ml or 20 l/ml.
Figure 9 shows the stability of the antibody-Fab fragments according to the invention after incubation for 4 hours in human plasma or PBS at 37 C.
The immune reactivity was determined in the ELISA test with concentrations in the range of 0.039 to 5 g/ml. The signal with 0.62 g/ml in human plasma without incubation was defined as a 100% value.
Figure 10 shows the therapeutic effectiveness of anti-ED-B-function-blocking Fab-antibody fragments in the example of MOR 03255.
Figure 11 shows the nucleotide sequences that code for the VH and VL regions of the antibodies according to the invention.
Example 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 produced according to standard procedures (e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbour Press).
Mouse antibodies against purified ED-B receptors were also produced according to standard processes by 50 g of purified receptor being administered in Freund's complete adjuvant to a mouse, 3 weeks later 25 g of purified receptor being administered in Freund's incomplete adjuvant, and another 3 weeks later all applications being administered intraperitoneally in incomplete adjuvant. Blood was then removed via the postorbital sinus 14 days after the 3d immunization.
Plasma fibronectin was ordered from Sigma.
1.2 Selection of ED-B-Specific Phages The selection was performed according to the HuCAL Gold Phage selection protocol.
The HuCAL Gold Library was subdivided into six framework-specific pools. The ED-B-specific phages were concentrated in three successive selection rounds of recombinant protein immobilized in 96-hole plates (Maxisorb, Nunc, Rochester, NY, USA). For panning A, the plate was coated with 1 g/hole of ED-B (Hisb) (10 g/ml in Ampuwa ) for 2 hours at 37 C.
For panning B, the plate was coated with ED-B in the same concentration in PBS
(pH 7.2 with 1 mmol of MgC12/1 mmol of CaC12) overnight at 4 C. The plates were blocked with 5% skim milk powder in PBS, 0.05% Tween20 (Sigma, St. Louis, MO, USA) (blocking buffer) and incubated with 1 x 1013 HuCAL Gold phages, which had been preincubated with a volume of blocking buffer. For panning B, the phages were pre-incubated in addition with 0.5 g/ml of fibronectin-domain 6 in order to reduce the selection of binding molecules that are specific to the conserved structure of a type-3 domain repetition of fibronectin. After an incubation with the phages for 2 hours at room temperature, the holes were washed 5x with PBS, 0.05% Tween20 and 5x with PBS. The remaining phages were eluted with 20 mmol of dithiothreitol (DTT) in 10 mmol of tris/HCl (pH 8.0) for 10 minutes at room temperature. Then, an incubation with E. coli TG-1 (Stratagene, OD600 = 0.5) was carried out for infection. Then, the holes were incubated with E.
coli TG-1 as an additional elution step.
1.3 Phagemid Recovery, Phage Amplification and Purification Die HuCAL Gold phages were amplified in 2x TY medium with 34 g/ml of chloramphenicol and 1% Glucose (2x TY-CG). After infection with a helper phage (VCSM13) at 37 C and an OD600 of about 0.5, centrifuging and resuspending in 2x TY-CG/50 g/ml of kanamycin, the cells were induced with 0.25 mmol of IPTG and cultivated overnight at 22 C.
The phages were precipitated from the supernatant with polyethylene glycol (Ausubel et al.
(1998), Current Protocols of Microbiology), resuspended in PBS and used for subsequent selection rounds.
1.4 Subcloning of Selected Fab Fragments and Expression of Soluble Fab Fragments The Fab-coding insertions of the selected HuCAL Gold phages were subcloned in the expression vector pMORPHx9-FS. The plasmid-DNA preparation of the selected HuCAL Gold clone was cleaved with the restriction enzymes XbalI/EcoRI, whereby the Fab-coding insertion was cut out (ompA-VL and phoA-Fd). Fab molecules that are expressed in this vector contain two C-terminal labelings (FLAGTM and Strep-taglI) for detection and for purification.
1.5 Expression and Purification of HuCAL Gold Antibodies in E.coli The expression of the TG-1 F cells on pMORPHx9-FS-coded Fab fragments in E.coli was performed in shaking bottle cultures with 0.75 1 of 2xTY medium and 34 g/ml of chloramphenicol. After induction with 0.75 mmol of IPTG, the cells were cultivated for 16 hours at 30 C. As an alternative, Fab clones, which had been obtained from the second maturation pool 2, were induced with 0.1 mmol of IPTG and then cultivated at 22 C.
Periplasmatic extracts from cell pellets were produced by osmotic shock, and the Fab fragments were isolated by Strep-Tactiri chromatography (IBA, Gottingen, Germany). The apparent molecular weights were determined by size-exclusion chromatography (SEC) with calibrating standards. The concentrations were determined by UV spectrometry.
1.6 Identification of ED-B-Binding Fab-Fragments by ELISA
96-Hole-Maxisorb ELISA plates were coated with 100 l of ED-B solution (10 g/ml in coating buffer (PBS pH 7.4, 1 mmol of CaC12, I mmol of MgC12) overnight at 4 C. Crude lysates or purified Fab molecules were added; non-binding Fab molecules were removed by 5x washing with washing buffer (PBS pH 7.4, 0.05% Tween20). The Fab fragments were detected 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 that express for ED-B-specific Fab molecules were identified by a positive ELISA signal on immobilized ED-B versus little or no signal at 6FN.
1.7 Determination of Plasma Stability Under Physiological Temperature Conditions by ELISA
The coating with ED-B (2.5 g/ml) was performed essentially as described above. The Fab fragments were incubated for 4 hours at 37 C and a concentration of 50 g/ml in human plasma (German Red Cross; Batch 9985550). After incubation, the Fab molecules were diluted for the ELISA Test to the concentrations of 5.0, 2.5, 1.25, 0.62, 0.31, 0.156, 0.078 and 0.039 g/ml in PBS with 1% bovine serum albumin to determine the linear area of the signal intensity.
Functional Fab molecules were determined with the anti-Flag M2 antibody (Sigma F3165) and a secondary anti-mouse-antibody-alkaline phase conjugate, followed by development with Attophos (Roche) and by measurement at 535 nm.
1.8 HDMVEc Cell Culture and Cell Adhesion Test Human dermal microvascular endothelial cells (HDMVEc), isolated from juvenile foreskin, were cultivated in EGM-MV medium (Clonetics, Inc.) with the addition of 10% fetal calf serum, 2 mmol of glutamine, 20 g/ml of heparin and 3 ng/ml of bFGF in vessels, coated with 0.1 % gelatin (Sigma). For adhesion tests, cells that had not been cultivated longer than passage 8 were used. ED-B (10 g/ml) or plasma fibronectin (2.5 g/ml) was immobilized overnight at 4 C in PBS, pH 7.4, 0.9 mmol of CaC12, 0.5 mmol of MgC12 in 96-hole ELISA
plates and blocked with 1% RSA in PBS, pH 7.4, 0.9 mmol of CaClz, 0.5 mmol of MgCl2 for 1 hour at 37 C. The cells were labeled with 0.15 g/ml of calcein for 30 minutes at 37 C and washed once in adhesion medium (MCDB 131, 2 mmol of glutamine, 0.1 % RSA, 20 g/ml of heparin). I x 105 cells were incubated for 20 minutes at 37 C with the antibody concentration that is indicated in each case, diluted in adhesion medium (final volume 100 l). The cell suspension was added to the ED-B-coated plates for 2 hours at 37 C. After the cells are washed 2x in PBS, pH 7.4, 0.9 mmol of CaC12, 0.5 mmol of MgC12, adherent cells were detected in a plate fluorimeter (excitation 485 nm; emission 530 nm).
As a control, (A) the cell binding to ligand-coated plates was determined without adding specific antibodies, and (B) the cell adhesion to holes was determined without ligand coating, whereby the value obtained in the last-mentioned control was defined as a minimum of cell adhesion in the test.
1.9 Affinity Maturation of Selected Fab Molecules by Exchange of CDR
Cassettes in Steps To increase the affinity and biological activity of selected antibody fragments, CDR
regions were optimized by cassette mutagenesis with use of trinucleotide-directed mutagenesis.
To this end, Fab fragments from the expression vector pMORPHx9 in the phagemid vector pMORPH-23 were cloned with use of the EcoRI/Xbal restriction sites. To optimize the affinity of the selected Fab fragments, two successive maturation steps for each maturation pool were performed. In the first step, an antibody fragment-phage library was produced in which the L-CDR3 region of the starting clone was varied by a repertoire of 7 x 108 (Pool 1) and 3.8 x 108 (Poo12) individual light chain-CDR sequences. Then, affinity-improved derivatives from the first maturation step were exposed to a second maturation round by the H-CDR-2 region being replaced by a library of diversified elements. In this way, two phage libraries with 1 x 10g individual clones in each case were produced.
The affinity maturation libraries were produced by transformation into E.coli TOP l OF.
The phages were produced as described above. The selection in Fab fragments with improved affinity was performed under stringent conditions for three rounds in the first maturation and for two rounds in a second step.
1.10 Determination of Affinity by Surface Plasmon Resonance (BIAcore ) To determine the KD values, monomer fractions (at least 80% monomer content, determined by analytical SEC; Superdex75, Amersham Pharmacia) of Fab fragments were used.
F1-Chips (Biacore, Sweden) were coated with about 200 RU of ED-B (30 g/ml, 10 mmol of acetate buffer, pH 4.0) and reference flow cells with a corresponding amount of human serum albumin (20 g/ml, 10 mmol of acetate buffer, pH 4.5) with use of standard EDC-NHS-amine-coupling chemistry. The antigen density was reduced to about 100 RU for the Fab characterization of the second maturation. The regeneration was carried out with 10 mmol of HCI. All kinetic measurements were carried out in PBS buffer (136 mmol of NaC1, 2.7 mmol of KCI, 10 mmol of Na2HPO4 , 1.76 mmol of KHZPO4, pH 7.4) with a flow rate of 20 l/minute with use of a Fab concentration range of 1.5-500 nmol. The injection period was 1 minute in each case. All sensograms were evaluated with use of BIA evaluation software 3.1.
Abbreviations: EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, NHS: N-Hydroxysuccinimide, RU: Resonance Units 2. Results 2.1 Production of Antibodies Against Human ED-B
Two different panning assays were implemented. The coating with ED-B for the first assay (panning A) was performed according to the conditions known from the biological test to present ED-B in a conformation that allows the adhesion of HDMVEc cells to the immobilized antigen. With the second panning (B), the phages were blocked in addition with excess 6FN to avoid a selection of binding molecules that cross-react with the fibronectin-domain 6.
Altogether, 23 different HuCAL-Fab molecules from 1315 primary hits were found that meet the criteria of binding to ED-B and not to 6FN. In addition, the antibodies were tested in their capacity for binding to a recombinantly expressed fibronectin, comprising the domains 7, 8 and 9 with and without the inserted ED-B domains in their natural arrangement (see Figure 3).
All tested antibodies showed a 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 of the ED-B Specific Binding Molecule The antibodies as specifically identified in ELISA were purified and tested in their capacity for blocking the adhesion of HDMVEc cells to ED-B-coated plates. The adhesion of HDMVEc cells in the presence of ED-B-specific Fab fragments in two concentrations was tested in comparison to an unspecific HuCAL-control-Fab-molecule and positive mouse control sera.
Twelve of the 23 Fab molecules described in Section 2.1 showed an inhibition of cell adhesion in immobilized ED-B. A representative selection of the six most effective Fab molecules is shown in Figure 4. At a concentration of 25 g/ml, the cell adhesion was inhibited by 50% to almost 100%. At a concentration of 0.4 g/ml, only a few Fab molecules, e.g., MOR02610, MOR02616, MOR02715 and MOR02718, showed a reproducible inhibition of the cell adhesion.
The function-blocking Fab-antibody fragments were characterized by immunohistochemistry with respect to their localization on F9-mous-teratocarcinoma cells and human IRM30 neuroblastoma-xenotransplant cryosections. All tested antibodies showed a vascular localization on two tissue sections, but no staining of vessels on normal tissue (e.g., normal and sclerotic liver and skin). In Figures 5B and 5D, a perivascular abluminal staining of vessels, proliferating endothelium and tumor stroma by MOR02616 is shown. A
collection of ED-B in the vicinity of neovascular structures of exponentially growing tumors, but not in normal vessels, can be detected. With a negative control antibody against lysozyme at the same concentration, no staining was visible (Figures 5A and 5C).
The properties of twelve selected binding molecules was determined by BlAcore analysis (see Table 1). The affinities (Koõ) were in the range of 15 to 500 nmol. The affinity of the bivalent L19 derivative AP39 (a biologically inactive comparison antibody against ED-B) was determined with 2.4 nmol. Table 1 also shows the CDR sequences (only L-CDR3 and H-CDR3) and the framework combinations. At least two independent measurements with Fab molecules from different batches were performed for protein expression and protein purification. The binding rate (Koõ) and the dissociating-off rate (Koff) of the Fab molecule are indicated in separate columns.
Table 1 Clone VH H-CDR3 VL L-CDR3 kon koff KD
[1/Ms] [1/ s] [nM]
AP 39 VH3 PF'PY--I-'L' K3 C" f~l -- PY 6,6E+05 1,5E-03 2,4 0,6 MOR02610 VH1B SPVYYKYD L1 QSYDKTSSTY 1,3E+06 9,1E-02 75 19 MOR02611 VH1B CLYYR-Fr,S L2 AAnl'G ---GW 1,5E+05 5,1E-02 332 23 MOR02613 VH3 YvNC--FDz L2 Q'rYAKKDYSL 7,8E+05 1,3E-01 164 14 MOR02614 VH3 A----- Yrw L1 AMFSP---EC 2,8E+05 4,2E-02 160 6 MOR02616 VH3 VivL--FDY K3 LQKYSi PF 5,4E+05 2,8E-02 59 25 MOR02618 VH3 NiwV--F-Z,Y L3 UsYnrJFtaDsV 4,3E+05 2,OE-01 477 177 MOR02619 VH4 F-----FDV L1 QSWDGAS-TG 7,8E+05 1,2E-02 15,4 0,6 MOR02622 VH3 GLVT--FDN L2 SSwTxSFq'DY 2,4E+05 5,1E-03 21,3 1,7 MOR02715 VH3 raxvc--FUV L2 QAwDNQcMKY 8,9E+05 4,7E-02 53,7 1,7 MOR02718 VH3 cwF---FAx K3 FQYSSV--PL 4,5E+05 3,9E-02 85 12,5 MOR02721 VH5 'rr3----c~,e L1 ~7, "'T TC; --s 4,3E+05 2,4E-02 56 11,6 MOR02722 VH3 e as K2 aVf, ~ raF P F 1,5E+05 1,4E 02 94 2,3 2.3 Affinity Maturations of Function-Blocking ED-B-Antibodies by Optimization of L-CDR3 and H-CDR2 Regions The affinity maturations of ttie Fab molecules were perfonned in two sections.
First, the L-CDR3 sequence was diversified, and then the H-CDR2 sequence of the improved Fab molecule, obtained in the first step, was exchanged.
Two affinity maturation libraries for each step were produced. The optimization was performed in two separate pools. The L-CDR3 library I with a degree of diversity of 7 x l Ox elements contained the four starting Fab molecules MOR02610., MOR02616, MOR02619 and ~ . .
MOR02622. The L-CDR3 library 2 with a degree of diversity of 3.8 x 108 elements contained only derivatives of MOR02715 and MOR02718. Here, the Fab molecules were combined into groups in each case according to their affinity and biological activity in the adhesion test.
The two libraries were kept separately during the selection procedure. In this case, two panning strategies were performed. Panning I was carried out with ED-B
immobilized on maxisorb plates for three rounds, as previously indicated. With Panning II, a selection of biotinylated ED-B in solution was carried out. In this case, the phage-antigen complex was recovered by streptavidin-coated particles in the first round and on neutravidin plates in the two subsequent rounds. Stringent conditions were used within the scope of the selection procedure.
The screening on optimized Fab molecules was carried out by determining the Koff values in the BlAcore system. Altogether, 11 derivatives of library I were characterized. The 5 derivatives with the highest affinity are indicated in Table 2. In this case, all improved clones are derivatives of MOR02619 with an increase in affinity of up to 7x. In clone MOR03075, an affinity of 2.4 nmol was found.
From library 2, two derivatives of MOR02715 were analyzed in detail. For clone MOR03062, a 15-fold affinity increase was found, so that a monomer affinity of 2.4 nm was obtained. The changes in the amino acid sequence of the L-CDR3 region are shown in Tables 2a and 2b.
j , . 31 Table 2a Pan- MORO Initial LCDR3 Kon Koff KD x-Fold Improve-ning Clone [1/Ms] [1/s] [nm] ment Relative to Pool 1 the Parent Clone Kon Koff KD
AP 39- QQTGRI - - 6.6E + 1.49E - 2.4 ~
Biotin PP 05 003 0.6 2619 QSWDGAS- 7.8E + 1.2E - 02 15.4 f TG 05 0.6 Solid 3055 2619 QAWTRAHR 1.8E+ 5.1E - 03 3.0 f 2.2 2.6 5.4 YP 06 0.5 Dis- 3066 2619 SSYD- 1.1E + 4.7E - 03 4.2 ~ 1.4 2.8 3.8 solved TQVTR 06 0.6 IL-Dis- j 3075 2619 QSWDP- 1.1E + 2.6E - 031 2.4 f I 1.4 5.0 6.8 solved RSFT 06 0.3 Dis-3069 2619 WTGM - - 1.2E + 4.1E - 03 3.3 f 1.5 3.2 4=8 solved]__L SYHF 06 0.2 Dis- 3071 2619 LAYIQS- 1.9E + 5.7E - 03 3.1 ~ I 2.3 2.3 5.1 solved KGH 06 1 ! 0.8 I- _- -__ Table 2b Pan- MORO Par- LCDR3 Kon Koff KD x-Fold ning ent [1/Ms] [1/s] [nm] Improvement Pool 2 clone Relative to the Parent Clone Kon Koff K1 AP 39- QQTGRI - - 6.75E + 1.49E - 2.4 I
Biotin PP 05 03 0.6 1 2715 QAWDNQG 9.9E + 4.7E - 02 53.7 f~
MKY 05 1.7 Solid 3064 2715 QSWDLLAPS 1.5E + 1.4E - 0210 f 3.5 1.7 3.5 5.3 Solid 3062 2715 QSWDLSVH, 3.5E + !1.1 E -02 3.4 ~ 4.0 4.2 15.81 0.6 0.8 _--1------L--1----~-- -The specificity for ED-B (determined by ELISA, immunohistochemistry and cell adhesion test) of all derivatives from the first maturation round were unchanged.
The Fab molecules shown in Tables 2a and 2b were selected for a subsequent H-maturation in two pools. In pool 1, five Fab molecules were subjected to a similar activity, but with different L-CDR3 regions of a diversification in H-CDR2 (library size of 7 x 107 eleinents).
For pool 2 (library size of 8.5 x 107 eleinents), two Fab molecules were selected. The libraries were selected separately as described above. Binding molecules with increased affinity were concentrated by two panning rounds in solution under stringent conditions.
Three Fab molecules were selected from pool 1. For derivatives MOR03243 and MOR03245, affinities of about 0.7 nrnol were found. For derivative MOR03246, an affinity of 0.6 nmol was found.
The H-CDR2 secluences of the corresponding clones and the affinity data are shown in Table 3a.
Table 3a MORO Parental Sequenz HCDR2 kon [1/Ms] koff [1/ s] KD [nM]
Clone MOR03243 3055 EIHRGGYTQYNPSLKS 2,9E+06 1,8E-03 0,7 0,2 MOR03245 3069 vtttKwGFTNYNPs7,xs 3,5E+06 1,6E-03 0,7 0,3 MOR03246 3075 YIFIKYGwTKYNPSLKS 4,8E+06 3,OE-03 0,6 0,1 [Key: Sequenz = Sequence]
Derivatives with improved propertics could also be selected from pool 2. In comparisoarl to the parent clones, the affinities of the derivatives were improved by a factor of 26 up to 250x.
The affinities of the clones MOR03255 and MOR03258 were determined with 30 pM
or 40 pM.
In Table 3b, 11-CDR2-amino acid sequences of the selected clones from pool 2 as well as the corresponding affinity data are shown.
Table 3b Parental MORO Sequenz HCDR2 kon [11Ms] koff [11 s] KD [nM] STDEV
Clone MOR03251 3062 V1SNMsY'111YYnUwYG 3,3E+06 2,OE-04 0,07 0,04 MOR03252 3062 vzsNY6wHryYI~ nsvxc 3,1E+06 6,1E-05 0,03 0,03 MOR03253 3062 Mut ~I~ta.-~cE'~IYYnDS:~KG 1,6E+06 2,OE-04 0,13 0,05 MOR03255 3062 V TraussYI 1 YtYPsiF,4,9E+06 8,6E-05 0,03 0,03 MOR03257 3062 1: Ntzc,[~YIYYr.n~~vr~~ 5,3E+06 3,OE-04 0,08 0,06 MOR03258 3064 2,7E+06 8,8E-05 0,04 0,02 [Key: Sequenz = Sequence]
2.4 Characterization of Highly Affine Function-Blocking ED-B-Antibodies To test whether the antibodies that are obtained by affinity maturation are still always specific for ED-B, specificity tests in the ELISA forinat (Figure 6), iminunohistocheinical studies (Figure 7) and adhesion tests (Figure 8) were performed. In all three tests, it was found that the specificity of the affinity-matured Fab inolecules for ED-B remained unchanged. In addition, it is evident from Figure 8 that both the parent clones, such as MOR 02715, and the clones that are 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 the Fab molecules in human plasma was tested. To this end, the Fab molecules were incubated with human plasma for 4 hours at 37 C without a significant loss in immune reactivity determined by ELISA (Figure 9) being found.
All selected Fab molecules are able to block the interaction of a receptor with human microvascular endothelial cells on ED-B in a dose-dependent way, a property that known ED-B-antibody L19 does not have. Since the adhesion of HDMVEc to the extracellular matrix is one of the early steps in neoangiogenesis, the blocking of this process by the binding molecules according to the invention produces a therapeutic agent for inhibiting the formation of new vessels and for inhibiting tumor growth.
2.5 In vivo Action F9-Teratocarcinoma cells (106/mouse in Matrigel with/without 100 g of Fab MOR
03255) were implanted s.c. in the flanks of hairless mice. After 3 days, the animals were treated for 7 successive days with, in each case, 100 g of MOR03255/mouse. In comparison to the solvent control, both treatment groups show an inhibition in tumor growth in the range of 42-64%. A summary of test results is shown in Figure 10.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.
The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
In the foregoing and in the examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated.
The entire disclosures of all applications, patents and publications, cited herein and of corresponding German application No. 102004050101.7, filed October 14, 2005 and U.S.
Provisional Application Serial No. 60/697,565, filed July 11, 2005, are incorporated by reference herein.
The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Literature (1) Brown, L. F.; Guidi, A. J.; Schnitt, S. J.; Van De Water, L.; Iruela-Arispe, M. L.; Yeo, T.-K.; Tognazzi, K.; Dvorak, H. F. (1999) Clin. Cancer Res. 5:
(2) Folkman, J. (1995) Nature (Med.) 1: 27-31 (3) Risau, W. and Lemmon, V. (1988) Develop. Biol. 125: 441-450 (4) Van Den Hoff, A. (1988) Adv. Cancer Res. 50: 159-196 (5) Folkman, J. and Shing, Y. (1992) J. Biol. Chem. 267: 10931-10934 (6) George, E. L.; Baldwin, H. S.; Hynes, R. O. (1997) Blood 90: 3073-3081 (7) Bowersox, J. C. and Sorgente, N. (1982) Cancer Res. 42: 2547-2551 (8) Madri, J. A.; Pratt, B. M.; Tucker, A. M. (1988) J. Cell Biol. 106: 1375-(9) Nicosia, R. F.; Bonnano, E.; Smith, M. (1993) J. Cell. Physiol. 154: 654-(10) Kornblihtt, A. R.; Vibe-Pedersen, K.; and Baralle, F. E., 1983. Isolation and Characterization of cDNA Clones for Human and Bovine Fibronectins. Proc. Natl.
Acad.
Sci. USA, 80, 3218-22 (11) Leahy, D. J.; Hendrickson, W. A.; Aukhil, I. and Erickson, H. P., 1992.
Structure of Fibronectin Type III Domain from Tenascin Phased by MAS Analysis of the Selenomethionyl Protein. Science, 258, 987-91 (12) Hynes, R. 0., 1987, Cell 48, 549-550 (13) Plow, E. F. et al. 2000, J. Biol. Chem., 275, 21785-21788 (14) Castellani, P.; Viale, G.; Dorcaratto, A.; Nicolo, G.; Kaczmarek, J.;
Querze, G.; Zardi, L. (1994) Int. J. Cancer 59: 612-618 . . .
The invention relates to recombinant polypeptides, in particular antibodies or antibody fragments that bind to the ED-B-isoform of fibronectin and can block their function. In addition, the diagnostic and pharmaceutical application of the polypeptides according to the invention is disclosed.
During tumor development and progression, the extracellular matrix (ECM), in which the tumor grows, is modified by proteolytic degradation of already existing ECM.
This process generates a tumor-induced matrix, which is distinguished from that of the ECM
that occurs in normal tissue. The tumor-induced ECM appears to be the optimum environment for tumor growth and tumor angiogenesis (1-4).
In tumor angiogenesis, new blood vessels are formed from already existing vessels. This process requires the proteolysis of ECM, the targeted growth and differentiation of endothelial cells in new vascular structures, which are essential for further tumor growth (5).
Fibronectins are an important class of matrix-glycoproteins. Their main role consists in facilitating the adhesion of cells to a number of different extracellular matrices. The presence of fibronectins on the surface of non-transformed cells in culture as well as their absence in the case of transformed cells resulted in the identification of fibronectins as important adhesion proteins.
They interact with numerous other different molecules, e.g., collagen, heparan sulfate-proteoglycans and fibrin, and thus regulate the cell shape and the creation of the cytoskeleton. In addition, they are responsible for cell migration and cell differentiation during embryogenesis. In addition, they are important for wound healing, in which they make possible the migration of macrophages and other immune cells in the field in question and in the formation of blood clots by making possible the adhesion of blood platelets to damaged regions of the blood vessels.
Fibronectins are dimers of two similar peptides, whereby each chain is approximately 60-70 nm long. At least 20 different fibronectin chains have been identified, of which all are produced by alternative splicing of the RNA transcript of a single fibronectin gene.
Fibronectins are high-molecular, adhesion-mediating glycoproteins that play an important role in the development of the vascular system. In addition, fibronectins act chemotactically on endothelial cells, modulate the action of growth factors and support the linear growth of endothelial cells during angiogenesis (6-9). An analysis of proteolytic fibronectin-cleavage products shows that the polypeptides consist of six heavily folded domains of which each domain in turn contains so-called repetition sequences ("repeats") whose similarities with respect to their amino acid sequence allow a classification in three types (types I, II, and III). The central region of both chains of the dimer consists of a section of so-called type-III
repetitions, which on average are 90 amino acids long (10). Structural studies have revealed that each type-III
repetition consists of seven beta-strands, which are folded into two antiparallel folded sheets, whereby short loop regions are exposed as potential protein-protein-interaction sites (11).
These type-III repetitions make it possible for fibronectins to act as adhesion molecules that interact with cell surface molecules, the so-called "integrins." The term "integrin" was used for the first time in 1987 in (12) to describe a related group of heterodimeric cell surface molecules that act as mediators between the extracellular matrix and the intracellular cytoskeleton and thus induce cell adhesion and migration. These heterodimeric receptors "integrate" or mediate signals from the extracellular environment with specific cellular functions.
Up until now, 17 beta-subunits have been known that can interact specifically and non-covalently with more than 20 alpha-subunits, particularly to forrn as 20 different families (13).
The sequence RGDS, which is found in the tenth repetition of type III of the fibronectin (111-10), in particular mediates the interaction of fibronectin with at least 8 different integrins. Moreover, it was shown that at least four integrins can interact specifically with fibronectin in an RGDS-independent way (13). In addition to the 1117-, 1118-, 1119- and III10 sequences, the group of repetition sequences of type III also comprises the EIIIB and EIIIA (ED-B and ED-A) repetitions.
The ED-B-fibronectin cannot be detected in the normal tissue of adults (sole exception:
proliferating endometrium), while it is strongly expressed in fetal tissue and in tumor growth, in addition to a stromal local expression of ED-B. Moreover, ED-B-fibronectin is localized perivascularly around blood vessels that have newly formed during the 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 component that consists of 91 amino acids. The degree of homology between humans and rats is 100%, between humans and chickens 96%. In literature, very little is known on the function of the ED-B-domains. A few publications (15-17) speculate on a general adhesion-mediating action for various cells. A specific action on endothelial cells was still not shown to date.
In WO 02/20563, it was shown that recombinant ED-B shows a specific pro-angiogenic action in vitro: (i) the proliferation of bFGF-stimulated human dermal microvascular endothelial cells (HDMVEc) is enhanced 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.
In addition, these ED-B-mediated effects could be specifically blocked by synthetic peptides that were derived from the ED-B domains. The peptide sequences thus represent the binding region for an ED-B-specific receptor on the endothelial cell surface that was identified by means of affinity chromatography as the a2P I -integrin. The specific interaction between the aZ(31-integrin and the ED-B domain was not previously described in the literature.
Also disclosed in WO 02/20563 are proteins that are regulated specifically by the ED-B-fibronectin domain, which comprises the a2p I -integrin, the focal adhesion kinase, the CD6 ligand (ALCAM), the alpha chain of the vitronectin receptor, the integrated alpha-8 subunit or the precursor of the follistatin-related protein.
A number of integrin receptors with partially overlapping properties are expressed by endothelial cells (lit.: D. G. Stupack and D. A. Cheresh, SciSTKE, 2002 Feb 12; 2002). These expression patterns show that different integrins (e.g., alphavbeta3 and alpha5betal) mediate similar biological phenomena (adhesion, migration and survival) and therefore represent redundant systems for the endothelial cells that safeguard their behavior and survival. It was previously known that alpha2betal interacts with its natural ligands, the collagens. The blocking of this interaction can result in an anti-angiogenic action (Y. Funahashi et al. Cancer Res. 62:
6116-6123, 2002).
One object of this invention was therefore the preparation of function-blocking binding molecules, such as, e.g., antibodies that specifically block receptor binding sites of the ED-B-domains. These binding molecules have an anti-angiogenic action on (tumor) endothelial cells.
In contrast to relatively broadly expressed a2(3I -integrin, the ED-B domain represents an ideal and specific target molecule for such binding molecules.
The structure of ED-B makes the development of monoclonal and polyclonal antibodies difficult since it thus can be expected that ED-B has a low immunogenicity in vivo. The antibody BC-1 (J. Cell Biol. 108 (1989), 1139-1148) thus reacts with a cryptic epitope of fibronectin, which is present only in the presence of ED-B and therefore not directly with ED-B. Antibody L19 (Tarli et al. Blood 94 (1999), 192-198 and WO 01/62800), which was produced by use of recombinant ED-B as an immunogen, is in turn biologically inactive, i.e., it cannot recognize cell adhesion to ED-B to a significant extent.
Surprisingly enough, it was now found that function-blocking ED-B-binding molecules can be produced, such as, for example, the ED-B-function-blocking antibody MOR03255, which greatly inhibit the adhesion of cells to ED-B and bring about in vivo a significant reduction of the tumor growth. The blocking of ED-B produced by the binding molecules according to the invention obviously cannot be compensated for by compensatory mechanisms of collagen-integrin.
By means of the HuCAL -GOLD antibody library -- a library with, for example, 1.6 x 10E10 different antibodies in the Fab-fragment format, which was generated 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. and Virnekas, B. (2000) J.
Mol. Biol. 296:57-86) by diversification corresponding to the diversity of human antibodies in all six CDR areas and is closely examined by means of CysDisplay (WO 01/05950) of a variant of the phage display process -- function-blocking Fab-antibody fragments that bind selectively to the ED-B-domain were identified in the tests leading to this invention. The effectiveness of these antibody fragments could be shown in an in vitro adhesion test that reflects the specific interaction between recombinant ED-B and isolated HDMVEc. The binding affinity to the ED-B-domain could be considerably improved by a specific change in the binding molecules. It was also possible to show in vivo that the binding molecules in an animal model inhibit the growth of tumors.
A first aspect of the invention is thus a polypeptide that (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 according to the invention is preferably an antibody or antibody fragment. The term "antibody" in terms of this invention comprises polyclonal, monoclonal, chimera, humanized or human antibodies as well as recombinant antibodies, e.g., single-chain antibodies, or antigen-binding antibody fragments, e.g., monovalent antibody fragments, such as, for example, Fab fragments or scFv fragments, or divalent antibody fragments, such as, for example, F(ab')2 fragments.
In this connection, it is essential to the invention that the antibodies contain one or more antigen binding sites that meet the above-mentioned requirements, i.e., specific binding to the ED-B domain and inhibition of the interaction between the ED-B domain and its receptor, especially its receptor on endothelial cells. These antigen binding properties are preferably achieved by combination of a VH and VL region, whereby these regions are built up from the thus-mentioned skeleton regions (FR1, FR2, FR3 and FR4) as well as the CDR
regions that mediate the antigen bond (H-CDRI, H-CDR2, H-CDR3 for the VH region and L-CDR1, L-CDR2, L-CDR3 for the VL region).
The polypeptide according to the invention preferably has an affinity to the ED-B domain corresponding to a Kp value of 5 1 m, preferably <_ 100 nM, especially preferably < 10 nM, still more preferably <- 1 nM, and most preferably <- 0.1 nM, whereby the affinity can be determined as indicated in the examples by a test on the BIAcore system.
The polypeptide according to the invention is distinguished in that it specifically binds to the ED-B domain of fibronectin, e.g., it binds with a significantly lower affinity to other fibronectin domains, in particular to fibronectin domain 6 (FN6) and/or fibronectin domains 7-8-9 (7-8-9). In a preferred embodiment, the polypeptide according to the invention and the fibronectin ED-B domain bind with an affinity that is higher by at least a factor of 2-- in particular by at least a factor of 5, and especially preferably by at least a factor of 10 -- than that of FN6 and/or 7-8-9, as can be determined, for example, by the binding test that is indicated in the examples.
In another preferred embodiment, the polypeptide according to the invention shows in vitro an inhibition of the adhesion of recombinant ED-B to HDMVEc cells, preferably an inhibition of at least 50% and especially preferably of at least 75% in a concentration of 10 g/ml in each case.
Moreover, it is preferred that the polypeptide according to the invention shows in vivo an inhibition of the growth of a tumor that is produced by implantation of F9-teratocarcinoma cells (ATCC CRL- 1720) in test animals, for example hairless mice.
The polypeptide according to the invention is preferably selected from antibodies and antibody fragments that comprise (a) a VH region (i) Coded 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- and/or H-CDR3 region of one of the above-mentioned VH regions, or (ii) Derived from a VH region according to (i) by a change in at least one H-CDR region and/or (b) A VL region (i) Coded 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-CDR 1-, L-CDR2- and/or L-CDR3- region of one of the above-mentioned VL regions or (ii) Derived from a VL region according to (i) by a change iri at least one L-CDR region.
Changes in the L-CDR3 region in the VL region and/or changes of the H-CDR2 region in the VH region are preferred.
For example, the polypeptide according to the invention exhibits a VL region that is derived from the VL region and that is coded by the nucleic acid sequence SEQ
ID NO. 27 (MOR 02619) or SEQ ID NO. 35 (MOR 02715). Especially preferred is a polypeptide that comprises a VH region (i) Coded by 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 and/or H-CDR3 region of one of the above-mentioned VH regions, or (ii) Derived from a VH region according to (i) by a change in at least one H-CDR
region.
The polypeptide preferably exhibits a VH region that is derived by a change in the H-CDR2 region and is coded by a VH region from the nucleic acid sequence SEQ ID
NO. 25 (MOR 02619) or SEQ ID NO. 33 (MOR 02715). Especially preferred in this case is a polypeptide, comprising (a) A VH region (i) Coded 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 the above-mentioned VH regions or (ii) Derived from a VH region according to (i) by a change in at least one H-CDR region and/or (b) a VL region (i) Coded 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 as well as MOR 03252, MOR 03253, MOR 03255 and MOR 03257), SEQ ID NO. 89 (MOR 03258) or at least the L-CDRI, L-CDR2 or L-CDR3 region of one of the above-mentioned VL regions, or (ii) Derived from a VL region according to (i) by a change in at least one L-CDR region.
Specific examples of the polypeptide according to the invention are as follows:
A polypeptide that comprises the VH region that is coded by SEQ ID NO. I and the VL
region that is coded 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 that comprises the VH region that is coded by SEQ ID NO. 5 and the VL
region that is coded by SEQ ID NO. 7 (MOR 02611) or at least one H-CDR 1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 9 and the VL
region that is coded by SEQ ID NO. 11 (MOR 02613) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 13 and the VL
region that is coded by SEQ ID NO. 15 (MOR 02614) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 17 and the VL
region that is coded by SEQ ID NO. 19 (MOR 02616) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 21 and the VL
region that is coded by SEQ ID NO. 23 (MOR 02618) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 27 (MOR 02619) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 29 and the VL
region.that is coded by SEQ ID NO. 31 (MOR 02622) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 33 and the VL
region that is coded by SEQ ID NO. 35 (MOR 02715) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 37 and the VL
region that is coded by SEQ ID NO. 39 (MOR 02718) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 41 and the VL
region that is coded by SEQ ID NO. 43 (MOR 02721) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 45 and the VL
region that is coded by SEQ ID NO. 47 (MOR 02722) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof. .
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 49 (MOR 03055) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 51 (MOR 03066) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR 1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 53 (MOR 03075) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 55 (MOR 03069) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 25 and the VL
region that is coded by SEQ ID NO. 57 (MOR 03071) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 33 and the VL
region that is coded by SEQ ID NO. 59 (MOR 03064) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 33 and the VL
region that is coded by SEQ ID NO. 61 (MOR 03062) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 63 and the VL
region that is coded by SEQ ID NO. 65 (MOR 03243) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 67 and the VL
region that is coded by SEQ ID NO. 69 (MOR 03245) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 71 and the VL
region that is coded by SEQ ID NO. 73 (MOR 03246) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 75 and the VL
region that is coded by SEQ ID NO. 77 (MOR 03251) or at least one H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 79 and the VL
region that is coded by SEQ ID NO. 77 (MOR 03252) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 81 and the VL
region that is coded by SEQ ID NO. 77 (MOR 03253) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 83 and the VR
region that is coded by SEQ ID NO. 77 (MOR 03255) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDRI, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 85 and the VR
region that is coded by SEQ ID NO. 77 (MOR 03257) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
A polypeptide that comprises the VH region that is coded by SEQ ID NO. 87 and the VR
region that is coded by SEQ ID NO. 89 (MOR 03258) or at least one H-CDRI, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3 region thereof.
The creation of the VH and VR chains of the polypeptides according to the invention is as follows:
VH Chain:
- The 1-region framework extends from nt 1-78 (that is, 26 aa); the last codon (aa) of FR1 is always: TCC (Cys).
- The "CDRI region" can be of 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 V142, VH4 and VH6).
- The 2-region framework always exhibits 33 nt (that is, 11 aa); the first codon (aa) of FR2 is always: TGG (Trp); the last two codons are always CTC.GAG (LeuGlu).
- The "CDR2 region" can be of three different lengths; either 57 nt (19 aa) (possible in families VH2 and VH4), 60 nt (20 aa) (possible in families VH1A, VHI B, 3 and 5); or 63 nt (21 aa) (possible in family V146).
- The 3-region framework always exhibits 96 nt (that is, 32 aa); the third 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 of several different lengths: from 12-69 nt (4-23 aa) (in all families VH2 and VH4), 60 nt (20 aa) (possible in families VH 1 A, VH 1 B, 3 and 5), or 63 nt (21 aa) (possible in family VH6).
- The 4-region framework always exhibits 33 nt (that is, 11 aa) and is identical in all 7 families: TGG.GGC.CAA.GGC.ACC.CTG.GTG.ACG.GTT.AGC.TCA
VL kappa Chain:
- The 1-region framework extends from nt 1-69 (that is, 23 aa); the last codon (aa) of FRI
is always: TGC (Cys).
- The "CDRI region" can be of four different lengths: either 24 nt (8 aa) (possible in families Vkl and Vk3), 27 nt (9aa) (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 CDRI region always: CTG (Leu).
- The 2-region framework always exhibits 33 nt (that is, 1 I aa); the first two codons (aa) of FR2 are always: TGG.TAC (Trp Tyr); the penultimate codon is always CCG (Pro).
- The "CDR2 region" always exhibits 33 nt (that is, 1 l aa), whereby the first three codons (aa) are always CTA.TTA.ATT (Leu Leu Ile).
- The 3-region framework always exhibits 96 nt (that is, 32 aa); the first 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 exhibits 24 nt (that is, 8 aa), whereby the second codon (aa) is always CAG (Gin).
- The 4-region framework always exhibits 39 nt (that is, 13 aa) and is identical in all four families: ACC.TTT.GGC.CAG.GGT.ACG.AAA.GTT.GAA.ATT.AAA.CGT.ACG.
VL lambda Chain:
- The 1-region framework extends from nt 1-66 (that is, 22 aa); the last codon (aa) of FRI
is always: TGT (Cys).
- The "CDRI region" can exhibit three different lengths: either 33 nt (11 aa) (possible in family V13), 39 nt (13 aa) (possible in family VI1), or 42 nt (14 aa) (possible in families Vll and V12).
- The 2-region framework always exhibits 33 nt (that is, 11 aa); the first two codons (aa) of FR2 are always: TGG.TAC (Trp Tyr); the third last codon is always GCG (Ala).
- The "CDR2 region" always exhibits 33 nt (that is, 11 aa), whereby the third codon (aa) is always ATT (Ile), and the last three codons are always CGT.CCC.TCA (Arg Pro Ser).
- The 3-region framework always exhibits 96 nt (that is, 32 aa), whereby 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 exhibit three different lengths: either 24 nt (8 aa), 27 nt (9 aa), or 30 nt (10 aa).
- The 4-region framework always exhibits 39 nt (that is, 13 aa) and is identical in all 3 families: GTG.TTT.GGC.GGC.GGC.ACG.AAG.TTA.ACC. GTT.CTT. GGC.CAG.
For therapeutic or diagnostic purposes, e.g., for an in vitro or in vivo diagnosis, the polypeptide according to the invention can be used.
For therapeutic applications, the polypeptide according to the invention can be present in the form of a conjugate with a therapeutic active ingredient, for example selected from radiotherapy agents or chemotherapy agents, e.g., low-molecular or biological cytostatic or cytotoxic active ingredients. The conjugation of the therapeutic active ingredient on the polypeptide can be carried out according to known methods, preferably via a covalent coupling to reactive amino, carboxy, hydroxy and/or thiol groups of the protein, optionally with use of homo-or hetero-bifunctional linkers, according to known methods.
Moreover, the polypeptide can also be present in the form of a fusion protein, which, in addition to the antibody, e.g., in the form of an IgG molecule or a fragment thereof, contains a cytokine fused thereto, e.g., IL2,1112 or TNFa-polypeptide. In addition, the fusion protein can be present in the form of a bispecific antibody, whereby in addition to the binding to the ED-B
domain, a binding to another antigen is preferred. Other antibody specificities of bispecific antibodies are binding domains against chelating agents for diagnostically and therapeutically relevant radionuclides, e.g., a-,13- or r-emitters, such as, for example, 90Y, diagnostic NIR (near-infrared)-dyes, therapeutically effective dyes, surface molecules on immunological effector cells (e.g., NK cells, cytotoxic T cells, or NKT cells), angiogenesis-relevant integrins, in particular binding domains that block their function (e.g., (11,133, aI133, a2131, a2132), inactivating anti VEGF-binding domains and inactivating binding domains against VEGF receptors 1, 2 and 3.
For diagnostic applications, the polypeptide can be present in the form of a conjugate of a diagnostically detectable labeling group, e.g., a labeling group for an in vitro or an in vivo diagnosis. Examples of labeling groups are radioactive labeling groups, NMR-, dye-, enzyme-and fluorescence- (e.g., fluorescence in the near-infrared range) labeling groups.
For therapeutic applications, the polypeptide is preferably formulated as a pharmaceutical composition that as active ingredient contains the polypeptide according to the invention, optionally additional active ingredients as well as pharmacologically common vehicles, adjuvants and/or diluents. The pharmaceutical composition contains the active ingredient in a therapeutically effective dose that can be determined by one skilled in the art in a simple way by in vitro tests, for example on suitable cell cultures, or in animal models.
The administration of the composition is preferably carried out by injection or infusion, but also other types of administration are conceivable. An intravenous and/or subcutaneous administration is preferably carried out. The dose of the administered active ingredient depends on the type and the severity of the disease as well as the condition of the patient to be treated. The therapeutic composition is preferably administered several times over an extended period of, for example, at least 2-4 weeks. In this connection, reference is made to known processes for administering antibodies or antibody conjugates, as described in, for example, Ferarra, N. et al., Nature Reviews Drug Discovery, Volume 3, May 2004, 391-400 and Salgaller, M. L., Current Opinion in Molecular Therapeutics 2003 5(6): 657-667, or to existing administration procedures for pharmaceutical antibodies, such as Rituximab, CAMPATH, Remicade, etc.
Another subject of the invention is a diagnostic composition that comprises a polypeptide according to the invention as a diagnostic reagent. In addition, the diagnostic composition can still contain additional diagnostically common reagents, vehicles, adjuvants and/or diluents. The diagnostic composition contains the polypeptide in a sufficient amount to make possible diagnostic detection in the respective format, e.g., in an in vivo or in vitro diagnostic format. In this connection, reference is made to known processes for in vivo and in vitro diagnosis with use of labeled antibodies.
The pharmaceutical and diagnostic compositions can for therapy and diagnosis of all diseases that are accompanied by an ED-B expression, for example a stromal and/or perivascular ED-B expression. Examples of such diseases are hyperproliferative diseases, e.g., diseases that are associated with angiogenesis, in particular cancer, ocular fundus diseases, hypertrophic scars, etc., diseases that are associated with a myofibroblast malfunction, e.g., endometriosis, arteriosclerotic plaque, etc., or inflammatory diseases, e.g., psoriasis, Crohn's disease, rheumatoid arthritis or multiple sclerosis.
The pharmaceutical or diagnostic composition according to the invention can contain one or more active polypeptides, e.g., a combination of polypeptides, which bind to different areas of the ED-B domain. The compositions are suitable for use in human and veterinary medicine.
Another subject of the invention is a nucleic acid that codes for a peptide or fusion polypeptide according to the invention. This nucleic acid can be a single-strand or double-strand DNA or an RNA. The nucleic acid is preferably in operative linkage with an expression monitoring sequence, which makes possible an expression in a suitable host cell or a suitable host organism. The nucleic acid can be present on a vector that is suitable for introduction into a host cell or a host organism. The vector can be, for example, a prokaryotic vector, suitable for introducing prokaryotic cells, e.g., a plasmid or bacteriophage. In contrast, the vector can also be 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 one 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.
Still another subject of the invention is a cell, e.g., a prokaryotic cell or a eukaryotic cell, such as, for example, a human cell, which is transformed with a nucleic acid according to the invention or a vector according to the invention. Another subject of the invention is a non- -human organism, e.g., a transgenic animal, which is transformed with a nucleic acid according to the invention or a vector according to the invention. In this case, the term "transformation"
within the, scope of this invention contains all possibilities for introducing foreign nucleic acids into a cell or an organism including transfection or infection.
The polypeptide according to the invention can be made by cultivating a cell according to the invention or a non-human organism according to the invention under conditions in which an expression of the polypeptide is accomplished, and then the expressed polypeptide is obtained, e.g., from the cell, the culture medium, the organism or excretion products of the organism.
In addition, the invention is to be explained by the figures and examples below:
Figure 1 shows the diagrammatic implementation of an inhibition test for identifying functionally active antibodies.
Human endothelial cells, which were obtained from microvessels of the skin (human dermal microvessel endothelial cells, HDMVEc), were incubated together with ED-B-specific Fab-antibody fragments. The number of bonded cells is made visible by means of crystal violet staining and measured in a photometer. High color intensity means many adherent cells and no binding-blocking antibodies. Low color intensity means little adhesion and a binding-blocking antibody.
Figure 2 shows a screening diagram for identifying ED-B-fibronectin-function-blocking antibodies by means of a Fab-fragment display.
In a first step, a panning procedure with recombinant ED-B is performed. The antibody fragments that are obtained in this way are subjected to a specificity test (ELISA), a functional adhesion test as well as an immunohistochemical study. Then, the affinity of the antibodies that carry out these tests is determined. Then, changes in the CDR domains are performed to improve the affinity, whereby affinity maturation I means a change in the L-CDR3 domain and affinity maturation 2 means a change in the H-CDR2 domain.
After the antibodies, obtained by affinity maturation, have passed through the above-mentioned tests, then the in vivo effectiveness is tested.
Figure 3 shows the results of a specificity test in ELISA format with the antibody-Fab fragments that are produced by panning with recombinant ED-B.
ED-B means the binding to recombinant ED-B, 6-FN means the binding to the recombinant fibronectin-domain 6, domain 7-8-9 means the binding to recombinant fibronectin domains 7-8-9 (without ED-B), and domain 7-EDB-8-9 means the binding to the recombinant fibronectin domains 7-8-9 with ED-B. The ratio of ED-B to 6-FN or 7-8-9 produces the specificity of the antibody under study. AP-39 means a covalent dimer of the biologically inactive anti-ED-B scFv antibody L19.
Figure 4 shows the result of an adhesion test with the antibodies under study.
The inhibition of the adhesion of HDMVEc cells to ED-B-coated plates by the indicated Fab fragments at concentrations of 25 g/ml or 0.4 g/ml Fab was examined. The indicated values are the result of 3x determination.
Figure 5 shows the immunohistochemical reaction pattern of function-blocking anti-ED-B-Fab fragments in the example of MOR 02616. In Figures 5A and 5C, the results with a negative control (negative HuCAL-Fab), and in Figures 5B and 5D, the results with the antibody MOR 02616 on human neuroblastoma cell-IRM xenotransplants (Figures 5A and 513) as well as on murine F9-teratocarcinoma cells (Figures 5C and 5D) are shown.
Cryosections of cells (thickness: 10 m) were air-dried and then set in ice-cold acetone for 10 minutes. Then, the sections were washed in PBS and incubated for 60 minutes with 2 g/ml of MOR 02616 or negative controls at room temperature. As a secondary antibody, a peroxidase-labeled polyclonal goat anti-human F(ab)2 antiserum (1:100 diluted in PBS, Dianova) was used in combination with diaminobenzidine (Sigma Chemicals) as a chromogenic substrate.
Figure 6 shows the results of a specificity test in ELISA format with the antibody Fab fragments that are optimized by maturation.
The execution of the test was as described in Figure 3.
Figure 7 shows the immunohistochemical reaction pattern of the antibody Fab fragments MOR 03255, optimized by maturation, on cryostat sections of F9-teratocarcinoma cells (Figure 7A) and on SKMEL-28 human-melanoma cell xenotransplants in hairless mice (Figure 7B).
The execution of the test was as described in Figure 5.
Figure 8 shows the result of an adhesion test with the antibody Fab fragments according to the invention relative to the comparison antibodies L19 and Ly6 0.3.
The execution of the test was described very much like in Figure 4. The antibody concentrations were I l/ml, 10 l/ml or 20 l/ml.
Figure 9 shows the stability of the antibody-Fab fragments according to the invention after incubation for 4 hours in human plasma or PBS at 37 C.
The immune reactivity was determined in the ELISA test with concentrations in the range of 0.039 to 5 g/ml. The signal with 0.62 g/ml in human plasma without incubation was defined as a 100% value.
Figure 10 shows the therapeutic effectiveness of anti-ED-B-function-blocking Fab-antibody fragments in the example of MOR 03255.
Figure 11 shows the nucleotide sequences that code for the VH and VL regions of the antibodies according to the invention.
Example 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 produced according to standard procedures (e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbour Press).
Mouse antibodies against purified ED-B receptors were also produced according to standard processes by 50 g of purified receptor being administered in Freund's complete adjuvant to a mouse, 3 weeks later 25 g of purified receptor being administered in Freund's incomplete adjuvant, and another 3 weeks later all applications being administered intraperitoneally in incomplete adjuvant. Blood was then removed via the postorbital sinus 14 days after the 3d immunization.
Plasma fibronectin was ordered from Sigma.
1.2 Selection of ED-B-Specific Phages The selection was performed according to the HuCAL Gold Phage selection protocol.
The HuCAL Gold Library was subdivided into six framework-specific pools. The ED-B-specific phages were concentrated in three successive selection rounds of recombinant protein immobilized in 96-hole plates (Maxisorb, Nunc, Rochester, NY, USA). For panning A, the plate was coated with 1 g/hole of ED-B (Hisb) (10 g/ml in Ampuwa ) for 2 hours at 37 C.
For panning B, the plate was coated with ED-B in the same concentration in PBS
(pH 7.2 with 1 mmol of MgC12/1 mmol of CaC12) overnight at 4 C. The plates were blocked with 5% skim milk powder in PBS, 0.05% Tween20 (Sigma, St. Louis, MO, USA) (blocking buffer) and incubated with 1 x 1013 HuCAL Gold phages, which had been preincubated with a volume of blocking buffer. For panning B, the phages were pre-incubated in addition with 0.5 g/ml of fibronectin-domain 6 in order to reduce the selection of binding molecules that are specific to the conserved structure of a type-3 domain repetition of fibronectin. After an incubation with the phages for 2 hours at room temperature, the holes were washed 5x with PBS, 0.05% Tween20 and 5x with PBS. The remaining phages were eluted with 20 mmol of dithiothreitol (DTT) in 10 mmol of tris/HCl (pH 8.0) for 10 minutes at room temperature. Then, an incubation with E. coli TG-1 (Stratagene, OD600 = 0.5) was carried out for infection. Then, the holes were incubated with E.
coli TG-1 as an additional elution step.
1.3 Phagemid Recovery, Phage Amplification and Purification Die HuCAL Gold phages were amplified in 2x TY medium with 34 g/ml of chloramphenicol and 1% Glucose (2x TY-CG). After infection with a helper phage (VCSM13) at 37 C and an OD600 of about 0.5, centrifuging and resuspending in 2x TY-CG/50 g/ml of kanamycin, the cells were induced with 0.25 mmol of IPTG and cultivated overnight at 22 C.
The phages were precipitated from the supernatant with polyethylene glycol (Ausubel et al.
(1998), Current Protocols of Microbiology), resuspended in PBS and used for subsequent selection rounds.
1.4 Subcloning of Selected Fab Fragments and Expression of Soluble Fab Fragments The Fab-coding insertions of the selected HuCAL Gold phages were subcloned in the expression vector pMORPHx9-FS. The plasmid-DNA preparation of the selected HuCAL Gold clone was cleaved with the restriction enzymes XbalI/EcoRI, whereby the Fab-coding insertion was cut out (ompA-VL and phoA-Fd). Fab molecules that are expressed in this vector contain two C-terminal labelings (FLAGTM and Strep-taglI) for detection and for purification.
1.5 Expression and Purification of HuCAL Gold Antibodies in E.coli The expression of the TG-1 F cells on pMORPHx9-FS-coded Fab fragments in E.coli was performed in shaking bottle cultures with 0.75 1 of 2xTY medium and 34 g/ml of chloramphenicol. After induction with 0.75 mmol of IPTG, the cells were cultivated for 16 hours at 30 C. As an alternative, Fab clones, which had been obtained from the second maturation pool 2, were induced with 0.1 mmol of IPTG and then cultivated at 22 C.
Periplasmatic extracts from cell pellets were produced by osmotic shock, and the Fab fragments were isolated by Strep-Tactiri chromatography (IBA, Gottingen, Germany). The apparent molecular weights were determined by size-exclusion chromatography (SEC) with calibrating standards. The concentrations were determined by UV spectrometry.
1.6 Identification of ED-B-Binding Fab-Fragments by ELISA
96-Hole-Maxisorb ELISA plates were coated with 100 l of ED-B solution (10 g/ml in coating buffer (PBS pH 7.4, 1 mmol of CaC12, I mmol of MgC12) overnight at 4 C. Crude lysates or purified Fab molecules were added; non-binding Fab molecules were removed by 5x washing with washing buffer (PBS pH 7.4, 0.05% Tween20). The Fab fragments were detected 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 that express for ED-B-specific Fab molecules were identified by a positive ELISA signal on immobilized ED-B versus little or no signal at 6FN.
1.7 Determination of Plasma Stability Under Physiological Temperature Conditions by ELISA
The coating with ED-B (2.5 g/ml) was performed essentially as described above. The Fab fragments were incubated for 4 hours at 37 C and a concentration of 50 g/ml in human plasma (German Red Cross; Batch 9985550). After incubation, the Fab molecules were diluted for the ELISA Test to the concentrations of 5.0, 2.5, 1.25, 0.62, 0.31, 0.156, 0.078 and 0.039 g/ml in PBS with 1% bovine serum albumin to determine the linear area of the signal intensity.
Functional Fab molecules were determined with the anti-Flag M2 antibody (Sigma F3165) and a secondary anti-mouse-antibody-alkaline phase conjugate, followed by development with Attophos (Roche) and by measurement at 535 nm.
1.8 HDMVEc Cell Culture and Cell Adhesion Test Human dermal microvascular endothelial cells (HDMVEc), isolated from juvenile foreskin, were cultivated in EGM-MV medium (Clonetics, Inc.) with the addition of 10% fetal calf serum, 2 mmol of glutamine, 20 g/ml of heparin and 3 ng/ml of bFGF in vessels, coated with 0.1 % gelatin (Sigma). For adhesion tests, cells that had not been cultivated longer than passage 8 were used. ED-B (10 g/ml) or plasma fibronectin (2.5 g/ml) was immobilized overnight at 4 C in PBS, pH 7.4, 0.9 mmol of CaC12, 0.5 mmol of MgC12 in 96-hole ELISA
plates and blocked with 1% RSA in PBS, pH 7.4, 0.9 mmol of CaClz, 0.5 mmol of MgCl2 for 1 hour at 37 C. The cells were labeled with 0.15 g/ml of calcein for 30 minutes at 37 C and washed once in adhesion medium (MCDB 131, 2 mmol of glutamine, 0.1 % RSA, 20 g/ml of heparin). I x 105 cells were incubated for 20 minutes at 37 C with the antibody concentration that is indicated in each case, diluted in adhesion medium (final volume 100 l). The cell suspension was added to the ED-B-coated plates for 2 hours at 37 C. After the cells are washed 2x in PBS, pH 7.4, 0.9 mmol of CaC12, 0.5 mmol of MgC12, adherent cells were detected in a plate fluorimeter (excitation 485 nm; emission 530 nm).
As a control, (A) the cell binding to ligand-coated plates was determined without adding specific antibodies, and (B) the cell adhesion to holes was determined without ligand coating, whereby the value obtained in the last-mentioned control was defined as a minimum of cell adhesion in the test.
1.9 Affinity Maturation of Selected Fab Molecules by Exchange of CDR
Cassettes in Steps To increase the affinity and biological activity of selected antibody fragments, CDR
regions were optimized by cassette mutagenesis with use of trinucleotide-directed mutagenesis.
To this end, Fab fragments from the expression vector pMORPHx9 in the phagemid vector pMORPH-23 were cloned with use of the EcoRI/Xbal restriction sites. To optimize the affinity of the selected Fab fragments, two successive maturation steps for each maturation pool were performed. In the first step, an antibody fragment-phage library was produced in which the L-CDR3 region of the starting clone was varied by a repertoire of 7 x 108 (Pool 1) and 3.8 x 108 (Poo12) individual light chain-CDR sequences. Then, affinity-improved derivatives from the first maturation step were exposed to a second maturation round by the H-CDR-2 region being replaced by a library of diversified elements. In this way, two phage libraries with 1 x 10g individual clones in each case were produced.
The affinity maturation libraries were produced by transformation into E.coli TOP l OF.
The phages were produced as described above. The selection in Fab fragments with improved affinity was performed under stringent conditions for three rounds in the first maturation and for two rounds in a second step.
1.10 Determination of Affinity by Surface Plasmon Resonance (BIAcore ) To determine the KD values, monomer fractions (at least 80% monomer content, determined by analytical SEC; Superdex75, Amersham Pharmacia) of Fab fragments were used.
F1-Chips (Biacore, Sweden) were coated with about 200 RU of ED-B (30 g/ml, 10 mmol of acetate buffer, pH 4.0) and reference flow cells with a corresponding amount of human serum albumin (20 g/ml, 10 mmol of acetate buffer, pH 4.5) with use of standard EDC-NHS-amine-coupling chemistry. The antigen density was reduced to about 100 RU for the Fab characterization of the second maturation. The regeneration was carried out with 10 mmol of HCI. All kinetic measurements were carried out in PBS buffer (136 mmol of NaC1, 2.7 mmol of KCI, 10 mmol of Na2HPO4 , 1.76 mmol of KHZPO4, pH 7.4) with a flow rate of 20 l/minute with use of a Fab concentration range of 1.5-500 nmol. The injection period was 1 minute in each case. All sensograms were evaluated with use of BIA evaluation software 3.1.
Abbreviations: EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, NHS: N-Hydroxysuccinimide, RU: Resonance Units 2. Results 2.1 Production of Antibodies Against Human ED-B
Two different panning assays were implemented. The coating with ED-B for the first assay (panning A) was performed according to the conditions known from the biological test to present ED-B in a conformation that allows the adhesion of HDMVEc cells to the immobilized antigen. With the second panning (B), the phages were blocked in addition with excess 6FN to avoid a selection of binding molecules that cross-react with the fibronectin-domain 6.
Altogether, 23 different HuCAL-Fab molecules from 1315 primary hits were found that meet the criteria of binding to ED-B and not to 6FN. In addition, the antibodies were tested in their capacity for binding to a recombinantly expressed fibronectin, comprising the domains 7, 8 and 9 with and without the inserted ED-B domains in their natural arrangement (see Figure 3).
All tested antibodies showed a 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 of the ED-B Specific Binding Molecule The antibodies as specifically identified in ELISA were purified and tested in their capacity for blocking the adhesion of HDMVEc cells to ED-B-coated plates. The adhesion of HDMVEc cells in the presence of ED-B-specific Fab fragments in two concentrations was tested in comparison to an unspecific HuCAL-control-Fab-molecule and positive mouse control sera.
Twelve of the 23 Fab molecules described in Section 2.1 showed an inhibition of cell adhesion in immobilized ED-B. A representative selection of the six most effective Fab molecules is shown in Figure 4. At a concentration of 25 g/ml, the cell adhesion was inhibited by 50% to almost 100%. At a concentration of 0.4 g/ml, only a few Fab molecules, e.g., MOR02610, MOR02616, MOR02715 and MOR02718, showed a reproducible inhibition of the cell adhesion.
The function-blocking Fab-antibody fragments were characterized by immunohistochemistry with respect to their localization on F9-mous-teratocarcinoma cells and human IRM30 neuroblastoma-xenotransplant cryosections. All tested antibodies showed a vascular localization on two tissue sections, but no staining of vessels on normal tissue (e.g., normal and sclerotic liver and skin). In Figures 5B and 5D, a perivascular abluminal staining of vessels, proliferating endothelium and tumor stroma by MOR02616 is shown. A
collection of ED-B in the vicinity of neovascular structures of exponentially growing tumors, but not in normal vessels, can be detected. With a negative control antibody against lysozyme at the same concentration, no staining was visible (Figures 5A and 5C).
The properties of twelve selected binding molecules was determined by BlAcore analysis (see Table 1). The affinities (Koõ) were in the range of 15 to 500 nmol. The affinity of the bivalent L19 derivative AP39 (a biologically inactive comparison antibody against ED-B) was determined with 2.4 nmol. Table 1 also shows the CDR sequences (only L-CDR3 and H-CDR3) and the framework combinations. At least two independent measurements with Fab molecules from different batches were performed for protein expression and protein purification. The binding rate (Koõ) and the dissociating-off rate (Koff) of the Fab molecule are indicated in separate columns.
Table 1 Clone VH H-CDR3 VL L-CDR3 kon koff KD
[1/Ms] [1/ s] [nM]
AP 39 VH3 PF'PY--I-'L' K3 C" f~l -- PY 6,6E+05 1,5E-03 2,4 0,6 MOR02610 VH1B SPVYYKYD L1 QSYDKTSSTY 1,3E+06 9,1E-02 75 19 MOR02611 VH1B CLYYR-Fr,S L2 AAnl'G ---GW 1,5E+05 5,1E-02 332 23 MOR02613 VH3 YvNC--FDz L2 Q'rYAKKDYSL 7,8E+05 1,3E-01 164 14 MOR02614 VH3 A----- Yrw L1 AMFSP---EC 2,8E+05 4,2E-02 160 6 MOR02616 VH3 VivL--FDY K3 LQKYSi PF 5,4E+05 2,8E-02 59 25 MOR02618 VH3 NiwV--F-Z,Y L3 UsYnrJFtaDsV 4,3E+05 2,OE-01 477 177 MOR02619 VH4 F-----FDV L1 QSWDGAS-TG 7,8E+05 1,2E-02 15,4 0,6 MOR02622 VH3 GLVT--FDN L2 SSwTxSFq'DY 2,4E+05 5,1E-03 21,3 1,7 MOR02715 VH3 raxvc--FUV L2 QAwDNQcMKY 8,9E+05 4,7E-02 53,7 1,7 MOR02718 VH3 cwF---FAx K3 FQYSSV--PL 4,5E+05 3,9E-02 85 12,5 MOR02721 VH5 'rr3----c~,e L1 ~7, "'T TC; --s 4,3E+05 2,4E-02 56 11,6 MOR02722 VH3 e as K2 aVf, ~ raF P F 1,5E+05 1,4E 02 94 2,3 2.3 Affinity Maturations of Function-Blocking ED-B-Antibodies by Optimization of L-CDR3 and H-CDR2 Regions The affinity maturations of ttie Fab molecules were perfonned in two sections.
First, the L-CDR3 sequence was diversified, and then the H-CDR2 sequence of the improved Fab molecule, obtained in the first step, was exchanged.
Two affinity maturation libraries for each step were produced. The optimization was performed in two separate pools. The L-CDR3 library I with a degree of diversity of 7 x l Ox elements contained the four starting Fab molecules MOR02610., MOR02616, MOR02619 and ~ . .
MOR02622. The L-CDR3 library 2 with a degree of diversity of 3.8 x 108 elements contained only derivatives of MOR02715 and MOR02718. Here, the Fab molecules were combined into groups in each case according to their affinity and biological activity in the adhesion test.
The two libraries were kept separately during the selection procedure. In this case, two panning strategies were performed. Panning I was carried out with ED-B
immobilized on maxisorb plates for three rounds, as previously indicated. With Panning II, a selection of biotinylated ED-B in solution was carried out. In this case, the phage-antigen complex was recovered by streptavidin-coated particles in the first round and on neutravidin plates in the two subsequent rounds. Stringent conditions were used within the scope of the selection procedure.
The screening on optimized Fab molecules was carried out by determining the Koff values in the BlAcore system. Altogether, 11 derivatives of library I were characterized. The 5 derivatives with the highest affinity are indicated in Table 2. In this case, all improved clones are derivatives of MOR02619 with an increase in affinity of up to 7x. In clone MOR03075, an affinity of 2.4 nmol was found.
From library 2, two derivatives of MOR02715 were analyzed in detail. For clone MOR03062, a 15-fold affinity increase was found, so that a monomer affinity of 2.4 nm was obtained. The changes in the amino acid sequence of the L-CDR3 region are shown in Tables 2a and 2b.
j , . 31 Table 2a Pan- MORO Initial LCDR3 Kon Koff KD x-Fold Improve-ning Clone [1/Ms] [1/s] [nm] ment Relative to Pool 1 the Parent Clone Kon Koff KD
AP 39- QQTGRI - - 6.6E + 1.49E - 2.4 ~
Biotin PP 05 003 0.6 2619 QSWDGAS- 7.8E + 1.2E - 02 15.4 f TG 05 0.6 Solid 3055 2619 QAWTRAHR 1.8E+ 5.1E - 03 3.0 f 2.2 2.6 5.4 YP 06 0.5 Dis- 3066 2619 SSYD- 1.1E + 4.7E - 03 4.2 ~ 1.4 2.8 3.8 solved TQVTR 06 0.6 IL-Dis- j 3075 2619 QSWDP- 1.1E + 2.6E - 031 2.4 f I 1.4 5.0 6.8 solved RSFT 06 0.3 Dis-3069 2619 WTGM - - 1.2E + 4.1E - 03 3.3 f 1.5 3.2 4=8 solved]__L SYHF 06 0.2 Dis- 3071 2619 LAYIQS- 1.9E + 5.7E - 03 3.1 ~ I 2.3 2.3 5.1 solved KGH 06 1 ! 0.8 I- _- -__ Table 2b Pan- MORO Par- LCDR3 Kon Koff KD x-Fold ning ent [1/Ms] [1/s] [nm] Improvement Pool 2 clone Relative to the Parent Clone Kon Koff K1 AP 39- QQTGRI - - 6.75E + 1.49E - 2.4 I
Biotin PP 05 03 0.6 1 2715 QAWDNQG 9.9E + 4.7E - 02 53.7 f~
MKY 05 1.7 Solid 3064 2715 QSWDLLAPS 1.5E + 1.4E - 0210 f 3.5 1.7 3.5 5.3 Solid 3062 2715 QSWDLSVH, 3.5E + !1.1 E -02 3.4 ~ 4.0 4.2 15.81 0.6 0.8 _--1------L--1----~-- -The specificity for ED-B (determined by ELISA, immunohistochemistry and cell adhesion test) of all derivatives from the first maturation round were unchanged.
The Fab molecules shown in Tables 2a and 2b were selected for a subsequent H-maturation in two pools. In pool 1, five Fab molecules were subjected to a similar activity, but with different L-CDR3 regions of a diversification in H-CDR2 (library size of 7 x 107 eleinents).
For pool 2 (library size of 8.5 x 107 eleinents), two Fab molecules were selected. The libraries were selected separately as described above. Binding molecules with increased affinity were concentrated by two panning rounds in solution under stringent conditions.
Three Fab molecules were selected from pool 1. For derivatives MOR03243 and MOR03245, affinities of about 0.7 nrnol were found. For derivative MOR03246, an affinity of 0.6 nmol was found.
The H-CDR2 secluences of the corresponding clones and the affinity data are shown in Table 3a.
Table 3a MORO Parental Sequenz HCDR2 kon [1/Ms] koff [1/ s] KD [nM]
Clone MOR03243 3055 EIHRGGYTQYNPSLKS 2,9E+06 1,8E-03 0,7 0,2 MOR03245 3069 vtttKwGFTNYNPs7,xs 3,5E+06 1,6E-03 0,7 0,3 MOR03246 3075 YIFIKYGwTKYNPSLKS 4,8E+06 3,OE-03 0,6 0,1 [Key: Sequenz = Sequence]
Derivatives with improved propertics could also be selected from pool 2. In comparisoarl to the parent clones, the affinities of the derivatives were improved by a factor of 26 up to 250x.
The affinities of the clones MOR03255 and MOR03258 were determined with 30 pM
or 40 pM.
In Table 3b, 11-CDR2-amino acid sequences of the selected clones from pool 2 as well as the corresponding affinity data are shown.
Table 3b Parental MORO Sequenz HCDR2 kon [11Ms] koff [11 s] KD [nM] STDEV
Clone MOR03251 3062 V1SNMsY'111YYnUwYG 3,3E+06 2,OE-04 0,07 0,04 MOR03252 3062 vzsNY6wHryYI~ nsvxc 3,1E+06 6,1E-05 0,03 0,03 MOR03253 3062 Mut ~I~ta.-~cE'~IYYnDS:~KG 1,6E+06 2,OE-04 0,13 0,05 MOR03255 3062 V TraussYI 1 YtYPsiF,4,9E+06 8,6E-05 0,03 0,03 MOR03257 3062 1: Ntzc,[~YIYYr.n~~vr~~ 5,3E+06 3,OE-04 0,08 0,06 MOR03258 3064 2,7E+06 8,8E-05 0,04 0,02 [Key: Sequenz = Sequence]
2.4 Characterization of Highly Affine Function-Blocking ED-B-Antibodies To test whether the antibodies that are obtained by affinity maturation are still always specific for ED-B, specificity tests in the ELISA forinat (Figure 6), iminunohistocheinical studies (Figure 7) and adhesion tests (Figure 8) were performed. In all three tests, it was found that the specificity of the affinity-matured Fab inolecules for ED-B remained unchanged. In addition, it is evident from Figure 8 that both the parent clones, such as MOR 02715, and the clones that are 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 the Fab molecules in human plasma was tested. To this end, the Fab molecules were incubated with human plasma for 4 hours at 37 C without a significant loss in immune reactivity determined by ELISA (Figure 9) being found.
All selected Fab molecules are able to block the interaction of a receptor with human microvascular endothelial cells on ED-B in a dose-dependent way, a property that known ED-B-antibody L19 does not have. Since the adhesion of HDMVEc to the extracellular matrix is one of the early steps in neoangiogenesis, the blocking of this process by the binding molecules according to the invention produces a therapeutic agent for inhibiting the formation of new vessels and for inhibiting tumor growth.
2.5 In vivo Action F9-Teratocarcinoma cells (106/mouse in Matrigel with/without 100 g of Fab MOR
03255) were implanted s.c. in the flanks of hairless mice. After 3 days, the animals were treated for 7 successive days with, in each case, 100 g of MOR03255/mouse. In comparison to the solvent control, both treatment groups show an inhibition in tumor growth in the range of 42-64%. A summary of test results is shown in Figure 10.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.
The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
In the foregoing and in the examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated.
The entire disclosures of all applications, patents and publications, cited herein and of corresponding German application No. 102004050101.7, filed October 14, 2005 and U.S.
Provisional Application Serial No. 60/697,565, filed July 11, 2005, are incorporated by reference herein.
The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Literature (1) Brown, L. F.; Guidi, A. J.; Schnitt, S. J.; Van De Water, L.; Iruela-Arispe, M. L.; Yeo, T.-K.; Tognazzi, K.; Dvorak, H. F. (1999) Clin. Cancer Res. 5:
(2) Folkman, J. (1995) Nature (Med.) 1: 27-31 (3) Risau, W. and Lemmon, V. (1988) Develop. Biol. 125: 441-450 (4) Van Den Hoff, A. (1988) Adv. Cancer Res. 50: 159-196 (5) Folkman, J. and Shing, Y. (1992) J. Biol. Chem. 267: 10931-10934 (6) George, E. L.; Baldwin, H. S.; Hynes, R. O. (1997) Blood 90: 3073-3081 (7) Bowersox, J. C. and Sorgente, N. (1982) Cancer Res. 42: 2547-2551 (8) Madri, J. A.; Pratt, B. M.; Tucker, A. M. (1988) J. Cell Biol. 106: 1375-(9) Nicosia, R. F.; Bonnano, E.; Smith, M. (1993) J. Cell. Physiol. 154: 654-(10) Kornblihtt, A. R.; Vibe-Pedersen, K.; and Baralle, F. E., 1983. Isolation and Characterization of cDNA Clones for Human and Bovine Fibronectins. Proc. Natl.
Acad.
Sci. USA, 80, 3218-22 (11) Leahy, D. J.; Hendrickson, W. A.; Aukhil, I. and Erickson, H. P., 1992.
Structure of Fibronectin Type III Domain from Tenascin Phased by MAS Analysis of the Selenomethionyl Protein. Science, 258, 987-91 (12) Hynes, R. 0., 1987, Cell 48, 549-550 (13) Plow, E. F. et al. 2000, J. Biol. Chem., 275, 21785-21788 (14) Castellani, P.; Viale, G.; Dorcaratto, A.; Nicolo, G.; Kaczmarek, J.;
Querze, G.; Zardi, L. (1994) Int. J. Cancer 59: 612-618 . . .
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Claims (50)
1. A polypeptide, characterized in that it has (a) a VH region encoded by a nucleic acid sequence SEQ ID NO.
1 (MOR 02610), SEQ ID NO. 17 (MOR 02616), SEQ
ID NO. 25 (MOR 02619), SEQ ID NO. 29 (MOR
02622), SEQ ID NO. 33 (MOR 02715), or SEQ ID
NO. 37 (MOR 02718) or the H-CDR1, H-CDR2 and H-CDR3 regions from any of said VH regions, and (b) a VL region encoded by a nucleic acid sequence SEQ ID NO.
3 (MOR 02610), SEQ ID NO. 19 (MOR 02616), SEQ
ID NO. 27 (MOR 02619), SEQ ID NO. 31 (MOR
02622), SEQ ID NO. 35 (MOR 02715), or SEQ ID
NO. 39 (MOR 02718) or the L-CDR1, L-CDR2 and L-CDR3 regions from any of said VL regions.
1 (MOR 02610), SEQ ID NO. 17 (MOR 02616), SEQ
ID NO. 25 (MOR 02619), SEQ ID NO. 29 (MOR
02622), SEQ ID NO. 33 (MOR 02715), or SEQ ID
NO. 37 (MOR 02718) or the H-CDR1, H-CDR2 and H-CDR3 regions from any of said VH regions, and (b) a VL region encoded by a nucleic acid sequence SEQ ID NO.
3 (MOR 02610), SEQ ID NO. 19 (MOR 02616), SEQ
ID NO. 27 (MOR 02619), SEQ ID NO. 31 (MOR
02622), SEQ ID NO. 35 (MOR 02715), or SEQ ID
NO. 39 (MOR 02718) or the L-CDR1, L-CDR2 and L-CDR3 regions from any of said VL regions.
2. The polypeptide as claimed in claim 1, characterized in that it has a VL region encoded by the nucleic acid sequence SEQ ID NO. 27 (MOR 02619) or SEQ ID NO.
35 (MOR 02715), or the L-CDR1, L-CDR2 and L-CDR3 regions from any of these VL regions.
35 (MOR 02715), or the L-CDR1, L-CDR2 and L-CDR3 regions from any of these VL regions.
3. The polypeptide as claimed in either of claims 1 and 2, characterized in that it has a VH region encoded by the nucleic acid sequence SEQ ID NO. 25 (MOR 02619) or SEQ ID NO.
33 (MOR 02715), or the H-CDR1, H-CDR2 and H-CDR3 regions from any of these VH regions.
33 (MOR 02715), or the H-CDR1, H-CDR2 and H-CDR3 regions from any of these VH regions.
4. The polypeptide as claimed in claim 1, characterized in that it has the VH region encoded by SEQ ID NO. 1 (MOR 02610) and the VL region encoded by SEQ ID
NO. 3 (MOR 02610), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 3 (MOR 02610), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
5. The polypeptide as claimed in claim 1, characterized in that it has the VH region encoded by SEQ ID NO. 17 (MOR 02616) and the VL region encoded by SEQ ID
NO. 19 (MOR 02616), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 19 (MOR 02616), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
6. The polypeptide as claimed in claim 1, characterized in that it has the VH region encoded by SEQ ID NO. 25 (MOR 02619) and the VL region encoded by SEQ ID
NO. 27 (MOR 02619), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 27 (MOR 02619), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
7. The polypeptide as claimed in claim 1, characterized in that it has the VH region encoded by SEQ ID NO. 29 (MOR 02622) and the VL region encoded by SEQ ID
NO. 31 (MOR 02622), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 31 (MOR 02622), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
8. The polypeptide as claimed in claim 1, characterized in that it has the VH region encoded by SEQ ID NO. 33 (MOR 02715) and the VL region encoded by SEQ ID
NO. 35 (MOR 02715), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 35 (MOR 02715), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
9. The polypeptide as claimed in claim 1, characterized in that it has the VH region encoded by SEQ ID NO. 37 (MOR 02718) and the VL region encoded by SEQ ID
NO. 39 (MOR 02718), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 39 (MOR 02718), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
10. A polypeptide, characterized in that it has (a) a VH region encoded by the nucleic acid sequence SEQ ID
NO. 25 (MOR 02619), or SEQ ID NO. 33 (MOR
02715), or the H-CDR1, H-CDR2 and H-CDR3 regions from any of said VH regions, and (b) a VL region encoded by 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), or SEQ ID NO. 61 (MOR 03062) or the L-CDR1, L-CDR2 and L-CDR3 regions from any of said VL regions.
NO. 25 (MOR 02619), or SEQ ID NO. 33 (MOR
02715), or the H-CDR1, H-CDR2 and H-CDR3 regions from any of said VH regions, and (b) a VL region encoded by 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), or SEQ ID NO. 61 (MOR 03062) or the L-CDR1, L-CDR2 and L-CDR3 regions from any of said VL regions.
11. The polypeptide as claimed in claim 10, characterized in that it has the VH region encoded by SEQ ID NO. 25 (MOR 03055) and the VL region encoded by SEQ ID
NO. 49 (MOR 03055), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 49 (MOR 03055), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
12. The polypeptide as claimed in claim 10, characterized in that it has the VH region encoded by SEQ ID NO. 25 (MOR 03066) and the VL region encoded by SEQ ID
NO. 51 (MOR 03066), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 51 (MOR 03066), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
13. The polypeptide as claimed in claim 10, characterized in that it has the VH region encoded by SEQ ID NO. 25 (MOR 03075) and the VL region encoded by SEQ ID
NO. 53 (MOR 03075), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 53 (MOR 03075), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
14. The polypeptide as claimed in claim 10, characterized in that it has the VH region encoded by SEQ ID NO. 25 (MOR 03069) and the VL region encoded by SEQ ID
NO. 55 (MOR 03069), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 55 (MOR 03069), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
15. The polypeptide as claimed in claim 10, characterized in that it has the VH region encoded by SEQ ID NO. 25 (MOR 03071) and the VL region encoded by SEQ ID
NO. 57 (MOR 03071), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 57 (MOR 03071), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
16. The polypeptide as claimed in claim 10, characterized in that it has the VH region encoded by SEQ ID NO. 33 (MOR 03064) and the VL region encoded by SEQ ID
NO. 59 (MOR 03064), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 59 (MOR 03064), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
17. The polypeptide as claimed in claim 10, characterized in that it has the VH region encoded by SEQ ID NO. 33 (MOR 03062) and the VL region encoded by SEQ ID
NO. 61 (MOR 03062), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 61 (MOR 03062), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
18. A polypeptide, characterized in that it has (a) a VH region 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 ), or SEQ ID
NO. 87 (MOR 03258) or the H-CDR1, H-CDR2 and H-CDR3 regions from any of said VH regions, and (b) a VL region 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), OR SEQ ID NO. 89 (MOR 03258) or the L-CDR1, L-CDR2 and L-CDR3 regions from any of said VL regions.
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 ), or SEQ ID
NO. 87 (MOR 03258) or the H-CDR1, H-CDR2 and H-CDR3 regions from any of said VH regions, and (b) a VL region 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), OR SEQ ID NO. 89 (MOR 03258) or the L-CDR1, L-CDR2 and L-CDR3 regions from any of said VL regions.
19. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 63 (MOR 03243) and the VR region encoded by SEQ ID
NO. 65 (MOR 03243), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 65 (MOR 03243), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
20. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 67 (MOR 03245) and the VR region encoded by SEQ ID
NO. 69 (MOR 03245), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 69 (MOR 03245), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
21. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 71 (MOR 03246) and the VL region encoded by SEQ ID
NO. 73 (MOR 03246), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 73 (MOR 03246), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
22. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 75 (MOR 03251) and the VL region encoded by SEQ ID
NO. 77 (MOR 03251), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 77 (MOR 03251), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
23. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 79 (MOR 03252) and the VL region encoded by SEQ ID
NO. 77 (MOR 03252), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 77 (MOR 03252), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
24. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 81 (MOR 03253) and the VL region encoded by SEQ ID
NO. 77 (MOR 03253), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 77 (MOR 03253), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
25. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 83 (MOR 03255) and the VL region encoded by SEQ ID
NO. 77 (MOR 03255), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 77 (MOR 03255), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
26. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 85 (MOR 03257) and the VL region encoded by SEQ ID
NO. 77 (MOR 03257), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 77 (MOR 03257), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
27. The polypeptide as claimed in claim 18, characterized in that it has the VH region encoded by SEQ ID NO. 87 (MOR 03258) and the VL region encoded by SEQ ID
NO. 89 (MOR 03258), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
NO. 89 (MOR 03258), or the H-CDR1, H-CDR2 and H-CDR3 regions from this VH
region and the L-CDR1, L-CDR2 and L-CDR3 regions from this VL
region.
28. The polypeptide as claimed in any of claims 1 to 27 in the form of a conjugate with a therapeutic drug.
29. The polypeptide as claimed in claim 28, characterized in that the therapeutic drug is selected from radio-therapeutic and chemotherapeutic agents.
30. The polypeptide as claimed in any of claims 1 to 27 in the form of a fusion protein.
31. The polypeptide as claimed in claim 30 as fusion protein with a cytokine or as bispecific antibody.
32. The polypeptide as claimed in any of claims 1 to 27 in the form of a conjugate with a diagnostically detectable labeling group.
33. The polypeptide as claimed in claim 32, characterized in that the diagnostically detectable labeling group is selected from radio-, NMR-, dye-, enzyme- and fluorescence-labeling groups.
34. A pharmaceutical composition, characterized in that it comprises as active compound a polypeptide as claimed in any of claims 1 to 31 and pharmacologically customary carriers, excipients or/and diluents.
35. The composition as claimed in claim 34 for preventing or treating hyperproliferative disorders.
36. The composition as claimed in claim 34 or 35 for preventing or treating cancer.
37. A diagnostic composition which comprises as diagnostic reagent a polypeptide as claimed in any of claims 1 to 27, 32 or 33.
38. The composition as claimed in claim 37 for diagnosing hyperproliferative disorders or a predisposition therefor.
39. The composition as claimed in claim 37 or 38 for diagnosing cancer or a predisposition thereof.
40. The composition as claimed in any of claims 34 to 39 for application in human medicine.
41. A nucleic acid coding for a polypeptide as claimed in any of claims 1 to 27 or 30 and 31.
42. The nucleic acid as claimed in claim 41, operatively linked to an expression control sequence.
43. A vector, characterized in that it comprises a nucleic acid as claimed in either of claims 41 and 42.
44. A cell, characterized in that it is transformed with a nucleic acid as claimed in either of claims 41 and 42 or a vector as claimed in claim 43.
45. A nonhuman organism, characterized in that it is transformed with a nucleic acid as claimed in either of claims 41 and 42 or a vector as claimed in claim 43.
46. A method of preparing a polypeptide as claimed in any of claims 1 to 27, characterized in that a cell as claimed in claim 44 or a nonhuman organism as claimed in claim 45 is cultured under conditions under which the polypeptide is expressed, and the expressed polypeptide is obtained.
47. The use of a polypeptide as claimed in any of claims 1 to 31 for preparing a medicament for preventing or treating ED-B-associated disorders.
48. The use as claimed in claim 47 for preventing or treating cancer.
49. The use of a polypeptide as claimed in any of claims 1 to 27, 32 or 33 for preparing a detection reagent for diagnosing ED-B-associated disorders or a predisposition therefor.
50. The use as claimed in claim 49 for diagnosing cancer or a predisposition therefor.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP2005/012185 WO2007054120A1 (en) | 2005-11-09 | 2005-11-09 | Identification and characterization of function-blocking anti-ed-b-fibronectin antibodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2628552A1 true CA2628552A1 (en) | 2007-05-18 |
Family
ID=36170357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002628552A Abandoned CA2628552A1 (en) | 2005-11-09 | 2005-11-09 | Identification and characterization of function-blocking anti-ed-b-fibronectin antibodies |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1945767B1 (en) |
| JP (1) | JP2009514540A (en) |
| CN (1) | CN101356271A (en) |
| AT (1) | ATE463512T1 (en) |
| BR (1) | BRPI0520677A2 (en) |
| CA (1) | CA2628552A1 (en) |
| DE (1) | DE502005009389D1 (en) |
| WO (1) | WO2007054120A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019212694A1 (en) * | 2018-04-30 | 2019-11-07 | Viktor Veniaminovich Tets | Tetz-proteins and prion-like proteins and associated methods |
| US11192943B2 (en) | 2017-09-30 | 2021-12-07 | Hefei Lifeon Pharmaceutical Co., Ltd. | Protein binding to fibronectin B domain |
| US11918610B2 (en) | 2018-06-29 | 2024-03-05 | Viktor Veniaminovich Tets | Methods for diagnosis and treatment of type 1 diabetes |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19932688B4 (en) | 1999-07-13 | 2009-10-08 | Scil Proteins Gmbh | Design of beta-sheet proteins of gamma-II-crystalline antibody-like |
| DE10324447A1 (en) | 2003-05-28 | 2004-12-30 | Scil Proteins Gmbh | Generation of artificial binding proteins based on ubiquitin |
| EP1925664A1 (en) | 2006-11-15 | 2008-05-28 | Scil proteins GmbH | Artificial binding proteins based on a modified alpha helical region of ubiquitin |
| WO2009102421A2 (en) | 2008-02-14 | 2009-08-20 | Bristol-Myers Squibb Company | Targeted therapeutics based on engineered proteins that bind egfr |
| TWI496582B (en) | 2008-11-24 | 2015-08-21 | 必治妥美雅史谷比公司 | Bispecific egfr/igfir binding molecules |
| CA2778871C (en) | 2009-12-14 | 2017-08-01 | Scil Proteins Gmbh | Modified ubiquitin proteins having a specific binding activity for the extradomain b of fibronectin |
| CN103180339B (en) | 2010-05-26 | 2016-04-27 | 百时美施贵宝公司 | There is the scaffold protein based on fibronectin of the stability of improvement |
| WO2012171541A1 (en) | 2011-06-15 | 2012-12-20 | Scil Proteins Gmbh | Human fusion proteins comprising interferons and hetero-dimeric modified ubiquitin proteins |
| US9492572B2 (en) | 2011-06-15 | 2016-11-15 | Scil Proteins Gmbh | Dimeric binding proteins based on modified ubiquitins |
| EP2861616A1 (en) | 2012-06-13 | 2015-04-22 | Scil Proteins GmbH | Human fusion proteins comprising single chain tnfalpha and targeting domains |
| WO2014094799A1 (en) | 2012-12-19 | 2014-06-26 | Scil-Proteins-Gmbh | Ubiquitin moieties as a means for prolonging serum half-life |
| CA2975362A1 (en) | 2015-02-06 | 2016-08-11 | Navigo Proteins Gmbh | Egfr binding proteins comprising ubiquitin muteins |
| CN107922483B (en) | 2015-07-16 | 2021-07-30 | 纳维格蛋白质有限公司 | Novel immunoglobulin-binding proteins and their use in affinity purification |
| JP2018520675A (en) | 2015-07-20 | 2018-08-02 | ナフィゴ プロテインズ ゲゼルシャフト ミット ベシュレンクテル ハフツングＮａｖｉｇｏ Ｐｒｏｔｅｉｎｓ ＧｍｂＨ | Novel binding protein based on diubiquitin mutant protein and production method thereof |
| US20180340936A1 (en) * | 2015-11-16 | 2018-11-29 | Hefei Lifeon Pharmaceutical Co. Ltd. | Use of ed-b protein in diagnosis of tissue hyperplasia |
| WO2017191252A1 (en) | 2016-05-04 | 2017-11-09 | Navigo Proteins Gmbh | Targeted compounds for the site-specific coupling of chemical moieties comprising a peptide linker |
| WO2018029158A1 (en) | 2016-08-11 | 2018-02-15 | Repligen Corporation | Alkaline stable fc—binding proteins for affinity chromatography |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2229043C (en) | 1995-08-18 | 2016-06-07 | Morphosys Gesellschaft Fur Proteinoptimierung Mbh | Protein/(poly)peptide libraries |
| TWI259837B (en) * | 1998-05-11 | 2006-08-11 | Eidgenossische Tech Hochscule | Specific binding molecules for scintigraphy, conjugates containing them and therapeutic method for treatment of angiogenesis |
| CA2347973C (en) | 1999-07-20 | 2010-06-22 | Morphosys Ag | Novel methods for displaying (poly)peptides/proteins on bacteriophage particles via disulfide bonds |
| WO2001051087A2 (en) * | 2000-01-12 | 2001-07-19 | Light Sciences Corporation | Novel treatment for eye disease |
| JP2003524018A (en) | 2000-02-24 | 2003-08-12 | アイトゲネーシシェ テクニシェ ホッホシューレ チューリッヒ | Antibodies specific for the ED-B domain of fibronectin, complexes containing said antibodies, and uses thereof for detecting and treating angiogenesis |
| BR0113737A (en) | 2000-09-07 | 2004-02-25 | Schering Ag | Edbfibronectin Domain Receptor (11) |
-
2005
- 2005-11-09 WO PCT/EP2005/012185 patent/WO2007054120A1/en active Application Filing
- 2005-11-09 CN CNA2005800524909A patent/CN101356271A/en active Pending
- 2005-11-09 BR BRPI0520677-4A patent/BRPI0520677A2/en not_active IP Right Cessation
- 2005-11-09 CA CA002628552A patent/CA2628552A1/en not_active Abandoned
- 2005-11-09 EP EP05811334A patent/EP1945767B1/en not_active Not-in-force
- 2005-11-09 JP JP2008539258A patent/JP2009514540A/en not_active Withdrawn
- 2005-11-09 AT AT05811334T patent/ATE463512T1/en not_active IP Right Cessation
- 2005-11-09 DE DE502005009389T patent/DE502005009389D1/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11192943B2 (en) | 2017-09-30 | 2021-12-07 | Hefei Lifeon Pharmaceutical Co., Ltd. | Protein binding to fibronectin B domain |
| US11970529B2 (en) | 2017-09-30 | 2024-04-30 | Hefei Lifeon Pharmaceutical Co., Ltd. | Protein binding to fibronectin B domain |
| WO2019212694A1 (en) * | 2018-04-30 | 2019-11-07 | Viktor Veniaminovich Tets | Tetz-proteins and prion-like proteins and associated methods |
| US11918610B2 (en) | 2018-06-29 | 2024-03-05 | Viktor Veniaminovich Tets | Methods for diagnosis and treatment of type 1 diabetes |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1945767A1 (en) | 2008-07-23 |
| BRPI0520677A2 (en) | 2009-05-19 |
| ATE463512T1 (en) | 2010-04-15 |
| JP2009514540A (en) | 2009-04-09 |
| CN101356271A (en) | 2009-01-28 |
| WO2007054120A1 (en) | 2007-05-18 |
| DE502005009389D1 (en) | 2010-05-20 |
| EP1945767B1 (en) | 2010-04-07 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |