DE10013286A1 - New human humanized antibody that specifically binds to fibroblasts activating protein alpha, useful for treating cancer or tumor, and for imaging tumors associated with activated stromal fibroblasts, e.g. lung or breast cancer - Google Patents

Abstract

Human or humanized antibody, which specifically binds to fibroblasts activating protein alpha (FAPalpha ), and which is fully human or contains not more than one murine complementarity-determining region (CDR region) of the monoclonal antibody F19 (ATCC accession number HB 8269), is new. Independent claims are also included for the following: (1) antibodies having a variable region of the heavy chain (VH) consisting of a 127 (VH13), 125 (VH46) or 134 (VH50) residue amino acid sequence, all fully defined in the specification, and the variable region of light chain (VL) comprises a 118 residue amino acid sequence (VLIII25), fully defined in the specification; (2) antibodies having a coding sequence of the variable region of VH consisting of a 381 (VH13), 375 (VH46) or 402 base pair sequence, (VH50) all fully defined in the specification, and the coding sequence of the variable region of the VLL consists of a 358 base pair sequence (VLIII25), fully defined in the specification; (3) an antibody having a variable region of VH consisting of a sequence selected from two amino acids sequences each comprising 132 amino acids (VH34 or VH18), and a variable region of VL consisting 118 amino acids (VLIII43); (4) antibodies having a coding sequence of the variable region of VH consisting of a defined sequence selected from two 396 base pair sequences (VH34 or VH18), and the variable region of the VL consists of 356 base pairs (VLIII43); (5) a nucleic acid coding for an antibody of (4); (6) a recombinant DNA vector containing the nucleic acid of (3); (7) a host containing a vector of (6); (8) a process for preparing the antibody by cultivating a host of (7) under conditions where antibody protein is expressed by the host cell, and isolating the antibody protein; (9) pharmaceutical preparations containing an antibody of (4), and one or more carrier; (10) process for detecting activated stromal fibroblasts in wound healing, inflammatory processes or tumor by contacting a probe that might possibly contain activated fibroblasts, with an antibody protein under conditions for the formation of a complex between the antibody protein and its antigen; (11) antibodies comprising or corresponding to a fully defined sequence of 255, 260, 260, 253 or 262 amino acids given in the specification, and their functional variants; and (12) antibodies encoded by a 767, 782, 782, 761 or 788 base pair sequence, fully defined in the specification. - ACTIVITY : Cytostatic. No biological data is given. - MECHANISM OF ACTION : Fibroblast activating protein-alpha modulator.

Description

The invention relates to antibody proteins, specifically fibroblast activating protein bind alpha (FAPα). The invention further relates to the use of said antibodies for diagnostic and therapeutic purposes and methods for Production of said antibodies.

Background of the invention

Massive growth of epithelial cancer is characteristic with a number of cellular and molecular changes in the surrounding stromal cells. On highly consistent feature of the reactive stroma of numerous types of Epithelial cancer is the induction of the fibroblast activating protein alpha (from now on as FAPα or FAP), a cell surface molecule of the reactive stromal Fibroblasts originally incubated with the monoclonal antibody F19 (Garin-Chesa P., Old L.J. and Rettig W.J .; 1990; Proc Natl. Acad. Sci. 87: 7235). Since that FAP is selectively expressed in stroma of a number of epithelial cell carcinomas, namely regardless of the location and histological type of carcinoma, it was desirable to one Therapy concept for the FAPα target molecule to develop imaging techniques, Diagnosis and treatment of epithelial cell cancer as well as many others Disease pictures. For this purpose, a monoclonal murine Developed antibody that specifically binds to FAP. This antibody has been fully described in U.S. Patents 5,059,523 and WO 93/05804 be incorporated by reference in this document. A serious problem occurs when looking at non-human antibodies for in vivo human applications used, namely, that they quickly directed against the foreign antigen Cause immune response. In the worst case, such an immune response against the Antibodies cause an anaphylactic shock. This will be the Efficiency of the antibody in the patient is drastically reduced and further application deteriorated or made impossible.

The humanization of non-human antibodies is usually achieved in one of two ways:

• 1. by the construction of non-human / human chimeric antibodies, wherein the non-human variable regions are linked to the human constant regions (Boulianne G.L., Hozumi N. and Shulman, M.J. (1984)) Nature 312: 643) or
• 2. By determining complementarity in human variable regions Regions (English: "complementarity determining regions" - CDRs) with those of the replaced by non-human variable region and then the reshaped humanized couples variable regions to human constant regions (Riechmann L., Clark M., Waldmann H. and Winter G. (1988) Nature 332: 323).

Chimeric antibodies are less than non-human antibodies Foreign protein sequences and therefore have a lower xenoantigenes potential. Nevertheless, such chimeric antibodies may be due to the non-human V regions in the Humans trigger an immune response (LoBuglio A.F., Wheeler R.H., Trang J., Haynes A., Roger K., Harvey E.B., Sun L., Ghrayeb J. and Khazaeli M.B. (1989) Proc. Natl. Acad. Sci. 86: 4220). CDR-transmitted or reshaped humanized Although antibodies contain fewer foreign protein sequences in the V regions, they are These humanized antibodies are still able to provide an immune response in humans cause. In WO99 / 57151 A2 are such FAPα-specific humanized Described antibodies in which humanisation was achieved by transferring all 6 CDR Regions (3 of the light, 3 of the heavy chain) from the corresponding F19 Mouse antibody was. These antibodies also contain parts of the murine ones Framework region.

The object of the present invention is to provide improved FAPα-specific antibodies to provide that overcome the above-mentioned disadvantages of the prior art.

Description of the invention

The task was within the scope of the claims and description of the present Invention solved.

The use of the singular or plural in the claims or the description should not be limiting in any way and also include the other form.

The invention enters new human or humanized antibody proteins which bind specifically to fibroblast activating protein alpha (FAPα), and either  are completely human or no more than a murine complementarity determining Region (CDR region) of the monoclonal antibody F19 (ATCC accession number HB 8269). Said antibodies according to the invention have the surprising advantageous property that they have a significantly reduced xenoantigenes potential and thus better suited for use in humans than those of the prior Art known antibody (see also description of the invention Procedure, infra). The antibodies of the invention advantageously have none or very small amounts of the murine amino acid sequence, namely at most one CDR Region. Also the framework regions (English: "Framework Region = FR") of the variable Region of the antibodies according to the invention correspond completely human Amino acid sequences. Despite the small murine proportions are the invention Antibody surprisingly nevertheless highly specific for the target antigen FAP.

In the context of this invention is under antibody one or more of in this Description of the described polypeptide (s) understood. Also included are humane Antibody proteins selected from fragments, allelic variants, functional variants, Variants due to the degenerative nucleic acid code, fusion proteins with a antibody protein according to the invention, chemical derivatives or a Glycosylation variant of the antibody proteins according to the invention.

The manufacturing methods of the prior art are insufficient to inventive to obtain human antibodies. With a method according to the invention as with the described below and illustrated in more detail in the examples may inventive human or humanized antibody with reduced xenoantigens Properties are obtained. In a preferred production process according to the invention For example, the following steps are performed:

1) PCR amplification of the human VL and VH repertoires

• a) For the production of VH and VL repertoires, the various V gene families separately with the respective family-specific primers by PCR from cDNA amplified (see Example 1).
• b) All forward / 3 'primers for VH and VL-PCR amplification are complementary to the Gene sequences of the constant immunoglobulin domains (IgG, IgD, IgM, κ, λ). This allows efficient isotype-specific amplification of the V regions with very few 3'-primers. In contrast, in known from the prior art method  Variety of different 3 'primer complementary to the J sections of the V regions (Marks et al., 1991; J. Mol. Biol. 222: 581).

2) Preparation and cloning of a human VH repertoire

In the prior art, only certain lymphoid tissues with very few have so far been various donors as sources of V repertoires (e.g., Vaughan et al. 1996; Nature Biotechnology 14: 309). To become a human V repertoire consisting of a large clone number with high diversity (details see example 1) as the basis for the Production of the antibodies of the invention to arrive, are not obvious Not only for the respective lymphoid organs far more different donors, namely about ten times more than recommended in the prior art, used, but Also, the fetal liver and thymus used as a source for V repertoires. also were in addition to the IgM and IgG in addition to a large Repertoirediversität the IgD repertoire amplified (see Example 1).

3) Preparation of a combination repertoire consisting of a human VH Repertoire and various human FAP-specific VL regions

To obtain an antibody according to the invention, for example, that of the monoclonal, FAP-specific mouse antibody F19 known VH region used and by means of a "guided selection" and a "guided selection" Phage display method (English: "phage display") a suitable human FAP specific VL region are selected. Then, for example, with said human VL region as a lead structure selected a human FAP-specific VH region become. Here, the technical problem of DNA contamination of the Combination repertoires with phagemid vectors occur that for already existing FAP encode specific scFv (eg, murine scFv from hybridoma F19 or the chimeric ones anti-FAP scFv with human VL and F19 VH). A "guided selection procedure" is in described the examples.

Combination repertoire includes the genetic combination of a V repertoire understood according to complementary V sequences. (Complementary to VH to VL and vice versa). The V sequences used for the combination can be selected from a V Sequence, several different V-sequences or a V-repertoire exist.

Preferably, an antibody protein according to the invention is characterized in that it a heavy chain (VH) of immunoglobulin class IgM comprises.  

Preferably, an antibody protein according to the invention is also characterized in that it comprises a heavy chain (V _H ) of the class IgG. Non-limiting examples are the fully human antibodies scFv # 13 and scFv # 46 (see examples).

Preferably, an antibody protein according to the invention is also characterized in that it comprises a heavy chain (V _H ) of the class IgD. A non-limiting example of this is the human antibody scFv # 50 according to the invention (see also Examples). In this antibody, the VH sequence is derived from a human IgD and is consistent with an amino acid exchange with the germ line sequence. This advantageously reduces the likelihood of an allogeneic immune response against this human VH region.

Further preferably, an antibody protein according to the invention is characterized in that it comprises a lambda-type light chain (V _L ) (λ).

Further preferably, an antibody protein according to the invention is characterized in that it comprises a kappa-type light chain (V _L ) (κ) (see example, eg III25, III43). For many applications of the antibodies according to the invention as small as possible antigen-binding, ie FAP-binding units are desirable. Therefore, in another preferred embodiment, an antibody protein of the invention is a Fab fragment (English: "Fragment antigen-binding = Fab"). These FAP-specific antibody proteins according to the invention consist of the variable regions of both chains, which are held together by the subsequent constant region. These can by protease digestion such. B. with papain from conventional antibodies, similar Fab fragments can also be produced by genetic engineering. In a further preferred embodiment, an antibody protein according to the invention is an F (ab ') ₂ fragment which can be prepared by proteolytic cleavage with pepsin.

Using genetic engineering methods, it is possible to shorten antibody fragments produce only from the variable regions of heavy (VH) and light Chain (VL) exist. These are called Fv fragments (English: "fragment variable" = Fragment of the variable part). In a further preferred embodiment is an inventive, FAP-specific antibody molecule such Fv fragment. Since these Fv fragments are the covalent linkage of both chains by the cysteines of the lacking constant chains, the Fv fragments are often stabilized. It is advantageous that  variable regions of the heavy and light chains through a short piece of peptide, For example, from 10 to 30 amino acids, preferably 15 amino acids to link. As a result, a single peptide strand of VH and VL, connected by a Peptide linker, obtained. Such an antibody protein is termed an Fv single chain or English: "single-chain Fv" (scFv). Examples of prior art for such scFv Antibody proteins are described in Huston et al. (1988, PNAS 16: 5879-5883). Therefore In yet another preferred embodiment, an inventive FAP specific antibody protein an Fv single chain protein (scFv).

Various strategies have been developed in recent years to make scFv multimers Produce derivatives. This should especially be done with recombinant antibodies improved pharmacokinetics and biodistribution properties as well as with increased Lead binding avidity. To achieve multimerization of the scFv, scFv was used as a Fusion proteins prepared with multimerization domains. As multimerization domains serve z. For example, the CH3 region of an IgG or coiled coil structures (helix structures) such as Leucine zipper domains. However, there are also strategies where the interaction between the VH / VL regions of the scFv are used for multimerization (eg di-, tri- and pentabodies). Therefore, in another preferred embodiment, a Antibody protein according to the invention a FAP-specific diabody antibody fragment. By diabody the skilled person understands a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883). Shortening of the linker in a scFv molecule to 5-10 Amino acids leads to the formation of homodimers in which an inter-chain VH / VL Assembly takes place. Diabodies can additionally by the installation of Stabilized disulfide bridges. Examples of the Prior Art for Diabody Antibody proteins are found in Perisic et al. (1994, Structure 2: 1217-1226).

By minibody, the skilled person understands a bivalent, homodimeric scFv derivative. It consists of a fusion protein containing the CH3 region of an immunoglobulin, preferably IgG, most preferably IgG1 contains as Dimerisierungsregion over a hinge region (eg also from IgG1) and a linker region with the scFv connected is. The disulfide bridges in the hinge region are mostly found in higher cells and not trained in prokaryotes. In a further preferred embodiment is a Antibody protein according to the invention a FAP-specific minibody antibody fragment.  

Examples of prior art minibody antibody proteins can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).

By tri-body, the skilled person understands a trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives in which VH-VL directly without Linker sequence are fused, lead to the formation of trimers.

The skilled person also known as Miniantibodies are a bi-, tri- or have tetravalent structure and are derived from scFv. The multimerization takes place by di-, tri- or tetrameric coiled-coil structures (Pack et al., 1993 Biotechnology 11: 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28-34).

Therefore, in a further preferred embodiment, an inventive Antibody protein A FAP-specific multimerized molecule based on the above mentioned antibody fragments and may for example be a tri-body, a tetravalent Mini antibody or a pentabody.

Particularly preferably, an antibody protein according to the invention is completely human. Another preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 1 (VH13).

Another preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 2 (VH46).

Another preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 3 (VH50).

Another preferred antibody protein of the present invention is characterized in that the light chain variable region (V _L ) comprises the amino acid sequence ID No. 4 (VLIII25).

Another preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID No. 5 (VH13) or by fragments or degenerate variants thereof.

Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID N ° 6 (VH46) or by fragments or degenerate variants thereof.

Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID No. 7 (VH50) or by fragments or degenerate variants thereof.

Another preferred antibody protein according to the invention is characterized in that the variable region of the light chain (V _L ) is encoded by the nucleotide sequence ID No. 8 (VLIII25) or by fragments or degenerate variants thereof.

A very preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 1 (VH13) and the light chain variable region (V _L ) contains the amino acid sequence ID no. 4 (VLIII25).

A still further preferred antibody protein of the present invention is characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 5 (VH13) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence ID No. 8 (VLIII25).

A still further preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 2 (VH46) and the light chain variable region (V _L ) contains the amino acid sequence ID no 4 (VLIII25).

A still further preferred antibody protein of the present invention is characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 6 (VH46) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence ID No. 8 (VLIII25).

A still further preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID NO. 3 (VH50) and the light chain variable region (V _L ) contains the amino acid sequence ID NO 4 (VLIII25).

A still further preferred antibody protein of the present invention is characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 7 (VH50) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence ID No. 8 (VLIII25).

Particularly preferably, an antibody protein according to the invention is humanized. The humanized antibody protein according to the invention has compared to those of the prior Technique, FAPα-specific antibody proteins have the advantageous property of that it does not contain all six murine CDR regions of F19, but only one murine CDR region as described in the following preferred embodiments. This antibody protein according to the invention advantageously has a lower one xenoantigenes potential as known from the prior art antibody proteins. Surprisingly, the inventors have succeeded in producing antibody molecules which only one murine CDR region contain the prevailing view that at least two murine CDR regions required for successful humanization (Rader et al 1998, Proc Natl Acad Sci USA, 95: 8910).

Another surprising feature in the case of humanized scFv 34 and scFv 18 is that this scFv has a higher apparent binding affinity to FAP ⁺ cells (EC ₅₀ 6 nM) compared to FAP-specific antibodies known from the prior art, such as e.g. B. scFv F19 (EC ₅₀ 20 nM) have.

A preferred process according to the invention for the production of inventive humanized antibodies can be exemplified by the following steps become:

1) Humanization of scFv F19 by the HCDR3 retaining guided selection method

Our experience has shown that through the guided selection process human Ak can be selected, which in comparison to the parental murine Ak a changed Have epitope specificity. To overcome this disadvantage in the prior art has been in an advantageous manner for the humanization of scFv F19 the HCDR3 F19 in Guided selection process as well as retained in the final humanized product. In the state of Technique (Rader et al., 1998, PNAS 95: 8910) are humanized only by Guided Selection Antibodies were described in which both the LCDR3 and the HCDR3 of the parent murine Ab were maintained (see Example 1).

2) Combination of a human VH gene segment repertoire with the murine HCDR3 (F19)

The VH segments of all known human VH families are associated with HCDR3 F19 combine to create the most complex combination repertoire possible. This succeed z. B. preferably in an advantageous manner in that an interface for the Restriction enzyme Pfl23II could be integrated into the HCDR3 F19 without encoding to change at the amino acid level. For the combination of PCR-amplified human VH gene segments, a phage display vector has been developed which has the following characteristics: Sequence sections contains: HCDR3 F19 with Pfl23II site, a human VH FR4 Region with high homology to the corresponding region from F19 as well as various selected human anti-FAP VL regions (see scheme in Example 1). The primers for PCR amplification of the VH gene segment repertoires are shown in Example 1.

This preferred method has the following advantages over the prior art for Combination of VH gene segment repertoires with defined CDR3 regions:

Schier et al. 1996: J. Mol. Biol. 255: 28: In this prior art, a Restriction site (BssHII) integrated in the 3 'region of VH FR3. The installation However, this interface via PCR is in different VH gene families with a linked to altered amino acid sequence. Thus, in Schier et al. only part of the TH Gene families could be included in the combination repertoire.

PCR overlap extension Rader et al. 1998: Although this procedure allows all VH To include gene families in the combination, but the disadvantages are low Linking efficiency and a high error rate. This increases the probability of inactive scFv mutants and in particular clones with an interrupted scFv reading frame which leads to genetically unstable combination repertoires.

3) Use of various human FAP-specific VL regions as a lead structure

To increase the likelihood of selecting an F19 analog scFv the human VH repertoire (see Figure 2) with the sequences of various human FAP specific VL regions combined. (Carrying out in analogy to human antibodies, supra).

4) Stringent washing step in phage display selection

This procedure was used to remove low affinity and polyreactive antibodies during of the selection process (execution see below).

5) Use of an efficient screening method to identify the selected ones humanized scFv

During the HCDR3 retaining guided selection procedure, a very large number of Enriched cloning. The scFv # 34 and # 18 can be advantageously obtained by the method described in Mersmann et al. 1998 (J. Immunol. Methods, 220: 51) become.

Another preferred antibody protein according to the invention is characterized in that it contains the murine light-chain CDR 1 (V _L ) of the monoclonal antibody F19.

Another preferred antibody protein according to the invention is characterized in that it contains the murine light chain CDR 2 (V _L ) of the monoclonal antibody F19.

Another preferred antibody protein according to the invention is characterized in that it contains the murine light chain CDR 3 (V _L ) of the monoclonal antibody F19.

Another preferred antibody protein according to the invention is characterized in that it contains the murine CDR 1 heavy chain (V _H ) of the monoclonal antibody F19.

Another preferred antibody protein according to the invention is characterized in that it contains the murine CDR 2 of the heavy chain (V _H ) of the monoclonal antibody F19.

Another preferred antibody protein according to the invention is characterized in that it contains the murine CDR 3 heavy chain (V _H ) of the monoclonal antibody F19.

Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 9 (VH34).

Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 10 (VH18).

Another preferred antibody protein of the present invention is characterized in that the light chain variable region (V _L ) comprises the amino acid sequence ID No. 11 (VLIII43).

Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID No. 12 (VH34) or by fragments or degenerate variants thereof.

Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID No. 13 (VH18) or by fragments or degenerate variants thereof.

Another preferred antibody protein of the invention is characterized in that the variable region of the light chain (V _L ) is encoded by the nucleotide sequence ID No. 14 (VLIII43) or by fragments or degenerate variants thereof.

A very preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 9 (VH34) and the light chain variable region (V _L ) contains the amino acid sequence ID no. 11 (VLIII43).

A still further preferred antibody protein of the present invention is characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 12 (VH34) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence ID No. 14 (VLIII43).

A still further preferred antibody protein according to the invention is characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 10 (VH18) and the light chain variable region (V _L ) contains the amino acid sequence ID no 11 (VLIII43).

A still further preferred antibody protein of the present invention is characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 13 (VH18) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence ID No. 14 (VLIII43).

Another preferred embodiment of the invention comprises a nucleic acid which encoded for an antibody protein according to the invention. Further preferred is a nucleic acid according to the invention characterized in that it is 5 'or 3' or 5 'and 3' contains untranslated regions. The nucleic acid according to the invention can be upstream and or downstream contain further untranslated regions. Said untranslated Region may have a regulatory element, such. B. a transcription initiation unit (Promoter) or enhancer. Said promoter can, for example, a  constitutive, inducible or developmentally driven promoter. Preferred, without To exclude other known promoters are the constitutive promoters of the human Cytomegalovirus (CMV) and Rous Sarcoma Virus (RSV), as well as simian virus 40 (SV40) and herpes simplex promoter. Inducible promoters of the invention include Antibiotic resistant promoter, heat shock promoter, hormone-inducible mammary Tumor virus promoter "and the metallothionein promoter Nucleic acid according to the invention characterized in that it is suitable for a fragment of the encoded antibody protein according to the invention. Below this will be part of understood polypeptide according to the invention.

Further preferably, a nucleic acid according to the invention is characterized in that they encoded a functional variant of the antibody protein according to the invention. These are understood to mean polypeptides that are largely similar to a Antibody protein according to the invention and have the same biological activity as a antibody protein according to the invention or an inhibitory activity for a have antibody protein according to the invention. A variant of an inventive Antibody protein can be obtained by substitution, deletion or addition of one or more Amino acids differ from an antibody protein according to the invention, preferably from 1 to 10 amino acids.

Further preferably, a nucleic acid according to the invention is characterized in that it codes for an allelic variant of the antibody protein according to the invention. Further preferably, a nucleic acid according to the invention is characterized in that it codes for variants of the antibody protein according to the invention on the basis of the degenerative code of the nucleic acids. A nucleic acid is furthermore preferably characterized in that it can hybridize to a nucleic acid according to the invention under stringent conditions. Stringent conditions are known to the person skilled in the art and can be found in particular in Sambrook et al. (1989). Molecular Cloning. A Laboratory Manual, ^2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it has an amino acid sequence by sequence ID No. 15 or a part thereof or a functional variant thereof.  

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it has an amino acid sequence by sequence ID No. 16 or a part thereof or a functional variant thereof.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it has an amino acid sequence by sequence ID No. 17 or a part thereof or a functional variant thereof.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it has an amino acid sequence by sequence ID No. 18 or a part thereof or a functional variant thereof.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it has an amino acid sequence by sequence ID No. 19 or part thereof or a functional variant thereof.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by a nucleotide sequence Sequence ID No. 20 or a part thereof or a functional variant thereof becomes.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by a nucleotide sequence Sequence ID No. 21 or a part thereof or a functional variant thereof becomes.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by a nucleotide sequence Sequence ID No. 22 or a part thereof or a functional variant thereof becomes.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by a nucleotide sequence Sequence ID No. 23 or a part thereof or a functional variant thereof becomes.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by a nucleotide sequence Sequence ID No. 24 or a part thereof or a functional variant thereof becomes.  

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is the amino acid sequence sequence ID No. 15 corresponds.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is the amino acid sequence sequence ID No. 16 corresponds.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is the amino acid sequence sequence ID No. 17 corresponds.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is the amino acid sequence sequence ID No. 18 corresponds.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is the amino acid sequence sequence ID No. 19 corresponds.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by the nucleotide sequence of Sequence ID No. 20 is encoded.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by the nucleotide sequence of Sequence ID No. 21 is encoded.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by the nucleotide sequence of Sequence ID No. 22 is encoded.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by the nucleotide sequence of Sequence ID No. 23 is encoded.

Another particularly preferred embodiment of the invention comprises a Antibody protein, characterized in that it is expressed by the nucleotide sequence of Sequence ID No. 24 is encoded.

Sequence ID No. refers to the number mentioned in the sequence listing under <400>, so that z. For example, the nucleotide sequence of SEQ ID NO: 24 is listed as <400 <24.  

Another aspect of the present invention relates to a recombinant DNA vector containing a nucleic acid according to the invention. Examples are viral Vectors such. Vaccinia, Semliki Forest virus and adenovirus. Vectors to Use in COS cells have the SV40 origin of replication and allow high Copy numbers of plasmids. For example, vectors for use in insect cells are E. coli transfer vectors and contain, for. B. as a promoter coding for polyhedrin DNA.

Another aspect of the present invention relates to an inventive recombinant DNA vector which is an expression vector.

Yet another aspect of the present invention is a host which is a subject of the invention Vector contains.

Another host according to the invention is a eukaryotic host cell. The Eucaryotic host cells of the invention include fungi, such as. Pichia pastoris. Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells (eg Spodoptera frugiperda Sf-9, with a baculovirus expression system), plant cells. z. From Nicotiana tabacum, mammalian cells, e.g. COS cells, BHK, CHO or Myeloma cells.

In descendants of cells of the immune system, in which also in our body Antibody proteins are formed, the antibody proteins of the invention particularly well folded and glycosylated. Therefore, a preferred host according to the invention a mammalian cell.

Most preferably, a host of the invention is a BHK, CHO or COS cell.

Another host of the invention is a bacteriophage.

Another host according to the invention is a prokaryotic host cell. examples for prokaryotic host cells are Escherichia coli, Bacillus subtilis, Streptomyces or also Proteus mirabilis.

The invention also relates to a process for the preparation of inventive Antibody proteins, comprising the following steps: a host according to the invention as described above, under conditions under which said antibody protein is expressed by said host cell, cultured and said antibody protein becomes isolated.  

The antibody proteins of the invention may be in any of the hosts described above are expressed.

The preparation with prokaryotic expression systems such as Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis is preferred for antibody fragments according to the invention, such as Fab, F (ab ') 2, scFv fragments, minibodies, Diabodies and multimers of said fragments. The antibody proteins of the invention are in an inventive method either intracellular, z. In Inclusion bodies, by secretion in cell-wall-free bacteria such as Proteus mirabilis or by periplasmic secretion in Gram-negative bacteria by means of this made of suitable vectors. In Example 2, the preparation according to the invention Antibody proteins in prokaryotes described by way of example. Examples from the state of Techniques for the production of scFv antibody proteins are described in Rippmann et al. (1998, Appl. Environ. Microbiol., 1998, 64: 4862-4869). Further examples are known in the art.

The antibody proteins according to the invention can be used in a method according to the invention also in mushrooms, such as. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma with vectors that lead to intracellular expression or secretion, getting produced.

Even with insect cells, the inventive method for producing the Antibody proteins are carried out, for. B. as transient or stable Expression system or baculovirus expression system. This z. B. Sf-9 Insect cells with z. Autographa californica nuclear polyhedrosis virus (AcNPV) or infected related viruses. There is also no risk of contamination with mammalian pathogenic viruses, therefore, therapeutic antibodies according to the invention can also be used can be advantageously prepared in insect cells. The E. coli described above Transfer vectors contain z. B. as a promoter encoding polyhedrin DNA, behind in which the DNA coding for the antibodies according to the invention is cloned into. To Identification of a correct transfer vector clone in E. coli is described together with incomplete baculovirus DNA transfected into an insect cell and recombined with the Baculovirus DNA to form functional baculoviruses. With the help of strong Insect cell promoters are in a process of the invention large amounts of the antibody protein according to the invention formed, which z. B. by fusion  eukaryotic signal sequences are secreted into the medium. Insect cell expression systems for the expression of antibody proteins commercially available. Insect cell expression systems are particularly suitable for the Inventive scFv fragments and Fab or F (ab ') 2 fragments and Antibody proteins or fragments thereof, which are fused with effector molecules, but also for complete antibody molecules.

An advantage of mammalian expression systems is that they are very good glycosylation and Convolution conditions allow z. B. transient expression systems, e.g. In COS cells or stable expression systems e.g. B. BHK, CHO, myeloma cells (see also Example 2). Mammalian cells are also z. B. with viral expression systems z. Vaccinia, Semi- Forest virus, adenovirus usable. Also transgenic animals z. Cows, goats, mice are suitable for a method according to the invention. Also transgenic plants like Nicotiana Tabacum (tobacco) can be used in a method according to the invention. These are suitable in particular for the production of antibody fragments according to the invention. To Genomic integration of the nucleic acid according to the invention, which for a antibody protein encoded according to the invention, which is fused to a signal sequence can the secretion of the antibody protein into the interstitial space can be achieved.

The invention relates in particular to a method according to the invention, in which said host is a mammalian cell, preferably a CHO or COS cell.

The invention relates in particular to a method according to the invention, in which said host cell is co-transfected with two plasmids expressing the expression units wear for the light or heavy chain.

The antibody proteins of the present invention are highly specific agents for therapeutic agents to the FAP antigen. Therefore, another preferred one is Antibody protein according to the invention, characterized in that said Antibody protein is coupled to a therapeutic agent.

This antibody protein according to the invention may preferably also be obtained by genetic engineering be coupled to a therapeutic agent or an effector molecule. Such Therapeutic agent includes according to the invention cytokines, such as. Interleukins (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL- 13, IL-14, IL 15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosis factor (TNF) TNF alpha and TNF beta, TRAIL  immunomodulating or immunostimulating protein, or apoptosis or necrosis inducing protein. Therefore, the antibody Effector molecule conjugates Antibody-cytokine fusion proteins, continue as well bispecific antibody derivatives and antibody-superantigen fusion proteins. These are preferred for activation of the body's own anti-tumoral defense mechanisms and thus suitable for therapeutic use. Another preferred inventive FAP specific antibody protein is characterized by being somatic Gene therapy is used. For example, this may be achieved by use as antibody toxin Fusion protein (as described in Chen et al., 1997, Nature 385: 78-80 for other targets (Target regions) exemplified) or as a fusion protein consisting of a antibody of the invention and a T-cell receptor or Fc receptor (transmembrane and intracellular region, s. z. Wels et al., 1995, Gene, 159: 73-80). The use for somatic gene therapy can also be achieved by expression of the present invention Nucleic acid in a "shuttle vector", a gene probe or a host cell done.

Another preferred antibody protein according to the invention is characterized in that said therapeutic agent from the group of radioisotopes, toxins or Immunotoxins, toxoids, fusion proteins, for example genetically engineered Fusion proteins, inflammatory agents and chemotherapeutic agents and elements that enable a neutron capture reaction, such as. Boron (boron Neutron capture reaction, BNC).

Another preferred antibody protein according to the invention is characterized in that characterized in that said radioisotope is a β-emitting radioisotope.

Another preferred antibody protein of the invention is characterized in that said radioisotope is selected from the group ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ¹³¹ iodine and ⁹⁰ yttrium, which have been found to be particularly suitable for coupling as therapeutic agents to the antibodies of the invention. An exemplary method for the radioiodination of the antibodies according to the invention is described in WO 93/05804.

Another preferred antibody protein according to the invention is characterized in that characterized in that it is marked.

Another preferred antibody protein according to the invention is characterized in that that it is marked with a detectable marker.  

Another preferred antibody protein according to the invention is characterized in that that the detectable marker from the group of enzymes, dyes, radioisotopes, Digoxygenin, streptavidin and biotin is selected.

Another preferred antibody protein according to the invention is characterized in that that it is coupled to an imageable agent.

Another preferred antibody protein according to the invention is characterized in that that the imageable agent is a radioisotope.

Another preferred antibody protein according to the invention is characterized in that that said radioisotope is a γ-emitting radioisotope.

Another preferred antibody protein of the invention is characterized in that said radioisotope is ¹²⁵ Iodine.

Another important aspect of the present invention relates to a Pharmaceutical preparation containing an antibody protein according to the invention and one or more contains several pharmaceutically acceptable carriers. Pharmaceutically acceptable Carriers or excipients in this invention may be physiologically acceptable Compounds, for example, the absorption of inventive Stabilize or improve antibody proteins. Such physiologically acceptable Compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, Antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight Compounds or other stabilizers or excipients (see, eg, Remington's Pharmaceutical Sciences, 18th Edition, Mack Publ., Easton.). The skilled person knows that the selection of a pharmaceutically acceptable carrier e.g. B. from the administration route of Connection depends. The said pharmaceutical composition may also have a vector for gene therapy according to the invention and can additionally as adjuvant a colloidal dispersion system or liposomes for a targeted application of pharmaceutical composition. Also a host or a host cell, the contains a vector of the invention can be in a pharmaceutical Composition used in the context of this invention, for example, for gene therapy become.

Another important aspect of the present invention relates to use a pharmaceutical preparation according to the invention for treatment or imaging  of tumors wherein said tumors are associated with activated stromal fibroblasts are.

Said use according to the invention refers in particular to the case in which said tumors are attributable to one of the following types of cancer or are based on and therefore from the group of colorectal cancer, non-small cell lung cancer, Breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, Pancreatic cancer and metastatic brain cancer are selected.

Yet another important aspect of the present invention relates to use an antibody protein according to the invention for the preparation of a pharmaceutical Preparation for the treatment of cancer.

Yet another important aspect of the present invention relates to use an antibody protein according to the invention for imaging activated stromal Fibroblasts.

An additional aspect of the present invention is a method for detecting activated stromal fibroblasts in wound healing, inflammatory processes or at a tumor which is characterized in that a sample in which possibly activated fibroblasts are contained with an antibody protein according to the invention under conditions suitable for a complex of said antibody protein is contacted with its antigen, and the formation of said complex and Thus, the presence of activated stromal fibroblasts in wound healing, inflammatory processes or in the case of a tumor.

The method according to the invention described in the previous paragraph is in particular characterized characterized in that said tumor is selected from the group of colorectal cancer, not small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, Lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer is selected. The invention further comprises a method for the detection of tumor stroma, wherein a suitable sample with an antibody protein according to the invention under suitable Conditions for the formation of an antibody-antigen complex is contacted, the so formed complex is detected and the presence of the complex thus formed in Relationship with the presence of tumor stroma is set.

The method according to the invention described in the previous paragraph is in particular characterized characterized in that said antibody is labeled with a detectable marker.  

The following examples are intended to aid the understanding of the invention and in no way as limiting the breadth of the invention.  

example 1 1) Cloning of a human VH repertoire for the guided selection method A) Development of anti-FAP antibodies with fully human V regions production method 1. Cloning of F19 VH 2. Production of human V repertoires

• Reverse transcription, PCR amplification of human VL (λ, κ) - Repertoires of peripheral blood lymphocytes an improved method after Persson et al. 1991, PNAS 88: 2432.
• Cloning of VL repertoires into phage display vector (pSEX81, DKFZ, Heidelberg, Breitling et al., 1991, Gene 104: 147)
  Repertoire size: VL 10 ⁷ clones
• - Reverse transcription, PCR amplification of human VH repertoire (IgG, IgD, IgM) from peripheral blood lymphocytes, thymus, spleen, bone marrow, Tonsils, lymph nodes, fetal liver (improved after Persson et al., 1991, PNAS 88: 2432)
• - Process improvement: Use of IgD and various lymphoid tissues
• Cloning of the VH repertoire into phage display vector (pSEX81, DKFZ, Heidelberg, Breitling et al., 1991, Gene 104: 147)
  Repertoire size: VH 3 × 10 ⁸ clones

3. Selection of human VL regions that functionally replace VL F19

• - phage display selection and guided selection strategy with VH F19 as lead structure
  (improved after McCafferty et al., 1990, Nature 348: 552 and Jespers et al., 1994, Bio / Technology 12: 899)
  → Isolation of human FAP-specific VL regions
  (Designation VL: III5, III10, III25, III43)

4. Selection of human VH regions which functionally replaced VH F19 or FAP Convey specificity

• - phage display selection and guided selection strategy with different VL as lead structures
  (improved after McCafferty et al., 1990, Nature 348: 552 and Jespers et al., 1994, Bio / Technology 12: 899)
  → Isolation of the following human FAP-specific scFv:
  scFv # 13: VH # 13, IgG; VL III25
  scFv # 46: VH # 46, IgG; VL III25
  scFv # 50: VH # 50, IgD, VL III25

Sequence of the selected VII and VL regions

(see pictures)

Antigen-binding properties

• - ELISA: Detection of antigen specificity for human FAP
• Competition of antigen binding by cF19 (detected for scFv # 13)
• - Binding studies on FAP ⁺ cells:
  scFv # 13 (as bivalent in minibody format) EC ₅₀ : 8-12 nM (see below)
  scFv # 50 (as bivalent in minibody format) EC ₅₀ : 32 nM
• - FAP-specific immunohistological staining of tumor biopsy material (detected for scFv # 13 in minibody format)

1) PCR amplification of the human VL and VH repertoires

• a) For the preparation of the VH and VL repertoires, the various V gene families separately with the respective family-specific primers by PCR from cDNA amplified (see below).
• b) All forward / 3 'primers for VH and VL-PCR amplification are complementary to the Gene sequences of the constant immunoglobulin domains (IgG, IgD, IgM, κ, λ). This allows efficient isotype-specific amplification of the V regions with very few 3'-primers. In contrast, Marks et al., 1991 (J. Mol. Biol. 222: 581) use a variety of different 3 'primers complementary to the J portions of the V regions.

2) Preparation and cloning of a human VH repertoire

Preparation and cloning of a human VH repertoire consisting of a large clone number (3 × 10 ⁸ ) with high diversity (for implementation see below).

• a) In order to ensure the high level of diversity, the starting material for the VH Repertoires in addition to freshly isolated peripheral blood lymphocytes commercially available cDNA / RNA of various lymphoid tissues from a very large number of donors used. By using bone marrow and fetal liver, naive V Repertoires and thus created the conditions for the isolation of autoantibodies become.

Lymphoid tissues used (donor number):

I) Commercial cDNA

• - Peripheral blood lymphocytes, PBL (550 donors)
• - spleen (5 donors)
• - Thymus (7 donors)
• - bone marrow (51 donors)
• - lymph nodes (59 donors)
• - tonsils (5 donors)
• - fetal liver (32 donors)

II) Commercial RNA, which was subsequently transcribed into cDNA in the laboratory (Execution see Example 1, A 1.2)

• - lymph nodes (25 donors)

III) PBL of fresh "Buffy coats" (10 donors) (see below for implementation)

In the prior art, only the following lymphoid tissues have so far been described as sources of V repertoires. (The tissue combinations and donor numbers are given):

• - PBL (15 donors), bone marrow (4 donors), tonsils (4 donors) (Vaughan et al., 1996; Nature Biotechnology 14: 309)
• Spleen (3 donors) and PBL (2 donors) (Sheets et al., 1998; PNAS 95: 6157
• Bone marrow (Williamson et al., 1993, PNAS 90: 4141)
• Lymph node (1 donor) (Clark et al., 1997; Clin. Exp. Immunol. 109: 166)
  • a) In addition, the IgD repertoire was amplified in addition to the IgM and IgG in terms of a large Repertoirediversität. For this purpose, an IgD-specific PCR primer was developed (see below).
  • b) It turned out to be very important to re-purify the PCR fragments of the human VH repertoire after treatment with restriction enzymes on an agarose gel. Subsequent cloning of this repertoire into a phage display vector enabled a very high proportion of clones to be obtained with a functional scFv expression cassette. This is an essential prerequisite for obtaining a genetically stable phage display repertoire (for execution, see Example 1, A 1.4).

3) Preparation of a combination repertoire consisting of a human VH repertoire and various human FAP-specific VL regions

Definition of combination repertoire:
Genetic combination of a V repertoire with correspondingly complementary V sequences. (Complementary with respect to VH to VL and vice versa). The V sequences used for combination may consist of a V sequence, several different sequences or a V repertoire.

• a) Cloning strategy: In a phage display vector was the human VH repertoire combined with a defined non FAP-specific VL region (dummy VL). These dummy VL region could be isolated very efficiently by means of restriction cleavage sites by FAP specific VL regions are replaced. This created the conditions created to effectively create a previously reviewed human VH repertoire with specific to combine human VL to thereby provide a diverse combination repertoire  ensure a very high proportion (<95%) of functional clones (in terms of the integrity of the scFv reading frame) (see below for procedure).
• b) To increase the probability of being an analogous to F19 and fully human scFv was selected, the human VH repertoire with the sequences of various human FAP-specific VL regions combined (VL: III10, III25, III5, III43,). These Human VL regions served as lead structures for the selection of human FAP-specific VH. The FAP-specific human VL itself were in a previous guided selection Step with F19 VH isolated from a human VL repertoire.
• c) DNA contamination of the combination repertoires with phagemid vectors already considered encode existing FAP-specific scFv (eg, murine scFv from hybridoma F19 or the chimeric anti-FAP scFv with human VL and F19 VH), represents a large technical problem. To prevent this, the following strategy proved to be necessary: After the guided selection step of the human anti-FAP VL sequences with the murine F19 VH as a leader, this human VL sequence lacked VH sequence first subcloned in a plasmid (pUCBM21). Subsequently, this human VL- Cut out the region by means of restriction enzymes and with the human VH repertoire, which was already present in a phage display vector combined. That way you could it is prevented that, apart from the VL sequences of the respective lead structure, no FAP specific V regions were included in the combination repertoire. (Implementation see below)

4) phage display selection

The phage display selection of the FAP specific human V regions required the Development of selective washing procedures to increase the accumulation of cross-reactive scFv prevent (see below for implementation).

B) Development of human anti-FAP antibodies containing the murine HCDR3 F19 contain (HCDR3 retaining guided selection) production method 1. Cloning of F19 VH 2. Production of human V repertoires

• Reverse transcription, PCR amplification of human VL (λ, κ) - Repertoires of peripheral blood lymphocytes (modified according to Persson et al., 1991, PNAS 88: 2432)
• - cloning of the VL repertoire in phage display vector (pSEX81, DKFZ, Heidelberg; Breitling et al, 1991, Gene 104: 147th), Repertoire Size: 10 ⁷ clones VL
• - reverse transcription, PCR amplification of human VH repertoire peripheral blood lymphocytes (improved by Persson et al., 1991, PNAS 88: 2432), PCR amplification of the VH segment consisting of FR1 + + CDR1 FR2 + CDR2 + FR3
• Cloning of a repertoire consisting of the VH segment (FR1 + CDR1 + FR2 + CDR2 + FR3) into phage display vector (pSEX81, DKFZ, Heidelberg, Breitling et al., 1991, Gene 104: 147), repertoire size: VH4 × 10 ⁷ clones

3. Selection of human VL regions that functionally replace VL F19

(see A) 3)

4. Selection of a human VH region containing HCDR3 from F19 and VH F19 functionally replaced

• - HCDR3 retaining guided selection strategy with VL III43 or VL III5 and HCDR3 F19 + human FR4 as lead structure (Own process development improved according to McCafferty et al., 1990, Nature 348: 552; Jespers et al., 1994, Bio / Technology 12: 899 and Rader et al., 1998, PNAS 95: 8910)
  → Isolation of the following human FAP-specific scFv containing the murine HCDR3 F19:
  scFv # 34: VH # 34, IgG; VL III43
  scFv # 18: VH # 18, IgG; VL III43

structure

(see pictures)

Antigen-binding properties

- ELISA: Detection of antigen specificity for human FAP

- Competition of antigen binding by cF19 and mAb F19

- Binding studies on FAP ⁺ cells:
scFv # 34 and # 18 (monovalent) EC ₅₀ : ca. 6 nM

FAP-specific immunohistological staining of tumor biopsy material (as scFv # 34 Minibody)

1.1 RNA isolation

The mRNA source was isolated total RNA from fresh lymphocytes of total 10 Buffy coats.

To isolate the lymphocytes from buffy coat, 15 ml of Ficoll (LYMPHOPREP) was placed in a 50 ml Falcon tube at room temperature and overlaid with 30 ml of buffy coat diluted 1: 4 in RPMI medium. After centrifugation for 30 min at 700 g, the interphase was removed and centrifuged after addition of 40 ml of RPMI medium for 5 min at 700 g. The cell pellet was then washed once more with RPMI medium and once with PBS. The cells were centrifuged off after the last washing step and 200 μl of RNA-Clean ™ solution (AGS, Heidelberg) were added to each 10 ⁶ cells. Immediately after addition of the denaturing solution, the cells were homogenized by repeated pulling on and off through a coarse cannula (size 1) and subsequently through a finer cannula (size 18). The low-viscosity lysate was mixed with 1/10 volume of chloroform (pa), shaken well and incubated on ice for 5 min. After centrifugation (15 min at 12000 g), the supernatant was removed generously and mixed with the same volume of isopropanol, incubated for 45 min at 4 ° C and then centrifuged off at 12000 g for 45 min. The supernatant was poured off gently and the pellet washed with ice-cold 70% ethanol. The RNA pellet was then washed again with components of the RNA Quick prep (Pharmacia). For this purpose, the pellet was taken up in a mix of 113 μl of extraction buffer, 263 μl of LiCl solution and 375 μl of Cs-trifluoroacetate, vortexed vigorously and centrifuged in an Eppendorf centrifuge tube (12000 g). The RNA pellet was washed again with 70% ethanol, air-dried for 10 min and adjusted with H ₂ O to a concentration of 1 μg / μl.

Alternatively, the total RNA isolation was also by the RNA isolation column of Company QIAGEN (Midi) according to the manufacturer.

The preparation of the mRNA from total RNA was carried out using the Oligotex kit (Midi) from QIAGEN. The implementation corresponded to the manufacturer's instructions. The isolated mRNA was treated with 1/10 volume of 2.5 M RNAase-free K acetate pH 5.2 and precipitated by addition of 2.5 volumes of ethanol pa at -20 ° C for 2 hours or overnight. After centrifugation (45 min, 13000 g, 4 ° C), the mRNA was washed twice with ice cold 70% ethanol (centrifugation for 5 min 12000 g, 4 ° C) and after brief air drying in 10-20 ul RNAse-free H ₂ O. solved. To estimate the concentration, the mRNA was compared to a total RNA standard dilution series. For this purpose, 1 .mu.l of the sample to be determined was mixed with 10 .mu.l Ethydiumbromidlösung (1 ug / ml), dropped onto a film and compared using a UV lamp with the standardized concentration. The mRNA was used immediately for cDNA synthesis or frozen at -80 ° C for storage.

1.2 cDNA synthesis of human VH regions

The production of IgG, IgM and IgD specific VH cDNA was carried out by mRNA using the cDNA synthesis kit from Boehringer-Mannheim and Amersham. The synthesis of the first cDNA strand was carried out with the Ig-specific primers HuIgG1-4 RT for the IgG library, HuIgM-RT for the IgM library or HuIgDelta for the IgD library. Optionally, oligo (dT) and oligo-hexa nucleotides were used. The cDNA synthesis was carried out with 100 ng mRNA according to the manufacturer, in order to solve the secondary structures, the mRNA had to be heated directly before use for 10 min at 70 ° C. The cDNA synthesis was carried out in a 20 μl batch with AMV reverse transcriptase in a thermocycler for 60 min at 42 ° C. The quality of the cDNA was checked by PCR amplification using the primer pair HuIgGFOR and HuVHB1 exemplified. For this purpose, 10 ⁿ dilutions of the cDNA were used as a template and the highest dilution was determined in which after 36 cycles, still a specific band of the PCR product in the agarose gel was detectable.

1.3 PCR amplification of the human VH repertoire

The cDNA of each human lymphatic organ served separately as a template for PCR amplification of the VH regions. From each lymphoid organ, six separate PCR approaches were performed, each one of the six VH-specific 5 'primers (HuVHB1 to HuVHB6) combined with one of the isotype-specific 3' primers HuIgGFOR, HuIgMFOR or HuIgDFOR. The amplification was carried out in a 50 μl reaction mixture with 1 μl template cDNA (200 μg), 25 mM MgCl ₂ , 5 μl Goldstar reaction buffer, 200 μM of each dNTP (Pharmacia) and 25 pmol of each primer. After 10 min at 95 ° C 0.6 U Goldstar polymerase was added and the batch coated with PCR wax. 36 amplification cycles were performed with 15 s denaturation at 94 ° C, 30 s annealing at 52-55 ° C and 30 s elongation at 72 ° C. After completion of the last amplification step, additional extension was carried out for 15 minutes at 72 ° C.

To introduce the restriction sites Nco I and Hind III to the amplified VH- Regions were subjected to a second PCR amplification with those around the restriction sites extended primer (HuIgGFORHINDIII, HuIgMFORHINDIII, HuIgDHINDIII as 3'- Primer and HuVHB1NCOI to HuVHB6NCOI as 5 'primer). For this was used as template in each case 1 .mu.l of the reaction solution of the first PCR assays. The second PCR amplification was performed with 15 cycles of 15 s denaturation at 94 ° C, 30 s annealing at 65 ° C and 30 s elongation at 72 ° C. The last amplification step followed by an additional Elongationsschritt for 5 min at 72 ° C. The amplicons, the were based on the same isotype and were combined to reduce the volume by adding of 1/10 volume of Na acetate, pH 5.2, and 2.5 volumes of ethanol, p. a. for 2 hours at -20 ° C precipitated and dissolved in TE buffer. To remove the primers, the precipitated PCR Separated on a 1.5% agarose gel and the 400 bp fragment of VH Region cut out. The isolation of the fragment was carried out according to the Manufacturer with the QIA exile kit from QIAGEN (Hilden). The elution was carried out with prewarmed elution buffer for 5 min at 50 ° C.

1.4 Digestion of PCR amplified VH regions with restriction enzymes

The gel-purified VH regions (of the three isotypes) were each in a 100 ul batch first digested with 70 U Hind III for 2 hours in buffer B and then by  Add 20 μl buffer H, 60 U NcoI and make up to 20 μl for another 2 hours incubated. Ablated overhangs were removed using the QIA-Quick PCR kit and the fragments are eluted with prewarmed EB buffer. The eluate was again over 1% Purified agarose gel and eluted with the QIA ExII kit in 25 ul EB buffer. It was found, that additional gel purification step is the percentage of functional insert significantly increased after ligation into the vector. The digested PCR fragments were aliquoted and stored at -20 ° C.

1.5 Ligation of the human VH repertoire into a phagemid vector

A phage display vector pSEX81 that already has the human VL sequence of a hapten specific Ab (dummy VL sequence) was used to clone the PCR used amplified VH repertoire.

20 μg of vector pSEX81 (VH & VLphox) were used in a total volume of 125 μl with 40 U NcoI (Boehringer-Mannheim) and 60 μl of Hind III (Boehringer-Mannheim) in buffer H for Digested for 2 hours at 37 ° C. After addition of 30 μl 6x concentrated Loading Buffer (30% glycerol, 30 mM EDTA), the digestion was heated at 65 ° C for 10 min. And slowly cooled at room temperature. Vector DNA was in a 1% agarose gel from Insert separately and isolated using the QIAGEN gel elution kit. The elution occurred twice with 50 μl of elution buffer (preheated to 50 ° C) for 5 min. The Elution fractions were pooled and the cut vector DNA was added by adding 1 / 10 volumes of sodium acetate pH 5.2 and 2.5 volumes of ethanol p. a. at -20 ° C for 2 hours like. If necessary, the vector DNA thus cut could also be stored at -20 ° C become. After centrifugation for 30 minutes (13000 g, 4 ° C) and washing at -20 ° C cold 70% ethanol, the DNA was dried and dissolved in 50 ul of 10 mM TRIS pH 7.9.

For exact quantitative estimation for the subsequent ligation, 2 μl of the vector DNA with standardized DNA fragments (High-Mass Ladder, Gibco Life Technologies) compared. For immediate comparison on the same gel as in Example 1. A 1.4 prepared VH-PCR fragments with standardized DNA fragments lower Molecular weight (low-mass ladder, Gibco Life Technologies).

A ligation approach with an equimolar insert to vector ratio proved to be optimal. In 40 μl final volume was 500 ng of vector DNA and 50 ng of insert DNA) with 1 μl of ligase and 4 μl of ligation buffer are incubated. The ligation was carried out overnight at 16 ° C using the T4  DNA ligase from Boehringer Mannheim. The ligation reaction was by addition of 60μl TE buffer stopped. The proteins were prepared by adding 100 μl Chloroform / phenol mixture (1: 1), short mixing (vortex) and subsequent centrifuge with 13000 g removed. The aqueous phase was removed and added to complete removal of the phenol extracted again with chloroform. 90 μl vector DNA solution was prepared by adding 9 μl of 3 M NaAcetate (pH 5.2), 225 μl of ethanol, p. a. and 1 μl of glycogen (Boehringer Mannheim) as carrier (see above) for 2 hours at -20 ° C like. After centrifugation at 12000 g (4 ° C) and washing with ice-cold 70% ethanol, the DNA air-dried and taken up in 25 μl of water.

Inefficient restriction digests in the vector preparation lead to not or simply cut vector DNA, which has the consequence that in the VH Repertoireklonierung the Repertoire size is distorted by religation of incomplete vector cut. For early control of completeness of restriction digestion, the prepared Vector compared with and without VH insert ligated, transformed into E. coli and the Clone number determined. In an efficient restriction digest of the vector was the Clone number in the vector sample without insert <1% compared to the approach in the vector with VH insert had been used.

2. Subcloning of the human FAP-specific VL regions, combination of the human VH Repertiores with various human FAP-specific VL

In order to avoid DNA contamination with already existing FAP-specific DNA sequences in the construction of the scFv gene libraries, the selected human VL chains were initially cloned into the expression vector pUCBM21 (Boehringer-Mannheim). For this purpose, the FAP-specific VL chains were cut out in each case from the phagemid vector (pSEX81) used for selection with MluI and NotI (Boehringer-Mannheim) and recloned into the appropriately cut pUCBM21. After transformation into E. coli, one clone was picked for each VL chain, amplified in LB _AT medium, and the vector DNA isolated using the Nucleobond kit (Malcherey & Nagel). The human VL chains were each excised in a 150 ul restriction mixture with MluI (60 U) and NotI (60 U) from 15 ug pUC plasmid and isolated in a 1% agarose gel. These human FAP-specific VLs were cloned into appropriately cut phage display vectors containing the VH repertoires. Methodically, the procedure for cloning the VH regions was as described above. The combination banks with the different VL regions were kept separate. Aliquots of these combination libraries were frozen and used for the selection of fully human FAP-specific scFv.

3. phage display selection Production of phage-associated scFv

To avoid possible growth advantages of the different VL chains in the first round of panning, the phage-associated scFv of the different combination libraries containing the different human VL regions (see point 2) were produced independently. In each case, 10 ml of 2YT _AT medium were inoculated in a baffled agitated flask with an aliquot of the VL / VH combination banks with an OD of 0.4 and cultured with shaking (180 rpm) at 37 ° C. until an OD of 0.8 was achieved , After infection with 10 ¹² helper phages (New England Biolabs), the incubation was carried out without shaking for 15 min at 37 °. After subsequent incubation with shaking and 37 ° C, the bacteria were centrifuged off (4000 g for 5 min) and the pellet resuspended in 50 ml of glucose-free kanamycin-containing (65 μg / ml) 2YT _AT medium. The production of the phage-associated scFv was carried out overnight with vigorous shaking (200 rpm) at 30 ° C. To harvest the phages, the bacteria were centrifuged off (9000 g) and the supernatant was treated with PEG and incubated for precipitation for one hour on ice. After centrifugation at 9000 g for 30 minutes at 4 ° C., the precipitated phages were resuspended in 45 ml of 4 ° C. cold PBS and used with 5 ml of 5 × PEG. After another 1 hour incubation on ice, centrifugation was again carried out at 9000 g and the phage pellet resuspended in 5 ml PBS. The phages were filtered through a 0.45 μm filter and 500 μl of each phage preparation were combined and added with 2 ml of 4% milk powder suspension in PBS (MPBS) for 15 min. The phage suspension was clarified by centrifuging twice with 14000 g in a bench top centrifuge. The thus pre-adsorbed phages had to be used on the same day.

Selection of antigen-specific scFv

For selection, immunotubes were immobilized with 5-30 μg CD8-FAP the day before (Nunc-Maxi-Sorb Immunotubes 3.5 ml). Immobilization was at 4 ° C Overnight in PBS, then the tubes were washed twice with PBS and for One hour with ROTI block (Roth) blocked the non-specific binding sites. To the To be able to investigate specificity of phage display selection was for control purposes used an immunotube without immobilized antigen. After washing three times PBS were pre-adsorbed in MPBS phage-associated scFv in the antigen coated tubes or the control tubes and placed on a roller for 2 Incubated for hours.

For the preparation of the plating bacteria, 20 ml of 2YTtet with a Aliquot of an XL-1-Blue overnight culture inoculated with an OD of 0.0125 and at 37 ° C cultured with shaking (180 rpm). After three hours of incubation, the platinum Bacteria had an OD of 0.8 and were then at that time for infection with the eluted phages available.

One hour before infection, the phage suspensions were removed from the immunotubes emptied. Subsequently, the immunotubes were used to remove non-specific and washed cross-reactive scFv. In the first panning round was 10 × with TPBS (0.1% Tween20) and then washed 10 × with PBS. The stringency was in the second and third panning rounds by extending the washes to 15x TBBS (2nd round of panning) and 20 × TPBS (3rd round of panning) and by increasing Tween20 Concentration increased to 0.5%. To further increase the stringency was also in the last two panning rounds when washing with TPBS for better mixing the wash solution used briefly a vortex.

The last washing solution was discarded and into the Immunotubes 1 ml of 1 M TEA (Triethylamine) was added. After incubation in a roll incubator for 5 minutes the eluted phages are neutralized with 0.5 ml of 1 M TRIS pH 7.4 and immediately to infection added to the 20 ml of plating bacteria.

After incubation for 15 min without shaking at 37 ° C, the bacteria were incubated for 45 min shaken and centrifuged with 3000 g for 10 min. The bacteria were incubated in 500 μl 2YT- Resuspend medium and place on large SOBGAT plates (15 cm) overnight at 37 ° C incubated. For harvest, the cells were scraped off the plate with LBAT medium, with  25% final glycerol concentration and aliquoted at -80 ° C frozen or to Inoculation of another Amplifikationsrunde used.

The phage titer of each panning round was confirmed by titration of 0.01-10 μl of the infected Plating bacteria determined. For the determination of the specific enrichment at each Selection round the number eluier 24314 00070 552 001000280000000200012000285912420300040 0002010013286 00004 24195th Phage of CD8-FAP immobilized immunotube compared with those of the corresponding control immunotubes without antigen. The Amount ratio of the eluted phages of the antigen-coated and the uncoated immunotubes gave the enrichment factor.

An increase of the enrichment factor after successive rounds of amplification indicated an accumulation of specific binding phages.

Example 2 Expression of human FAP-specific scFv derivatives Microtiter scale screening method for phage display- selected scFv

Both for screening and for screening as well as for the analysis of phage Display banks selected scFv clones can express the scFv expressed by pSEX81. pIII fusion proteins are used.

Bacterial production of seFv-pIII fusion protein in microtiter scale

300 μl of 2YT _GAT were inoculated with colonies scattered on LB _GAT plates and incubated overnight in a 96-well microtiter plate (Beckman) at 37 ° C. and shaking at 300 rpm overnight. If the colonies to be analyzed were not stored frozen, this initial incubation was carried out in U-form 96-well tissue culture plates (Greiner). The next morning, 10 .mu.l of these üN cultures each were transferred to fresh 100 .mu.l 2YT and incubated again shaking at 37.degree. C. in U-form 96-well tissue culture plates in a moist chamber. The remainder of the cultures remaining in the Beckman microtiter plates could be mixed to 20% with glycerol and frozen at -80 ° C. If necessary, the growth of the 100 μl cultures could be checked with an ELISA reader at a filter wavelength of 630 nm. After about 6-8 h, the cultures were centrifuged off at 1200 rpm (5 min RT), the supernatants were removed with a multichannel pipette. The pelleted bacteria were resuspended in 100 .mu.l of 2YT _AT (without glucose) incl. 50 .mu.M IPTG and incubated üN shaking in the humid chamber at 30 ° C and 300 rpm. After üN incubation, the cultures each with 25 ul 0.5% Tween were added and incubated for partial lysis shaking a further 3-4 h. Finally, the cultures were centrifuged for 10 min at 1200 rpm and the supernatants were carefully removed. These could be used directly for Western blot analyzes or after pre-adsorption in the ELISA.

Production of scFv-pIII fusion protein in ml scale

If only small clone numbers are to be examined for their expression and / or functionality of the expressed scFv-pIII fusion protein, then the U-preforming and the main cultivation of the bacteria in a volume of 3-10 ml were carried out in test tubes or in 50 ml PP- When the bacterial growth had reached its logarithmic phase (OD _{600 nm} of about 0.7), the cultures were centrifuged off (2500 rpm, 5 min RT) and in the same volume of fresh SB _AT or 2YT _AT including 50 μM IPTG resuspended for induction. After incubation at 25-30 ° C., the cultures were either mixed with Tween 20 (ad 0.1%) and the supernatants were removed after 3 h of further incubation. However, to increase the concentration of the fusion protein, the bacterial pellet could also be disrupted (see below).

The scFv-gIII fusion proteins were used to demonstrate the integrity of the reading frame of the scFv coding region (Western blot) and to study the FAP specificity of the selected scFv in the ELISA on immobilized FAP or in the cell analyzer on FAP ⁺ cells. To detect the scFv-gIII fusion protein, an anti-gIII-specific monoclonal antibody in combination with a peroxidase or alkaline phosphatase-conjugated detection antibody (Western Blot and ELISA) was used. In the case of cell binding studies with the scFv-gIII proteins in the cell analyzer, a FITC-labeled detection antibody was used.

Prokaryotic expression media

All data refer to a final volume of 1 L, the pH was adjusted to 7.0. The following media additives were sterile filtered and added to the autoclaved medium if necessary. G: 100 mM glucose (stock solution: 2 M), A: ampicillin 100 μg / ml, T: tetracycline 12.5 μg / ml, K kanamycin 50 μg / ml

Liquid media for bacterial culture

AL = L <BHI Brain Heart Infusion (DIFCO) 35 g yeast extract 5 g AL = L <dYT peptone 17 g yeast extract 10 g NaCl 5 g AL = L <LB peptone 10 g yeast extract 10 g NaCl 5 g AL = L <SB peptone 30 g yeast extract 10 g PUG 10 g AL = L <SOC peptone 20 g yeast extract 5 g NaCl 10mM KCl 2.5mM

after autoclaving, add sterile MgCl ₂

u. _MgSO4

, 10 mM each ad and 20 mM sterile glucose ad

agar plates

AL = L <BHI AL = L CB = 3 <(data per Petri dish) @ BHI (without yeast) 30 ml Agar Agar 1% Sucrose (60%) 0.5 ml horse serum 2.5 ml Yeast extract (20%) 1 ml Glucose (20%) 0.5 ml

Sucrose, serum, yeast extract, glucose are each added sterile

AL = L <LB AL = L CB = 3 <LB medium + 1.5% (w / v) Agar Agar @ AL = L CB = 3 <SOB @ peptone 20 g yeast extract 5 g NaCl 0.5 g Agar Agar 15 g

after autoclaving, add sterile MgCl ₂

ad 10mM

Further abbreviations: G: glucose, A: ampicillin, T: tetrazyclin, K: kanamycin

Bacterial expression of scFv in E. coli

To prepare a simple soluble scFv derivative with cmyc and HIS ₆ tag in E. coli, pOPE vectors and derivatives derived therefrom were used (Dubel et al., 1993, Gene 128: 97-101). The scFv expression in E. coli and its purification are based on the methods of Moosmayer et al., 1995 (Ther. Immunol., 2: 31-40).

The scFv production was carried out in E. coli XLT-Blue in volumes of 3-100 ml. The Incubation was either shaken into test tubes or shaking in 50 ml PP tubes about 200 rpm or in Erlenmeyer baffled flasks at 180 rpm in LB or 2YT medium. The Media were buffered with 1/10 vol. MOPS (pH 7) and always for strain XILI-Blue with tetracycline (12, 5 ug / ml) added.

2YT _GAT or LB _GAT was inoculated to a preculture with colonies spotted on LB _GAT plates and incubated shaking at 37 ° C. The following day the main culture was inoculated 1:50 and incubated at 37 ° C. For induction, the centrifuged bacteria (2500 rpm, 1000 × g, 10 min RT) were taken up in the same volume of medium (without glucose) with 50 μM IPTG and shaken for 2-3 h at 22-25 ° C and 220 rpm. The bacterial pellet was harvested after centrifugation at 1000 x g (10 min RT) and digested as follows. The harvested pellets of the induced E. coli cultures were taken up in 1 / 20-1 / 30 vol of ice-cold PBS and thoroughly resuspended, incubated with occasional mixing for about 30 min on ice and dissolved in liquid nitrogen or in an ethanol dry ice. Blend frozen. The frozen sample could now be stored at -80 ° C. For digestion, the sample was thawed slowly and subjected to sonication (25-30 cycles with ice-water cooling) until homogeneous and clear. In order to obtain the entire soluble fraction of bacterial protein, the sample was centrifuged off for 20 min at 13000 rpm, the ÜS was carefully removed and the pellet discarded. For longer storage, if necessary, the supernatants were flash-frozen with BSA (ad 1%) and stored at -80 ° C.

In the production of scFv F19 in E. coli, a drastic impairment of the Functionality of the recombinant protein to reach when using (SB medium) or unbuffered culture media.

Expression of scFv derivatives in Proteus mirabilis (LVI)

In Proteus mirabilis monomeric scFv and dimeric scFv (minibodies) were expressed. The expression and purification process was carried out in the same way as we used for soluble monovalent scFv have already been published (Rippmann et al., 1998, Applied and Environmental Microbiology 64: 4862-4869).

Transformation of plasmid DNA into P. mirabilis LVI

The incubation of P. mirabilis LVI was carried out in Erlenmeyer flasks (without baffles) at <200 rpm. For the transformation of the LVI bacteria they were needed in the stationary growth phase (OD ₅₅₀ ≈ 6). For this purpose, 20 ml of a BHI _K culture were inoculated 1:20 from a 4 ° C culture and incubated shaking at 37 ° C üN. Each 100 μl of the above culture was mixed with 20 μl each of the prepared plasmid and 150 μl of PEG (including 0.4 M sucrose) and stored on ice for 10 min. The temperature shock was for 5 min with occasional gentle shaking in a 37 ° C water bath. The transformed LVI bacteria were taken up in 1 ml BYS medium (1 ml BHI, 0.5% yeast extract, 1% sucrose) and shaking vigorously for 3 h in a small steep wall vessel at 37 ° C. From each transformation mixture, 100 μl each were plated on a BHI _K plate. After 24-48 h incubation (37 ° C.), significantly large colonies were punched out using a sterile spatula and transferred into 20 ml of BHI _K medium. After growth and microscopic control for the presence of L-form bacteria, this culture could be treated with cryomedium and frozen at -80 ° C. Non-frozen transformed P. mirabilis cultures remained viable for at least 4 weeks when stored at 4 ° C.

In order to induce the expression in transformed P. mirabilis, two successive 11- or 11-12 h precultures (20 ml each) were inoculated and incubated at 30 ° C, the first of which was from a 4 ° C culture. Depending on the achieved density of the respective preculture and on the incubation length of the following culture, 1: 10 or 1:20 was always inoculated. The BHI _K -Induction cultures (including 0.5 mM IPTG) had a volume of 20-50 ml and were also inoculated, then shaking at 30 ° C for at least 11 h incubated. Before harvesting the bacteria, the OD ₅₅₀ (≧ 4), the pH (7.5-8.5) and the visual appearance of the L-forms were examined under the microscope. The expression culture was centrifuged off (5000 rpm, 3800 × g, 4 ° C.) and the pellet discarded. The ÜS could be used directly for ELISA or Western blot analysis or purified.

The minibodies were cleaned in this work by means of IMAC (immobilized metal affinity chromatography). For this, 1 ml HiTrap columns from Pharmacia Biotech used. As a second purification step, gel exclusion chromatography was performed carried out.

Before starting the induction supernatant, it was thoroughly dialysed against 5 l of cold PBS (pH 8), then ultracentrifuged for at least 30 min (113000 × g, 4 ° C., rotor: Beckman 45 Ti). The column had to be loaded with Zn ²⁺ ions prior to each purification: The solutions used were previously sterile filtered to prevent particulate clogging. Residues of metal ions were removed with 5 ml of 50 mM EDTA. After rinsing with 10 ml H ₂ O _bid , the loading was carried out with 10 ml of 100 mM ZnSO ₄ . After rinsing again with 20 ml H ₂ O _bid , the column was equilibrated with 10 ml PBS (pH 8). The UAS was added to the column by means of a peristaltic pump (1.5 ml / min), followed by a washing step (10 ml PBS incl. 5-20 mM imidazole). Elution was done in 1 ml fractions with 10 ml PBS incl. 300 mM imidazole. The elution fractions were stored on ice.

For gel exclusion chromatography, a Superdex 200 column (10/30) from Pharmacia Biotech used in conjunction with a FPLC facility of the same manufacturer. The IMAC-cleaned sample was centrifuged off for 5 min prior to injection (13000 rpm, 4 ° C).

After equilibration of the pump system and column with the selected running buffer (PBS, pH 8), 500 μl (equivalent to 0.75-1 mg) of IMAC-purified MB # 34 was added to the System injected, pumped at a flow rate of 0.5 ml / min, using a UV detector detected and automatically collected in 500 μl fractions.

Structure of recombinant human antibodies

The pro- and eukaryotic expression of the human recombinant anti-FAP antibodies was carried out as monovalent scFv and bivalent scFv (so-called minibodies). The structure of the produced minibodies and the expression cassettes used for this purpose is comparable to those described by Hu et al. 1996 (Cancer Res. 56: 3055-61). In addition, the minibodies we prepared at the C-terminus have a c-myc domain for immunological detection (with monoclonal antibody 9E10) and a HIS ₆ domain for chromatographic purification. The cmyc- and HIS ₆ -coding sequences correspond to those from pOPE 101 (S. Dübel, University of Heidelberg).

Construction of the minibody

N-signal sequence scFv (VH-left VL) -hinge-left-CH ₃ -cmyc-HIS ₆ -C

Prokaryotic expression of antibody proteins according to the invention

The expression vectors used and the methods for expression and purification monovalent scFv derivatives in E. coli (Moosmayer et al., 1995, Ther. Immunol., 2: 31-40). and Proteus mirabilis LVI (Rippmann et al., 1998, Applied and Environmental Microbiology 64: 4862-4869) are known in the art. For the production and purification of a minibody in Proteus mirabilis LVI, were also the vector pACK02scKan and the methods of Rippmann et al., 1998.

Eukaryotic expression of the antibody proteins of the invention

The production of the minibodies described was also carried out in mammalian cells. When Expression vectors for the minibody expression cassettes served: pAD-CMV-1 and a pg1d105 derivative.

Transient expression in COS cells

For transfection of COS7 cells, the expression vector was previously incubated in E. coli (XL1). Blue) and then purified. The vector DNA was under sterile Conditions adjusted to a concentration of 1 μg / μl and stored at -20 ° C.

The day before transfection, 5x10 ⁵ COS7 cells were seeded in a cell culture petri dish (8 cm diameter, Greiner) in DEMEM 10% FCS and incubated for 16 h at 37 ° C in a CO ₂ incubator. On the day of transfection, a suspension was prepared per Petri dish consisting of 1 ml of OptiMEM (Gibco), 35 μl of Lipofectamine (Gibco Life Science) and 10 μg of expression vector DNA. After incubation at room temperature for 45 min, an additional 4 ml of OptMEM were added and the suspension was carefully pipetted over the cells previously washed with PBS. The solution was spread by gentle swirling and incubated for 5 hours at 37 ° C. The Petri dish was filled with preheated 5 ml DEMEM 20% FCS and incubated at 37 ° C for 16 h. Subsequently, the incubation medium was carefully aspirated and replaced with 10 ml of OptiMEM. After a further 48 hours incubation at 37 ° C, the supernatant was harvested and the cells removed by centrifugation at 700 g. Another centrifugation step at 12000 g pelleted the remaining cell debris. The supernatant was either ultracentrifuged for 30 min (60,000 x g for 30 min) and then placed on an IMAC column (Amersham-Phamacia) or in 1/30 centrifugal concentrators with 30 kDa cut-off (Fugisept-Midi, Maxi-tube, Intersept) concentrated to 1/80 volume. The centrifugation was according to the manufacturer at 6000 g and took usually 6 hours. The concentrated protein solution was mixed with 1% BSA, aliquoted into 100 μl and stored after rapid freezing in N ₂ at -80 ° C.

Stable expression in CHO cells

Stable transfectants of CHO DG44 were prepared for the expression of FAP specific minibodies.

transfection

1st day: 2 × 10 ⁵

Cells were seeded in a well of a 6-well plate.
2nd day: Carefully aspirate the cell culture supernatant and then add 800 μl of CHO-SFM II medium plus HT supplement (Gibco BRL).
Preparation of transfection suspension: 6 μl Lipofectamine + 200 μl CHO-SFM II with HT supplement + 3 μl (3 μg) expression vector. The suspension was mixed and gently added to the cells.
3rd day: Medium change: Addition of CHO-SFM II without HT supplement.

The medium change was repeated regularly. For gene amplification and enhancement The foreign gene expression was measured in the medium from the period 10-14 days after Transfection methotrexate added. The methotrexate concentration became slow increased, the concentrations were between 10 and 1000 nM.

Minibodies were produced in T-bottles or bioreactors.

Determination of the apparent cell binding affinity of the recombinant anti-FAP antibodies

FAP ⁺ cells were incubated in parallel runs with various concentrations of mono- or bivalent scFv derivatives. The binding of these recombinant antibodies was determined by means of a FITC-labeled detection antibody in a cell analyzer (Coulter). As a measure of the apparent affinity, the concentration of the scFv derivatives was chosen at which the half-maximal saturation of the binding signal was achieved.

Example 3 sequences

The sequences are exemplified here. Minor mutations, such as the Exchange one to a few amino acids or the nucleotides coding for them are included in the invention.

VH13 protein sequence as described, for. B. may be present in the minibody vector. The first amino acid may also be an E (glutamate).

VH13 corresponding nucleotide sequence.

VH46 protein sequence.

VH46 corresponding nucleotide sequence as described, for. B. may be present in the minibody. The sixth nucleotide may also be an A instead of a G - a silent mutation, therefore no effect on the amino acid sequence.

VH50 protein sequence as found in the minibody. Again, the same applies to VH13: The first amino acid can also be an E.

VH50 corresponding nucleotide sequence as z. B. present in the minibody.

VLIII25 protein.

VLIII25 corresponding nucleotide sequence.

Protein VH34 with the first 8 amino acids of CH1 inclusive.

VH34 corresponding nucleic acid sequence.

VH18 with some amino acids of CH1.

VH18 corresponding nucleic acid sequence.

VL chain III43.

III43 corresponding nucleic acid sequence.

VH13 YOL VL III25 Protein sequence of the complete antibody protein as present in the minibody.

VH13 YOL VL III25 corresponding nucleotide sequence.

VH46 YOL VL III25 protein sequence of the complete antibody protein as described e.g. B. occurs in the minibody.

VH46 YOL VL III25 corresponding nucleotide sequence.

VH50 YOL VL III25 protein sequence of the complete antibody protein as described e.g. B. in the minibody (possible variation see VH50, above).

VH50 YOL VL III25 corresponding nucleotide sequence.

VH34 YOL III43 protein sequence of the complete antibody protein.

VH34 YOL III43 corresponding nucleotide sequence.

VH18 YOL III43 protein sequence of the complete antibody protein.

VH18 YOL III43 corresponding nucleotide sequence.

Legends to the pictures

Illustration

1: HCDR3 obtained guided selection (HCDR3-retaining guided selection)

Illustration

2: Schematic representation of the HCDR3 sequence with the integrated SplI (Pfl23II)

Illustration

3: binding of scFv # 13 (mini-body format) to FAP ⁺

Cells (FACS analyzes)

Illustration

4: Primers used for PCR amplification of human V repertoires

Illustration

Figure 5: Primer for amplification of the human VH gene segment repertoire for the HCDR3 retaining guided selection procedure

Illustration

Figure 6: Sequences of the selected human FAP-specific VL regions

SEQUENCE LISTING

Claims (100)

A human or humanized antibody protein which specifically binds to fibroblast activating protein alpha (FAPα), characterized in that it is either completely human or no more than a murine complementarity determining region (CDR region) of monoclonal antibody F19 (ATCC Accession No. HB 8269 ) contains. 2. Antibody protein according to claim 1, characterized in that it comprises a heavy chain (V _H ) of the class IgM. 3. Antibody protein according to claim 1 or 2, characterized in that a heavy chain (V _H ) of the class IgG comprises. 4. Antibody protein according to one of claims 1 to 3, characterized in that it comprises a heavy chain (V _H ) of the class IgD. 5. Antibody protein according to one of claims 1 to 4, characterized in that it comprises a light chain (V _L ) of the lambda type (λ). 6. Antibody protein according to one of claims 1 to 5, characterized in that it comprises a light chain (V _L ) of the kappa type (κ). 7. Antibody protein according to one of claims 1 to 6, characterized in that it is a Fab fragment. 8. Antibody protein according to one of claims 1 to 7, characterized in that it is an F (ab) ₂ fragment. 9. Antibody protein according to one of claims 1 to 8, characterized in that it is an Fv single chain protein (scFv). 10. Antibody protein according to one of claims 1 to 9, characterized in that it a diabody antibody fragment. 11. Antibody protein according to one of claims 1 to 10, characterized in that it is a minibody antibody fragment. 12. Antibody protein according to one of claims 1 to 11, characterized in that it is a multimerized antibody fragment. 13. Antibody protein according to one of claims 1 to 12, characterized in that it is completely human.   Antibody protein according to one of Claims 1 to 13, characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 1 (VH13). Antibody protein according to one of Claims 1 to 14, characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 2 (VH46). Antibody protein according to one of Claims 1 to 15, characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 3 (VH50). Antibody protein according to any one of claims 1 to 16, characterized in that the variable region of the light chain (V _L ) comprises the amino acid sequence ID No. 4 (VLIII25). Antibody protein according to one of Claims 1 to 17, characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID No. 5 (VH13) or by fragments or degenerate variants thereof. Antibody protein according to one of Claims 1 to 18, characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID No. 6 (VH46) or by fragments or degenerate variants thereof. Antibody protein according to one of Claims 1 to 19, characterized in that the variable region of the heavy chain (V _H ) is encoded by the nucleotide sequence ID No. 7 (VH50) or by fragments or degenerate variants thereof. Antibody protein according to one of Claims 1 to 20, characterized in that the variable region of the light chain (V _L ) is encoded by the nucleotide sequence ID No. 8 (VLIII25) or by fragments or degenerate variants thereof. Antibody protein, characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 1 (VH13) and the light chain variable region (V _H ) contains the amino acid sequence ID No. 4 ( VLIII25). Antibody protein, characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 5 (VH13) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence Sequence ID No. 8 (VLIII25). Antibody protein, characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 2 (VH46) and the light chain variable region (V _L ) contains the amino acid sequence ID No. 4 ( VLIII25). Antibody protein, characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 6 (VH46) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence Sequence ID No. 8 (VLIII25). 26. Antibody protein, characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 3 (VH50) and the light chain variable region (V _L ) contains the amino acid sequence ID No. 4 (FIG. VLIII25). 27. An antibody protein, characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 7 (VH50) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence Sequence ID No. 8 (VLIII25). 28. Antibody protein according to one of claims 1 to 12 and 14 to 21, characterized characterized in that it is humanized. 29. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28, characterized in that it contains the murine light-chain CDR 1 (V _L ) of the monoclonal antibody F19. 30. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28 to 29, characterized in that it contains the murine light chain CDR 2 (V _L ) of the monoclonal antibody F19. 31. An antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 30, characterized in that it contains the murine light-chain CDR 3 (V _L ) of the monoclonal antibody F19. 32. An antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 31, characterized in that it contains the murine CDR 1 heavy chain (V _H ) of the monoclonal antibody F19. 33. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28 to 32, characterized in that it contains the murine CDR 2 heavy chain (V _H ) of the monoclonal antibody F19. 34. An antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 33, characterized in that it contains the murine CDR 3 heavy chain (V _H ) of the monoclonal antibody F19. 35. An antibody protein according to any one of claims 1 to 34, characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 9 (VH34). 36. Antibody protein according to one of claims 1 to 35, characterized in that the variable region of the heavy chain (V _H ) comprises the amino acid sequence ID No. 10 (VH18). 37. Antibody protein according to one of claims 1 to 36, characterized in that the light chain variable region (V _L ) comprises the amino acid sequence ID No. 11 (VLIII43). 38. Antibody protein according to one of claims 1 to 37, characterized in that the variable region of the heavy chain (V _H ) by the nucleotide sequence ID No. 12 (VH34) or by fragments or degenerate variants thereof is encoded. 39. Antibody protein according to one of claims 1 to 38, characterized in that the variable region of the heavy chain (V _H ) by the nucleotide sequence ID No. 13 (VH18) or by fragments or degenerate variants thereof is encoded. 40. Antibody protein according to one of claims 1 to 39, characterized in that the variable region of the light chain (V _L ) by the nucleotide sequence ID No. 14 (VLIII43) or by fragments or degenerate variants thereof is encoded. 41. Antibody protein, characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 9 (VH34) and the light chain variable region (V _H ) contains the amino acid sequence ID No. 11 (FIG. VLIII43). 42. Antibody protein, characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 12 (VH34) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence Sequence ID No. 14 (VLIII43). 43. Antibody protein, characterized in that the variable region of the heavy chain (V _H ) contains the amino acid sequence ID No. 10 (VH18) and the light chain variable region (V _L ) contains the amino acid sequence ID No. 11 (FIG. VLIII43). 44. Antibody protein, characterized in that the heavy chain variable region coding sequence (V _H ) contains the nucleotide sequence ID No. 13 (VH18) and the light chain variable region coding sequence (V _L ) contains the nucleotide sequence Sequence ID No. 14 (VLIII43). 45. Nucleic acid, characterized in that it is suitable for an antibody protein according to one of the Claims 1 to 44 encoded. 46. Recombinant DNA vector, characterized in that it detects a nucleic acid Claim 45 contains. 47. Recombinant DNA vector according to claim 46, characterized in that it has a Is expression vector. 48. Host, characterized in that it contains a vector according to claim 46 or 47. 49. A host according to claim 48, characterized in that it comprises a eukaryotic host cell is. 50. Host according to claim 48 or 49, characterized in that it is a mammalian cell. 51. Host according to one of claims 48 to 50, characterized in that it has a BHK, CHO or COS cell is. 52. Host according to claim 48, characterized in that it is a bacteriophage. 53. Host according to claim 48, characterized in that it comprises a prokaryotic host cell is. 54. Method for the production of antibody proteins according to one of claims 1 to 44, characterized in that it comprises the following steps: a host according to a of claims 48 to 51 is subjected to conditions under which Antibody protein is expressed by said host cell, cultured and said Antibody protein is isolated. 55. The method according to claim 54, characterized in that said host a Mammalian cell, preferably a CHO or COS cell. 56. The method according to claim 54 or 55, characterized in that said host cell is co-transfected with two plasmids expressing the expression units for the light or wear the heavy chain. 57. Antibody protein according to one of claims 1 to 44, characterized in that said antibody protein is coupled to a therapeutic agent.   58. Antibody protein according to claim 57, characterized in that said therapeutic agent from the group of radioisotopes, toxins, toxoids, boron, Fusion proteins, inflammatory agents and chemotherapeutic agents Agents is selected. 59. Antibody protein according to claim 57 or 58, characterized in that said Radioisotope is a β-emitting radioisotope. 60. Antibody protein according to claim 59, characterized in that said radioisotope is selected from the group ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ¹³¹ iodine and ⁹⁰ yttrium. 61. Antibody protein according to one of claims 1 to 44, characterized in that it is marked. 62. Antibody protein according to claim 61, characterized in that it contains a detectable marker is marked. 63. Antibody protein according to claim 61 or 62, characterized in that the detectable markers from the group of enzymes, dyes, radioisotopes, Digoxygenin, streptavidin and biotin is selected. 64. Antibody protein according to one of claims 1 to 44, characterized in that it coupled to an imageable agent. 65. An antibody according to claim 64, characterized in that the imageable agent Radioisotope is. 66. An antibody according to claim 64 or 65, characterized in that said Radioisotope is a γ-emitting radioisotope. 67. An antibody protein according to claim 66, characterized in that said radioisotope is ¹²⁵ iodine. 68. Pharmaceutical preparation, characterized in that it is an antibody protein according to any one of claims 1 to 44 and one or more pharmaceutically acceptable ones Contains carrier substances. 69. Pharmaceutical preparation, characterized in that it is an antibody protein according to any of claims 57 to 60 and one or more pharmaceutically acceptable ones Contains carrier substances.   70. Pharmaceutical preparation, characterized in that it is an antibody protein according to any one of claims 64 to 67 and one or more pharmaceutically acceptable ones Contains carrier substances. 71. Use of a pharmaceutical preparation according to one of claims 68 to 70, characterized in that they are for the treatment or imaging of tumors wherein said tumors are activated with activated stromal fibroblasts are associated. Use according to claim 71, wherein said tumors are selected from the group of colorectal cancer, non-small cell lung cancer, breast cancer, and head Nephrotic cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer are selected. 73. Use of an antibody protein according to any one of claims 1 to 44 for Preparation of a pharmaceutical preparation for the treatment of cancer. 74. Use of an antibody protein according to any one of claims 57 to 60 for Preparation of a pharmaceutical preparation for the treatment of cancer. 75. Use of an antibody protein according to any one of claims 64 to 67 for Illustration of activated stromal fibroblasts. 76. Method for detecting activated stromal fibroblasts in wound healing, inflammatory processes or in a tumor, characterized in that a Sample containing possibly activated fibroblasts, with a An antibody protein according to any one of claims 1 to 44 or 61 to 64 below Conditions that are suitable for a complex of said antibody protein to form contact with its antigen and the formation of said complex and thus the presence of activated stromal fibroblasts in wound healing, inflammatory processes or in the case of a tumor. 77. The method according to claim 76, characterized in that said tumor from the Group of colorectal cancer, non-small cell lung cancer, breast cancer, head and cervical cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer is selected. 78. The method according to claim 76 or 77, characterized in that said Antibody protein is a protein according to any one of claims 61 to 63.   79. A method for the detection of tumor stromal, characterized in that a suitable A sample with an antibody protein according to any one of claims 1 to 44 below suitable conditions for the formation of an antibody-antigen complex is contacted, the complex thus formed is detected and the presence of the so formed complex in relation to the presence of tumor stroma. 80. The method according to claim 79, characterized in that said antibody with a detectable marker is marked. 81. Antibody protein, characterized in that it has an amino acid sequence according to Sequence ID No. 15 or a part thereof or a functional variant thereof contains. 82. Antibody protein, characterized in that it has an amino acid sequence according to Sequence ID No. 16 or a part thereof or a functional variant thereof contains. 83. Antibody protein, characterized in that it has an amino acid sequence according to Sequence ID No. 17 or a part thereof or a functional variant thereof contains. 84. Antibody protein, characterized in that it has an amino acid sequence according to Sequence ID No. 18 or a part thereof or a functional variant thereof contains. 85. Antibody protein, characterized in that it has an amino acid sequence according to Sequence ID No. 19 or a part thereof or a functional variant thereof contains. 86. Antibody protein, characterized in that it by a nucleotide sequence according to Sequence ID No. 20 or a part thereof or a functional variant thereof is encoded. 87. Antibody protein, characterized in that it by a nucleotide sequence according to Sequence ID No. 21 or a part thereof or a functional variant thereof is encoded. 88. Antibody protein, characterized in that it by a nucleotide sequence according to Sequence ID No. 22 or a part thereof or a functional variant thereof is encoded.   89. Antibody protein, characterized in that it by a nucleotide sequence according to Sequence ID No. 23 or a part thereof or a functional variant thereof is encoded. 90. Antibody protein, characterized in that it by a nucleotide sequence according to Sequence ID No. 24 or a part thereof or a functional variant thereof is encoded. 91. Antibody protein, characterized in that it according to the amino acid sequence Sequence ID No. 15 corresponds. 92. Antibody protein, characterized in that it according to the amino acid sequence Sequence ID No. 16 corresponds. 93. Antibody protein, characterized in that it according to the amino acid sequence Sequence ID No. 17 corresponds. 94. Antibody protein, characterized in that it according to the amino acid sequence Sequence ID No. 18 corresponds. 95. Antibody protein, characterized in that it according to the amino acid sequence Sequence ID No. 19 corresponds. 96. Antibody protein, characterized in that it by the nucleotide sequence according to Sequence ID No. 20 is encoded. 97. Antibody protein, characterized in that it by the nucleotide sequence according to Sequence ID No. 21 is encoded. 98. Antibody protein, characterized in that it by the nucleotide sequence according to Sequence ID No. 22 is encoded. 99. Antibody protein, characterized in that it by the nucleotide sequence according to Sequence ID No. 23 is encoded. 100. Antibody protein, characterized in that it by the nucleotide sequence coded according to sequence ID No. 24.