JP2003530092A - Human FAP-α-specific antibody - Google Patents

Abstract

(57) [Summary] The present invention relates to an antibody protein that specifically binds to fibroblast activation protein α (FAPα). The invention further relates to the use of said antibodies for diagnostic and therapeutic purposes, as well as to methods for preparing said antibodies.

Description

Detailed Description of the Invention

[0001] The present invention relates to an antibody protein that specifically binds to fibroblast activation protein α (FAPα). The invention further provides for the use of said antibody for diagnostic and therapeutic purposes,
It relates to the antibody preparation method.

BACKGROUND OF THE INVENTION Bulk growth of epithelial cell carcinoma is associated with many characteristic cellular and molecular alterations of the surrounding stromal cells. One of the highly consistent features of reactive stroma of various epithelial cell carcinomas is the cell surface molecule of reactive stromal fibroblasts, originally identified as monoclonal antibody F19, fibroblast activating protein α ( (Hereinafter referred to as FAPα or FAP) (Garin-Chesa P., Old LJ and Rettig WJ
., Proc Natl. Acad. Sci. 87: 7235 (1990)). FAP is selectively expressed in the stroma of many epithelial cell carcinomas, allowing imaging, diagnosis and treatment of epithelial cell carcinoma and many other syndromes, regardless of the site and histological type of the carcinoma In order to achieve this, it was desired to develop a therapeutic concept for the FAPα target molecule. To this end, a mouse monoclonal antibody designated F19 has been developed that specifically binds to FAP. This antibody is described in US Pat. No. 5,059,523 and WO 93/05.
No. 804, which is incorporated herein by reference in its entirety. The use of non-human antibodies for in vivo applications in humans poses a significant problem, ie they elicit a rapid immune response against foreign antigens. In the worst case, such an immune response against the used antibody may trigger an anaphylactic shock. This dramatically reduces the efficacy of the antibody in patients and has a deleterious effect on further use or renders further use impossible.

Humanization of non-human antibodies is usually performed by one of two methods: (1) a non-human / human chimera in which the non-human variable region is linked to a human constant region. By antibody construction (Boulianne GL, Hozumi N. and Shulman, MJ, Nature, 31
2: 643 (1984)), or (2) the complementarity determining region (CDR) within the human variable region, exchanged for that of the non-human variable region, followed by the newly formed humanized variable region. By binding to human constant regions (Riechmann L., Clark M., Waldmann H. and Winter G., Nature, 332: 323 (1
988)).

Chimeric antibodies consist of less foreign protein sequences than non-human antibodies and, as a result, have less heterologous antigenic potency. However, this type of chimeric antibody
Can trigger an immune response in humans to non-human V-regions (LoBuglio AF
., Wheeler RH, Trang J., Haynes A., Roger K., Harvey EB, Sun L., Ghr
ayeb J. and Khazaeli MB, Proc. Natl. Acad. Sci., 86: 4220 (1989)). CDR-transduced or newly formed humanized antibodies, which are widely accepted, contain fewer foreign protein sequences in the V-region, although these humanized antibodies are still immunogenic in humans. It is possible to trigger a response. WO 99/57151 A2 shows that all six CDR regions (3
FAPα-of this species as realized by the transfer of a light chain and three heavy chains.
Disclosed are specific humanized antibodies. These antibodies still contain part of the mouse framework regions. The object of the present invention is to provide an improved FAPα-, which overcomes the aforementioned drawbacks of the prior art.
It is to provide a specific antibody.

DESCRIPTION OF THE INVENTION This problem has been solved within the scope of the claims and the description of the invention. Use of the singular or plural in the claims or in the specification is in no way meant to be limiting, and also includes the other forms.

The present invention relates to a novel human or humanized antibody protein that specifically binds to fibroblast activating protein alpha (FAPα), which is either fully human or monoclonal antibody F19 (ATCC Deposit No. HB 8269) or a mouse complementarity determining region (CDR region) that does not exceed one. The antibodies of the present invention have the surprisingly advantageous property of having significantly reduced heterologous antigenic potency and, as a result, are more suitable for use in humans than the antibodies known in the prior art (below, the method of the present invention. (See also the explanation of). The antibodies of the present invention advantageously do not contain or have a minor portion of the mouse amino acid sequence, ie at most one CDR region. Similarly, the entire framework region (FR) of the variable region of the antibody of the present invention corresponds to the human amino acid sequence. Despite the few mouse components, the antibodies of the invention are surprisingly highly specific for the target antigen FAP.

Within the scope of the present invention, the term “antibody” refers to 1 as described herein.
By more than one polypeptide is meant. It is further derived from fragments, allelic variants, functional variants, variants based on the degenerate nucleic acid code, fusion proteins with the antibody proteins of the invention, chimeric derivatives or glycosylation variants of the antibody proteins of the invention. Also included are selected human antibody proteins. The preparation methods known in the prior art are not suitable for obtaining the human antibodies of the invention. By the methods of the present invention as more fully described and illustrated in the Examples below, it is possible to obtain human or humanized antibodies of the present invention having reduced xenoantigenicity. In the preferred preparation method of the present invention, for example, the following steps are carried out:

1) PCR amplification of human VL- and VH-repertoires : a) For the preparation of VH and VL repertoires, various V-gene families have cDNAs.
From the above, PCR is performed to individually amplify using a primer specific to each family (see Example 1). b) All Forward / 3'-primers for VH- and VL-PCR amplifications are complementary to the gene sequences of the constant immunoglobulin domain (IgG, IgD, IgM, kappa, lambda). This allows efficient isotype-specific amplification of the V region with very few 3'-primers. In contrast, methods known in the prior art use multiple different 3'-primers complementary to the J-section of the V region (Marks et al., J. Mol. Bio
L., 222: 581 (1991)).

2) Preparation and cloning of the human VH-repertoire : In the prior art, to date only certain lymphoid tissues have been described as sources of the V repertoire by very few different donors (eg Vaugh.
an et al., Nature Biotechnology, 14: 309 (1996)). To obtain a human V-repertoire consisting of a large number of clones with a high degree of diversity as the basis of antibody preparation of the present invention (
(See Example 1 for details), more or less different donors are used, i.e. about 10 times more than recommended in the prior art, but in an unclear way, only for the lymphoid organ in question. However, it is also used as a source of V repertoire for fetal liver and thymus. Furthermore, in addition to the IgM and IgG repertoires, the IgD repertoire is also amplified to achieve large repertoire diversity (see Example 1).
.

3) A combinatorial reper consisting of a human VH repertoire and various human FAP-specific VL regions
Preparation of Trees: To obtain the antibodies of the invention, for example, the monoclonal FAP-specific mouse antibody F1
Known VH regions from 9 can be used, and appropriate human FAP-specific VL regions can be selected using the guided selection method and the phage display method. The human VL region can then be used as a guide structure to select, for example, the human FAP-specific VH region. FAP-specific scFv present (e.g., mouse hybridoma strain F19
There may be technical problems with DNA contamination of the combinatorial repertoire with the scFv of origin or the phagemid vector encoding the chimeric anti-FAP scFv with human VL and F19 VH). Guided selection is described in the examples below. A combinatorial repertoire is a genetically engineered V repertoire of corresponding complementary V
-Means a combination with a sequence (complementarity to VL with respect to VH or vice versa). The V-sequences used in this combination can consist of one V-sequence, many different V-sequences or V repertoires.

Preferably, the antibody protein of the invention is characterized in that it comprises a heavy chain (V _H ) of immunoglobulin class IgM. Preferably, the antibody protein of the present invention is an immunoglobulin class IgG heavy chain (V _H ).
It is characterized by including. A non-limiting example of these is the fully human antibody scFv #
13 and scFv # 46 (see Examples). Preferably, the antibody protein of the present invention is characterized in that it also contains a heavy chain (V _H ) of class IgD. A non-limiting example of this is the human antibody scFv # 50 of the present invention.
(See also Example). In this antibody, the VH-sequence is identical to the germline sequence except that it originates from human IgD and has a single amino acid exchange. This advantageously reduces the probability of an alloimmune response against this VH region in humans.

Also preferably, the antibody protein of the invention is characterized in that it comprises a lambda (λ) type light chain (V _L ). Also preferably, the antibody protein of the invention is characterized in that it comprises a kappa (κ) type light chain ( _VL ) (see Examples eg III25, III43). For many uses of the antibodies of the present invention, it is desirable to have a minimal potential antigen-binding, or FAP-binding unit. Therefore, in another preferred embodiment, the antibody protein of the invention comprises a Fab fragment (antigen-binding fragment =
Fab). These FAP-specific antibody proteins of the invention consist of the variable regions of both chains which are held together by the adjacent constant regions. These can be formed from conventional antibodies by protease digestion, eg with papain, while similar Fab fragments can also be generated by genetic engineering. In another preferred embodiment, the antibody protein of the invention is F (ab
') 2 fragment, which can be prepared by proteolytic cleavage with pepsin.

Using genetic engineering techniques, it is possible to produce truncated antibody fragments that consist only of the variable regions of the heavy (VH) and light (VL) chains. These are referred to as Fv fragments (fragment variable part = fragment of variable part). In another preferred embodiment, the FAP-specific antibody molecule of the present invention is such an Fv fragment. these
These Fv fragments are often stabilized because Fv fragments lack the covalent attachment of these two chains by the constant chain cysteines. It is an advantage to link the variable regions of the heavy and light chains by short peptide fragments, eg 10-30 amino acids, preferably 15 amino acids. In this way, a single peptide chain consisting of V _H and V _L linked by a peptide linker is obtained. This type of antibody protein is known as single-chain-Fv (scFv). Examples of this type of scFv-antibody protein known in the prior art can be found in the article by Huston et al. (PNAS, 16: 5879-5883 (1
988)). Therefore, in another preferred embodiment, the FAP of the invention
-The specific antibody protein is a single-chain-Fv protein (scFv).

In recent years, various strategies have been developed for preparing scFv as multimeric derivatives.
This is intended to lead to recombinant antibodies that are associated with increased avidity, as well as particularly improved pharmacokinetic and biodistribution properties. In order to carry out scFv multimerization, scFv was prepared as a fusion protein with a multimerization domain. The multimerization domain can be, for example, a CH3 region of IgG or a coiled coil structure (helix structure) such as a leucine-zipper domain. But scFv
There are also strategies such as using the interaction between the VH / VL regions of E. coli for multimerization (eg bi-, tri- and penta-specific antibodies). Thus, in another preferred embodiment, the antibody protein of the invention is a FAP-specific bispecific antibody fragment. Those skilled in the art refer to bispecific antibodies as bivalent homodimeric scFv derivatives (Hu et al., PNAS, 16: 5879-58.
83 (1996)). Shortening of the linker in the scFv molecule to 5-10 amino acids results in inner-chain VH / V
This leads to the formation of homodimers with L-superimposition occurring. Bispecific antibodies can be further stabilized by the incorporation of disulfide bridges. Examples of prior art bispecific antibody proteins are found in the article by Perisic et al. (Structure, 2: 1217-122.
6 (1994)).

To the person skilled in the art, a minibody means a divalent homodimeric scFv derivative. This includes the CH3 region of an immunoglobulin, preferably IgG, most preferably IgG1, as a dimerization region as linked to the scFv via a hinge region (e.g. also derived from IgG1) and a linker region. It consists of a fusion protein. This disulfide bridge in the hinge region is mainly formed in higher cells and not in prokaryotic cells. In another preferred embodiment, the antibody protein of the invention is a FAP-specific minibody antibody fragment. Examples of prior art minibody-antibody proteins can be found in the article by Hu et al. (Cancer Res., 56: 3055-61 (1996)).

To the person skilled in the art, trispecific antibody means a trivalent homotrimeric scFv derivative (K
ortt et al., Protein Engineering, 10: 423-433 (1997)). A scFv derivative in which VH-VL was directly fused without a linker sequence leads to trimerization. Those skilled in the art are also familiar with so-called mini-antibodies, which have a di-, tri- or tetravalent structure and are derived from scFv. This multimerization is carried out by di-, tri- or tetrameric coiled-coil structures (Pack et al., Biotechnology, 11: 1271-1277 (1993); Lovejoy
Et al., Science, 259: 1288-1293 (1993); Pack et al., J. Mol. Biol., 246: 28-34 (199).
Five)). Therefore, in another preferred embodiment, the antibody protein of the present invention is a FAP-specific multimerized molecule based on the aforementioned antibody fragment, and, for example, a trispecific antibody, a tetravalent mini antibody or It can be a penta-specific antibody.

Particularly preferably, the antibody protein of the invention is wholly human. Another preferred antibody protein of the present invention is characterized in that the variable region of the heavy chain ( _VH ) comprises the amino acid sequence SEQ ID NO: 1 (VH13). Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) comprises the amino acid sequence SEQ ID NO: 2 (VH46). Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) comprises the amino acid sequence SEQ ID NO: 3 (VH50). Another preferred antibody protein of the invention is characterized in that the variable region of the light chain ( _VL ) comprises the amino acid sequence SEQ ID NO: 4 (VLIII25). Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) is encoded by nucleotide sequence SEQ ID NO: 5 (VH13) or fragments thereof or degenerate variants thereof. Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) is encoded by nucleotide sequence SEQ ID NO: 6 (VH46) or fragments thereof or degenerate variants thereof. Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) is encoded by nucleotide sequence SEQ ID NO: 7 (VH50) or a fragment thereof or a degenerate variant thereof. Another preferred antibody protein of the invention is characterized in that the variable region of the light chain ( _VL ) is encoded by nucleotide SEQ ID NO: 8 (VLIII25) or a fragment thereof or a degenerate variant thereof.

In a particularly preferred antibody protein of the present invention, the variable region of the heavy chain (V _H ) comprises amino acid SEQ ID NO: 1 (VH13) and the variable region of the light chain (V _L ) comprises amino acid SEQ ID NO: 4 ( VLIII25
) Is included. Another particularly preferred antibody protein of the invention has a heavy chain (V _H ) variable region coding sequence comprising nucleotides SEQ ID NO: 5 (VH13), and a light chain (V _L ) variable region coding sequence comprising nucleotides It is characterized by containing SEQ ID NO: 8 (VLIII25). Another particularly preferred antibody protein of the invention has the variable region of the heavy chain (V _H ) comprising amino acid SEQ ID NO: 2 (VH46) and the variable region of the light chain (V _L ) amino acid SEQ ID NO: 4 (VLI).
II25) is included. Another particularly preferred antibody protein of the invention has the heavy chain (V _H ) variable region coding sequence comprising nucleotide SEQ ID NO: 6 (VH46), and the light chain (V _L ) variable region coding sequence comprises nucleotides It is characterized by containing SEQ ID NO: 8 (VLIII25). Another particularly preferred antibody protein of the invention has the heavy chain (V _H ) variable region comprising amino acid SEQ ID NO: 3 (VH50) and the light chain (V _L ) variable region comprising amino acid SEQ ID NO: 4 (VLI).
II25) is included. Another particularly preferred antibody protein of the invention has a heavy chain (V _H ) variable region coding sequence comprising nucleotide SEQ ID NO: 7 (VH50), and a light chain (V _L ) variable region coding sequence comprising nucleotides It is characterized by containing SEQ ID NO: 8 (VLIII25).

Particularly preferably, the antibody protein of the present invention is humanized. The humanized antibody protein of the present invention, as described in the preferred embodiment below, does not contain all six CDR regions of mouse F19, but in the prior art such that it contains only one mouse CDR region. It has advantages over known FAP α-specific antibody proteins. This antibody protein of the invention advantageously has less heterologous antigenic capacity than the antibody proteins known in the prior art. Surprisingly, we have a dominant opinion that at least two mouse CDR regions are required for successful humanization.
(Rader et al., Proc. Natl. Acad. Sci. USA, 95: 8910 (1998)) and only 1
We have succeeded in producing an antibody molecule containing mouse CDR regions. Another surprising property of humanized scFv 34 and scFv 18 is that these scFv
Shows a higher apparent binding affinity for FAP + cells (EC ₅₀ 6 nM) than FAP-specific antibodies known in the art, eg scFv F19 (EC ₅₀ 20 nM).

A preferred method of the invention for preparing a humanized antibody of the invention can be illustrated, for example, by the following steps: 1) Humanization of scFv F19 by a guided selection method that maintains HCDR3 . Have shown that by using the "Guided selection" method, human Abs with different epitope specificities than the Ab of the parental mouse can be selected. To overcome this drawback of the prior art, HCDR3 F19 was advantageously retained in a guided selection method for the humanization of scFv F19, as well as in the final humanized product. Prior art (Rader et al., PNAS, 95: 8910 (1998))
Describe only antibodies that have been humanized by "guided selection" such that both LCDR3 and also HCDR3 of the parental mouse Ab are maintained (see Example 1).

2) Combining the human VH-gene segment repertoire with mouse HCDR3 (F19) All known human VH family VH segments are combined with HCDR3 F19 to create the most complex combinatorial repertoire possible. Advantageously, this does not change the code at the amino acid level, for example in the HCDR3 F19 the restriction enzyme P
It is preferably carried out by incorporating the cleavage site of fl23II. For the PCR-amplified human VH-gene segment combination, a phage display vector was developed containing the following Ab-sequence segments: HCDR3 F19 with a Pfl23II cleavage site, humans with high homology to the region corresponding to F19. VH FR4 region, as well as various selected human anti-FAP VL regions (see schematic in Example 1). Primers for PCR amplification of the VH-gene segment repertoire are shown in Example 1.

This preferred method has the advantage over the following prior art on the combination of the VH-gene segment repertoire with the restricted CDR3 region: Schier et al., J. Mol. Biol., 255: 28 (1996) : In this prior art, the restriction cleavage site (BssHII) is integrated in the 3'region of VH FR3. However, PCR of this cleavage site
Has led to altered amino acid sequences of various VH-gene families. The reason for this is described in the paper by Schier et al. Some VH-
Only gene families can be included in this combinatorial repertoire. PCR overlap extension, Rader et al., 1998 : This method actually allows to include the entire VH-gene family in the combination, but its drawbacks are low linkage efficiency and high error rate. This increases the probability of inactive scFv mutants and especially clones with disrupted scFv reading frames, leading to a genetically unstable combinatorial repertoire.

3) Use of various human FAP-specific VL regions as guide structures To increase the selection probability of ScFvs similar to F19, human VH repertoire (see Section 2) was added to various human FAP-specific. Combined with the sequence of the sex VL region (performed in the same manner as the human antibody described above). 4) Stringent washing step in phage display selection Using this technique, low-affinity antibodies and polyreactive antibodies were removed in the selection process (see below for method).

5) Use of Efficient Screening Method for Identification of Selected Humanized scFvs During the "Guided Selection Method to Maintain HCDR3", a very large number of clones were enriched. Advantageously, scFv # 34 and # 18 were found in the article by Mersmann et al. (J. Immunol. Me
thods, 220: 51 (1998)). Another preferred antibody protein of the invention is the light chain (V _L ) of monoclonal antibody F19.
It is characterized by including mouse CDR1. Another preferred antibody protein of the invention is the light chain (V _L ) of monoclonal antibody F19.
It is characterized by including mouse CDR2. Another preferred antibody protein of the invention is the light chain (V _L ) of monoclonal antibody F19.
It is characterized by including mouse CDR3. Another preferred antibody protein of the invention is the heavy chain (V _H ) of monoclonal antibody F19.
It is characterized by including mouse CDR1. Another preferred antibody protein of the invention is the heavy chain (V _H ) of monoclonal antibody F19.
It is characterized by including mouse CDR2. Another preferred antibody protein of the invention is the heavy chain (V _H ) of monoclonal antibody F19.
It is characterized by including mouse CDR3.

Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) comprises the amino acid sequence SEQ ID NO: 9 (VH34). Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) comprises the amino acid sequence SEQ ID NO: 10 (VH18). Another preferred antibody protein of the invention is characterized in that the variable region of the light chain ( _VL ) comprises the amino acid sequence SEQ 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 nucleotide sequence SEQ ID NO: 12 (VH34) or a fragment or degenerate variant thereof. There is. Another preferred antibody protein of the invention is characterized in that the variable region of the heavy chain ( _VH ) is encoded by nucleotide sequence SEQ ID NO: 13 (VH18) or a fragment or degenerate variant thereof. Another preferred antibody protein of the invention is characterized in that the variable region of the light chain ( _VL ) is encoded by the nucleotide sequence SEQ ID NO: 14 (VLIII43) or a fragment or degenerate variant thereof.

In a particularly preferred antibody protein of the present invention, the variable region of the heavy chain (V _H ) contains amino acid sequence number: 9 (VH34), and the variable region of the light chain (V _L ) has amino acid sequence number: SEQ ID NO: 11 (VL
It is characterized by including III43). Another particularly preferred antibody protein of the present invention is that the coding sequence for the variable region of the heavy chain (V _H ) comprises nucleotide SEQ ID NO: 12 (VH34), and the coding sequence for the variable region of the light chain (V _L ) is , Nucleotide sequence number: 14 (VLIII43).
Another particularly preferred antibody protein of the present invention is that the variable region of the heavy chain (V _H ) comprises amino acid SEQ ID NO: 10 (VH18) and the variable region of the light chain (V _L ) comprises amino acid SEQ ID NO: 11
(VLIII43) is included. Another particularly preferred antibody protein of the present invention has a heavy chain (V _H ) variable region coding sequence comprising nucleotide sequence number: 13 (VH18) and a light chain (V _L ) variable region coding sequence. , Nucleotide sequence number: 14 (VLIII43).

Another particularly preferred embodiment of the invention comprises a nucleic acid encoding the antibody protein of the invention. Preferably, also the nucleic acid of the invention is characterized in that it comprises 5'or 3'or 5'and 3'untranslated regions. The nucleic acid of the present invention may contain another untranslated region upstream and / or downstream. The untranslated region can include regulatory elements such as transcription initiation units (promoters) or enhancers. The promoter may be, for example, constitutive, inducible or developmentally regulated (de
velopment-controlled) promoter. Although not excluding other known promoters, constitutive promoters of human cytomegalovirus (CMV) and Rous sarcoma virus (RSV), as well as the simian virus 40 (SV40) and herpes simplex promoters are preferred. Inducible promoters of the present invention include antibiotic resistance promoters, heat shock promoters, hormone inducible "breast cancer virus promoters" and metallothionein promoters. Preferably also
The nucleic acid of the present invention is also characterized in that it encodes a fragment of the antibody protein of the present invention. This means part of the polypeptide of the invention.

Also preferably, the nucleic acid of the present invention is characterized in that it encodes a functional variant of the antibody protein of the present invention. This means a polypeptide which is largely identical to the antibody protein of the invention and which has the same biological activity as the antibody protein of the invention or has an inhibitory effect on the antibody protein of the invention. Variants of antibody proteins of the invention include one or more amino acids, preferably 1-10.
It may differ from the antibody protein of the present invention by the substitution, deletion or addition of individual amino acids.

Preferably also, the nucleic acid of the invention is characterized in that it encodes an allelic variant of the antibody protein of the invention. Also preferably, the nucleic acid of the present invention is also characterized in that it encodes a variant of the antibody protein of the present invention based on the degenerate code of the nucleic acid. Also preferably, the nucleic acid is also characterized in that it can hybridize under stringent conditions to the nucleic acid according to the invention. Stringent conditions are known to those skilled in the art, and in particular, Sambrook et al., "Molecular Cloning: A Laboratory Manual," Second Edition (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (1989)).

Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it comprises the amino acid sequence of SEQ ID NO: 15 or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it comprises the amino acid sequence of SEQ ID NO: 16 or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it comprises the amino acid sequence of SEQ ID NO: 17, or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it comprises the amino acid sequence of SEQ ID NO: 18, or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it comprises the amino acid sequence of SEQ ID NO: 19, or a part thereof or a functional variant thereof.

Another particularly preferred embodiment of the present invention provides an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 20 or a part thereof or a functional variant thereof. Including. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 21, or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 22 or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 23, or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 24, or a part thereof or a functional variant thereof.

Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it corresponds to the amino acid sequence of SEQ ID NO: 15. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it corresponds to the amino acid sequence of SEQ ID NO: 16. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it corresponds to the amino acid sequence of SEQ ID NO: 17. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it corresponds to the amino acid sequence of SEQ ID NO: 18. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it corresponds to the amino acid sequence of SEQ ID NO: 19.

Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 20. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 21. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 22. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 23. Another particularly preferred embodiment of the invention comprises an antibody protein, characterized in that it is encoded by the nucleotide sequence according to SEQ ID NO: 24.

The SEQ ID NO means the number specified next to <400> in the sequence list, and for example, the nucleotide sequence of SEQ ID NO: 24 is represented as <400> 24. Another aspect of the present invention relates to a recombinant DNA vector containing the nucleic acid of the present invention. Examples include viral vectors such as vaccinia, semliki forest virus and adenovirus. Vectors for use in COS-cells have the SV40 origin of replication and high plasmid copy numbers are achievable. Vectors for use in insect cells are, for example, E. coli delivery vectors and contain as a promoter, for example, DNA encoding polyhedrin. Another aspect of the invention relates to the recombinant DNA vector of the invention, which is an expression vector.

Yet another aspect of the present invention is a host containing the vector of the present invention. Another host of the invention is a eukaryotic host cell. The eukaryotic host cell of the present invention is a fungus,
For example, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces,
Insect cells, such as Trichoderma (e.g., with a baculovirus expression system, Sp
odoptera frugiperda Sf-9), plant cells such as Nicotiana tabacum, and mammalian cells such as COS cells, BHK, CHO or myeloma cells.

The antibody proteins of the invention are particularly well folded and glycosylated in the progeny of cells of the immune system in which they are also formed in the human body. Therefore, the preferred host of the present invention is a mammalian cell. Particularly preferred hosts according to the invention are BHK, CHO or COS cells. Another host of the invention is a bacteriophage. Another host of the invention is a prokaryotic host cell. An example of a prokaryotic host cell is Escherichi
a coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

The present invention relates to a method of preparing an antibody protein of the invention comprising the steps of: subjecting a host of the invention as described above to conditions under which the antibody protein is expressed by the host cell. And a step of isolating the antibody protein. The antibody protein of the present invention can be expressed in any of the aforementioned hosts. Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis
The preparation of prokaryotic expression systems such as is particularly suitable for antibody fragments of the invention, such as Fab-, F (ab ') 2-, scFv fragments, minibodies, bispecific antibodies and multimers of said fragments. There is. The antibody proteins of the present invention may be expressed in cells such as inclusion bodies, by secretion into cell-free bacteria such as Proteus mirabilis, or into the periplasmic space of Gram-negative bacteria using vectors suitable for this purpose. Is prepared according to the methods of the present invention. The preparation of the antibody protein of the invention in prokaryotic cells is described in Example 2 for purposes of illustration.
Prior art examples for the preparation of scFv-antibody proteins are described in Rippmann et al. (Appl.
Environ. Microbiol., 64: 4862-4869 (1998)). Other examples are known to those skilled in the art. The antibody protein of the present invention is a vector that brings about intracellular expression or secretion,
For example, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces,
Fungi such as Trichoderma can also be prepared by the method of the present invention.

The method of preparing the antibody protein of the present invention can also be carried out by insect cells, for example as a transient or stable expression system or a baculovirus expression system. Here, for example, Sf-9 insect cells are, for example, Autographa cal
Infected by ifornica nuclear polyhedrosis virus (AcNPV) or related viruses. There is no risk of contamination with viruses that are pathogens for mammals,
As a result, the therapeutic antibody of the present invention can be advantageously prepared in insect cells. The aforementioned E. coli delivery vector contains, for example, a DNA encoding polyhedrin as a promoter, and then the DNA encoding the antibody of the present invention is cloned. After identification of the correct delivery vector clone in E. coli, it was transfected into insect cells with incomplete baculovirus DNA and recombined with baculovirus DNA, resulting in the production of viable baculovirus. To be done. Using the strong insect cell promoter, the method of the invention produces large amounts of the antibody protein of the invention, which is secreted into the medium, for example by fusion with a eukaryotic signal sequence. Non-functional expression of antibody protein (d
Insect cell expression systems for ie expression) are commercially available. The insect cell expression system is particularly suitable for scFv and Fab or F (ab ') 2 fragments of the invention and antibody proteins or fragments thereof fused to effector molecules, but also for complete antibody molecules. .

One of the advantages of mammalian expression systems is that they are very good in transient expression systems such as COS-cells or stable expression systems such as BHK, CHO, myeloma cells etc. Glycosylation and folding conditions (see also Example 2). Mammalian cells can also be used with viral expression systems such as vaccinia, semliki forest virus and adenovirus. Transgenic animals such as cows, goats and mice are also suitable for the method of the invention. Transgenic plants such as Nicotiana tabacum (tobacco) are also suitable for use in the method of the invention. These are particularly suitable for the preparation of the antibody fragments of the invention. After genomic integration of a nucleic acid of the invention encoding an antibody protein of the invention fused to a signal sequence, secretion of the antibody protein into the interstitial space can be achieved.

The invention especially relates to the method of the invention, wherein said host is a mammalian cell, preferably a CHO or COS cell. The invention especially relates to the method of the invention wherein the host cell is co-transfected with two plasmids carrying the expression units for the light or heavy chains. The antibody protein of the present invention is a substance with high specificity for guiding a therapeutic substance to the FAP antigen. Therefore, another preferred antibody protein of the present invention is characterized in that the antibody protein is bound to a therapeutic substance.

It is also preferred that the antibody protein of the present invention can be further linked to a therapeutic substance or effector molecule by genetic engineering. In the present invention, this type of therapeutic substance is a cytokine, for example, 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,
Interleukins (IL) such as IL-18, interferon (IFN) α, IFNβ, IFNγ,
There are IFNω or IFNτ, tumor necrosis factor (TNF) TNFα and TNFβ, TRAIL, immunomodulatory or immunostimulatory proteins, or apoptosis- or necrosis-inducible proteins. Thus, the antibody-effector molecule complex of the present invention comprises an antibody-cytokine fusion protein and further comprises a bispecific antibody derivative and an antibody-superantigen fusion protein. They are preferably used for the activation of the body's own anti-tumor defense mechanisms, so that they are suitable for therapeutic use. Another preferred invention
The FAP-specific antibody protein is characterized in that it is used in somatic gene therapy. For example, this can be as an antibody toxin-fusion protein (e.g., for other targets, see Chen et al., Nature, 385: 78-80 (1997)), or with antibodies of the invention and T-cells. As a fusion protein consisting of receptor or Fc-receptor (
Transmembrane region and intracellular region, for example, see Wels et al., Gene, 159: 73-80 (1995)). The use of somatic gene therapy can also be carried out by expression of the nucleic acids of the invention in shuttle vectors, gene probes or host cells.

In another preferred antibody protein of the present invention, said therapeutic substance is a radioisotope, a toxin or an immunotoxin, a toxoid, a fusion protein such as a genetically engineered fusion protein, an inflammatory substance and a chemotherapeutic agent and boron. It is characterized by being selected from the elements (boron-neutron capture reaction, BNC) that enable the neutron capture reaction such as. Another preferred antibody protein of the present invention is characterized in that the radioisotope is a β-emitting radioisotope. Another preferred antibody proteins of the invention, the radioisotopes, such as to be particularly suitable for coupling to the antibodies of the present invention has been demonstrated as a therapeutic substance, ^{18 6} rhenium, ¹⁸⁸ rhenium, ¹³¹ It is characterized by being selected from iodine and ⁹⁰ yttrium. The radioactive iodine labeling method of the antibody of the present invention is described in WO 93/058.
It is disclosed in No. 04.

Another preferred antibody protein of the present invention is characterized in that it is labeled. Another preferred antibody protein of the invention is characterized in that it is labeled with a detectable marker. Another preferred antibody protein of the invention is characterized in that the detectable marker is selected from enzymes, dyes, radioisotopes, digoxigenin, streptavidin and biotin. Another preferred antibody protein of the invention is characterized in that it is linked to an imageable substance. Another preferred antibody protein of the present invention is characterized in that the imageable substance is a radioisotope. Another preferred antibody protein of the present invention is characterized in that said radioisotope is a γ-ray emitting radioisotope. Another preferred antibody protein of the invention is characterized in that the radioisotope is ¹²⁵ iodine.

Another important aspect of the invention relates to a pharmaceutical preparation containing the antibody protein of the invention and one or more pharmaceutically acceptable carrier substances. The pharmaceutically acceptable carrier or adjuvant in the present invention is, for example, a physiologically acceptable compound that stabilizes or improves the absorption of the antibody protein of the present invention. Such physiologically acceptable compounds are, for example, sugars such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight compounds or other stabilizers or adjuvants ( Similarly, "Remingt
on's Pharmaceutical Sciences ", 18th edition, edited by Mack Publ., East on.). Those skilled in the art will know that the choice of pharmaceutically acceptable carrier will depend, for example, on the route of administration of the compound. The pharmaceutical composition may also include a vector of the invention for gene therapy and may additionally include a colloidal dispersion system or liposomes as an adjuvant for targeted administration of the pharmaceutical composition. Hosts or host cells containing the vectors of the invention can also be used in pharmaceutical compositions within the scope of the invention, eg, for gene therapy.

Another important aspect of the invention relates to the use of the pharmaceutical preparation of the invention for the treatment or imaging of tumors, wherein the tumor is associated with activated stromal fibroblasts. . This use of the present invention is particularly relevant when the tumor is classified into one of the following types of cancer or a form based on them, and as a result: colorectal cancer, non-small cell lung cancer, breast cancer, It is selected from head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain tumor.

Yet another important aspect of the invention relates to the use of the antibody protein of the invention for the preparation of a pharmaceutical preparation for the treatment of cancer. Yet another important aspect of the invention relates to the use of the antibody protein of the invention for imaging activated stromal fibroblasts. An additional aspect of the invention is that a probe, which may comprise activated fibroblasts, is contacted with an antibody protein of the invention under conditions suitable for the formation of a complex of said antibody protein and its antigen. And the formation of said complex and the resulting detection of the presence of activated stromal fibroblasts in the wound healing, inflammatory process or tumor, in wound healing, inflammatory process or tumor, This is a method for detecting activated stromal fibroblasts.

The method of the invention as described in the preceding paragraph, in particular that the tumor is selected from colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain tumors. It is characterized by The invention further provides that a suitable probe is contacted with an antibody protein of the invention under conditions suitable to form an antibody-antigen complex, the complex thus formed is detected and thus formed Including methods of detecting tumor stroma, such that the presence of the complex correlates with the presence of tumor stroma. The method of the invention described in the preceding paragraph is particularly characterized in that the antibody is labeled with a detectable marker. The following examples are intended to aid the understanding of the present invention and are not intended to limit the scope of the invention in any way.

Example 1 1. Cloning of human VH repertoire for guided selection A) Development of anti-FAP antibody with complete human V region Preparation: 1. Cloning of F19 VH 2. Human V-repertoire Preparation of the method: Reverse transcription, PCR amplification of human VL (λ, κ) repertoire from peripheral blood lymphocytes by an improved method of the method of Persson et al. (PNAS, 88: 2432 (1991)).・ Phage display vector (pSEX81, DKFZ, Heidelberg; Breitling et al., Ge
ne, 104: 147 (1991)), cloning of the VL repertoire. Repertoire size: VL 10 ⁷ clones ・ Human VH (I) derived from peripheral blood lymphocytes, thymus, spleen, bone marrow, tonsils, lymph nodes, fetal liver
Reverse transcription of the gG, IgD, IgM) repertoire, PCR amplification (Persson et al. (PNAS, 88: 2432
(1991)) improved method). -Improved method: use of IgD and various lymphoid tissues-Phage display vector (pSEX81, DKFZ, Heidelberg; Breitling et al., Ge
ne, 104: 147 (1991)) VH repertoire cloning Repertoire size: VH 3x10 ⁸ clones 3. Selection of human VL region functionally exchanging VL F19: by VH F19 as phage display selection and guide structure Guided selection strategies (improved McCafferty et al. (Nature, 348: 552 (1990)) and Jespers et al. (B
io / Technology, 12: 899 (1994)) => Isolation of human FAP-specific VL region (known as VL: III5, III10, III25, III43) 4. Functional replacement of VH F19 or FAP- Selection of human VH regions to confer specificity: Phage display selection with various VL as guide structures and guided selection strategies (improved McCafferty et al. Method (Nature, 348: 552 (1990)) and Jespers. Et al. Method (B
io / Technology, 12: 899 (1994)) => 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 selected VH and VL regions: (see figure)

Antigen binding properties-ELISA: Detection of antigen specificity of human FAP-Competition of antigen binding by cF19 (detection of scFv # 13) -Study of binding to FAP + cells: scFv # 13 (2 in minibody constructs) EC ₅₀ : 8-12 nM (see below) scFv # 50 (as bivalent in minibody constructs) EC ₅₀ : 32 nM FAP-specific immunohistological staining of tumor biopsy specimens (minibody constructs) (Detected in scFv # 13 inside)

1) PCR amplification of human VL- and VH repertoires : a) To prepare VH and VL repertoires, various V-gene families were cloned from cDNA by PCR using appropriate family-specific primers. Amplify individually (
See below). b) All forward / 3'-primers for VH- and VL-PCR amplifications are complementary to the gene sequences of the constant immunoglobulin domain (IgG, IgD, IgM, kappa, lambda). This allows efficient isotype-specific amplification of the V region with very few 3'-primers. In contrast, the method of Marks et al. (J. Mol. Biol., 222: 581 (1991).
)) Uses multiple different 3'-primers complementary to the J-section of the V region.

2) Preparation and cloning of human VH repertoire: Preparation and cloning of human VH repertoire consisting of a large number of highly diverse clones (3x10 ⁸ ) (see method below). a) To ensure high diversity, in addition to freshly isolated peripheral blood lymphocytes, commercially available cDNAs / RNAs from a variety of lymphoid tissues from a large number of donors were used for the VH repertoire. Used as starting material. By using bone marrow and fetal liver,
A naive V repertoire is obtained, which forms the precondition for autoantigen isolation.

Lymphoid tissue (number of donors): I) Commercially available 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 circumscribed in the cDNA in the laboratory. (See Example 1, A 1.2 for method) -Lymph nodes (25 donors) III) PBL from fresh'buffy coat '(10 donors) (see below for method)

In the prior art, only the following lymphoid tissues have been described so far as sources of the V repertoire. (Shows the combination of tissue and donor number):-PBL (15 donors), bone marrow (4 donors), tonsils (4 donors) (Vaughan et al. Nature Biote
chnology, 14: 309 (1996))-spleen (3 donors) and PBL (2 donors) (Sheets et al., PNAS, 95: 6157 (1998))-bone marrow (Williamson et al., PNAS, 90: 4141 (1993))- Lymph node (1 donor) (Clark et al., Clin. Exp. Immunol., 109: 166 (1997))

B) Furthermore, in addition to the IgM and IgG repertoires, the IgD repertoire was additionally amplified in order to obtain large repertoire diversity. For this, IgD-specific PCR primers were developed (see below). c) Purification of the PCR fragment of the human VH repertoire on agarose gel after treatment with restriction enzymes proved to be very important. Subsequent cloning of this repertoire into phage display vectors resulted in functional sc
It is possible to achieve a very high proportion of clones with Fv expression cassettes.
This is an essential prerequisite for obtaining a genetically stable phage display repertoire (see Example 1, A 1.4 for method). 3) Preparation of combination repertoire consisting of human VH repertoire and various human FAP- specificity VL regions:

Definition of a combinatorial repertoire: A combination of V repertoires with the corresponding complementary V-sequences by genetic engineering (complementarity to VL with respect to VH and vice versa). The V-array used for this combination is one V
-Can consist of sequences, multiple different sequences or V repertoire. a) Cloning strategy: In the phage display vector, the human VH repertoire was combined with a defined non-FAP-specific VL region (dummy-VL). This dummy-V
The L region uses the restriction cleavage site and can be very efficiently replaced by the FAP-specific VL region. This was compared to previously tested human VH repertoires and Conditions were created for effective combination with VL (see below for method).

B) In order to increase the establishment of the selection of a complete human scFv similar to F19, the human VH repertoire was sequenced with various human FAP-specific VL region sequences (VL: III10, III25, III5, III43).
). These human VL regions served as guide structures for human FAP-specific VH selection. FAP-specific human VL has itself been isolated from the human VL repertoire in a preceding "guided selection" step with F19 VH.

C) a combination repertoire of existing FAP-specific scFvs (eg hybridoma strains)
DNA contamination with a phagemid vector encoding F19-derived mouse scFv or human VL and chimeric anti-FAP scFv with F19 VH is a serious technical problem. To overcome this, the following strategy has been demonstrated to be necessary: after the "guided selection" step of human anti-FAP VL-sequence with mouse F19 VH as guide structure, this VH-sequence The human VL-sequence without is first sub-cloned in the plasmid (pUCBM21). This human VL region is then cleaved with restriction enzymes and combined with the human VH repertoire already present in the phage display vector. This prevents the FAP-specific V regions from being introduced into the combinatorial repertoire apart from the VL-sequence of the relevant guide structure (see below for method).

4) Phage display selection : Phage display selection of FAP-specific human V regions requires the development of selective washing methods to prevent accumulation of cross-reactive scFv (see below for method). .

B) Development of human anti-FAP antibody containing mouse HCDR3 F19 (guided to maintain HCDR3
Selection) : Preparation method: 1. Cloning of F19 VH 2. Preparation of human V-repertoire-Reverse transcription of human VL (λ, κ) repertoire derived from peripheral blood lymphocytes, PCR amplification (modified Persson et al. PNAS, 88: 2432 (1991)) Phage display vector (pSEX81, DKFZ, Heidelberg; Breitling et al., Ge
ne, 104: 147 (1991)), cloning of the VL repertoire. Repertoire size: VL 10 ⁷ clones ・ Reverse transcription of human VH repertoire derived from peripheral blood lymphocytes, PCR amplification (improved Pers
Son et al. (PNAS, 88: 2432 (1991))), PCR amplification of VH segment consisting of FR1 + CDR1 + FR2 + CDR2 + FR3.・ Phage display vector (pSEX81, DKFZ, Heidelberg; Breitling et al., G
ene, 104: 147 (1991)) cloning of repertoire consisting of VH segment (FR1 + CDR1 + FR2 + CDR2 + FR3), repertoire size: VH 4x10 ⁷ clones

3. Selection of human VL region functionally exchanging VL F19: (see Section A) 3) 4. Selection of human VH region containing HCDR3 derived from F19 and functionally exchanging VH F19 HCD with VL III43 or VL III5 and HCDR3 F19 + human FR4 as a guide structure
A guided selection strategy that maintains 3 (the development of our method was McCafferty et al. (Nature, 348: 552 (1990)) and Jespers et al. (Bio / Technology, 12: 899 (1994) and Rader's method, PNAS,
95: 8910 (1998) was improved. ) => Isolation of the following human FAP-specific scFv containing mouse HCDR3 F19: scFv # 34: VH # 34, IgG; VL III43 scFv # 18: VH # 18, IgG; VL III43

Structure (see figure) Antigen binding properties-ELISA: Detection of antigen specificity of human FAP-Competition of antigen binding by cF19 and mAb F19-Study on binding to FAP + cells: scFv # 34 and # 18 (monovalent) ) EC ₅₀ : about 6 nM ・ FAP-specific immunohistological staining of tumor biopsy specimens (as scFv # 34 minibody)

1.1 RNA isolation The mRNA source used was total RNs from a total of 10 buffy coat fresh lymphocytes.
A was isolated. Ficoll (LYMPHOPREP) 15 ml for isolation of lymphocytes from buffy coat
Were placed in a 50 ml Falcon Tube, left at ambient temperature and coated with 30 ml of buffy coat diluted 1: 4 in RPMI medium. After centrifuging at 700 g for 30 minutes, the interphase was removed, and 40 ml of RPMI medium was added, followed by centrifugation at 700 g for 5 minutes. The cell pellet was then washed again with RPMI medium and once with PBS. These cells were centrifuged after the final washing step, and 200 μl of RNA-Clean (registered trademark) solution (AGS, Heidelberg) was added to 10 ⁶ cells. Immediately after the addition of the denaturing solution, the cells were homogenized by repeatedly passing them up and down a coarse cannula (size 1) and then a thin cannula (size 18). The thin liquid lysate was mixed with 1/10 volume chloroform (pa), shaken vigorously and incubated on ice for 5 minutes. After centrifugation (15 minutes at 12000g
), The supernatant was roughly removed and mixed with an equal volume of isopropanol, incubated at 4 ° C. for 45 minutes and then centrifuged at 12000 g for 45 minutes. Carefully pour off the supernatant,
And the pellet was washed with ice-cold 70% ethanol. Then RNA-Quick-Prep (Pha
The RNA pellet was rewashed with the components of rmacia. To do this, the pellet was dissolved in a mixture of 113 μl extraction buffer, 263 μl LiCl solution and 375 μl Cs-trifluoroacetic acid, mixed thoroughly (Vortex) and placed in an Eppendorf centrifuge tube and centrifuged ( 12000g). Wash the RNA pellet again with 70% ethanol, air dry for 10 minutes,
The concentration was adjusted with H ₂ 0 to 1 μg / μl.

Alternatively, total RNA was isolated using an RNA isolation column manufactured by QIAGEN (Midi) according to the manufacturer's instructions. From the total RNA, Oligotex-Kit (Midi) manufactured by QIAGEN was prepared. The method used followed the manufacturer's instructions. Isolate isolated mRNA in 1/10 volume of 2.5M RNA
Mix with se-free potassium acetate (pH 5.2) and add 2.5 volumes of ethanol pa at -20 ° C.
At room temperature for 2 hours or overnight and precipitated. After centrifugation (45 min, 13000 g, 4 ° C), the mRNA was washed twice with ice-cold 70% ethanol (12000 g, 5 min centrifugation at 4 ° C) and briefly air-dried, then RNAse-free H It was dissolved in ₂ 0 _10~20μl. The total RNA standard dilution series was compared to estimate the concentration of mRNA. To do this, 1 μl of the sample to be measured was combined with 10 μl of ethidium bromide solution (1 μg / ml), hung on a film and compared with a standardized concentration using a UV lamp. This mRNA is cD
Used directly for NA synthesis and frozen for storage at -80 ° C.

1.2 cDNA synthesis of human VH region IgG, IgM and IgD specific VH-cDNAs were produced by Boehringer-Mannheim and Amersham.
Prepared with mRNA using a cDNA synthesis kit. The first cDNA strand is Ig-specific primer HuIgG1-4RT for IgG library, HuIgM-RT for IgM library.
Alternatively, it was synthesized by HuIgDelta for IgD library. Optionally oligo (dT) and oligo-hexa-nucleotides were used. For cDNA synthesis, follow the manufacturer's instructions.
Performed with 100 ng NA; mRNA was depleted immediately before use to detach secondary structure.
Was heated at 70 ° C. for 10 minutes. The cDNA was labeled with AMV-reverse transcriptase in 4 μl in 20 μl of the mixture.
Synthesis was carried out in a Thermocycler at 2 ° C for 60 minutes. The quality of this cDNA was checked by PCR amplification using the primer pair HuIgGFOR and HuVHB1 by way of example. For this purpose, a 10 ^n- fold dilution of cDNA was used as template and after 36 cycles,
The maximum dilution was determined such that the specific band of the PCR product was still detectable on the agarose gel.

1.3 PCR amplification of the human VH repertoire The cDNA of each human lymphoid organ was used individually as a template for PCR amplification of the VH region. Six individual PCR batches were prepared from each lymphoid organ and six VH-specific 5 '
Use one of the primers (HuVHB1 to HuVHB6) as the isotype-specific 3'primer H
Combined with one of uIgGFOR, HuIgMFOR or HuIgDFOR. Amplification, template cDNA (200pg)
It was carried out in 50 μl of reaction mixture containing 1 μl, 25 mM MgCl ₂ , 5 μl Goldstar reaction buffer, 200 μM each dNTP (Pharmacia) and 25 pmol of each primer. After 10 minutes at 95 ° C, 0.6 U of Goldstar-polymerase was added and the preparation was coated with PCR wax. Thirty-six amplification cycles were carried out, each with 15 seconds of denaturation at 94 ° C, 30 seconds of addition at 52-55 ° C, and 30 seconds of extension at 72 ° C. After the final amplification step,
An additional extension was performed at 72 ° C for 15 minutes.

In order to introduce the restriction cleavage sites NcoI and HindIII above the amplified VH region, a second PCR amplification was performed with primers extended by the restriction cleavage sites (HuIgGF
ORHINDIII, HuIgMFORHINDIII, HuIgDHINDIII as 3'primer, and HuVHB1
NCOI to HuVHB6NCOI as 5'primer). 1 μl of the reaction solution of the first PCR mixture was used as a template. The second PCR amplification was carried out for 15 cycles, in each case with denaturation at 94 ° C. for 15 seconds, binding at 65 ° C. for 30 seconds, and extension at 72 ° C. for 30 seconds. After the last amplification step was completed, another extension step was performed again at 72 ° C. for 5 minutes. Amplified materials based on the same isotype were combined and added to 1/10 volume of sodium acetate (pH 5.2) and 2.5 volumes of ethanol pa to reduce the volume for 2 hours at -20 ° C. It was precipitated over time and dissolved in TE buffer. To remove these primers, the precipitated PCR fragments were separated on a 1.5% agarose gel and the 400 bp fragment of the VH region was cut out. This fragment was isolated using the QIA ExII-Kit manufactured by QIAGEN (Hilden) according to the manufacturer's instructions. Elution was performed with preheated elution buffer
It was performed at 50 ° C. for 5 minutes.

1.4 Digestion of PCR-amplified VH region with restriction enzyme Gel purified VH region (3 isotypes) was first prepared in buffer B with HindIII 70U.
Digest for 2 hours in 100 μl of the mixture containing 20 μl of buffer H, NcoI 60U
Was added and the mixture was further incubated for 2 hours to make 200 μl. Any digested overhangs were removed using the QIA-Quick PCR-Kit and these fragments were eluted with preheated EB buffer. The eluent was again purified on a 1% agarose gel and eluted with the QIA ExII Kit in 25 μl EB buffer. This additional gel purification step was found to significantly increase the percentage of functional inserts after ligation into the vector. The digested PCR fragment was aliquoted and stored at -20 ° C.

1.5 Ligation of Human VH Repertoire to Phagemid Vector The phage display vector pSEX81, which already contains the human VL-sequence of the hapten-specific Ab (dummy VL-sequence), was cloned into a PCR-amplified VH repertoire. 20 μg of vector pSEX81 (VH & VLphox) was added to NcoI (Boehringe
r-Mannheim) 40 U and HindIII (Boehringer-Mannheim) 60 μl in a total volume of 125 μl was digested at 37 ° C. for 2 hours. 6-fold concentrated loading buffer (30% glycerol, 30%
After the addition of 30 μl of mM EDTA), the digestion mixture was heated at 65 ° C. for 10 minutes and gradually cooled to ambient temperature. Vector DNA was separated from the insert on a 1% agarose gel and isolated using the QIAGEN gel elution kit. Elution was performed twice with 50 μl of elution buffer (preheated to 50 ° C.) for 5 minutes each time. These elution fractions were combined and the digested vector DNA was treated with 1/10 volume of sodium acetate (pH 5.2) and 2.5 volumes of ethanol p.
Addition of .a. caused precipitation at -20 ° C over 2 hours. If necessary, the vector DNA thus cut can also be stored at -20 ° C. After centrifugation for 30 minutes (13000g, 4 ° C) and washing with 70% ethanol cooled to -20 ° C, the DNA is dried and 10mM TRIS (
It was dissolved in 50 μl of pH 7.9).

To estimate the exact amount of subsequent ligation, 2 μl of the vector DNA
l was compared to a standardized DNA fragment (High-Mass Ladder, Gibco Life Technologies). For direct comparison, the VH-PCR fragment prepared in Example 1, A 1.4 was compared to a low molecular weight standardized DNA fragment on the same gel (Low-Mass Ladder, Gibco.
Life Technologies). A ligation mixture such that the insert to vector ratio was equimolar proved ideal. 500 ng of vector DNA in a final volume of 40 μl
And 50 ng of insert DNA was incubated with 1 μl of ligase and 4 μl of ligation buffer. The ligation was performed overnight at 16 ° C. using T4 DNA-ligase manufactured by Boehringer Mannheim. The ligation reaction was stopped by the addition of 60 μl TE buffer. Chloroform /
Phenol mixture (1: 1) was removed by addition of 100 μl, mixed briefly (Vortex) and subsequently centrifuged at 13000 g. The aqueous phase was removed and again extracted with chloroform to completely remove the phenol. 90 μl of vector DNA solution was added to 3M sodium acetate (pH5.2
) 9 μl, ethanol 225 μl and glycogen 1 μl as carrier (Boehringer
(Mannheim) added to precipitate at -20 ° C for 2 hours. After centrifuging at 12000 g (4 ° C) and washing with ice-cold 70% ethanol, the DNA was air-dried and dissolved in 25 µl of water. Insufficient restriction digestion during vector preparation is caused by uncut or once-cut vector DNA.
And consequently in VH repertoire cloning, repertoire size is altered by religation of the incompletely cleaved vector.
The prepared vectors were lightly ligated, transformed in E. coli and the number of clones determined, with or without VH inserts, for early monitoring of the integrity of the restriction digests. Due to efficient restriction digestion of this vector, the number of clones in the vector sample without insert was <1% compared to the mixture where the vector with VH insert was used.

2. Human FAP-specificity by combining various human FAP-specific VL and human VH-repertoires.
Subcloning of the heterologous VL region To avoid DNA contamination with the FAP-specific DNA-sequences present in the construction of the scFv gene library, the selected human VL-chain was first expressed in the expression vector pUCBM21 (Boehb
ringer-Mannheim). To do this, FAP-specificity
The VL-chain was excised from the phagemid vector (pSEX 81) used for selection with MluI and NotI (Boehringer-Mannheim), respectively, and the corresponding cut pUCBM21.
Re-cloned into. After transformation in E. coli, clones were picked for each VL-chain, grown in LB _AT -medium and the vector DNA was cloned into Nucleobond K
It was isolated using it (Macherey & Nagel). Human VL chain has MluI (60U) and NotI (6
15 μg of pUC-plasmid in 150 μl of restriction mixture containing
Isolated on a% agarose gel. These human FAP-specific VLs were cloned into the corresponding truncated phage display vector containing the VH repertoire. The method used to clone the VH region has been previously described. This various VL
The combination bank with the region was kept separate. Aliquots of these combination banks were frozen and used for selection of fully human FAP-specific scFv.

3. Phage Display Selection Generation of Phage-Associated scFvs : Different human VL regions were included (see point 2) to avoid potential growth benefits for different VL-chains in the first round of panning. Different combination banks of phage-associated scFv were generated independently of each other. To do this, 10 ml of 2YT _AT medium in a chicane shake flask was inoculated with one aliquot of the VL / VH combination bank to OD 0.4 and agitated at 37 ° C until OD 0.8 (180 rpm). Cultured. After infection with 10 ¹² helper phages (New England Biolabs), incubation was carried out at 37 ° C for 15 minutes without stirring. Following incubation with agitation at 37 ° C., the bacteria are removed by centrifugation (5 min at 4000 g) and the pellet is
Resuspended in 50 ml glucose-free 2YT _AT medium containing kanamycin (65 μg / ml). Phage-associated scFv were made by vigorous agitation (200 rpm) at 30 ° C. overnight.
To collect these phages, the bacteria were removed by centrifugation (9000 g) and the supernatant was mixed with PEG and incubated on ice for 1 hour, which was precipitated. After subsequent centrifugation at 4 ° C for 9000g for 30 minutes, the precipitated phages were treated with 45 ml of cold PBS at 4 ° C.
Resuspended in and mixed with 5 ml of 5x PEG. After a further incubation on ice for several hours, the mixture was centrifuged again at 9000 g and the phage pellet was washed with PBS 5
Resuspended in ml. These phage were filtered through a 0.45 μm filter,
And combine 500 μl of each phage preparation, and 2 ml of 4% milk powder suspension (MPBS) in PBS
And mixed for 15 minutes. This phage suspension at 14000 g in a bench centrifuge.
It was clarified by centrifugation twice. The phage thus preadsorbed was to be used within the day.

Selection of antigen-specific scFv : For selection, immunotubes (Nunc-M) immobilized with 5 to 30 μg of CD8-FAP the day before were selected.
axi-Sorb-Immunotubes, 3.5 ml) were used. This immobilization was done in PBS overnight at 4 ° C, then the tubes were washed twice with PBS and the non-specific binding sites were labeled with ROTI-B.
Blocked at lock (Roth) for 1 hour. To test the specificity of phage display selection, immunotubes without immobilized antigen were used as controls. After washing 3 times with PBS, the phage-associated scFvs pre-adsorbed in MPBS were placed in antigen-coated test tubes or control test tubes and incubated on rollers for 2 hours.

For the preparation of inoculated bacteria, 20 ml of 2YTtet per mixture was inoculated with 1 aliquot of an overnight culture of XL-1-Blue to an OD of 0.0125 and at 37 ° C. with stirring (180 rpm). Cultured. After 3 hours of incubation, the inoculated bacteria had an OD of 0.8 and could therefore be used for infection by the phage eluted at this point.

One hour before infection, the phage suspension was emptied from the immunotube. The immunotubes were then washed to remove any non-specific and cross-reactive scFv.
In the first round of panning, the preparation was washed with 10x TPBS (0.1% Tween 20) and then 10x PBS. In addition to extending the wash steps to 15x TBBS (second round of panning) and 20x TPBS (third round of panning) in the second and third rounds of panning, stringency increased the concentration of Tween20 to 0.5%. Rising rather than increasing. To further increase stringency, a brief vortex was used during the TPBS wash to more thoroughly mix the wash in the last two rounds of panning.

Discard the final wash solution and add 1M TEA (triethylamine) 1m to this immunotube.
l was added. After 5 minutes of incubation in a rotating incubator,
Eluted phage were neutralized with 0.5 ml of 1 M TRIS (pH 7.4) and added directly to 20 ml of inoculated bacteria for infection. After a 15 minute incubation at 37 ° C without agitation, the bacteria were agitated for 45 minutes and removed by centrifugation at 3000g for 10 minutes. Bacteria were resuspended in 500 μl of 2YT medium,
And incubated on a large SOBGAT plate (15 cm) overnight at 37 ° C. For harvesting, cells were scraped from plates containing LBAT medium, mixed with glycerol to a final concentration of 25% and frozen in aliquots at -80 ° C and used for another round of inoculation of amplification.

The phage titer of each panning round was determined by titrating 0.01-10 μl of infected inoculated bacteria. To determine the specific concentration, the number of phage eluted from the CD8-FAP immobilized on the immunotubes was compared to the number of the corresponding control immunotubes without antibody in each selection round. The ratio of the amount of uncoated immunotubes to the amount of phage eluted from the antigen-coated immunotubes gave a concentration factor. The increase in concentration factor after successive rounds of amplification indicated the concentration of specifically bound phage.

Example 2 Expression of Human FAP-Specific scFv Derivatives Phage Display- Screen on a microquantitative scale for evaluation of selected scFvs.
ScFv-pIII-fusion protein expressed using the leaning method pSEX81, "screening",
ScFv selected for sampling and from phage display banks
It can be used for both analysis of clones.

Bacterial production of scFv-pIII-fusion proteins on a microquantitative scale 300 μl aliquots of 2YT _GAT were inoculated into colonies placed individually on LB _GAT plates and in 96-well microtiter plates (Beckman). At 37 ° C and 300 rpm
Incubate overnight with agitation. If the colonies to be analyzed were not stored frozen, they were first incubated in U-type 96-well tissue culture plates (Greiner). The following morning, 10 μl aliquots of these overnight cultures were transferred to 100 μl of fresh 2YT and again incubated at 37 ° C. in a humidified chamber in U-type 96-well tissue culture plates with agitation. Be
The residue of these cultures left in the ckman microtiter plate could be mixed with glycerol at 20% and frozen at -80 ° C. Growth of 100 μl cultures was checked using an ELISA Reader with filter wavelength 630 nm, if necessary. About 6
After -8 hours, the culture was centrifuged at 1200 rpm (5 minutes, RT) and the supernatant removed with a multichannel pipette. The pelleted bacteria were resuspended in 100 μl aliquots of 2YT _AT containing 50 μM IPTG (without glucose) and incubated overnight in a humidified chamber with agitation at 30 ° C. and 300 rpm. After overnight incubation, each of these cultures was mixed with 25 μl of 0.5% Tween and incubated for an additional 3-4 hours with agitation to achieve partial lysis. Finally the culture was centrifuged at 1200 rpm for 10 minutes and the supernatant was carefully removed. These were used directly for Western blot analysis or in pre-sphered ELISA.

Production of scFv-pIII-fusion proteins on ml scale Only a few clones were expressed and / or expressed
To investigate the functionality of the scFv-pIII-fusion protein, overnight pre-cultures of bacteria plus main cultures were performed in tubes or 50 ml PP-tubes with a volume of 3-10 ml with agitation at about 200 rpm. . Once the bacterial growth reached its exponential phase (OD600nm, about 0.7), the culture was centrifuged (2500 rpm, 5 min, RT) and fresh SB _AT or 2YT _AT containing 50 μM-IPTG for induction. Resuspend in volume. After overnight incubation at 25-30 ° C, either culture was mixed with Tween 20 (ad 0.1%) and incubated for a further 3 hours before removing the supernatant. However, the bacterial pellet was further opened up to increase the concentration of the fusion protein (see below).

These scFv-gIII-fusion proteins were used to reveal the integrity of the reading frame of the scFv-coding region (Western blot) and to select on immobilized FAP in an ELISA or in a cell analyzer for FAP + cells. ScF
The FAP specificity of v was investigated. ScFv-gIII-fusion proteins were detected using anti-gIII-specific monoclonal antibodies in combination with peroxidase- or alkaline phosphatase-coupled detection antibodies (Western blot and ELISA). FITC-labeled detection antibody was used when performing a cell binding test with scFv-gIII protein in a cell analyzer.

Prokaryotic Expression: Medium All data are for a final volume of 1 L and pH adjusted to 7.0. The following media additives were added to media that had been filter sterilized and optionally autoclaved. G: 100 mM glucose (preservative: 2M), A: ampicillin 100 μg / ml, T: tetracycline 12.5 μg / ml, K: kanamycin 50 μg / ml

Liquid medium for bacterial culture: BHI Brain Heart Infusion (DIFCO) 35g yeast extract 5g dYT peptone 17g yeast extract 10g NaCl 5g LB peptone 10g yeast extract 10g NaCl 5g SB peptone 30g yeast extract 10g MOPS 10g SOC peptone 20 g Yeast extract 5 g NaCl 10 mM KCl 2.5 mM After autoclaving, ad 10 mM each of sterilized MgCl ₂ and MgSO ₄ and further ad 20 mM sterilized glucose were added.

Agar plate BHI (amount 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 and glucose were all sterilized.

LB LB medium + 1.5% (w / v) agar agar SOB peptone 20 g yeast extract 5 g NaCl 0.5 g agar agar 15 g After autoclaving, sterilized MgCl ₂ was added to ad 10 mM. Other abbreviations: G
: Glucose, A: Ampicillin, T: Tetracycline, K: Kanamycin

Bacterial expression of scFv in E. coli: The pOPE vector and the derivatives obtained from it were transformed into cmyc- and HI in E. coli.
Involving S ₆ -Tag, was used for the preparation of simple soluble scFv derivative (Dubel et al., Gene, 128:
97-101 (1993)). Expression of scFv in E. coli and their purification were performed according to the method of Moosmayer et al. (Ther. Immunol., 2: 31-40 (1995)).

ScFv were produced in E. coli XL1-Blue in a volume of 3-100 ml. Incubations were performed in either LB or 2YT medium in test tubes or 50 ml PP-test tubes with agitation at about 200 rpm, at 180 rpm in Erlenmeyer flasks with chicane.
The medium was buffered with 1/10 volume MOPS (pH 7) and mixed with tetracycline (12.5 μg / ml) for strain XL1-Blue.

2YTG _AT or LB _GAT were inoculated with the colonies separated on LB _GAT plates to form precultures and incubated overnight at 37 ° C. with agitation. The next day, the main culture was inoculated thereon 1:50 and incubated at 37 ° C. For induction, the centrifuged bacteria (2500 rpm, 1000 xg, 10 min, RT) are taken up in an equal volume of medium (without glucose) containing 50 μM-IPTG for 2 to 3 hours at 22 to 25 ° C and 220 rpm. It was stirred. Bacterial pellets were collected after centrifugation at 1000 xg (10 min, RT) and disrupted as follows. The harvested pellet of induced E. coli culture was placed in 1/20 to 1/30 volume ice-cold PBS and completely resuspended and incubated on ice for about 30 minutes,
Mixing and flash freezing optionally in liquid nitrogen or a mixture of ethanol and dry ice. The frozen sample was then stored at -80 ° C. To break the sample, it was slowly thawed and sonicated until homogeneous and clear (25-30 cycles with ice water cooling). Samples were centrifuged for 20 minutes at 13000 rpm, the supernatant was carefully removed and the pellet discarded, in order to obtain a fraction of total solubilization of bacterial proteins. For relatively long-term storage, if desired, decant the supernatant with BSA (ad 1%).
Were mixed, flash frozen and stored at -80 ° C. A dramatic deterioration of the function of the recombinant protein was obtained when an over-rich (SB medium) or unbuffered culture medium was used in the preparation of scFv F19 in E. coli.

Expression of scFv Derivatives in Proteus mirabilis (LVI) : Monomeric scFv plus dimeric scFv (minibodies) were expressed in Proteus mirabilis. This expression and purification method was similar to what we previously published for soluble monovalent scFv (Rippmann et al., Applied and Environmental Microbiol).
ogy, 64: 4862-4869 (1998)).

Transformation of plasmid DNA in P. mirabilis LVI: Incubation of P. mirabilis LVI was performed at> 200 rpm in Erlenmeyer flasks (without chicane). Due to transformation of LVI bacteria, stationary growth phase (OD550, <6). To do this, 20 ml of BHI _K medium were inoculated 1:20 with a 4 ° C. culture and incubated overnight at 37 ° C. with stirring. Every 100 μl of overnight culture,
20 μl of the prepared plasmid and 150 μl of PEG (containing 0.4 M-sucrose) were mixed and left on ice for 10 minutes. This temperature shock is kept at 37 ° C with occasional gentle stirring.
In a water bath for 5 minutes. The transformed LVI-bacteria was transformed into BYS medium (1 ml BHI, 0.
5% yeast extract, 1% sucrose) in 1 ml and in a container with a small steep wall
Incubated at 37 ° C for 3 hours with vigorous stirring. Each transformant mixture 1
00 μl was plated on BHI _K plates. After 24-48 hours incubation (37 ° C), significantly larger colonies were picked using a sterile spatula and BH
Transferred to 20 ml of I _K medium. After overnight growth and microscopic examination for the presence of L-type bacteria, the culture was mixed with freezing medium and frozen at -80 ° C. Non-frozen transformed P. mi
The rabilis cultures survived for at least 4 weeks when stored at 4 ° C.

To induce the expression of transformed P. mirabilis, two consecutive overnight or 11-12 h precultures were inoculated (20 ml each) and the first from 4 ° C. cultures. One was incubated at 30 ° C. Depending on the density of the preculture achieved and the length of subsequent culture incubations, this was always inoculated in excess of 1:10 or 1:20. BHI _K induction cultures (containing 0.5 mM-IPTG) had a volume of 20-50 ml and were also inoculated and then incubated at 30 ° C. for at least 11 hours with agitation. Prior to collection of this bacterium, OD ₅₅₀ (≧ 4), pH (7.5-8.5) and optical appearance L-form
It was verified under a microscope. The expression culture was centrifuged (5000 rpm, 3800 xg, 4 ° C) and the pellet discarded. The supernatant can be used directly for ELISA or Western blot analysis or purified.

In the present study, these minibodies were purified by IMAC (immobilized metal affinity chromatography). For this, 1m manufactured by Pharmacia Biotech
l HiTrap column was used. Gel chromatography was performed as the second purification step.

Prior to applying the induction supernatant, it was thoroughly dialyzed against 5 L of cold PBS (pH 8) and then ultracentrifuged for at least 30 minutes (113000 xg, 4 ° C, rotor: Beckman 45 Ti). Prior to each purification, the column was charged with Zn ²⁺ ions: the solution used was pre-sterilized to prevent agglomeration of particles. The metal ion residue was removed with 5 ml of 50 mM EDTA. After rinsing with 10 ml H ₂ 0 _bid , it was charged with 10 ml 100 mM ZnSO ₄ . Again H ₂ 0 _bid
After rinsing with 20 ml, the column was equilibrated with 10 ml PBS (pH 8). Transfer the supernatant to a peristaltic pump (1
0.5 ml / min), followed by a washing step (10 ml PBS containing 5-20 mM imidazole). Elution was performed with 10 ml of PBS containing 300 mM imidazole in 1 ml fractions. The eluted fractions were stored on ice.

For gel chromatography, Superde manufactured by Pharmacia Biotech
A x200 column (10/30) was used. It was connected to an FPLC device manufactured by the same manufacturer. IM
The AC-purified sample was centrifuged for 5 minutes (13000 rpm, 4 ° C) and then injected. After equilibrating the pump system and column with the selected elution buffer (PBS, pH 8),
500 μl of IMAC-purified MB # 34 (corresponding to 0.75 to 1 mg) was injected into this system, pumped at a flow rate of 0.5 ml / min, detected by UV-detector and automatically 500 μl fractions were collected. Collected.

Structure of Recombinant Human Antibodies Prokaryotic and eukaryotic expression of human recombinant anti-FAP-antibodies was determined by monovalent scFv and divalent scF.
I went as v (so-called mini body). The structure of these minibodies created and the expression cassette used for this purpose was described by Hu et al. (Cancer Res. 56: 3055-61 (1
996)). In addition, the minibodies prepared by us had a c-myc domain at the C-terminus for immunological detection (by monoclonal antibody 9E10) and a HIS ₆ domain for chromatographic purification. These cmy
The c- and HIS ₆ -coding sequences correspond to those from pOPE 101 (S. Dubel, University of Heidelberg). Minibody structure: N-signal sequence-scFv (VH-linker-VL) -hinge-linker-CH3-cmyc-HIS ₆ -C

Prokaryotic Expression of Antibody Proteins of the Invention : The expression vector used and the monovalent scFv derivative E. coli (Moosmayer et al., Ther.
Immunol., 2: 31-40 (1995)) and Proteus mirabilis LVI (Rippmann et al., Applied.
and Environmental Microbiology, 64: 4862-4869 (1998)), methods for expression and purification are known from the prior art. Vector pACK02scKan and Rippman
The method from n et al. (1998) was also used to prepare and purify minibodies in Proteus mirabilis LVI.

Eukaryotic Expression of Antibody Proteins of the Invention : The minibodies described were also prepared in mammalian cells. The expression vectors used for the minibody expression cassette are: pAD-CMV-1 and pg1d1
05 derivative.

Transient expression in COS cells : For transfection of COS7 cells, the expression vector was first transformed into E. c.
Amplified in oli (XL1-Blue) and then purified. Vector DNA was adjusted to a concentration of 1 μg / μl under sterile conditions 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) containing DEMEM 10% FCS and incubated at 37 ° C in a CO ₂ heated cupboard. And incubated for 16 hours. On the day of transfection, 1 ml OptiMEM (Gibco), 35 μl Lipofectamine (Gibco Life) per Petri dish.
A suspension containing 10 μl of expression vector DNA was prepared. 45 at ambient temperature
After a minute of incubation, another 4 ml of OptMEM was added and the suspension was pipetted carefully onto the cells previously washed with PBS. The solution was gently tilted and distributed over the entire surface, and incubated at 37 ° C. for 5 hours. The petri dish was filled with 5 ml of preheated DEMEM 20% FCS and incubated at 37 ° C for 16 hours. The incubation medium is then carefully filtered by suction and OptiMEM 10 ml
I replaced it with. After a further 48 hours incubation at 37 ° C, the supernatant was removed for collection and the cells were removed by centrifugation at 700g. An additional centrifugation step at 12000 g pelleted the remaining cell fragments. The supernatant was ultracentrifuged for 30 minutes (30 minutes at 60000 xg), then added to an IMAC column (Amersham-Pharmacia), or a centrifugal concentrator with a 30 kDa separation threshold (Fugisept-Midi or Maxi Rohrchen, Intersept). Capacity at 1 /
Either by evaporation to 40 to 1/80. This centrifugation was performed at 6000 g according to the manufacturer's instructions and usually took 6 hours. The concentrated protein solution was mixed with 1% BSA, divided into 100 μl aliquots and snap frozen in N ₂ before storage at −80 ° C.

Stable expression in CHO cells : Stable transfectants of CHO DG44 were prepared for expression of FAP-specific minibodies. Transfection: Day 1: 2x10 ⁵ cells were seeded in one well of a 6-well plate. Day 2: Carefully aspirate the cell culture supernatant followed by 800 μl CHO-SFM II medium.
+ Add HT auxiliary (Gibco BRL). Preparation of transfection suspension: 6 μl lipofectamine + 200 μl CHO-SFM II with HT auxiliary + 3 μl expression vector (3 μg). This suspension was mixed and added carefully to the cells. Day 3: Medium change: CHO-SFM II without HT supplement.

The medium exchange was repeated periodically. Due to gene amplification and increased expression of foreign genes, methotrexate was added to the medium for a period of 10-14 days post transfection. The methotrexate concentration was titrated; this concentration was between 10 and 1000 nM. These minibodies were produced in T-culture flasks or bioreactors.

Determination of Apparent Cell Binding Affinity of Recombinant Anti-FAP Antibodies FAP + cells were incubated in parallel batches with various concentrations of monovalent or divalent scFv derivatives. Binding of these recombinant antibodies was performed using a cell analyzer (C
oulter) using a FITC-labeled detection antibody. The concentration of scFv derivative that reaches half maximal saturation of the binding signal was chosen as a measure of apparent affinity.

Example 3: Sequence An example sequence is shown here. Smaller mutations, eg substitutions of one or several amino acids or the nucleotides encoding them, are also included in the invention.

[0103] For example, as can be seen in the mini body vector, VH13 protein sequence. The first amino acid may be E (glutamic acid). QVQLVESGGTLVQPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWVSGISASGGYIDYADSVKGRVTISR
DNSKNMAY LQMSSLRAEDTALYYCAKGGNYQMLLDHWGQGTLVTVSSASTKGPKL

Nucleotide sequence corresponding to VH13 CAGGTACAGCTGGTGGAGTCTGGGGGAACCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC
TCTGGATT CACCTTTAGCAGCTATGCCATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTAT
TAGTGCTA GTGGTGGTTATATAGACTATGCCGATTCCGTGAAGGGCCGGGTCACCATCTCCAGAGACAATTCCAAGAACA
TGGCATAT CTACAAATGAGCAGCCTGAGAGCCGAGGACACGGCCCTTTATTACTGTGCGAAAGGAGGCAACTACCAGATG
CTATTGGA CCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAAGCTT

VH 46 protein sequence QVQLVQSGAEVKKDGASVKVSCKATGGTFSGHAISWVRQAPGQRLEWMGEISPMFGTPNYAQSFQGRVTITA
DESTSYME VSSLRSEDTATYYCARGANYRALLDYWGQGTLVTVSSASTKGPKL Nucleotide sequence corresponding to VH46 , such as occurs in minibodies .
This 6th nucleotide may be A instead of G-silent mutation, here with no effect on the amino acid sequence.

CAGGTACAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGGATGGGGCCTCAGTGAAGGTCTCCTGCAAGGCT
ACTGGAGG CACTTTCAGCGGTCACGCTATCAGTTGGGTGCGACAGGCCCCTGGGCAAAGACTTGAGTGGATGGGGGAGAT
CAGCCCTA TGTTTGGAACACCAAACTACGCACAGAGCTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCTACGAGTT
ACATGGAG GTGAGCAGCCTGAGATCTGAGGACACGGCCACTTATTACTGTGCGAGAGGTGCGAACTACCGGGCCCTCCTT
GATTACTG GGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAAGCTT

VH50 protein sequence as it occurs in a minibody . Again, apply the same as VH13: the first amino acid may be E. QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISR
DNAKNSLY LQMNSLRAEDTAVYYCARGSLCTDGSCPTIGPGPNWGQGTLVTVSSAPTKAPKL

For example, a nucleotide sequence corresponding to VH50 as occurs in a minibody . CAGGTACAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC
TCTGGATT CACCTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACAT
AAAGCAAG ATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGTTCACTCTGTACTGAT
GGTAGCTG CCCCACCATAGGGCCTGGGCCAAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCACCCACCAAGGC
TCCGAAGC TT

VLIII25 protein DIQMTQSPSSLSASTGDRVTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFT
LTITSLQS EDFATYYCQQYYIYPPTFGQGTRVEIKRTVAAPSVFAA

[0110] The nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAGGAGACAGAGTCACCATCACTTGTCGG corresponding to VLIII25
GCGAGTCA AGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAAGCCCCTCATCTCCTGATGTCTGGAGC
AACCACTT TACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACGTCCC
TGCAGTCT GAAGATTTTGCAACTTATTACTGTCAACAGTATTATATTTACCCTCCGACGTTCGGCCAAGGGACCAGGGTG
GAAATCAA ACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC

Protein VH34 with the first 8 amino acids of CH1 including: QVQLQQSGAEVKKPGSSVKVSCKASGGTFSTHTINWVRQAPGQGLEWMGGIAPMFGTANYAQKFQGRVTITA
DKSTSTAY MEMSSLRSDDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKL

[0112] VH34 to the corresponding nucleic acid sequence: CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCT
TCTGGAGG CACCTTCAGCACCCATACTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGAT
CGCCCCTA TGTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACAATTACCGCGGACAAATCCACGAGCA
CAGCCTAC ATGGAGATGAGCAGCCTGAGATCTGACGACACGGCTGTGTATTACTGTGCAAGAAGAAGAATCGCGTACGGT
TACGACGA GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAGCCTCCACCAAGGGGCCAAA
GCTT

[0113] with a part of the amino acid of CH1 VH18: QVQLVQSGAELKKPGSSMKVSCKASGDTFSTYSINWVRQAPGQGLEWMGVINPSGGSTSYAQKFQGRVTMTR
DTSTSTVY MELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKL

Nucleic acid sequence corresponding to VH18 : CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGTTGAAGAAGCCTGGGTCCTCGATGAAGGTCTCCTGCAAGGCT
TCTGGAGA CACCTTCAGCACCTATTCTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTAAT
CAACCCTA GTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCA
CAGTTTAC ATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTGCGAGAAGAAGAATCGCGTACGGT
TACGACGA GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAA
GCTT

VL chain III43 DIQMTQSPSSLSASTGDRVTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFT
LTISSLQA EDVAVYYCQQYYRTPFTFGQGTKLEIKRTVAAPSVFAA

[0116] III43 to the corresponding nucleic acid sequence: GACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAGGAGACAGAGTCACCATCACTTGTCGG
GCGAGTCA AGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAAGCCCCTCATCTCCTGATGTCTGGAGC
AACCACTT TACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCC
TGCAGGCT GAAGATGTGGCAGTTTATTACTGTCAGCAATATTATCGTACTCCGTTTACTTTTGGCCAGGGGACCAAGTTG
GAGATCAA ACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC

VH 13 YOL VL III25 protein sequence of total antibody protein, as occurs in minibodies : QVQLVESGGTLVQPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWVSGISASGGYIDYADSVKGRVTISR
DNSKNMAY LQMSSLRAEDTALYYCAKGGNYQMLLDHWGQGTLVTVSSASTKGPKLEEGEFSEARVDIQMTQSPSSLSAST
GDRVTITC RASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTITSLQSEDFATYYCQQYYIYP
PTFGQGTR VEIKRTVAAPSVFAA

Nucleotide sequence corresponding to VH 13 YOL VL III 25 : CAGGTACAGCTGGTGGAGTCTGGGGGAACCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC
TCTGGATT CACCTTTAGCAGCTATGCCATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTAT
TAGTGCTA GTGGTGGTTATATAGACTATGCCGATTCCGTGAAGGGCCGGGTCACCATCTCCAGAGACAATTCCAAGAACA
TGGCATAT CTACAAATGAGCAGCCTGAGAGCCGAGGACACGGCCCTTTATTACTGTGCGAAAGGAGGCAACTACCAGATG
CTATTGGA CCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAAGCTTGAAGAAGGTGA
ATTTTCAG AAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAGGAGACAGAGTCACCA
TCACTTGT CGGGCGAGTCAAGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAAGCCCCTCATCTCCTG
ATGTCTGG AGCAACCACTTTACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCAC
CATCACGT CCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATATTTACCCTCCGACGTTCGGCCAAG
GGACCAGG GTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC

For example, the VH46 YOL VL III2 5 protein sequence QVQLVQSGAEVKKDGASVKVSCKATGGTFSGHAISWVRQAPGQRLEWMGEISPMFGTPNYAQSFQGRVTITA of the total antibody protein as occurs in minibodies.
DESTSYME VSSLRSEDTATYYCARGANYRALLDYWGQGTLVTVSSASTKGPKLEEGEFSEARVDIQMTQSPSSLSASTGD
RVTITCRA SQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTITSLQSEDFATYYCQQYYIYPPT
FGQGTRVE IKRTVAAPSVFAA

Nucleotide sequence corresponding to VH46 YOL VL III25 CAGGTACAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGGATGGGGCCTCAGTGAAGGTCTCCTGCAAGGCT
ACTGGAGG CACTTTCAGCGGTCACGCTATCAGTTGGGTGCGACAGGCCCCTGGGCAAAGACTTGAGTGGATGGGGGAGAT
CAGCCCTA TGTTTGGAACACCAAACTACGCACAGAGCTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCTACGAGTT
ACATGGAG GTGAGCAGCCTGAGATCTGAGGACACGGCCACTTATTACTGTGCGAGAGGTGCGAACTACCGGGCCCTCCTT
GATTACTG GGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAAGCTTGAAGAAGGTGAATTTTC
AGAAGCAC GCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAGGAGACAGAGTCACCATCACTT
GTCGGGCG AGTCAAGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAAGCCCCTCATCTCCTGATGTCT
GGAGCAAC CACTTTACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAC
GTCCCTGC AGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATATTTACCCTCCGACGTTCGGCCAAGGGACCA
GGGTGGAA ATCAAACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC

For example, the VH 50 YOL VL III 25 protein sequence of the total antibody protein as it occurs in minibodies (see VH50 above for potential variants) QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISR
DNAKNSLY LQMNSLRAEDTAVYYCARGSLCTDGSCPTIGPGPNWGQGTLVTVSSAPTKAPKLEEGEFSEARVDIQMTQSP
SSLSASTG DRVTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTITSLQSEDFATYYC
QQYYIYPP TFGQGTRVEIKRTVAAPSVFAA

Nucleotide sequence corresponding to VH 50 YOL VL III 25 CAGGTACAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC
TCTGGATT CACCTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACAT
AAAGCAAG ATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGTTCACTCTGTACTGAT
GGTAGCTG CCCCACCATAGGGCCTGGGCCAAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCACCCACCAAGGC
TCCGAAGC TTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCAT
CTACAGGA GACAGAGTCACCATCACTTGTCGGGCGAGTCAAGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCC
GGGAAAGC CCCTCATCTCCTGATGTCTGGAGCAACCACTTTACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAG ATTTCACTCTCACCATCACGTCCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATATTT
ACCCTCCG ACGTTCGGCCAAGGGACCAGGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC

VH34YOLIII43 protein sequence of total antibody protein: QVQLQQSGAEVKKPGSSVKVSCKASGGTFSTHTINWVRQAPGQGLEWMGGIAPMFGTANYAQKFQGRVTITA
DKSTSTAY MEMSSLRSDDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKLEEGEFSEARVDIQMTQSPSS
LSASTGDR VTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ
YYRTPFTF GQGTKLEIKRTVAAPSVFAA

[0124] VH34YOLIII43 to the corresponding nucleotide sequence: CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCT
TCTGGAGG CACCTTCAGCACCCATACTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGAT
CGCCCCTA TGTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACAATTACCGCGGACAAATCCACGAGCA
CAGCCTAC ATGGAGATGAGCAGCCTGAGATCTGACGACACGGCTGTGTATTACTGTGCAAGAAGAAGAATCGCGTACGGT
TACGACGA GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAGCCTCCACCAAGGGGCCAAA
GCTTGAAG AAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAG
GAGACAGA GTCACCATCACTTGTCGGGCGAGTCAAGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAA
GCCCCTCA TCTCCTGATGTCTGGAGCAACCACTTTACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGAC
AGATTTCA CTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATCGTACTCCGT
TTACTTTT GGCCAGGGGACCAAGTTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC

VH18 YOL III43 protein sequence of total antibody protein: QVQLVQSGAELKKPGSSMKVSCKASGDTFSTYSINWVRQAPGQGLEWMGVINPSGGSTSYAQKFQGRVTMTR
DTSTSTVY MELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKLEEGEFSEARVDIQMTQSPSS
LSASTGDR VTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ
YYRTPFTF GQGTKLEIKRTVAAPSVFAA

Nucleotide sequence corresponding to VH18 YOL III43 : CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGTTGAAGAAGCCTGGGTCCTCGATGAAGGTCTCCTGCAAGGCT.
TCTGGAGA CACCTTCAGCACCTATTCTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTAAT
CAACCCTA GTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCA
CAGTTTAC ATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTGCGAGAAGAAGAATCGCGTACGGT
TACGACGA GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAA
GCTTGAAG AAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAG
GAGACAGA GTCACCATCACTTGTCGGGCGAGTCAAGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAA
GCCCCTCA TCTCCTGATGTCTGGAGCAACCACTTTACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGAC
AGATTTCA CTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATCGTACTCCGT
TTACTTTT GGCCAGGGGACCAAGTTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC

Example 4 Materials and Methods V-Gene Library Construction Total RNA was isolated from peripheral blood lymphocytes (buffy coat) of two naive donors. mRNA was prepared by mRNA isolation kit (Qiagen, Germany). cDNA is oligo
Synthesized by dT-priming.

For amplification of kappa and lambda light chains, Marks et al. (1991) reported in "human V _kappa and V _lambda Back.
Primary PCR was used, applying 5'-oligonucleotides described as "primers" and 3'-oligonucleotides described as constant kappa and constant lambda primers in Welschof et al. (1995). Thirty cycles of annealing at 56 ° C were selected. Secondary PCR (maximum 14 cycles) was used to add a _VL 5'side cloning site MluI and a 3'side site NotI to the first amplicon. Here, using 5'extended TAC AGG ATC C AC GCG T A, added 5'side cloning site MluI to Back primer, and 5'extended TGA CAA GCT T GC GGC CGC Added to _L primer. The resulting secondary PCR _VL amplificate was run on an agarose gel, and the QiaEx kit (Qiagen,
Purified by Germany. To clone the V _L repertoire, the phagemid vector pSEX 81 (essentially described in Breitling et al. (1991)) was cloned into Ml.
Overdigested with uI and NotI. The restricted DNA was purified using QiaQuick (Qiagen, Germany) and ligated overnight with _VL PCR product overdigested with the same endonuclease. Using ethanol-precipitation ligation, E.
E. coli XL1-Blue (Stratagene, California) was transformed. Transformants were plated on 2YT plates containing 100 mM-glucose, 100 μg / ml ampicillin, 12.5 μg / ml tetracycline and grown overnight at 30 ° C. The diversity of the cloned library was tested by BstNI-digestion of PCR-amplified V regions and analysis on polyacrylamide gels.

For amplification of the heavy chain, the N-terminus of V _H was described in Marks et al. (1991) “Human V _H B
The 5'-oligonucleotide already described as `` ack primer '', as well as the 3'-oligonucleotide below for the C-terminus of the FR3 region within the functionally rearranged gene segment family, primary PCR, Used: HU VG VH1 / 3/4: TCT CGC ACA GTA ATA CAC GGC HU VG VH2: TCT GTG TGC ACA GTA ATA TCT GGC HU VG VH5: TCT CGC ACA GTA ATA CAT GGC HU VG VH6: TCT TGC ACA GTA ATA CAC AGC

The annealing temperature was 55-58 ° C. and 30 cycles were performed. Utilizing secondary PCR (up to 14 cycles), and add the V _H 5 'side cloning site NcoI and 3' site SplI, the latter, promotes binding to FR3 gene segments with parent (patental) HCDR3 of FR1 To do. 2,
3 μl of primary PCR was used as template for the above primers and the following 5 ′ sequences were added: 5 ′ primer: GAA TAG G CC ATG G CG; 3 ′ primer: GGG GGC GGG CGT A CG CGA TTC TTC T. PCR of a new SplI site without changing its codesense
Was inserted into the parent HCDR3. This site allowed cloning of all V _H -gene segment families that were found to be functionally rearranged (Figure 9).

Phage preparation and selection To obtain phage-associated antibodies (phabs), exponentially growing E. co
li superinfection was performed according to Schier's paper (1996). After overnight growth at 30 ° C., the bacteria were pelleted and the phage were precipitated twice with 20% polyethylene glycol in 2.5M NaCl. Maxisor spinning 1-20 μg FAP for selection
b Coated in immunotube (Nunc) overnight at 4 ° C. After washing twice with PBS, the coated tube was treated with 3% fat-free dry milk in PBS or Roti-Block (Roth,
Blocked in Germany. Immediately before panning, the tube was washed twice with PBS. 10 ¹⁰ to 10 ¹² cfu was pre-adsorbed in 6% fat-free dry milk (working concentration) and used for tumbling selection for 2 hours at room temperature. In selection rounds 1 and 2, 10-15 washing steps with PBS followed by the same number of PBS-Tween 20 (0.1%)
Was performed. In the latter round, wash up to 25 times with PBS-Tw
een and up to the same number of pure PBS. To increase the stringency of the wash, the Tween concentration was raised to 0.5% and a significant swirl was introduced into the immunotube. Elution of phage was performed using 100 mM triethylamine or 0.1 M HCl (pH 2.2).
). Eluted phage were immediately neutralized with Tris and used to infect XL-1 Blue. After growing overnight at 30 ° C., the bacteria were scraped from agar plates and used for either rounds of further selection or glycerol freezing.

Screening for Specific Phage This screening of selected phabs was performed as described elsewhere (Mersmann.
Et al., 1998). Briefly, we induced expression of the scFv-pIII fusion protein without producing intact phage. These fusion proteins are
Purified FAP and irrelevant Ag were tested in an ELISA. Binders that revealed FAP-specificity were analyzed in a competitive ELISA, utilizing varying amounts of the chimeric bivalent construct of the parental F19 for synchronous competition. DNA-sequencing was performed using fluorescent dideoxynucleotides and ALFexpress (Amersham Pharmacia, Sweden) or by commercial service.

Affinity measurements To estimate the functional affinities of Ab constructs, half of their maximum saturating concentrations were calculated :
It was determined for fibrosarcoma cells overexpressing FAP (HT1080). 10 ⁵ FAP + cells or control cells were incubated with serially diluted Ab constructs for 90 minutes. Detection was performed with anti-c-myc Ab 9E10 followed by FITC-labeled goat anti-mouse specific serum (for scFv) or FITC-labeled goat anti-human specific serum (for Mb). Incubations and washes were performed on ice except for labeled Abs at room temperature. The bound Ab construct (contruct) was labeled with FACStar (Becton Dickinson
) Or EPICS flow cytometer (Coulter). Mean fluorescence was measured for 10 ⁴ cells in each dilution. The concentration of Ab derivative applied was determined by iterative approximation to the scFv or Mb standards used in SDS-PAGE and Western blot.

Cloning, Expression and Purification of Minibodies (Mb) The scFv cassettes of selected clones 18 and 34 were cut from the scFv expression vector pOPE101 (Dubel et al., 1992) by restriction with NcoI / NotI and published. Equivalently prepared Mb-vector pD1 which is a derivative of vector pACK02scKan- (Pack et al., 1993).
Inserted into. According to a paper by Rippmann et al. (1998), E. coli XL1-Blue was transformed as usual, followed by transformation of Proteus mirabilis cell wall and outer membrane deficient strain LVI, and expression was induced overnight. . After dialysis against PBS, ultracentrifuge Mb (
Purified by IMAC on HiTrap column (Pharmacia, Sweden) loaded with Zn ²⁺ (113,000 xg, 4 ° C, 30 minutes). Fractions were tested by SDS-PAGE followed by Coomassie staining.

Mb Stability Assay The thermostability of Mb # 34 in RPMI medium containing 5% FCS was examined by incubating purified freshly thawed Mb at 37 ° C. for up to 72 hours. After incubation, the solution was centrifuged (20,000 xg, 4 ° C, 10 minutes) and fixed with FAP.
Used in the ELISA above. Previous experiments were used to determine the appropriate dilution to reach a discriminating but not saturated ELISA signal for each Mb preparation.

Immunohistochemistry Fresh frozen sections of tumor tissue fixed with acetone were used. The tissue sections were incubated with recombinant antibody (10 μg / ml) at 4 ° C (16 hours), followed by anti-cm2.
Incubated with yc Mab 9E10 for 1 hour at room temperature. Then, biotinylated horse anti-mouse serum was applied. The Ag / Ab complex was detected by the avidin-biotin immunoperoxidase method. As a negative control, sections were treated only with biotinylated serum antibody, followed by color reaction. Harris hematoxylin was used for counterstaining the sections.

Results 1. Selection of human VL : A guided selection method based on scFv constructs was first performed on mice of FAP-specific antibody F19.
_VL substitutions were selected for subsequent F19 V _H humanization. Using the vector pSEX81, wherein the V _L repertoires from naïve human donors, combined with V _H F19, about 3 x
A combinatorial library of 10 ⁶ different clones was obtained.

This library contains immobilized FAP for the isolation of human V _L F19 analogs.
The phage display selected above. After 3 rounds of selection, screening of binders by ELISA yielded several FAP binding clones. To confirm the diversity of these isolated chimeric scFvs (mouse V _H / human V _L ), their phagemid DNA was analyzed by restriction enzymes and sequenced. Is to guide the V _H and matter another parent scFv F19 (itemized) consisting of human V _L, were identified various chimeric scFv (referred in short V _L NICHINAN here) (III5, III10, III25, III43). Table 1 shows
Amino acid sequence homology of selected light chains III5 and III43 compared to the exchanged VLF19. Kabat both germline genes with V _L listed belonging to human V _L subgroup κI accordance (https://immuno.bme.nwu.edu/), and a close homology, subgroup Vκ1 family (III5 : Ve; III43: Ve). Focusing on the amino acid sequence, clone III5 had as much as 64% identity in the FR position and 59% identity in the CDR position compared to the parental F19. III43 had 69% identity for the FR position and 59% identity for the CDR position compared to F19. In addition, III5 and III43 showed a high degree of mutation compared to their putative germline genes. III5 differs from the closest germline sequence by 14 amino acid positions and III43 shows 17 differences (ImMunoGeneTics database:
https://imgt.cnusc.fr:8104; and Cox et al., 1994).

Regarding the binding properties, the chimeric scFv is highly specific for FAP (Figure 7). Binding competition with cF19 in the ELISA revealed that the chimeric bivalent antibody containing the F19 variable fragment and the human constant domain exhibited a common epitope specificity of the selected chimeric scFv and parental Ab (FIG. 8). To assess the functional affinity of selected scFvs, a sandwich ELISA using the c-myc tag to detect the concentration that results in half-maximal saturation of binding (SC ₅₀ ).
(Table 2). Using this assay, the parental scFv F19 has a functional affinity of 20 nM.
, ScFv III5 had 45 nM and scFv III43 had 20 nM. This suggests that the guided selection of V _L carried out results in a chimeric scFv with retention of epitope specificity and with functional affinities in the nanomolar range.

2. Selection of humanized VH: To avoid the epitope shift during humanization of _VH by the guided selection previously reported (Watzka et al., 1998), the parental HCDR3 of mouse mAb F19 was Retained for subsequent selection. For this method, HCDR3 F19, human FR4 (Kabat subgroup I, II
(Recognized in III and III), and a new restriction site were constructed and introduced into HCDR3 without changing the amino acid sequence (FIG. 9).
In this vector, the V _L III5 and V _L III43 selected, respectively, inserted, and encodes the specificity of guided structure. In the next step, cDNA from a V _H segment library covering the rearranged sequences of all known V _H germline families spanning the FR1 to FR3 heavy chain segments was cloned into a phagemid. Built in. Resulting V _H segment library: (size 4 x 10 ⁷ cells clones), and with either V _L III5 or V _L III43, and were selected phage display on a fixed FAP.

Selection of scFvs in the phage-associated form is often associated with strong non-specific binding,
The result was a complex data analysis, and various selection strategies were applied (data not shown). Only high stringency wash conditions during the panning step resulted in the isolation of two highly antigen-specific FAP-binding clones after 5 consecutive selection rounds. In Table 3, the amino acid sequences of _VH clone # 18 and _VH clone # 34,
Compared to the parent V _H F19 and V _H OS4 (CDR grafted variants of F19). Confirming the comparison with the gene segment regions from FR1 to FR3, the selected clone # 18 was scFv in FR.
It showed 66% identity with the amino acid sequence of F19 and 50% identity in CDR1 and CDR2. For selected clone # 34, the FR identity is 67% and CDR1 + 2
About 55%. According to Kabat, both isolated V _H chains are _VL as complement.
III43 is used and belongs to human V _H subgroup I. For both V _H, the closest germline gene segments, indicates that the VH1 segment family belongs, this is all human V _H - represents about 12% of the gene segments (Guigou et al, 1990
Brezinschek et al., 1995). Compared to the VH1 family (# 18: DP-7, # 34: DP-88), VH
# 18 and # 34 showed differences of 10 and 9 amino acids, respectively.

FIG. 10 shows the strict FAP-specificity of humanized scFv # 18 and # 34 in ELISA. However, given the potential clinical use of selected human scFvs, their binding properties to native cell membrane expressed FAPs are of particular importance. By flow cytometry, we show that scFv # 18 and # 34 bind to the human fibrosarcoma cell line HT1080 expressing FAP in a manner similar to the parental scFv F19. (Fig. 11). Saturation studies yielded a functional cell binding affinity (SC ₅₀ ) of 6 nM for each of scFv # 18 and scFv # 34. Parental scF in parallel assay
The SC ₅₀ for vF19 and its CDR-grafted derivative scFv OS4 is 20 nM each.
And 4.6 nM, which is the selected scFv compared to the original Ab.
Showing a higher affinity for (Table 2). Furthermore, the binding competition of scFv # 18 and # 34 with cF19 was dose-dependent for cells overexpressing FAP measured in ELISA (data not shown) and by flow cytometry, which was selective. Was done
It is shown that the epitope specificity of cFvs is maintained (Fig. 12).

Given the potential clinical use, the scFv selected was L- from Proteus mirabilis.
Expressed as a minibody (Mb) using type strain LVI (Gumpert and Taubeneck, 1983
). This Ab construct is advantageous for tumor targeting due to its bivalent properties, high tumor uptake and rapid blood clearance, resulting in selective accumulation in tumors (H
u et al., 1996). As expected, Mb # 18 and Mb # 34 show high antigen specificity,
And retains F19 epitope specificity as shown in the antigen binding assay and competition with cF19 (data not shown). Furthermore, after affinity and size exclusion chromatography, the functional affinity of Mb # 34 on FAP-overexpressing cells was determined to be 2 nM (Fig. 13), which is a minibody of CDR-grafted scFv OS4. Exactly equal to the affinity evaluated for the species (Mb OS4). Furthermore, Mb # 34
It became highly stable at 37 ° C. in serum-containing medium; after 72 hours of incubation, there was only a 20% loss of binding activity (FIG. 14).

Immunohistochemical analysis with Mb # 34 on cryo-sections of various human tumors resulted in specific staining of tumor stroma in breast, lung and colon cancers. Furthermore, malignant cells of tendonoma and malignant fibrous histiocytoma can be specifically detected by Mb # 34
(Figure 15). Thus, for both epithelial tumors and tumors of mesenchymal origin, this human Mb exhibits an immunohistological staining pattern that is indistinguishable from F19 and MbOS4.

[0145]

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[Brief description of drawings]

Figure 1: HCDR3-guided selection to maintain.

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

FIG. 3: Binding of scFv # 13 (minibody construct) to FAP + cells (FACS analysis).

FIG. 4: Primers used for PCR amplification of human V repertoire.

FIG. 5: Primers for human VH-gene segment repertoire amplification for the “HCDR3-maintaining guided selection” method.

FIG. 6 Sequences of selected human FAP-specific VL regions

FIG. 7. Ag specificity of selected chimeric scFv . ELISA wells were coated with FAP or irrelevant Ag. TTX: Tetanus toxin; BSA: Bovine serum albumin; HSA: Human serum albumin; TF: Transferrin; CHY: Chymotrypsinogen; LYS: Lysozyme; Detection was performed with 9E10- and POD-labeled goat anti-mouse serum. Data were derived from triplicate values.

FIG. 8. Epitope specificity of selected chimeric scFv . Various concentrations of competitor were mixed with each scFv and added to FAP-coated ELISA wells. The competitors used were: cF19 (including chimeric F19, mouse variable region and human constant region); hu IgG (non-specific human IgG serum). The detection was performed as shown in FIG. Data were obtained from duplicate values.

9 sustained HCDR3 F19 by construction of a human VH- gene segment library. Schematic of the final construct of V _H , linker, V _L and phage protein gpIII. Due to the creation of new restriction sites, the V _H segment repertoire already exists
It can be ligated to HCDR3 F19 and then ligated to selected human V _L.

FIG. 10. Ag specificity of selected humanized scFv . Coating and detection of ELISA wells was performed as in Figure 1. PLA: Plastic

[11] coupled to the flow cytometer FAP bound to the humanized scFv and Mb of cell surface analyzed by cytometry. A) Binding of scFv # 18 and # 34 to FAP + cells. Cells were incubated with scFv 100-200 nM from E. coli extract. B) Mb #
Binding of 18 and # 34 to FAP + cells. Supernatant of P. mirabilis LVI containing 20 nM MB. C
) Control binding of scFv F19 (purified by IMAC) to FAP + cells. The binding area to FAP-control cells is shown in grey. scFv is 9E10 and FITC-labeled Fc-specific anti-mouse serum, Mb
Was detected by FITC-labeled Fc-specific anti-human serum. Each curve represents a pre-defined and measured cytometer value at 5,000 events.

FIG. 12. Epitope specificity of humanized scFv for cell-bound FAP . Various concentrations of competitor were mixed with each scFv and added to FAP + cells. cF19: chimeric F19 (chimeric F19, murine variable region and human constant region); hu IgG: non-specific human IgG serum. Detected by 9E10 and FITC-labeled Fc-specific anti-mouse serum. The data represent cytometer values at 10,000 events that were previously defined and measured.

FIG. 13. Evaluation of apparent affinity of Mb # 34 on FAP + cells . Mb # 34 is IMAC
And purified by size exclusion chromatography. Data are FITC-labeled F
Derived from cytometer values at 10,000 events defined and measured for each Ab concentration after detection with c-specific anti-human serum.

FIG. 14 Long-term stability of Mb # 34 at 37 ° C. In 10 times the capacity of RPMI (5% FCS)
After 0-42 h incubation, dilute IMAC purified Mb and anti-FAP ELIS
Used in A. Detection was performed with POD-labeled anti-human serum. The data are based on triplicate values.

FIG. 15. Immunohistological staining of biopsy specimen material from FAP + tumor sections with Mb # 34 . Cryo-sections of A) breast cancer, B) colon cancer, C) lung cancer, D) tendonoma, E) malignant fibrous histiocytoma were stained with Mb # 34. Bound Mb was subsequently sectioned with anti-c-myc mAb (9E10)
, Biotinylated horse anti-mouse serum and avidin-biotin immunoperoxidase complex. As a negative control, F) freeze-sections were treated only with detection antibody and avidin-biotin immunoperoxidase complex.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 19/00 C12N 7/00 C12N 1/15 C12P 21/02 C 5/10 G01N 33/574 A 7 / 00 D C12P 21/02 C12N 15/00 ZNAA G01N 33/574 5/00 B A61K 49/02 Z (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE, TR), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AU, BG, BR, CA, CN, CO, CZ, EE, HR, HU, ID, IL, IN, JP, KR, LT, LV, MX, NO, NZ, PL, RO, SG, SI, SK, A, US, UZ, VN, YU, ZA (72) Inventor Fiezen Meier Klaus, Federal Republic of Germany 75223 Tiefenbronn Saehausstraße 7 (72) Inventor, Mossmeier Dieter, Federal Republic of Germany 10179 Berlin Heinrich-Heine-Strasse 24 (72) Inventor Melsmann Michael Germany 38118 Braunschweig Amselstraße 9 (72) Inventor Schmid Alexei Germany 71229 Leonberg Wengertgasse 29 F term (reference) 4B024 AA01 AA11 AA12 BA41 BA44 BA61 CA04 CA07 DA02 DA06 DA11 EA03 EA04 GA01 GA11 HA08 4B064 AG26 AG27 CA02 CA05 CA10 CA19 CA20 CC24 CE07 DA05 DA14 4B065 AA26X AA72X AA90X AA93Y AA98X AB01 AB02 AB08 BA02 BA08 CA24 CA25 CA44 CA46 4C085 AA26 KA03 KA04 KB18 LL18 4H045 AA11 AA20 AA30 BA09 BA41 BA51 BA63 BA71 DA75 DA76 EA20 EA50 FA72 FA73 FA74

Claims (100)

[Claims] 1. A fully human or monoclonal antibody F19 (ATCC
Deposited number HB 8269), characterized in that it contains one or more mouse complementarity determining regions (CDR regions), specifically binds to fibroblast activating protein α (FAPα), human Or a humanized antibody protein. 2. The antibody protein according to claim 1, which comprises a heavy chain (V _H ) of class IgM. 3. The antibody protein according to claim 1, which comprises a heavy chain (V _H ) of class IgG. 4. A heavy chain of class IgD (V _H ), characterized in that it comprises:
The antibody protein according to any one of 3 above. 5. The antibody protein according to any one of claims 1 to 4, which comprises a λ-type light chain ( _VL ). 6. The antibody protein according to any one of claims 1 to 5, which comprises a kappa type light chain ( _VL ). 7. The antibody protein according to any one of claims 1 to 6, which is a Fab fragment. 8. The method according to claim 1, which is a F (ab ′) 2 fragment.
7. The antibody protein according to any one of 7. 9. The antibody protein according to claim 1, which is a single chain-Fv protein (scFv). 10. A bispecific antibody fragment, which is characterized in that:
The antibody protein according to any one of 1. 11. A minibody antibody fragment, which is characterized in that
10. The antibody protein according to any one of 10. 12. The antibody protein according to claim 1, which is a multimerized antibody fragment. 13. The antibody protein according to any one of claims 1 to 12, characterized in that it is completely human. 14. The antibody protein according to any one of claims 1 to 13, wherein the variable region of the heavy chain ( _VH ) comprises the amino acid sequence number: 1 (VH13). 15. The antibody protein according to any one of claims 1 to 14, wherein the variable region of the heavy chain ( _VH ) comprises the amino acid sequence number: 2 (VH46). 16. The antibody protein according to any one of claims 1 to 15, characterized in that the variable region of the heavy chain ( _VH ) comprises the amino acid sequence number: 3 (VH50). Variable region of 17. The light chain (V _L) comprises the amino acid SEQ ID NO: 4, characterized in that it comprises a (VLIII25), set forth in any one antibody protein of claim 1 to 16. 18. The variable region of the heavy chain (V _H ) has the nucleotide sequence SEQ ID NO: 5 (VH13).
Alternatively, the antibody protein according to any one of claims 1 to 17, which is encoded by a fragment or a degenerate variant thereof. 19. The variable region of the heavy chain (V _H ) has the nucleotide sequence SEQ ID NO: 6 (VH46).
Alternatively, the antibody protein according to any one of claims 1 to 18, which is encoded by a fragment or a degenerate variant thereof. 20. The variable region of the heavy chain (V _H ) has the nucleotide sequence SEQ ID NO: 7 (VH50).
Alternatively, the antibody protein according to any one of claims 1 to 19, which is encoded by a fragment or a degenerate variant thereof. 21. The variable region of the light chain ( _VL ) has the nucleotide sequence SEQ ID NO: 8 (VLIII2
5) or characterized by being encoded by a fragment or degenerate variant thereof,
The antibody protein according to any one of claims 1 to 20. 22. The variable region of the heavy chain (V _H ) contains the amino acid sequence number: 1 (VH13), and the variable region of the light chain (V _L ) contains the amino acid sequence number: 4 (VLIII25). An antibody protein characterized by: 23. The variable region coding sequence of the heavy chain (V _H ) comprises nucleotide SEQ ID NO: 5 (VH13), and the variable region coding sequence of the light chain (V _L ) comprises nucleotide SEQ ID NO: 8. (VLIII25) is contained, The antibody protein characterized by the above-mentioned. 24. The variable region of the heavy chain (V _H ) contains amino acid sequence number: 2 (VH46), and the variable region of the light chain (V _L ) contains amino acid sequence number: 4 (VLIII25). An antibody protein characterized by: 25. The coding sequence for the variable region of the heavy chain (V _H ) comprises nucleotide SEQ ID NO: 6 (VH46) and the coding sequence for the variable region of the light chain (V _L ) comprises nucleotide SEQ ID NO: 8. (VLIII25) is contained, The antibody protein characterized by the above-mentioned. 26. The variable region of the heavy chain (V _H ) comprises amino acid sequence number: 3 (VH50) and the variable region of the light chain (V _L ) comprises amino acid sequence number: 4 (VLIII25). An antibody protein characterized by: 27. The coding sequence for the variable region of the heavy chain (V _H ) comprises nucleotide SEQ ID NO: 7 (VH50) and the coding sequence for the variable region of the light chain (V _L ) comprises nucleotide SEQ ID NO: 8. (VLIII25) is contained, The antibody protein characterized by the above-mentioned. 28. The method according to claims 1 to 12 and 14, characterized in that it is humanized.
22. The antibody protein according to any one of 21 to 21. 29. The antibody protein according to any one of claims 1 to 12, 14 to 21 or 28, which comprises mouse CDR1 of the light chain (V _L ) of monoclonal antibody F19. 30. The antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 29, which comprises mouse CDR2 of the light chain (V _L ) of monoclonal antibody F19. 31. The antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 30, which comprises mouse CDR3 of the light chain (V _L ) of monoclonal antibody F19. 32. The antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 31, which comprises mouse CDR1 of the heavy chain (V _H ) of monoclonal antibody F19. 33. The antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 32, characterized in that it comprises mouse CDR2 of the heavy chain (V _H ) of monoclonal antibody F19. 34. The antibody protein according to any one of claims 1 to 12, 14 to 21 or 28 to 33, characterized in that it comprises mouse CDR3 of the heavy chain (V _H ) of monoclonal antibody F19. 35. The antibody protein according to any one of claims 1 to 34, wherein the variable region of the heavy chain ( _VH ) comprises the amino acid sequence number: 9 (VH34). 36. The antibody protein according to any one of claims 1-35, characterized in that the variable region of the heavy chain ( _VH ) comprises the amino acid sequence SEQ ID NO: 10 (VH18). 37. The variable region of the light chain ( _VL ) has the amino acid sequence SEQ ID NO: 11 (VLIII43).
The antibody protein according to any one of claims 1 to 36, which comprises: 38. The variable region of the heavy chain ( _VH ) has the nucleotide sequence SEQ ID NO: 12 (VH34).
Alternatively, the antibody protein according to any one of claims 1 to 37, characterized by being encoded by a fragment or a degenerate variant thereof. 39. The variable region of the heavy chain (V _H ) has the nucleotide sequence SEQ ID NO: 13 (VH18).
Alternatively, the antibody protein according to any one of claims 1 to 38, characterized by being encoded by a fragment or a degenerate variant thereof. 40. The variable region of the light chain ( _VL ) has the nucleotide sequence SEQ ID NO: 14 (VLIII
43) or an antibody protein according to any one of claims 1 to 39, characterized in that it is encoded by a fragment or a degenerate variant thereof. 41. The variable region of the heavy chain (V _H ) comprises amino acid sequence number: 9 (VH34), and the variable region of the light chain (V _L ) comprises amino acid sequence number: 11 (VLIII43). An antibody protein characterized by: 42. The coding sequence for the variable region of the heavy chain (V _H ) comprises nucleotide SEQ ID NO: 12 (VH34) and the coding sequence for the variable region of the light chain (V _L ) comprises nucleotide SEQ ID NO: 14 (VLIII43) is contained, The antibody protein characterized by the above-mentioned. 43. The variable region of the heavy chain (V _H ) comprises amino acid sequence number: 10 (VH18), and the variable region of the light chain (V _L ) comprises amino acid sequence number: 11 (VLIII43). An antibody protein characterized by: 44. The coding sequence for the variable region of the heavy chain (V _H ) comprises nucleotide SEQ ID NO: 13 (VH18), and the coding sequence for the variable region of the light chain (V _L ) comprises nucleotide sequence SEQ ID NO: 14 (VLIII43) is contained, The antibody protein characterized by the above-mentioned. 45. A nucleic acid, which encodes the antibody protein according to any one of claims 1 to 44. 46. Recombinant DNA comprising the nucleic acid according to claim 45.
vector. 47. The recombinant DNA vector according to claim 46, which is an expression vector. 48. A vector comprising the vector according to claim 46 or 47,
Host. 49. The host according to claim 48, which is a eukaryotic host cell. 50. The host according to claim 48 or 49, characterized in that it is a mammalian cell. 51. A BHK, CHO or COS cell, which is characterized in that
51. The host according to any one of 50 to 50. 52. The host according to claim 48, characterized in that it is a bacteriophage. 53. The host according to claim 48, characterized in that it is a prokaryotic host cell. 54. A method for preparing the antibody protein according to any one of claims 1 to 44, characterized in that it comprises the following steps: The host according to any one of claims 48 to 51, Culturing under conditions such that the antibody protein is expressed by the host cell, and isolating the antibody protein. 55. The method according to claim 54, characterized in that the host is a mammalian cell, preferably a CHO or COS cell. 56. The method according to claim 54 or 55, characterized in that the host cell is cotransfected with two plasmids carrying expression units for the light or heavy chains. 57. The antibody protein according to any one of claims 1 to 44, characterized in that the antibody protein is combined with a therapeutic substance. 58. The antibody protein according to claim 57, wherein the therapeutic substance is selected from radioisotopes, toxins, toxoids, boron, fusion proteins, inflammatory substances and chemotherapeutic agents. 59. The antibody protein according to claim 57 or 58, wherein the radioisotope is a β-ray-emitting radioisotope. 60. The antibody protein according to claim 59, characterized in that said radioisotope is selected from ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ¹³¹ iodine and ⁹⁰ yttrium. 61. The antibody protein according to any one of claims 1 to 44, which is labeled. 62. Labeled with a detectable marker,
62. The antibody protein of claim 61. 63. The antibody protein according to claim 61 or 62, characterized in that the detectable marker is selected from enzymes, dyes, radioisotopes, digoxigenin, streptavidin and biotin. 64. The antibody protein according to any one of claims 1 to 44, which is bound to an imageable substance. 65. The imageable substance is a radioisotope,
The antibody according to claim 64. 66. The antibody according to claim 64 or 65, wherein the radioactive isotope is a γ-ray emitting radioactive isotope. 67. The antibody protein of claim 66, wherein the radioisotope is ¹²⁵ iodine. 68. A pharmaceutical preparation, characterized in that it comprises an antibody protein according to any one of claims 1 to 44 and one or more pharmaceutically acceptable carrier substances. 69. A pharmaceutical preparation, characterized in that it comprises an antibody protein according to any one of claims 57 to 60 and one or more pharmaceutically acceptable carrier substances. 70. A pharmaceutical preparation, characterized in that it comprises an antibody protein according to any one of claims 64-67 and one or more pharmaceutically acceptable carrier substances. 71. The method according to any one of claims 68 to 70, characterized in that it is used in the treatment or imaging of a tumor, wherein the tumor is associated with activated stromal fibroblasts. Use of pharmaceutical preparations. 72. The use according to claim 71, wherein the tumor is selected from colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain tumor. 73. For preparing a pharmaceutical preparation for the treatment of cancer, 1.
44. Use of an antibody protein according to any one of -44. 74. For preparing a pharmaceutical preparation for the treatment of cancer, 57.
Use of the antibody protein according to any one of -60. 75. Use of an antibody protein according to any of claims 64-67 for imaging activated stromal fibroblasts. 76. A probe, which may comprise activated fibroblasts,
65. Contacting the antibody protein of any one of claims 1 to 44 or 61 to 64 under conditions suitable to form a complex from the antibody protein with its antigen, and formation of said complex and Detecting activated stroma fibroblasts in wound healing, inflammatory process or tumor, characterized by detecting the presence of activated stromal fibroblasts in wound healing, inflammatory process or tumor Method. 77. The tumor according to claim 76, wherein the tumor is selected from colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain tumor. The method described. 78. The method according to claim 76 to 77, characterized in that the antibody protein is the protein according to any one of claims 61 to 63. 79. A suitable probe is contacted with the antibody protein according to any one of claims 1 to 44 under conditions suitable for forming an antibody-antigen complex, and the complex thus formed is detected. And a method of detecting tumor stroma, characterized by correlating the presence of the complex thus formed with the presence of tumor stroma. 80. The method of claim 79, wherein the antibody is labeled with a detectable marker. 81. An antibody protein comprising the amino acid sequence of SEQ ID NO: 15 or a part thereof or a functional variant thereof. 82. An antibody protein comprising the amino acid sequence of SEQ ID NO: 16 or a part thereof or a functional variant thereof. 83. An antibody protein, which comprises the amino acid sequence of SEQ ID NO: 17 or a part thereof or a functional variant thereof. 84. An antibody protein comprising the amino acid sequence of SEQ ID NO: 18 or a part thereof or a functional variant thereof. 85. An antibody protein comprising the amino acid sequence of SEQ ID NO: 19 or a part thereof or a functional variant thereof. 86. An antibody protein characterized by being encoded by the nucleotide sequence of SEQ ID NO: 20 or a part thereof or a functional variant thereof. 87. An antibody protein characterized by being encoded by the nucleotide sequence of SEQ ID NO: 21 or a part thereof or a functional variant thereof. 88. An antibody protein characterized by being encoded by the nucleotide sequence of SEQ ID NO: 22 or a part thereof or a functional variant thereof. 89. An antibody protein characterized by being encoded by the nucleotide sequence of SEQ ID NO: 23 or a part thereof or a functional variant thereof. 90. An antibody protein characterized by being encoded by the nucleotide sequence of SEQ ID NO: 24 or a part thereof or a functional variant thereof. 91. An antibody protein, which corresponds to the amino acid sequence of SEQ ID NO: 15. 92. An antibody protein, which corresponds to the amino acid sequence of SEQ ID NO: 16. 93. An antibody protein, which corresponds to the amino acid sequence of SEQ ID NO: 17. 94. An antibody protein, which corresponds to the amino acid sequence of SEQ ID NO: 18. 95. An antibody protein, which corresponds to the amino acid sequence of SEQ ID NO: 19. 96. An antibody protein, characterized in that it is encoded by the nucleotide sequence of SEQ ID NO: 20. 97. An antibody protein, characterized in that it is encoded by the nucleotide sequence of SEQ ID NO: 21. 98. An antibody protein characterized by being encoded by the nucleotide sequence of SEQ ID NO: 22. 99. An antibody protein, characterized in that it is encoded by the nucleotide sequence of SEQ ID NO: 23. 100. An antibody protein characterized by being encoded by the nucleotide sequence of SEQ ID NO: 24.