EP1268550A2 - Human fap-alpha-specific antibodies - Google Patents

Human fap-alpha-specific antibodies

Info

Publication number
EP1268550A2
EP1268550A2 EP01929604A EP01929604A EP1268550A2 EP 1268550 A2 EP1268550 A2 EP 1268550A2 EP 01929604 A EP01929604 A EP 01929604A EP 01929604 A EP01929604 A EP 01929604A EP 1268550 A2 EP1268550 A2 EP 1268550A2
Authority
EP
European Patent Office
Prior art keywords
antibody protein
antibody
sequence
protein according
heavy chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01929604A
Other languages
German (de)
French (fr)
Inventor
John-Edward Park
Pilar Garin-Chesa
Klaus Pfizenmaier
Dieter Moosmayer
Michael Mersmann
Alexej Schmidt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim Pharma GmbH and Co KG
Original Assignee
Boehringer Ingelheim Pharma GmbH and Co KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10013286A external-priority patent/DE10013286A1/en
Priority claimed from GB0022216A external-priority patent/GB0022216D0/en
Application filed by Boehringer Ingelheim Pharma GmbH and Co KG filed Critical Boehringer Ingelheim Pharma GmbH and Co KG
Publication of EP1268550A2 publication Critical patent/EP1268550A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1075—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody the antibody being against an enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to antibody proteins which specifically bind fibroblast activating protein alpha (FAP ⁇ ).
  • the invention further relates to the use of said antibodies for diagnostic and therapeutic purposes as well as processes for preparing said antibodies.
  • FAP Since the FAP is selectively expressed in stroma of a number of epithelial cell carcinomas, irrespective of the site and histological type of the carcinoma, it was desirable to develop a treatment concept for the FAP ⁇ target molecule in order to allow imaging techniques, the diagnosis and treatment of epithelial cell cancer and many other syndromes.
  • a monoclonal murine antibody named F19 was developed which specifically binds to FAP. This antibody was described in US patents 5,059,523 and WO 93/05804 which are included in their entirety in this document by reference. A serious problem arises when non-human antibodies are used for in vivo applications in humans, i.e. they rapidly elicit an immune response to the foreign antigen.
  • Chimeric antibodies consist of fewer foreign protein sequences than non-human antibodies and therefore have a lesser xenoantigenic potential. Nevertheless, chimeric antibodies of this kind may trigger an immune reaction on account of the non-human V-regions in humans (LoBuglio A.F., Wheeler R.H., Trang J., Haynes A., Roger K., Harvey E.B., Sun L., Ghrayeb J. and Khazaeli M.B. (1989) Proc.Natl.Acad.Sci.86:4220).
  • WO99/57151 A2 describes FAP ⁇ -specific humanised antibodies of this kind in which the humanisation has been achieved by transferring all 6 CDR regions (3 from the light chain, 3 from the heavy) from the corresponding F19 murine antibody. These antibodies still contain parts of the murine framework region.
  • the problem of the present invention is to provide improved FAP ⁇ -specific antibodies which overcome the above disadvantages of the prior art.
  • the invention relates to new human or humanised antibody proteins which specifically bind to fibroblast activating protein alpha (FAP ⁇ ), and are either completely human or contain not more than one murine complementarity-determining region (CDR region) of the monoclonal antibody F19 (ATCC accession number HB 8269).
  • the antibodies according to the invention have the surprisingly advantageous property of having a significantly reduced xenoantigenic potential and consequently being better suited for use in humans than the antibodies known from the prior art (cf. also description of the process according to the invention, infra).
  • the antibodies according to the invention advantageously have no or very few parts of the murine amino acid sequence, namely at most one CDR region.
  • the framework regions ( FR) of the variable region of the antibodies according to the invention also correspond entirely to human amino acid sequences.
  • the antibodies according to the invention are nevertheless surprisingly highly specific for the target antigen FAP.
  • the term antibodies denotes one or more of the polypeptide(s) described in this specification. It also includes human antibody proteins selected from fragments, allelic variants, functional variants, variants based on the degenerative nucleic acid code, fusion proteins with an antibody protein according to the invention, chemical derivatives or a glycosylation variant of the antibody proteins according to the invention.
  • VH- and VL-PCR amplification are separately amplified with the respective family-specific primers by PCR from cDNA (see Example 1).
  • All Forward! 3 '-primers for VH- and VL-PCR amplification are complementary to the gene sequences of the constant immunoglobulin domains (IgG, IgD, IgM, K, ⁇ ). This enables efficient isotype-specific amplification of the V regions with very few 3 '-primers.
  • a plurality of different 3 '-primers complementary to the J-sections of the V regions are used (Marks et al., 1991; J. Mol. Biol. 222: 581).
  • the VH region known, for example, from the monoclonal, FAP-specific murine antibody F19 may be used and a suitable human FAP-specific VL region may be selected using a guided selection method and a phage display method. Then, using said human VL region as a guiding structure, for example, a human FAP-specific VH region may be selected.
  • a human FAP-specific VH region may be selected using a guided selection method and a phage display method.
  • a human FAP-specific VH region may be selected.
  • the technical problem of the DNA contamination of the combination repertoires with phagemid vectors which code for existing FAP-specific scFv, e.g. murine scFv from the hybridoma line F19 or the chimeric anti-FAP scFv with human VL and F19 VH
  • a guided selection process is described in the Examples.
  • combination repertoire is meant the combination, by genetic engineering, of a V repertoire with correspondingly complementary V-sequences. (Complementary with respect to VH to VL and vice versa).
  • the V-sequences used for the combination may consist of one V-sequence, a number of different V-sequences or a V repertoire.
  • an antibody protein according to the invention is characterised in that it comprises a heavy chain (V H ) of the immunoglobulin class IgM.
  • an antibody protein according to the invention is also characterised in that it contains a heavy chain (VH) of the class IgG.
  • V H heavy chain
  • VH heavy chain
  • Non-limiting examples of these are the completely human antibodies scFv #13 and scFv #46 (see Examples).
  • an antibody protein according to the invention is also characterised in that it comprises a heavy chain (VH) of the class IgD.
  • VH heavy chain
  • a non-limiting example of this is the human antibody according to the invention scFv #50 (see also Examples).
  • the VH-sequence originates from a human IgD and is identical to the germline sequence apart from one amino acid exchange. This advantageously reduces the probability of an allogenic immune response to this VH region in humans.
  • an antibody protein according to the invention is characterised in that it comprises a light chain (VL) of the lambda type ( ⁇ ).
  • an antibody protein according to the invention is characterised in that it comprises a light chain (VL) of the kappa type (K) (see Example, e.g. ILT25, III43).
  • VL light chain
  • K kappa type
  • an antibody protein according to the invention is an F(ab')2 fragment, which may be prepared by proteolytic cleaving with pepsin.
  • an FAP-specific antibody molecule according to the invention is such an Fv fragment. Since these Fv-fragments lack the covalent bonding of the two chains by the cysteines of the constant chains, the Fv fragments are often stabilised. It is advantageous to link the variable regions of the heavy and of the light chain by a short peptide fragment, e.g. of 10 to 30 amino acids, preferably 15 amino acids.
  • an antibody protein of this kind is known as a single-chain-Fv (scFv).
  • scFv single-chain-Fv
  • Examples of scFv- antibody proteins of this kind known from the prior art are described in Huston et al. (1988, PNAS 16: 5879-5883). Therefore, in another preferred embodiment an FAP-specific antibody protein according to the invention is a single-chain-Fv protein (scFv).
  • scFv single-chain-Fv protein
  • various strategies have been developed for preparing scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity.
  • an antibody protein according to the invention is an FAP-specific diabody antibody fragment.
  • diabody the skilled person means a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883).
  • Diabodies may additionally be stabilised by the incorporation of disulphide bridges. Examples of diabody-antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226).
  • minibody the skilled person means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerisation region which is connected to the scFv via a Hinge region (e.g.
  • an antibody protein according to the invention is an FAP-specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).
  • triabody By triabody the skilled person means a: trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein VH-VL are fused directly without a linker sequence lead to the formation of trimers.
  • miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv.
  • the multimerisation is carried out by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology 11 :, 1271- 1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28- 34).
  • an antibody protein according to the invention is an FAP-specific multimerised molecule based on the abovementioned antibody fragments and may be, for example, a triabody, a tetravalent miniantibody or a pentabody. Particularly preferably, an antibody protein according to the invention is totally human. Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (V H ) contains the amino acid sequence ID No. 1 (VH13). Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (V H ) contains the amino acid sequence LD No. 2 (VH46).
  • variable region of the heavy chain contains the amino acid sequence ID No. 3 (VH50).
  • variable region of the light chain (V ) contains the amino acid sequence ID No. 4 (VLIII25).
  • variable region of the heavy chain (V H ) is coded by the nucleotide sequence ED No. 5 (VH13) or by fragments or degenerate variants thereof.
  • variable region of the heavy chain (V H ) is coded by the nucleotide sequence ID No. 6 (VH46) or by fragments or degenerate variants thereof.
  • variable region of the heavy chain (V H ) is coded by the nucleotide sequence ID No. 7 (VH50) or by fragments or degenerate variants thereof.
  • variable region of the light chain (V L ) is coded by the nucleotide sequence ED No. 8 (VLLI125) or by fragments or degenerate variants thereof.
  • variable region of the heavy chain contains the amino acid sequence ED No. 1 (VH13) and the variable region of the light chain (V L ) contains the amino acid sequence ED No. 4 (VLEII25).
  • Another particularly preferred antibody protein according to the invention is characterised in that the coding sequence of the variable region of the heavy chain (V H ) contains the nucleotide sequence ED No. 5 (VH13) and the coding sequence of the variable region of the light chain (V L ) contains the nucleotide sequence ED No. 8 (VLIEL25).
  • Another particularly preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (V H ) contains the amino acid sequence ED No. 2 (VH46) and the va ⁇ able region of the light chain (V L ) contains the amino acid sequence ED No 4 (VLIII25)
  • Another particularly prefened antibody protein according to the invention is characte ⁇ sed in that the coding sequence of the va ⁇ able region of the heavy chain (V H ) contains the ! nucleotide sequence ID No 6 (VH46) and the coding sequence of the va ⁇ able region of the light chain (V L ) contains the nucleotide sequence ID No 8 (VLIII25)
  • Another particularly prefened antibody protein according to the invention is characte ⁇ sed in that the variable region of the heavy chain (V H ) contains the amino acid sequence ID No
  • VH50 va ⁇ able region of the light chain
  • VLEII25 amino acid sequence 7 ID No 4
  • Another particularly preferred antibody protein according to the invention is characte ⁇ sed in that the coding sequence of the va ⁇ able region of the heavy chain (V H ) contains the nucleotide sequence ED No 7 (VH50) and the coding sequence of the va ⁇ able region of the light chain (V L ) contains the nucleotide sequence ED No 8 (VLEII25)
  • an antibody protein according to the invention is humanised
  • the humanised antibody protein according to the invention has the advantage, over the FAP ⁇ - specific antibody proteins known from the p ⁇ or art, that it does not contain all six mu ⁇ ne CDR regions of F19, but only one mu ⁇ ne CDR region, as desc ⁇ bed in the following preferred embodiments
  • This antibody protein according to the invention advantageously
  • VH segments of all known human VH families are to be combined with HCDR3 F19 o in order to generate as complex a combination repertoire as possible.
  • this is preferably done e.g. by integrating a cutting site for the restriction enzyme P ⁇ 23ll in the HCDR3 F19 without altering the coding at the amino acid level.
  • a Phage display vector For combining the PCR- amplified human VH-gene segments a Phage display vector was developed which contains the following Ab-sequence sections: HCDR3 F19 with a P ⁇ 23U cutting site, a human VH FR4 region with high homology with the corresponding region from F19 as well as various selected human anti-FAP VL regions (see the diagram in Example 1). The primers for PCR amplification of the VH-gene segment repertoires are shown in Example 1. This preferred process has the following advantages over the prior art for combining VH- o gene segment repertoires with defined CDR3 regions:
  • Another prefened antibody protein according to the invention is characterised in that it contains murine CDR 1 of the light chain (V L ) of the monoclonal antibody F19.
  • Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 2 of the light chain (V L ) of the monoclonal antibody F19.
  • Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 3 of the light chain (V L ) of the monoclonal antibody F19.
  • Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 1 of the heavy chain (V H ) of the monoclonal antibody F19.
  • Another preferred antibody protein according to the invention is characterised in that it o contains murine CDR 2 of the heavy chain (V H ) of the monoclonal antibody F19.
  • Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 3 of the heavy chain (V H ) of the monoclonal antibody F19.
  • Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (V H ) contains the amino acid sequence ED No. 9
  • variable region of the heavy chain contains the amino acid sequence ED No. 10
  • Another preferred antibody protein according to the invention is characterised in that the o variable region of the light chain (V L ) contains the amino acid sequence ED No. 11 (VLEEE43).
  • Another preferred antibody protein according to the invention is characte ⁇ sed in that the va ⁇ able region of the heavy chain (V H ) is coded by the nucleotide sequence ED No 12 (VH34) or by fragments or degenerate va ⁇ ants thereof
  • V H va ⁇ able region of the heavy chain
  • Another preferred antibody protein according to the invention is characte ⁇ sed m that the va ⁇ able region of the light chain (V ) is coded by the nucleotide sequence ED No 14 (VLIII43) or by fragments or degenerate va ⁇ ants thereof
  • An especially preferred antibody protein according to the invention is characte ⁇ sed in that the va ⁇ able region of the heavy chain (V H ) contains the amino acid sequence ID No 9 (VH34) and the va ⁇ able region of the light chain (V ) contains the amino acid sequence ED No 1 1 (VLIII43)
  • Another particularly preferred antibody protein according to the invention is characte ⁇ sed in that the coding sequence of the va ⁇ able region of the heavy chain (V H ) contains the nucleotide sequence ED No 12 (VH34) and the coding sequence of the va ⁇ able region of the light chain (V L ) contains the nucleotide sequence ED No 14 (VLEII43)
  • Another particularly preferred antibody protein according to the invention is characte ⁇ sed in that the va ⁇ able region of the heavy chain (V H ) contains the amino acid sequence ED No 10 (VH18) and the va ⁇ able region of the light chain (V L ) contains the amino acid sequence ED No 11 (VLEII43)
  • Another particularly preferred antibody protein according to the invention is characte ⁇ sed in that the coding sequence of the va ⁇ able region of the heavy chain (V H ) contains the nucleotide sequence ED No 13 (VH18) and the coding sequence of the va ⁇ able region of the light chain (V L ) contains the nucleotide sequence ED No 14 (VLEEI43)
  • Another preferred embodiment of the invention composes a nucleic acid which codes for an antibody protein according to the invention
  • a nucleic acid according to the invention is characte ⁇ sed in that it contains 5' or 3' or 5' and 3' untranslated regions
  • the nucleic acid according to the invention may contain other untranslated regions upstream and/or downstream
  • the untranslated region may contain a regulatory element, such as e g a transc ⁇ ption initiation unit (promoter) or enhancer Said promoter may, for example, be a constitutive, inducible or development-controlled promoter Preferably, without ruling out other known promoter
  • Inducible promoters according to the invention comprise antibiotic-resistant promoters, heat-shock promoters, hormone-inducible maybeMammary tumour virus promoter" and the metallothioneine promoter.
  • a nucleic acid according to the invention is characterised in that it codes for a fragment of the antibody protein according to the invention. This refers to part of the polypeptide according to the invention.
  • a nucleic acid according to the invention is characterised in that it codes for a functional variant of the antibody protein according to the inventions.
  • a variant of an antibody protein according to the invention may differ from an antibody protein according to the invention by substitution, deletion or addition of one or more amino acids, preferably by 1 to 10 amino acids.
  • a nucleic acid according to the invention is characterised in that it codes for an allelic variant of the antibody protein according to the inventions.
  • a nucleic acid according to the invention is characterised in that it codes for variants of the antibody protein according to the inventions on the basis of the degenerative code of the nucleic acids.
  • a nucleic acid is characterised in that it is able to hybridise onto a nucleic acid according to the invention under stringent conditions. Stringent conditions are known to those skilled in the art and are found particularly in Sambrook et al. (1989). Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 15 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 16 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 17 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ID No. 18 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ID No. 19 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ID No. 20 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 21 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 22 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 23 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 24 or a part thereof or a functional variant thereof.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it corresponds to the amino acid sequence according to sequence ED No. 15.
  • Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it corresponds to the amino acid sequence according to sequence ID No. 16.
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, characte ⁇ sed in that it corresponds to the amino acid sequence according to sequence ID No. 17
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, characterised in that it corresponds to the amino acid sequence according to sequence ID No. 18
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, characte ⁇ sed in that it corresponds to the amino acid sequence according to sequence ED No 19
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, charactensed in that it is coded by the nucleotide sequence according to sequence ED No 20.
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, characte ⁇ sed in that it is coded by the nucleotide sequence according to sequence ED No 21.
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, characte ⁇ sed in that it is coded by the nucleotide sequence according to sequence ED No. 22.
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, characte ⁇ sed in that it is coded by the nucleotide sequence according to sequence ED No 23.
  • Another particularly preferred embodiment of the invention comp ⁇ ses an antibody protein, characte ⁇ sed in that it is coded by the nucleotide sequence according to sequence ED No 24.
  • Sequence ED No. refers to the No specified under ⁇ 400> in the Sequence Listing, so that e.g. the nucleotide sequence according to sequence ED No. 24 is listed as ⁇ 400> 24
  • a recombinant DNA vector which contains a nucleic acid according to the invention.
  • viral vectors such as e g Vaccinia, Semhki-Forest-Virus and Adenovirus.
  • Vectors for use in COS-cells have the SV40 o ⁇ gin of replication and make it possible to achieve high copy numbers of the plasmids.
  • Vectors for use in insect cells are, for example, E. colt transfer vectors and contain e g the DNA coding for polyhed ⁇ n as promoter
  • Another aspect of the present invention relates to a recombinant DNA vector according to the invention which is an expression vector
  • Yet another aspect of the present invention is a host which contains a vector according to the invention
  • Another host according to the invention is a eukaryotic host cell
  • the eukaryotic host cells according to the invention include fungi, such as e g Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomvces Tnchoderma insect cells (e g from Spodoptera frug ⁇ erda Sf-9, with a Baculovirus expression system), plant cells, e g from Nicotiana tabacum.
  • mammalian cells e g COS cells, BHK, CHO or myeloma cells
  • the antibody proteins according to the invention are particularly well folded and glycosylated Therefore a prefened host according to the invention is a mammalian cell
  • a host according to the invention is a BHK, CHO or COS cell
  • Another host according to the invention is a bacte ⁇ ophage
  • Another host according to the invention is a prokaryotic host cell
  • prokaryotic host cells are Escherichia coli, Bacillus subtihs, Streptomyces or Proteus mirab ⁇ is
  • the invention relates to a process for prepanng antibody protein according to the invention, which comp ⁇ ses the following steps a host according to the invention as desc ⁇ bed above is cultivated under conditions in which said antibody protein is expressed by said host cell and said antibody protein is isolated
  • the antibody proteins according to the invention may be expressed in any of the hosts descnbed above
  • antibody proteins according to the invention are prepared by a process according to the invention either intracellularly, e g in inclusion bodies, by secretion into bactena with no cells walls such as, for example, Proteus mirabihs or by pe ⁇ plasmatic secretion into Gram-negative bactena using suitable vectors for this purpose
  • the preparation of the antibody proteins according to the invention in prokaryotes is desc ⁇ bed by way of example Examples from the p ⁇ or art for the preparation of scFv-antibody proteins are descnbed in Rippmann et al. (1998, Appl. Environ. Microbiol., 1998, 64 4862-48
  • the antibody proteins according to the invention may also be prepared in a process according to the invention in fungi, such as e g Pichia pasto ⁇ s, Saccharomyces cerevisiae, Schizosaccharomvces. Tnchoderma with vectors which lead to intracellular expression or secretion
  • fungi such as e g Pichia pasto ⁇ s, Saccharomyces cerevisiae, Schizosaccharomvces. Tnchoderma with vectors which lead to intracellular expression or secretion
  • the process according to the invention for prepanng the antibody proteins may also be earned out with insect cells, e g as a transient or stabile expression system or Baculovirus expression system.
  • insect cells e g as a transient or stabile expression system or Baculovirus expression system.
  • Sf-9 insect cells for example, are infected with e.g. Autographa cahfornica nuclear polvhedrosis virus (AcNPV) or related viruses
  • AcNPV Autographa cahfornica nuclear polvhedrosis virus
  • the E coli transfer vectors descnbed above contain, for example, as promoters, the DNA which codes for polyhed ⁇ n, behind which the DNA coding for the antibodies according to the invention is cloned.
  • Insect cell expression systems are particularly suitable for the scFv fragments according to the invention and Fab or F(ab')2 fragments and antibody proteins or fragments thereof which are fused with effector molecules, but are also suitable for complete antibody molecules
  • mammalian expression systems give nse to very good glycosylation and folding conditions, e.g transient expression systems, e.g. in COS-cells or stable expression systems e.g. BHK, CHO, myeloma cells (cf. also Example 2).
  • Mammalian cells may also be used, for example, with viral expression systems e.g. Vaccinia, Semhki-Forest-Virus and Adenovirus.
  • Transgemc animals such as cows, goats and mice are also suitable for a process according to the invention.
  • Transgenic plants such as Nicotiana tabacum (tobacco) may also be used in a process according to the invention They are particularly suitable for the preparation of antibody fragments according to the invention.
  • genomic integration of the nucleic acid according to the invention which codes for an antibody protein according to the invention which is fused to a signal sequence secretion of the antibody protein into the interstitial space can be achieved.
  • the invention relates in particular to a process according to the invention wherein said host is a mammalian cell, preferably a CHO or COS cell.
  • the invention relates in particular to a process according to the invention wherein said host cell is co-transfected with two plasmids which carry the expression units for the light or the heavy chain.
  • the antibody proteins of the present invention are highly-specific agents for guiding therapeutic agents to the FAP antigen. Therefore another prefened antibody protein according to the invention is characterised in that said antibody protein is coupled to a therapeutic agent.
  • a therapeutic agent of this kind includes cytokines, such as for example interleukins (IL) such as EL-1, EL-2, EL-3, EL-4, IL-5, IL-6, EL-7, EL-8, EL-9, EL-10, IL-11, EL-12, IL-13, EL-14, IL-15, EL-16, EL-17, EL-18, interferon (EFN) alpha, EFN beta, EFN gamma, EFN omega or EFN tau, tumour necrosis factor (TNF) TNF alpha and TNF beta, TRAIL, an immunomodulatory or immunostimulant protein, or an apoptosis- or necrosis- inducing protein.
  • IL interleukins
  • the antibody-effector molecule conjugates according to the invention comprise antibody-cytokine fusion proteins, and also bispecific antibody derivatives and antibody-superantigen fusion proteins. These are preferably used for activating the body's own anti-tumoral defence mechanisms and are thus suitable for therapeutic use.
  • Another prefened FAP-specific antibody protein according to the invention is characterised in that it is used for somatic gene therapy. For example, this may be achieved by use as an antibody toxin-fusion protein (as described for example in Chen et al. 1997, Nature 385: 78-80 for other targets) or as a fusion protein consisting of an antibody according to the invention and a T-cell receptor or Fc-receptor (transmembrane and intracellular region, cfi.
  • somatic gene therapy may also be carried out by expression of the nucleic acid according to the invention in a shuttle vector, a gene probe or a host cell.
  • Another prefened antibody protein according to the invention is charactensed in that said therapeutic agent is selected from among the radioisotopes, toxins or lmmunotoxins, toxoids, fusion proteins, for example, genetically engineered fusion proteins, inflammatory agents and chemotherapeutic agents and elements which allow a neutron captunng reaction, such as e g boron (boron-neutron captunng reaction, BNC)
  • a neutron captunng reaction such as e g boron (boron-neutron captunng reaction, BNC)
  • Another prefened antibody protein according to the invention is characte ⁇ sed in that said radioisotope is a ⁇ -emitting radioisotope
  • Another prefened antibody protein according to the invention is characte ⁇ sed in that said radioisotope is selected from among 6 rhen ⁇ um, 188 rhen ⁇ um, 'iodine and 90 ytt ⁇ um which have proved particularly suitable for linking to the antibodies according to the invention as therapeutic agents
  • a process for radio-iodine labelling of the antibodies according to the invention is desc ⁇ bed in WO 93/05804
  • Another prefened antibody protein according to the invention is characte ⁇ sed in that it is labelled with a detectable marker
  • Another prefened antibody protein according to the invention is charactensed in that the detectable marker is selected from among the enzymes, dyes, radioisotopes, digoxygenme, streptavidine and biotin
  • Another prefened antibody protein according to the invention is characte ⁇ sed in that it is coupled to an imageable agent
  • Another prefened antibody protein according to the invention is characte ⁇ sed in that the imageable agent is a radioisotope
  • Another prefened antibody protein according to the invention is characte ⁇ sed in that said radioisotope is a ⁇ -emitting radioisotope
  • Another prefened antibody protein according to the invention is charactensed in that said radioisotope is 125 ⁇ od ⁇ ne
  • compositions which contains an antibody protein according to the invention and one or more pharmaceutically acceptable earner substances
  • Pharmaceutically acceptable earners or adjuvants in this invention may be physiologically acceptable compounds which stabilise or improve the absorption of antibody protein according to the invention, for example
  • physiologically acceptable compounds include , for example, carbohydrates such as glucose, sucrose or dextrane, antioxidants such as ascorbic acid or glutathione, chelating agents, lower-molecular compounds or other stabilisers or adjuvants (see also Remington's Pharmaceutical Sciences, 18th Edition, Mack PubL, Easton.).
  • the skilled person knows that the choice of a pharmaceutically acceptable carrier depends, for example, on the route of administration of the compound.
  • the said pharmaceutical composition may also contain a vector according to the invention for gene therapy and may additionally contain, as adjuvant, a colloidal dispersion system or liposomes for targeted administration of the pharmaceutical composition.
  • a host or a host cell which contains a vector according to the invention may also be used in a pharmaceutical composition within the scope of this invention, for example, for gene therapy.
  • Another important aspect of the present invention relates to the use of a pharmaceutical preparation according to the invention for treating or imaging tumours, wherein said tumours are associated with activated stromal fibroblasts.
  • tumours can be categorised as one of the following types of cancer or form the basis thereof and are therefore selected from among colorectal cancer, non-small-cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer.
  • Yet another important aspect of the present invention relates to the use of an antibody protein according to the invention for preparing a pharmaceutical preparation for treating cancer.
  • An additional aspect of the present invention is a process for detecting activated stromal fibroblasts in wound healing, inflammatory processes or in a tumour which is characterised in that a probe, which might possibly contain activated fibroblasts, is contacted with an antibody protein according to the invention under conditions which are suitable for forming a complex from said antibody protein with its antigen and the formation of said complex and hence the presence of activated stromal fibroblasts in wound healing, inflammatory processes or in a tumour is detected.
  • tumour is selected from among colorectal cancer, non-small-cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer.
  • the invention further includes a process for detecting tumour stroma wherein a suitable probe is contacted with an antibody protein according to the invention under suitable conditions for the formation of an antibody-antigen complex, the complex thus formed is detected and the presence of the complex thus formed is conelated with the presence of tumour stroma.
  • scFv #13 VH #13, IgG; VL ITI25 scFv #46: VH #46, IgG; VL III25 scFv #50: VH #50, IgD, VL III25
  • tumour biopsy material detected for scFv #13 in the minibody format
  • VH and VL-PCR amplification are separately amplified from cDNA with the appropriate family-specific primers by PCR (see below).
  • All Forward! 3 '-primers for VH- and VL-PCR amplification are complementary to the gene sequences of the constant immunoglobulin domains (IgG, IgD, IgM, K, ⁇ ). This allows efficient isotype-specific amplification of the V regions with very few 3 '-primers.
  • Marks et al., 1991 J. Mol. Biol. 222: 581) use a plurality of different 3'- primers complementary to the J-sections of the V regions.
  • Preparation and cloning of a human VH repertoire Preparation and cloning of a human VH repertoire consisting of a large number of clones (3 x 10 8 ) with high diversity (for method see below).
  • PBL 550 donors
  • spleen 5 donors
  • s -thymus gland 7 donors
  • lymphoid tissues have hitherto been described as sources of V repertoires. (The combinations of the tissues and the numbers of donors are shown):
  • the IgD repertoire was additionally amplified, as well as the IgM and IgG repertoires, to obtain a great repertoire diversity.
  • an IgD-specific PCR primer was developed (see below).
  • V- sequences used for the combination may consist of one V-sequence, a plurality of different sequences or a V repertoire.
  • Cloning strategy En a Phage display vector the human VH repertoire was combined with a defined, non- FAP-specific VL region (dummy- L). This dummy-VL region could very efficiently be replaced by FAP-specific VL regions using restriction cutting sites. This created the conditions for effectively combining a previously tested human VH repertoire with specific human VL, in order to guarantee a diverse combination repertoire which contains a very high proportion (>95 %) of functional clones (in relation to the integrity of the scFv reading frame) (for method see below).
  • Phage display selection of the FAP-specific human V regions required the development of selective washing methods to prevent the accumulation of cross-reactive scFv (for method see below).
  • HCDR3 F19 scFv #34- VH #34, IgG; VL EII43 scFv #18: VH #18, IgG; VL EEI43
  • the mRNA source used was isolated total RNA from fresh lymphocytes from a total of 10 Buffy coats
  • the total RNA was isolated using an RNA isolation column made by QIAGEN (Midi) according to the manufacturer's instructions
  • the mRNA was prepared from total RNA using the Ohgotex-Kit (Midi) made by QIAGEN The method used was in accordance with the manufacturer's instructions
  • the isolated mRNA was mixed with 1/10 volume of 2 5 M RNAse-free K-acetate, pH 5 2, and precipitated by the addition of 2 5 volumes of ethanol p a at -20°C for 2 hours or overnight After cent ⁇ fuging (45 min, 13000g , 4°C) the mRNA was washed twice with ice-cold 70 % ethanol (centnfugation for 5 mm at 12000g, 4°C) and after b ⁇ ef air-drying dissolved in 10-20 ⁇ l of RNAse-free H 2 0.
  • the mRNA was compared with a total RNA standard dilution se ⁇ es. In order to do this, l ⁇ l of the sample to be measured was combined with 10 ⁇ l of ethidium bromide solution (l ⁇ g ml), dnpped onto a film and compared with the standardised concentration using a UV lamp The mRNA was used directly for the cDNA synthesis or frozen for storage at -80°C.
  • IgG, IgM and IgD specific VH-cDNA was prepared with mRNA using the cDNA Synthesis Kit produced by Boeh ⁇ nger-Mannheim and Amersham.
  • the first cDNA strand was synthesised with the Ig-specific pnmers HuIgGl-4 RT for the IgG library, HulgM-RT for the IgM library or HulgDelta for the IgD library
  • ohgo(dT) and ohgo-hexa- nucleotides were used.
  • the cDNA synthesis was earned out with 100 ng of mRNA according to the manufacturer's instructions; to detach the secondary structures the mRNA had to be heated to 70 °C for 10 min immediately before use.
  • the cDNA was synthesised in a 20 ⁇ l mixture with AMV-Reverse transcnptase in a Thermocycler for 60 min at 42°C.
  • the quality of the cDNA was checked by PCR amplification using the pair of pnmers HulgGFOR and HuVHBl, by way of example.
  • 10" dilutions of the cDNA were used as template and the maximum dilution at which a specific band of the PCR product was still detectable in agarose gel after 36 cycles was determined.
  • the cDNA of each human lymphatic organ was used separately as a Template for the PCR amplification of the VH regions.
  • Six separate PCR batches were set up from each lymphatic organ, one of the six VH-specific 5 'pnmers (HuVHBl to HuVHB6) being combined with one of the isotype-specific 3 'pnmers HulgGFOR, HulgMFOR or HulgDFOR.
  • the amplification was earned out in a 50 ⁇ l reaction mixture with 1 ⁇ l of Template cDNA (200pg), 25mM MgCl 2 , 5 ⁇ l of Goldstar reaction buffer, 200 ⁇ M of each dNTP (Pharmacia) and 25pmol of each pnmer.
  • a second PCR amplification was carried out with the primers extended by the restriction cutting sites (HuIgGFORHENDIII, HuIgMFORHENDIII, HuIgDHINDI I as the 3' primers and HuVHBlNCOI to HuVHB ⁇ NCOI as the 5 ' primers), l ⁇ l of the reaction solution of the first PCR mixtures were used as the template.
  • the second PCR amplification was carried out over 15 cycles with in each case 15 s denaturing at 94°C, 30 s addition at 65°C and 30 s elongation at 72 °C.
  • the final amplification step is followed once again by an additional elongation step for 5 min at 72 °C.
  • the amplified materials which were based on the same isotype were combined and, in order to reduce the volume, precipitated by the addition of 1/10 volume of Na-acetate, pH 5.2, and 2.5 volumes of ethanol p. a. for 2 hours at -20°C and dissolved in TE buffer.
  • En order to eliminate the primers the precipitated PCR fragments were separated on a 1.5% agarose gel and the 400 Bp fragment of the VH region was excised. The fragment was isolated according to the manufacturer's instructions using the QIA ExEI-Kit made by QIAGEN (Hilden). Elution was performed with preheated elution buffer for 5 min at 50 °C.
  • the gel-purified VH regions (of the three isotypes) were first digested in a lOO ⁇ l mixture with 70 U of Hind EH for 2 hours in buffer B and then incubated for a further 2 hours by the addition of 20 ⁇ l of buffer H, 60 U of Ncol and topping up to 200 ⁇ l. Any digested overhangs were eliminated using the QIA-Quick PCR-Kit and the fragments were eluted with preheated EB buffer. The eluate was purified once more over a 1 % agarose gel and eluted with the QIA ExII Kit in 25 ⁇ l of EB buffer. Et was found that this additional gel purification step significantly increases the percentage of functional inserts after ligation into the vector.
  • the digested PCR fragments were divided into aliquots and stored at -20°C.
  • a ligation mixture with an equimolar insert to vector ratio proved to be ideal In 40 ⁇ l of final volume, 500 ng of vector DNA and 50ng Insert DNA) were incubated with l ⁇ l of hgase and 4 ⁇ l of ligation buffer The ligation was earned out overnight at 16°C using the T4 DNA-hgase made by Boehnnger Mannheim The ligation reaction was stopped by the addition of 60 ⁇ l of TE buffer The proteins were eliminated by the addition of 100 ⁇ l of chloroform/phenol mixture (1 1), b ⁇ ef mixing (Vortex) and subsequent centnfuging at 13000 g The aqueous phase was removed and extracted again with chloroform to eliminate the phenol completely 90 ⁇ l of vector DNA solution were precipitated by the addition of 9 ⁇ l of 3 M Na acetate (pH 5 2), 225 ⁇ l of ethanol p a.
  • the human VL-chains selected were first cloned in the expression vector pUCBM21 (Boehnnger-Mannheim) To do this, the FAP-specific VL-chains were each excised from the phagemid vector (pSEX 81 ) used for the selection with Mlul and Notl (Boehnnger-Mannheim ) and recloned into the conespondingly cut pUCBM21.
  • VL-chain After transformation in E coli a clone was picked for each VL-chain, amplified in LB A ⁇ -medium and the vector DNA was isolated using the Nucleobond Kit (Macherey & Nagel). The human VL chains were excised from 15 ⁇ g of pUC-plasmid in 150 ⁇ l of rest ⁇ ction mixture with Mlul (60U) and Notl (60U) and isolated in a 1% agarose gel.
  • the phage-associated scFv of the vanous combination banks which contain the different human VL regions were produced independently of one another.
  • 10ml of 2YT A ⁇ medium in a chicane shaking flask were inoculated with one aliquot of the VL/VH combination banks with an OD of 0.4 and cultivated, with agitation (180 rpm) at 37°C until an OD of 0.8 was reached.
  • 10 12 helper phages New England Biolabs
  • the bacteria were removed by centrifuging (4000g for 5 min) and the pellet was resuspended in 50 ml of glucose- free 2YT AT medium containing kanamycin (65 ⁇ g/ml).
  • the phage-associated scFv was produced overnight with vigorous agitation (200rpm) at 30°C.
  • the bacteria were removed by centrifuging (9000 g) and the supernatant was mixed with PEG and incubated on ice for one hour in order to precipitate it.
  • the phages precipitated were resuspended in 45 ml of 4°C cold PBS and mixed with 5 ml of 5x PEG. After a further hour's incubation on ice, the mixture was again centrifuged at 9000 g and the phage pellet was resuspended in 5 ml PBS. The phages were filtered through a 0.45 ⁇ m filter and 500 ⁇ l of each phage preparation were combined and mixed with 2 ml of 4% milk powder suspension in PBS (MPBS) for 15 min. The phage suspension was clarified by centrifuging twice with 14000 g in a bench centrifuge. The phages thus preadsorbed had to be used the same day.
  • Immunotubes (N nc-Maxi-So ⁇ b-Immunotubes 3.5 ml ) immobilised with 5-30 ⁇ g CD8- FAP the day before were used for the selection. The immobilisation was carried out at 4°C overnight in PBS, then the tubes were washed twice with PBS and the unspecific binding sites were blocked for one hour with ROTI-Block (Roth). In order to investigate the specificity of the phage display selection, an immunotube without immobilised antigen was used for control purposes. After washing three times with PBS, the phage-associated scFv preadsorbed in MPBS were placed in the antigen-coated test tubes or the control test tubes and incubated on a roller for 2 hours.
  • Plating bacteria 20 ml of 2YTtet per mixture were inoculated with one aliquot of an XL- 1 -Blue overnight culture with an OD of 0.0125 and cultivated at 37°C with agitation (180 rpm). After three hours' incubation the Plating bacteria reached an OD of 0.8 and were then available for this time for infection with the eluted phages. One hour before infection, the phage suspensions were emptied out of the Immunotubes. Then the Immunotubes were washed to eliminate any unspecific and cross-reactive scFv.
  • the first round of panning the preparations were washed lOx with TPBS (0.1% Tween 20) and then lOx with PBS.
  • the stringency was increased in the second and third rounds of panning by extending the washing steps to 15x TBBS (2 nd round of panning) and 20x TPBS (3rd round of panning) as well as by increasing the concentration of Tween20 to 0.5%).
  • a vortex was briefly used during the washing with TPBS in order to mix the washing solution more thoroughly.
  • the final washing solution was discarded and 1 ml of 1 M TEA (triethylamine) was added to the immunotubes. After five minutes' incubation in a roll incubator, the eluted phages were neutralised with 0.5 ml of 1 M TRIS, pH 7.4, and added directly to the 20 ml of plating bacteria for infection.
  • 1 M TEA triethylamine
  • the bacteria After incubation for 15 min without agitation at 37°C the bacteria were agitated for 45 min and removed by centrifuging at 3000g for 10 min. The bacteria were resuspended in 500 ⁇ l of 2YT medium and incubated on large SOBGAT plates (15cm) overnight at 37°C. For harvesting, the cells were scraped from the plate with LBAT medium, mixed with 25%> final concentration of glycerol and frozen in aliquots at -80°C or used for inoculation of another round of amplification.
  • the phage titre of each round of panning was determined by titration of 0.01-10 ⁇ l of the infected plating bacteria.
  • the number of eluted phages from CD8-FAP immobilised immunotubes was compared with that of the conesponding control immunotubes without an antigen.
  • the ratio of quantities of the eluted phages from the antigen-coated immunotubes and the uncoated immunotubes yielded the concentration factor.
  • the scFv-pIII- fusion proteins expressed using pSEX81 may be used both for Screening, i.e. sampling, and for analysis of scFv clones selected from phage display banks,.
  • the residue of the cultures left in the Beckman microtitre plates was able to be mixed with glycerol at 20 %> and frozen at -80°C.
  • the growth of the 100 ⁇ l of cultures could be checked if necessary with an ELISA Reader at a filter wavelength of 630 nm. After about 6-8 h the cultures were centrifuged at 1200 rpm (5 min, RT) and the supernatants were removed with a multichannel pipette. The pelleted bacteria were resuspended in 100 ⁇ l aliquots of 2YT A ⁇ (without glucose) incl. 50 ⁇ M EPTG and incubated o-n with agitation in the damp chamber at 30°C and 300 rpm.
  • the cultures were each mixed with 25 ⁇ l of 0.5 % Tween and incubated with agitation for a further 3-4 h to achieve partial lysis. Finally, the cultures were centrifuged for 10 min at 1200 rpm and the supernatants were carefully removed. These were used directly for Western-Blot analysis or after preadsorption used in the ELISA.
  • the overnight precultivation as well as the main cultivation of the bacteria were carried out in a volume of 3-10 ml in test tubes or in 50 ml PP-test tubes with agitation at about 200 rpm. If the bacterial growth had reached its logarithmic phase (O.D. ⁇ oonm about 0.7) the cultures were centrifuged (2500 rpm, 5 min, RT) and resuspended in an equal volume of fresh SB A ⁇ or 2YT A ⁇ incl. 50 ⁇ M- EPTG for induction.
  • the cultures were mixed with Tween 20 (ad 0.1 %) and the supernatants were removed after 3 h of further incubation.
  • the bacte ⁇ al pellet could also be opened up (see below)
  • the scFv-glll-fusion proteins were used to demonstrate the integ ⁇ ty of the reading frames of the scFv-coding region (Western blot) and to investigate the FAP specificity of the scFv selected in the ELISA on immobilised FAP or in the cell analyser on FAP+ cells.
  • An anti- glll-specific monoclonal antibody combined with a peroxidase- or alkaline phosphatase- conjugated detection antibody was used to detect the scFv-glll- fusion proteins.
  • an FITC-labelled detection antibody was used.
  • G 100 mM glucose (stock solution.: 2 M), A: ampicilhn 100 ⁇ g/ml, T- tetracychne 12,5 ⁇ g/ml, K: kanamycin 50 ⁇ g/ml
  • Liquid media for the bactenal culture Liquid media for the bactenal culture:
  • stenle MgCl 2 and MgSO 4 are added ad 10 mM in each case, as well as stenle glucose ad 20 mM
  • BHI (without yeast)30 ml agar agar 1 % saccharose (60 %) 0.5 ml horse serum 2.5 ml yeast extract (20 %) 1 ml glucose (20 %) 0 5 ml saccharose, serum, yeast extract, glucose are all added stenle
  • the scFv was produced in E coli XLl-Blue in volumes of 3-100 ml.
  • the incubation took place wither in test tubes or in 50 ml PP-test tubes with agitation at about 200 rpm or in Erlenmeyer chicane flasks at 180 rpm in LB or 2YT medium
  • the media were buffered with 1/10 vol MOPS (pH 7) and mixed with tetracycline (12 5 ⁇ g/ml) for the strain XLl- Blue
  • the transformed LVI-bacteria were taken up in 1 ml of BYS medium ( 1 ml BHI, 0.5 % yeast extract, 1 % saccharose) and incubated for 3 h with vigorous agitation in a small steep-walled container at 37°C. 100 ⁇ l of each transformation mixture were plated out on a BHI K plate. After 24 - 48 h incubation (37°C) significantly large colonies were pricked out using a sterile spatula and transfened into 20 ml of BHI K medium. After o-n growth and microscopic monitoring for the presence of L-form bacteria, this culture was mixed with cryomedium and frozen at -80°C. Unfrozen transformed P.
  • mirabilis cultures remained viable for at least 4 weeks if they were stored at 4°C.
  • two successive o-n or 11 - 12 h preliminary cultures were inoculated ( 20 ml each) and incubated at 30°C, the first of them from a 4°C culture. Depending on the density of the preliminary culture achieved and the length of incubation of the following culture, it was always overinoculated 1 :10 or 1:20.
  • the BHI K induction cultures (incl. 0.5 mM-EPTG) had a volume of 20 - 50 ml and were also inoculated, then incubated at 30°C with agitation for at least 11 h.
  • the OD550 ( ⁇ 4), the pH (7.5 - 8.5) and the optical appearance of the L forms were examined under the microscope.
  • the expression culture was centrifuged (5000 rpm, 3800 x g, 4°C) and the pellet was discarded. The supernatant could be used directly for ELISA or Western Blot analysis or it could be purified.
  • the minibodies were purified by EMAC (immobilized metal affinity chromatography). 1 ml HiTrap columns made by Pharmacia Biotech were used for this. Gel chromatography was carried out as the second purification step.
  • the column was equilibrated with 10 ml of PBS (pH 8). The supernatant was applied to the column using a peristaltic pump (1.5 ml/min), followed by a washing step (10 ml PBS incl. 5 - 20 mM imidazole). Elution was carried out in 1 ml fractions with 10 ml PBS incl. 300 mM imidazole. The elution fractions were stored on ice.
  • minibodies monovalent scFv and bivalent scFv (so-called minibodies).
  • the structure of the minibodies produced and the expression cassettes used for this purpose is comparable with those described by Hu et al. 1996 (Cancer Res. 56: 3055-61).
  • the minibodies we prepared have a c-myc domain at the C-terminus for immunological detection (with the monoclonal antibody 9E10) and a HIS 6 domain for chromatographic purification.
  • the cmyc- and HIS 6 -coding sequences conespond to those from pOPE 101 (S. Dubel, University of Heidelberg).
  • Prokaryotic expression of antibody proteins according to the invention The expression vectors used and the processes for the expression and punfication of monovalent scFv denvatives in E coli (Moosmayer et al., 1995, Ther. Immunol. 2 ⁇ 31-40) and Proteus mirabilis LVI (Rippmann et al, 1998, Applied and Environmental Microbiology 64 4862-4869) are known from the pnor art
  • the vector pACK02scKan and the processes from Rippmann et al , 1998 were also used to prepare and pu ⁇ fy a minibody in Proteus mirabilis L VI
  • minibodies desc ⁇ bed were also prepared in mammalian cells
  • the expression vectors used for the minibody expression cassettes were p AD-CM V-l and a pgldl05 de ⁇ vative
  • the expression vector was first amplified in E coli (XLl- Blue) and then punfied The vector DNA was adjusted to a concentration of l ⁇ g/ ⁇ l under ste ⁇ le conditions and stored at -20°C
  • the solution was dist ⁇ ubbed by gentle tilting and incubated for 5 hours at 37°C.
  • the Pet ⁇ dish was filled with 5 ml of preheated DEMEM 20%FCS and incubated for 16 h at 37°C. Then the incubation medium is carefully suction filtered and replaced by 10 ml of OptiMEM After another 48 hours' incubation time at 37°C the supernatant was removed for harvesting and the cells were removed by centnfuging at 700 g A further centnfugation step at 12000g pelleted the remaining cell fragments The supernatant was either ultracent ⁇ fuged for 30 mm (60000 xg for 30 min) and then added to an EMAC column (Amersham-Pharmacia) or evaporated down to 1/ 40 to 1/80 volume in cent ⁇ fugal concentrators with a 30 kDa separation threshold (Fugisept-Midi or MaxiRohrchen, Intersept). The centnfugation was earned out according to the manufacturer's instructions at
  • Stable transfectants of CHO DG44 were prepared for the expression of FAP-specific minibodies.
  • methotrexate was added to the medium from a period 10 - 14 days after the transfection.
  • the methotrexate concentration was slowly increased; the concentrations were between 10 and 1000 nM.
  • the minibodies were produced in T-culture flasks or in a bioreactor.
  • FAP+ cells were incubated in parallel batches with various concentrations of mono- or bivalent scFv derivatives.
  • the binding of these recombinant antibodies was determined using an FETC-labelled detection antibody in a cell analyser (Coulter).
  • the concentration of the scFv derivatives at which half the maximum saturation of the binding signal was achieved was chosen as a measurement of the apparent affinity.
  • Example 3 Sequences The sequences are shown here by way of example. Smaller mutations, e.g. the substitution of one or a few amino acids or the nucleotides coding therefor are also included in the invention.
  • the first amino acid may also be an E (glutamate).
  • CAGGTACAGCTGGTGGAGTCTGGGGGAACCTTGGTACAGCCTGGGGGGTCCCT is GAGACTCTCCTGTGCAGCCTCTGGATT
  • Nucleotide sequence conesponding to VH46 such as may occur in the minibody, for example.
  • the sixth nucleotide may also be an A instead of a G - a silent mutation, hence having no effect on the amino acid sequence.
  • the first amino acid may also be an E
  • VH46 YOL VL III25 Protein sequence of the total antibody protein as occurs in the minibody, for example
  • VH 50 YOL VL III25 Protein sequence of the total antibody protein as occurs in the minibody, for example (for possible variation see VH50, above)
  • VH34YOLIII43 Protein sequence of the total antibody protein:
  • VH18 YOL III43 Protein sequence of the total antibody protein:
  • the restricted D ⁇ A was purified using QiaQuick (Qiagen, Germany) and ligated overnight with VL PCR products, overdigested with the same endonucleases.
  • the ethanol-precipitated ligations were used to transform E. coli XLl-Blue (Stratagene, California). Transformands were plated on 2YT plates containing 100 mM-glucose, 100 ⁇ g/ml ampicillin, 12.5 ⁇ g/ml tetracylin and grown overnight at 30 °C. Diversity of the cloned libraries was tested by Zfat ⁇ I-digests of PCR- amplified V regions and analysis on polyacrylamid gels.
  • phage associated antibodies To obtain phage associated antibodies (phabs), the overinfection of exponentially growing E. coli was carried out following Schier et al. (1996). After growth at 30 °C overnight bacteria were pelleted and phages were precipitated twice with 20 %> polyethylene glycol in 2.5 M- ⁇ aCl. For selection 1-20 ⁇ g FAP were coated in Maxisorb immunotubes ( ⁇ unc) rotating overnight at 4°C. After washing twice with PBS the coated tubes were blocked with 3 % non fat dry milk in PBS or with Roti-Block (Roth, Germany). Immediately before the panning, the tubes were washed twice with PBS.
  • Eluted phages were immediately neutralized with Tris and used for infection of XL-1 Blue. After overnight growth at 30 °C, the bacteria were scraped from the agar plates and either used for a further round of selection or frozen with glycerol.
  • FAP overexpressing fibrosarcoma cells 10 5 FAP+ or control cells were incubated for 90 min with serial dilutions of the Ab construct. Detection was carried out by the anti-c-myc Ab 9E10 followed by an FITC labeled goat anti-mouse specific serum (in case of scFv) or by an FITC labeled goat anti-human specific serum (in the case of Mb). Incubations and washings were done on ice except for the labeled Abs which were applied at RT.
  • Bound Ab contructs were detected in a FACStar (Becton Dickinson) or in an EPICS Flow Cytometer (Coulter). The mean fluorescence was measured for 10 cells in each dilution. The concentration of the applied Ab derivatives were determined in repeated estimations against a scFv or Mb standard used in SDS-PAGE and western blotting. Cloning, expression and purification of minibody (Mb)
  • the scFv cassettes of the selected clones 18 and 34 were excised from the scFv expression vector pOPElOl (Dubel et al , 1992) by rest ⁇ ction with Ncol! Notl and inserted into an equally prepared Mb-vector, pDl , a de ⁇ vative of the published vector pACK02scKan- (Pack et al , 1993) E coli XLl-Blue were transformed as usual, subsequently, the cell wall and outer membrane deficient strain LVI of Proteus mirabilis was transformed and induced to overnight expression according to Rippmann et al (1998) After dialysis against PBS the Mb was ultracent ⁇ fuged ( 1 13,000 xg, 4°C, 30 min) and purified by means of IMAC with a Zn 2+ loaded HiTrap column (Pharmacia, Sweden) Fiactions wered tested by SDS-PAGE and subsequent Coomassie staining
  • the thermal stability of Mb #34 in RPMI medium containing 5 % FCS was by incubation of purified, freshly thawed Mb at 37 °C for up to 72 h After incubation the solution was centnfuged (20,000 xg, 4 °C, 10 min) and used on immobilized FAP in an ELISA. A preceding expenment was used to determine an approp ⁇ ate dilution for each of the Mb preparations to reach distinct but non-saturated ELISA signals
  • This library was phage display selected on immobilized FAP to isolate human VL F19 analogues After three rounds of selection, the screening for binders by ELISA yielded several FAP binding clones. To ensure the diversity of these isolated chime ⁇ c scFv (murine VH/ human VI ) their phagemid DNA was analyzed by rest ⁇ ction enzymes and sequenced Various chime ⁇ c scFv (now shortly named after their VL) could be identified (III5, III10, 11125, III43), consisting of the guiding VH of the paternal scFv F19 and the itemized human VLs.
  • Table 1 shows the aa sequence homology of the selected light chains IEI5 and III43 compared to the replaced VL F19. Both listed VLs belong to the human VL subgroup kappa I according to Kabat (httpV/immuno.bme nwu edu/), and the germline gene with the closest homology is a member of the subgroup V ⁇ l family (IH5: Ve; III43 Ve). Looking at the aa sequence, clone 1115 had as much as 64 % identity in FR positions compared to the parental F19, and 59 % identity in CDR positions. EII43 had 69 % identity in FR positions and, again, 59 % identity in CDR positions compared to F19.
  • E ⁇ 5 and IEI43 showed a high degree of mutations compared to their putative germline genes III5 differed in 14 aa positions from the sequence of the closest germline, IXI43 showed 17 differences (ImMunoGeneTics database : http://imgt.cnusc.fr.8104, and Cox et al., 1994)
  • a phagemid vector was constructed containing HCDR3 F19, a human FR4 (found in Kabat subgroups I, II and III), and a new restriction site, which was introduced in HCDR3 without changing the aa sequence (fig. 9).
  • the selected VL III5 and VL EEE43 were inserted, respectively, to encode the specific guiding structures.
  • the resulting VH segment library (size: 4 x 10 7 clones) was combined with either VL III5 or VL EII43 and phage display selected on immobilized FAP.
  • the selected clone #18 showed 66 % identity with the aa sequence of scFv F19 in the FRs, and 50 % identity in the CDRs 1 and 2.
  • the FR identity was 67 % > , and 55 % in CDR 1 plus 2.
  • Both isolated VH chains use VL EEI43 as complement and belong to the human VH subgroup I, according to Kabat.
  • the closest germline gene segments were shown to belong to the VHl segment family, which represents about 12 % of all human VH gene segments (Guigou et al., 1990; Brezinschek et al., 1995).
  • VH #18 and #34 showed 10 and 9 amino acids differences, respectively.
  • Figure 10 shows the stnct FAP-specificity of the humanized scFv #18 and #34 in ELISA But in view of a potential clinical application of the selected human scFv, their binding characte ⁇ stics to natural cell membrane expressed FAP is of particular importance
  • Saturation studies yielded in a functional cell binding afffinity (SC 50 ) of 6 nM for scFv #18 and scFv #34, each In a parallel assay the SCso for the parental scFv F19 and its CDR grafted derivative, scFv OS4.
  • Fig. 1 HCDR3-retaining guided selection
  • Fig. 2 Schematic representation of the HCDR3 sequence with the integrated Spll (Pfl23ll)
  • Fig. 3 Binding of scFv #13 (minibody format) to FAP+-cells (FACS analyses)
  • Fig. 4 Primers used for PCR amplification of the human V repertoire
  • Fig. 5 Primers for amplifying the human VH-gene segment repertoire for the HCDR3 retaining guided selection process
  • Fig. 6 Sequences of the selected human FAP-specific VL regions
  • Fig. 7 Ag specificity of selected chimeric scFv.
  • ELISA wells were coated with FAP or inelevant Ag.
  • TTX tetanus toxoid
  • BSA bovine serum albumin
  • HSA human serum albumin
  • TF transfemn
  • CHY chymotrypsinogen
  • LYS lysozyme
  • Detection was done with 9E10 and POD-labeled goat anti-mouse serum. Data are derived from triplicate values.
  • Fig. 8 Epitope specificity of selected chimeric scFv. Different concentrations of competitor were mixed with the respective scFv and added to FAP coated ELISA wells. The applied competitors were: cF19 (chimeric F19, with murine variable and constant human regions); hu IgG (unspecific human IgG serum). Detection was done as in figure 1. Data are from double values.
  • Fig. 9 Construction of the human VH gene segment library with retained HCDR3 F19. Schematic drawing of the final construct of VH, linker, VL and phage protein gpfll. By creation of a new restriction site the VH segment repertoire could be ligated to the preexisting HCDR3 F19, linked later to the selected human VLs.
  • Fig. 10 Ag specificity of selected humanized scFv. Coating of ELISA wells and detection was earned out as in fig. 1. PLA' plastic
  • Fig. 11 Binding of humanized scFv and Mb to cell surface-bound FAP analysed by flow cytometry.
  • Supernatants of P mirabilis LVI containg 20 nM MB C Control binding of scFv F19 (punfied by IMAC) to FAP + cells Area for binding to FAP control cells is gray.
  • scFv were detected by 9E10 and FITC-labeled Fc-specific anti-mouse serum, Mb by FITC-labeled Fc- specific anti-human serum. Each curve represents cytometer values of 5,000 predefined and measured events.
  • Fig. 12 Epitope specificity of humanized scFv for cellbound FAP. Different concentrations of competitor were mixed with the respective scFv and added to FAP + cells.
  • Fig. 13 Assessment of apparent affinity for Mb #34 on FAP + cells.
  • Mb #34 was purified by EMAC and size exclusion chromatography. Data are de ⁇ ved from the cytometer with values of 10,000 events for each Ab concentration after detection with FITC-labeled Fc- specific anti-human serum.
  • Fig. 14 Long term stability of Mb #34 at 37°C. After incubation in a tenfold volume of RPMI (5% FCS) for 0 to 42 h, the IMAC punfied Mb was diluted and used in an anti-FAP ELISA. Detection was carried out with POD-labeled anti-human serum. Data are based on triplicate values.
  • Fig. 15 lmmunohistological staining of biopsy material from FAP + tumor sections with Mb #34. Cryo-sections of A) breast carcinoma B) colon carcinoma C) lung carcinoma D) desmoid tumor E) malignant fibrous histiocytoma were stained with Mb #34.
  • Bound Mb was detected by subsequent treatment of the section with an anti-c-myc mAb (9E10), a biotinylated horse anti-mouse serum and the avidin-biotin immunoperoxidase complex.
  • an anti-c-myc mAb 9E10
  • a biotinylated horse anti-mouse serum 9E10
  • the avidin-biotin immunoperoxidase complex 9E10
  • a cryo-section was only treated with the detection antibodies and the avidin-biotin immunoperoxidase complex.

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Abstract

The invention relates to antibody proteins which specifically bind fibroblast activating protein alpha (FAPα). The invention further relates to the use of said antibodies for diagnostic and therapeutic purposes as well as processes for preparing said antibodies.

Description

Human FAP-α-specific antibodies
The invention relates to antibody proteins which specifically bind fibroblast activating protein alpha (FAPα). The invention further relates to the use of said antibodies for diagnostic and therapeutic purposes as well as processes for preparing said antibodies.
Background to the invention
Massive growth of epithelial cell cancer is associated with a number of characteristic cellular and molecular changes in the surrounding stroma cells. One highly consistent feature of the reactive stroma of numerous types of epithelial cell cancer is the induction of the fibroblast activating protein alpha (from now on referred to as FAPα or FAP), a cell surface molecule of the reactive stromal fibroblast which was originally identified with the monoclonal antibody F19 (Garin-Chesa P., Old L.J. and Rettig W.J.; 1990; Proc Natl. Acad. Sci. 87: 7235). Since the FAP is selectively expressed in stroma of a number of epithelial cell carcinomas, irrespective of the site and histological type of the carcinoma, it was desirable to develop a treatment concept for the FAPα target molecule in order to allow imaging techniques, the diagnosis and treatment of epithelial cell cancer and many other syndromes. For this purpose a monoclonal murine antibody named F19 was developed which specifically binds to FAP. This antibody was described in US patents 5,059,523 and WO 93/05804 which are included in their entirety in this document by reference. A serious problem arises when non-human antibodies are used for in vivo applications in humans, i.e. they rapidly elicit an immune response to the foreign antigen. In the worst case such an immune response against the antibody used may trigger anaphylactic shock. This drastically reduces the efficiency of the antibody in the patient and has an adverse effect on further use or makes any further use impossible. The humanisation of non-human antibodies is usually achieved by one of two methods:
(1) By the construction of non-human / human chimeric antibodies in which the non- human variable regions are coupled to the human constant regions (Boulianne G.L., Hozumi N. and Shulman, M.J. (1984)) Nature 312:643) or
(2) By replacing the complementarity determining regions (CDRs) in human variable regions with those of the non-human variable region and then coupling the newly formed humanised variable regions to human constant regions (Riechmann L., Clark
M, Waldmann H. and Winter G. (1988) Nature 332:323). Chimeric antibodies consist of fewer foreign protein sequences than non-human antibodies and therefore have a lesser xenoantigenic potential. Nevertheless, chimeric antibodies of this kind may trigger an immune reaction on account of the non-human V-regions in humans (LoBuglio A.F., Wheeler R.H., Trang J., Haynes A., Roger K., Harvey E.B., Sun L., Ghrayeb J. and Khazaeli M.B. (1989) Proc.Natl.Acad.Sci.86:4220). CDR- transmitted or newly formed humanised antibodies admittedly contain fewer foreign protein sequences in the V-regions, but these humanised antibodies are still capable of triggering an immune response in humans. WO99/57151 A2 describes FAPα-specific humanised antibodies of this kind in which the humanisation has been achieved by transferring all 6 CDR regions (3 from the light chain, 3 from the heavy) from the corresponding F19 murine antibody. These antibodies still contain parts of the murine framework region.
The problem of the present invention is to provide improved FAPα-specific antibodies which overcome the above disadvantages of the prior art.
Description of the invention
The problem was solved within the scope of the claims and specification of the present invention.
The use of the singular or plural in the claims or specification is in no way intended to be limiting and also includes the other form.
The invention relates to new human or humanised antibody proteins which specifically bind to fibroblast activating protein alpha (FAPα), and are either completely human or contain not more than one murine complementarity-determining region (CDR region) of the monoclonal antibody F19 (ATCC accession number HB 8269). The antibodies according to the invention have the surprisingly advantageous property of having a significantly reduced xenoantigenic potential and consequently being better suited for use in humans than the antibodies known from the prior art (cf. also description of the process according to the invention, infra). The antibodies according to the invention advantageously have no or very few parts of the murine amino acid sequence, namely at most one CDR region. The framework regions ( FR) of the variable region of the antibodies according to the invention also correspond entirely to human amino acid sequences. In spite of the few murine components the antibodies according to the invention are nevertheless surprisingly highly specific for the target antigen FAP. Within the scope of this invention the term antibodies denotes one or more of the polypeptide(s) described in this specification. It also includes human antibody proteins selected from fragments, allelic variants, functional variants, variants based on the degenerative nucleic acid code, fusion proteins with an antibody protein according to the invention, chemical derivatives or a glycosylation variant of the antibody proteins according to the invention.
The preparation methods known from the prior art are unsuitable for obtaining human antibodies according to the invention. With a process according to the invention as hereinafter described and illustrated more fully in the Examples it is possible to obtain a human or humanised antibody according to the invention with reduced xenoantigenic properties. In a preferred preparation process according to the invention the following steps are carried out, for example:
1) PCR amplification of the human VL- and VH-repertoires: a) In order to prepare the VH and VL repertoires the various V-gene families are separately amplified with the respective family-specific primers by PCR from cDNA (see Example 1). b) All Forward! 3 '-primers for VH- and VL-PCR amplification are complementary to the gene sequences of the constant immunoglobulin domains (IgG, IgD, IgM, K, λ). This enables efficient isotype-specific amplification of the V regions with very few 3 '-primers. By contrast, in processes known from the prior art a plurality of different 3 '-primers complementary to the J-sections of the V regions are used (Marks et al., 1991; J. Mol. Biol. 222: 581).
2) Preparation and cloning of a human VH-repertoire:
In the prior art, up till now, only certain lymphoid tissues have been described with very few different donors as sources of V repertoires (e.g. Vaughan et al., 1996; Nature Biotechnology 14: 309). In order to obtain a human V-repertoire consisting of a large number of clones with high diversity (for details see Example 1) as a basis for the preparation of the antibodies according to the invention, far more different donors are used, i.e. about ten times more than are recommended in the prior art, in non-obvious manner, not only for the lymphoid organs in question, but also the foetal liver and thymus gland are used as a source of V repertoires. Moreover, the IgD repertoire was also amplified, in addition to the IgM and IgG repertoires, in order to achieve great repertoire diversity (see Example 1).
3) Preparation of a combination repertoire consisting of a human VH repertoire and various human FAP-specific VL regions:
In order to obtain an antibody according to the invention, the VH region known, for example, from the monoclonal, FAP-specific murine antibody F19 may be used and a suitable human FAP-specific VL region may be selected using a guided selection method and a phage display method. Then, using said human VL region as a guiding structure, for example, a human FAP-specific VH region may be selected. The technical problem of the DNA contamination of the combination repertoires with phagemid vectors which code for existing FAP-specific scFv, (e.g. murine scFv from the hybridoma line F19 or the chimeric anti-FAP scFv with human VL and F19 VH) may arise. A guided selection process is described in the Examples.
By combination repertoire is meant the combination, by genetic engineering, of a V repertoire with correspondingly complementary V-sequences. (Complementary with respect to VH to VL and vice versa). The V-sequences used for the combination may consist of one V-sequence, a number of different V-sequences or a V repertoire. Preferably, an antibody protein according to the invention is characterised in that it comprises a heavy chain (VH) of the immunoglobulin class IgM. Preferably, an antibody protein according to the invention is also characterised in that it contains a heavy chain (VH) of the class IgG. Non-limiting examples of these are the completely human antibodies scFv #13 and scFv #46 (see Examples). Preferably, an antibody protein according to the invention is also characterised in that it comprises a heavy chain (VH) of the class IgD. A non-limiting example of this is the human antibody according to the invention scFv #50 (see also Examples). In this antibody the VH-sequence originates from a human IgD and is identical to the germline sequence apart from one amino acid exchange. This advantageously reduces the probability of an allogenic immune response to this VH region in humans.
Preferably, also, an antibody protein according to the invention is characterised in that it comprises a light chain (VL) of the lambda type (λ). Preferably, also, an antibody protein according to the invention is characterised in that it comprises a light chain (VL) of the kappa type (K) (see Example, e.g. ILT25, III43). For many uses of the antibodies according to the invention it is desirable to have the smallest possible antigen-binding, i.e. FAP -binding units. Therefore in another preferred embodiment an antibody protein according to the invention is a Fab fragment (Fragment antigen-binding = Fab). These FAP-specific antibody proteins according to the invention consist of the variable regions of both chains which are held together by the adjacent constant region. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced in the mean time by genetic engineering. In another preferred embodiment an antibody protein according to the invention is an F(ab')2 fragment, which may be prepared by proteolytic cleaving with pepsin.
Using genetic engineering methods it is possible to produce shortened antibody fragments which consist only of the variable regions of the heavy (VH) and of the light chain (VL). These are referred to as Fv fragments (Fragment variable = fragment of the variable part). In another preferred embodiment an FAP-specific antibody molecule according to the invention is such an Fv fragment. Since these Fv-fragments lack the covalent bonding of the two chains by the cysteines of the constant chains, the Fv fragments are often stabilised. It is advantageous to link the variable regions of the heavy and of the light chain by a short peptide fragment, e.g. of 10 to 30 amino acids, preferably 15 amino acids. In this way a single peptide strand is obtained consisting of VH and VL, linked by a peptide linker. An antibody protein of this kind is known as a single-chain-Fv (scFv). Examples of scFv- antibody proteins of this kind known from the prior art are described in Huston et al. (1988, PNAS 16: 5879-5883). Therefore, in another preferred embodiment an FAP-specific antibody protein according to the invention is a single-chain-Fv protein (scFv). In recent years, various strategies have been developed for preparing scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity. In order to achieve multimerisation of the scFv, scFv were prepared as fusion proteins with multimerisation domains. The multimerisation domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucin-zipper domains. However, there are also strategies in which the interaction between the VH/NL regions of the scFv are used for the multimerisation (e.g. di-, tri- and pentabodies). Therefore in another preferred embodiment an antibody protein according to the invention is an FAP-specific diabody antibody fragment. By diabody the skilled person means a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883). The shortening of the Linker in an scFv molecule to 5- 10 amino acids leads to the formation of homodimers in which an inter-chain VH/VL-superimposition takes place. Diabodies may additionally be stabilised by the incorporation of disulphide bridges. Examples of diabody-antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226). By minibody the skilled person means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from IgGl ) and a Linker region. The disulphide bridges in the Hinge region are mostly formed in higher cells and not in prokaryotes. In another preferred embodiment an antibody protein according to the invention is an FAP-specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).
By triabody the skilled person means a: trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein VH-VL are fused directly without a linker sequence lead to the formation of trimers.
The skilled person will also be familiar with so-called miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv. The multimerisation is carried out by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology 11 :, 1271- 1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28- 34).
Therefore in another preferred embodiment an antibody protein according to the invention is an FAP-specific multimerised molecule based on the abovementioned antibody fragments and may be, for example, a triabody, a tetravalent miniantibody or a pentabody. Particularly preferably, an antibody protein according to the invention is totally human. Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ID No. 1 (VH13). Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence LD No. 2 (VH46).
Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ID No. 3 (VH50).
Another preferred antibody protein according to the invention is characterised in that the variable region of the light chain (V ) contains the amino acid sequence ID No. 4 (VLIII25).
Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) is coded by the nucleotide sequence ED No. 5 (VH13) or by fragments or degenerate variants thereof.
Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) is coded by the nucleotide sequence ID No. 6 (VH46) or by fragments or degenerate variants thereof.
Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) is coded by the nucleotide sequence ID No. 7 (VH50) or by fragments or degenerate variants thereof.
Another preferred antibody protein according to the invention is characterised in that the variable region of the light chain (VL) is coded by the nucleotide sequence ED No. 8 (VLLI125) or by fragments or degenerate variants thereof.
An especially preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 1 (VH13) and the variable region of the light chain (VL) contains the amino acid sequence ED No. 4 (VLEII25).
Another particularly preferred antibody protein according to the invention is characterised in that the coding sequence of the variable region of the heavy chain (VH) contains the nucleotide sequence ED No. 5 (VH13) and the coding sequence of the variable region of the light chain (VL) contains the nucleotide sequence ED No. 8 (VLIEL25). Another particularly preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 2 (VH46) and the vaπable region of the light chain (VL) contains the amino acid sequence ED No 4 (VLIII25)
Another particularly prefened antibody protein according to the invention is characteπsed in that the coding sequence of the vaπable region of the heavy chain (VH) contains the ! nucleotide sequence ID No 6 (VH46) and the coding sequence of the vaπable region of the light chain (VL) contains the nucleotide sequence ID No 8 (VLIII25) Another particularly prefened antibody protein according to the invention is characteπsed in that the variable region of the heavy chain (VH) contains the amino acid sequence ID No
3 (VH50) and the vaπable region of the light chain (V ) contains the amino acid sequence 7 ID No 4 (VLEII25)
Another particularly preferred antibody protein according to the invention is characteπsed in that the coding sequence of the vaπable region of the heavy chain (VH) contains the nucleotide sequence ED No 7 (VH50) and the coding sequence of the vaπable region of the light chain (VL) contains the nucleotide sequence ED No 8 (VLEII25)
; Particularly preferably, an antibody protein according to the invention is humanised The humanised antibody protein according to the invention has the advantage, over the FAPα- specific antibody proteins known from the pπor art, that it does not contain all six muπne CDR regions of F19, but only one muπne CDR region, as descπbed in the following preferred embodiments This antibody protein according to the invention advantageously
. has a lesser xenoantigenic potential than the antibody proteins known from the pπor art Surpπsingly, the inventors have succeeded in producing antibody molecules which contain only one muπne CDR region, against the prevailing opinion that at least two muπne CDR regions are necessary for successful humanisation (Rader et al, 1998, Proc Natl Acad Sci USA, 95 8910) s Another surpnsing property in the case of humanised scFv 34 and scFv 18 is that these scFv exhibit a higher apparent binding affinity for FAP+-cells (EC50 6 nM) than the FAP- specific antibodies such as e g scFv F19 (EC50 20 nM) known from the pπor art A preferred process according to the invention for prepanng humanised antibodies according to the invention may be descπbed by the following steps, for example
1) Humanisation of scFv F19 by the HCDR3 retaining Guided selection method Our experience has shown that by using the Guided selection process human Ab can be selected which have a different epitope specificity from the parental murine Ab. In order to overcome this disadvantage in the prior art, the HCDR3 F19 was advantageously retained in the Guided selection process for humanising scFv F19 as well as in the final humanised product. The pπor art (Rader et al., 1998, PNAS 95: 8910) describes only antibodies humanised by Guided selection in which both the LCDR3 and also the HCDR3 of the parental murine Ab are retained (see Example 1).
2) Combination of a human VH-gene segment repertoire with murine HCDR3 (F19) The VH segments of all known human VH families are to be combined with HCDR3 F19 o in order to generate as complex a combination repertoire as possible. Advantageously, this is preferably done e.g. by integrating a cutting site for the restriction enzyme Pβ23ll in the HCDR3 F19 without altering the coding at the amino acid level. For combining the PCR- amplified human VH-gene segments a Phage display vector was developed which contains the following Ab-sequence sections: HCDR3 F19 with a Pβ23U cutting site, a human VH FR4 region with high homology with the corresponding region from F19 as well as various selected human anti-FAP VL regions (see the diagram in Example 1). The primers for PCR amplification of the VH-gene segment repertoires are shown in Example 1. This preferred process has the following advantages over the prior art for combining VH- o gene segment repertoires with defined CDR3 regions:
Schier et al. 1996; J. Mol. Biol. 255: 28: En this prior art a restriction cutting site (BssHIT) was integrated in the 3' region of VH FR3. The incorporation of this cutting site via PCR is, however, connected with an altered amino acid sequence in various VH-gene families. For this reason, in Schier et al. Only some of the VH-gene families were able to be j included in the combination repertoire.
PCR overlap extension Rader et al. 1998: This process does indeed make it possible to include all VH-gene families in the combination, but the disadvantages are a low linking efficiency and a high error rate. This increases the probability of inactive scFv mutants and especially clones with an interrupted scFv reading frame, leading to genetically unstable o combination repertoires.
3) Use of different human FAP-specific VL regions as a guide structure En order to increase the probability of selecting an ScFv analogous to F19, the human VH repertoire (see 2) was combined with the sequences of different human FAP-specific VL regions. (Carried out analogously to human antibodies, supra).
4) Stringent washing step in Phage display selection s This procedure was used to eliminate low-affinity and polyreactive antibodies during the selection process (for method see below).
5) Use of an efficient screening process for identifying the selected humanised scFv During the HCDR3 retaining guided selection process a very large number of clones were concentrated. The scFv #34 and #18 can advantageously be identified by the screening o process described in Mersmann et al. 1998 (J. Immunol. Methods, 220: 51).
Another prefened antibody protein according to the invention is characterised in that it contains murine CDR 1 of the light chain (VL) of the monoclonal antibody F19. Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 2 of the light chain (VL) of the monoclonal antibody F19. s Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 3 of the light chain (VL) of the monoclonal antibody F19. Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 1 of the heavy chain (VH) of the monoclonal antibody F19. Another preferred antibody protein according to the invention is characterised in that it o contains murine CDR 2 of the heavy chain (VH) of the monoclonal antibody F19.
Another preferred antibody protein according to the invention is characterised in that it contains murine CDR 3 of the heavy chain (VH) of the monoclonal antibody F19. Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 9
- (VH34).
Another preferred antibody protein according to the invention is characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 10
(VH18).
Another preferred antibody protein according to the invention is characterised in that the o variable region of the light chain (VL) contains the amino acid sequence ED No. 11 (VLEEE43). Another preferred antibody protein according to the invention is characteπsed in that the vaπable region of the heavy chain (VH) is coded by the nucleotide sequence ED No 12 (VH34) or by fragments or degenerate vaπants thereof
Another preferred antibody protein according to the invention is characteπsed in that the vaπable region of the heavy chain (VH) is coded by the nucleotide sequence ID No 13 (VH 18) or by fragments or degenerate vaπants thereof
Another preferred antibody protein according to the invention is characteπsed m that the vaπable region of the light chain (V ) is coded by the nucleotide sequence ED No 14 (VLIII43) or by fragments or degenerate vaπants thereof
An especially preferred antibody protein according to the invention is characteπsed in that the vaπable region of the heavy chain (VH) contains the amino acid sequence ID No 9 (VH34) and the vaπable region of the light chain (V ) contains the amino acid sequence ED No 1 1 (VLIII43)
Another particularly preferred antibody protein according to the invention is characteπsed in that the coding sequence of the vaπable region of the heavy chain (VH) contains the nucleotide sequence ED No 12 (VH34) and the coding sequence of the vaπable region of the light chain (VL) contains the nucleotide sequence ED No 14 (VLEII43) Another particularly preferred antibody protein according to the invention is characteπsed in that the vaπable region of the heavy chain (VH) contains the amino acid sequence ED No 10 (VH18) and the vaπable region of the light chain (VL) contains the amino acid sequence ED No 11 (VLEII43)
Another particularly preferred antibody protein according to the invention is characteπsed in that the coding sequence of the vaπable region of the heavy chain (VH) contains the nucleotide sequence ED No 13 (VH18) and the coding sequence of the vaπable region of the light chain (VL) contains the nucleotide sequence ED No 14 (VLEEI43) Another preferred embodiment of the invention composes a nucleic acid which codes for an antibody protein according to the invention Preferably, too, a nucleic acid according to the invention is characteπsed in that it contains 5' or 3' or 5' and 3' untranslated regions The nucleic acid according to the invention may contain other untranslated regions upstream and/or downstream The untranslated region may contain a regulatory element, such as e g a transcπption initiation unit (promoter) or enhancer Said promoter may, for example, be a constitutive, inducible or development-controlled promoter Preferably, without ruling out other known promoters, the constitutive promoters of the human Cytomegalovirus (CMV) and Rous sarcoma virus (RSV), as well as the Simian virus 40 (SV40) and Herpes simplex promoter. Inducible promoters according to the invention comprise antibiotic-resistant promoters, heat-shock promoters, hormone-inducible „Mammary tumour virus promoter" and the metallothioneine promoter. Preferably, too, a nucleic acid according to the invention is characterised in that it codes for a fragment of the antibody protein according to the invention. This refers to part of the polypeptide according to the invention.
Preferably, too, a nucleic acid according to the invention is characterised in that it codes for a functional variant of the antibody protein according to the inventions. This denotes polypeptides which are largely identical to an antibody protein according to the invention and which have the same biological activity as an antibody protein according to the invention or have an inhibiting effect on an antibody protein according to the invention. A variant of an antibody protein according to the invention may differ from an antibody protein according to the invention by substitution, deletion or addition of one or more amino acids, preferably by 1 to 10 amino acids.
Preferably, too, a nucleic acid according to the invention is characterised in that it codes for an allelic variant of the antibody protein according to the inventions. Preferably, too, a nucleic acid according to the invention is characterised in that it codes for variants of the antibody protein according to the inventions on the basis of the degenerative code of the nucleic acids. Preferably, too, a nucleic acid is characterised in that it is able to hybridise onto a nucleic acid according to the invention under stringent conditions. Stringent conditions are known to those skilled in the art and are found particularly in Sambrook et al. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 15 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 16 or a part thereof or a functional variant thereof. Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 17 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ID No. 18 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it contains an amino acid sequence according to sequence ID No. 19 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ID No. 20 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 21 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 22 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 23 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 24 or a part thereof or a functional variant thereof.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it corresponds to the amino acid sequence according to sequence ED No. 15.
Another particularly preferred embodiment of the invention comprises an antibody protein, characterised in that it corresponds to the amino acid sequence according to sequence ID No. 16. Another particularly preferred embodiment of the invention compπses an antibody protein, characteπsed in that it corresponds to the amino acid sequence according to sequence ID No. 17
Another particularly preferred embodiment of the invention compπses an antibody protein, characterised in that it corresponds to the amino acid sequence according to sequence ID No. 18
Another particularly preferred embodiment of the invention compπses an antibody protein, characteπsed in that it corresponds to the amino acid sequence according to sequence ED No 19
Another particularly preferred embodiment of the invention compπses an antibody protein, charactensed in that it is coded by the nucleotide sequence according to sequence ED No 20.
Another particularly preferred embodiment of the invention compπses an antibody protein, characteπsed in that it is coded by the nucleotide sequence according to sequence ED No 21.
Another particularly preferred embodiment of the invention compπses an antibody protein, characteπsed in that it is coded by the nucleotide sequence according to sequence ED No. 22.
Another particularly preferred embodiment of the invention compπses an antibody protein, characteπsed in that it is coded by the nucleotide sequence according to sequence ED No 23.
Another particularly preferred embodiment of the invention compπses an antibody protein, characteπsed in that it is coded by the nucleotide sequence according to sequence ED No 24.
Sequence ED No. refers to the No specified under <400> in the Sequence Listing, so that e.g. the nucleotide sequence according to sequence ED No. 24 is listed as <400> 24 Another aspect of the present invention relates to a recombinant DNA vector which contains a nucleic acid according to the invention. Examples are viral vectors such as e g Vaccinia, Semhki-Forest-Virus and Adenovirus. Vectors for use in COS-cells have the SV40 oπgin of replication and make it possible to achieve high copy numbers of the plasmids. Vectors for use in insect cells are, for example, E. colt transfer vectors and contain e g the DNA coding for polyhedπn as promoter Another aspect of the present invention relates to a recombinant DNA vector according to the invention which is an expression vector
Yet another aspect of the present invention is a host which contains a vector according to the invention
Another host according to the invention is a eukaryotic host cell The eukaryotic host cells according to the invention include fungi, such as e g Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomvces Tnchoderma insect cells (e g from Spodoptera frugψerda Sf-9, with a Baculovirus expression system), plant cells, e g from Nicotiana tabacum. mammalian cells, e g COS cells, BHK, CHO or myeloma cells In descendants of the cells of the immune system in which antibody proteins are also formed in our body, the antibody proteins according to the invention are particularly well folded and glycosylated Therefore a prefened host according to the invention is a mammalian cell
Particularly preferably, a host according to the invention is a BHK, CHO or COS cell Another host according to the invention is a bacteπophage
Another host according to the invention is a prokaryotic host cell Examples of prokaryotic host cells are Escherichia coli, Bacillus subtihs, Streptomyces or Proteus mirabύis The invention relates to a process for prepanng antibody protein according to the invention, which compπses the following steps a host according to the invention as descπbed above is cultivated under conditions in which said antibody protein is expressed by said host cell and said antibody protein is isolated
The antibody proteins according to the invention may be expressed in any of the hosts descnbed above
Preparation with prokaryotic expression systems such as Escherichia coli, Bacillus subtilis, Streptomvces or Proteus mirabύis is especially suitable for antibody fragments according to the invention, such as Fab-, F(ab')2-, scFv fragments, mimbodies, diabodies and multimers of said fragments The antibody proteins according to the invention are prepared by a process according to the invention either intracellularly, e g in inclusion bodies, by secretion into bactena with no cells walls such as, for example, Proteus mirabihs or by peπplasmatic secretion into Gram-negative bactena using suitable vectors for this purpose In Example 2 the preparation of the antibody proteins according to the invention in prokaryotes is descπbed by way of example Examples from the pπor art for the preparation of scFv-antibody proteins are descnbed in Rippmann et al. (1998, Appl. Environ. Microbiol., 1998, 64 4862-4869). Other examples are known to those skilled in the art.
The antibody proteins according to the invention may also be prepared in a process according to the invention in fungi, such as e g Pichia pastoπs, Saccharomyces cerevisiae, Schizosaccharomvces. Tnchoderma with vectors which lead to intracellular expression or secretion
The process according to the invention for prepanng the antibody proteins may also be earned out with insect cells, e g as a transient or stabile expression system or Baculovirus expression system. Here, Sf-9 insect cells, for example, are infected with e.g. Autographa cahfornica nuclear polvhedrosis virus (AcNPV) or related viruses There is no πsk of contamination with viruses which are pathogen to mammals, therefore therapeutic antibodies according to the invention may also advantageously be prepared in insect cells. The E coli transfer vectors descnbed above contain, for example, as promoters, the DNA which codes for polyhedπn, behind which the DNA coding for the antibodies according to the invention is cloned. After identification of a conect transfer vector clone in E. coh this is transfected together with incomplete Baculovirus DNA into an insect cell and recombined with the Baculovirus DNA so as to form viable Baculoviruses. Using powerful insect cell promoters, in a process according to the invention large amounts of the antibody protein according to the invention are formed which is secreted into the medium e.g. by fusion with eukaryotic signal sequences Insect cell expression systems for die expression of antibody proteins are commercially obtainable. Insect cell expression systems are particularly suitable for the scFv fragments according to the invention and Fab or F(ab')2 fragments and antibody proteins or fragments thereof which are fused with effector molecules, but are also suitable for complete antibody molecules One advantage of mammalian expression systems is that they give nse to very good glycosylation and folding conditions, e.g transient expression systems, e.g. in COS-cells or stable expression systems e.g. BHK, CHO, myeloma cells (cf. also Example 2). Mammalian cells may also be used, for example, with viral expression systems e.g. Vaccinia, Semhki-Forest-Virus and Adenovirus. Transgemc animals such as cows, goats and mice are also suitable for a process according to the invention. Transgenic plants such as Nicotiana tabacum (tobacco) may also be used in a process according to the invention They are particularly suitable for the preparation of antibody fragments according to the invention. After genomic integration of the nucleic acid according to the invention which codes for an antibody protein according to the invention which is fused to a signal sequence, secretion of the antibody protein into the interstitial space can be achieved. The invention relates in particular to a process according to the invention wherein said host is a mammalian cell, preferably a CHO or COS cell.
The invention relates in particular to a process according to the invention wherein said host cell is co-transfected with two plasmids which carry the expression units for the light or the heavy chain.
The antibody proteins of the present invention are highly-specific agents for guiding therapeutic agents to the FAP antigen. Therefore another prefened antibody protein according to the invention is characterised in that said antibody protein is coupled to a therapeutic agent.
This antibody protein according to the invention may preferably also be coupled to a therapeutic agent or an effector molecule by genetic engineering. According to the invention, a therapeutic agent of this kind includes cytokines, such as for example interleukins (IL) such as EL-1, EL-2, EL-3, EL-4, IL-5, IL-6, EL-7, EL-8, EL-9, EL-10, IL-11, EL-12, IL-13, EL-14, IL-15, EL-16, EL-17, EL-18, interferon (EFN) alpha, EFN beta, EFN gamma, EFN omega or EFN tau, tumour necrosis factor (TNF) TNF alpha and TNF beta, TRAIL, an immunomodulatory or immunostimulant protein, or an apoptosis- or necrosis- inducing protein. Therefore the antibody-effector molecule conjugates according to the invention comprise antibody-cytokine fusion proteins, and also bispecific antibody derivatives and antibody-superantigen fusion proteins. These are preferably used for activating the body's own anti-tumoral defence mechanisms and are thus suitable for therapeutic use. Another prefened FAP-specific antibody protein according to the invention is characterised in that it is used for somatic gene therapy. For example, this may be achieved by use as an antibody toxin-fusion protein (as described for example in Chen et al. 1997, Nature 385: 78-80 for other targets) or as a fusion protein consisting of an antibody according to the invention and a T-cell receptor or Fc-receptor (transmembrane and intracellular region, cfi. e.g. Wels et al., 1995, Gene, 159: 73-80). The use for somatic gene therapy may also be carried out by expression of the nucleic acid according to the invention in a shuttle vector, a gene probe or a host cell. Another prefened antibody protein according to the invention is charactensed in that said therapeutic agent is selected from among the radioisotopes, toxins or lmmunotoxins, toxoids, fusion proteins, for example, genetically engineered fusion proteins, inflammatory agents and chemotherapeutic agents and elements which allow a neutron captunng reaction, such as e g boron (boron-neutron captunng reaction, BNC) Another prefened antibody protein according to the invention is characteπsed in that said radioisotope is a β-emitting radioisotope
Another prefened antibody protein according to the invention is characteπsed in that said radioisotope is selected from among 6rhenιum, 188rhenιum, 'iodine and 90yttπum which have proved particularly suitable for linking to the antibodies according to the invention as therapeutic agents A process for radio-iodine labelling of the antibodies according to the invention is descπbed in WO 93/05804
Another prefened antibody protein according to the invention is charactensed in that it is labelled
Another prefened antibody protein according to the invention is characteπsed in that it is labelled with a detectable marker
Another prefened antibody protein according to the invention is charactensed in that the detectable marker is selected from among the enzymes, dyes, radioisotopes, digoxygenme, streptavidine and biotin
Another prefened antibody protein according to the invention is characteπsed in that it is coupled to an imageable agent
Another prefened antibody protein according to the invention is characteπsed in that the imageable agent is a radioisotope
Another prefened antibody protein according to the invention is characteπsed in that said radioisotope is a γ-emitting radioisotope
Another prefened antibody protein according to the invention is charactensed in that said radioisotope is 125ιodιne
Another important aspect of the present invention relates to a pharmaceutical preparation which contains an antibody protein according to the invention and one or more pharmaceutically acceptable earner substances Pharmaceutically acceptable earners or adjuvants in this invention may be physiologically acceptable compounds which stabilise or improve the absorption of antibody protein according to the invention, for example Such physiologically acceptable compounds include , for example, carbohydrates such as glucose, sucrose or dextrane, antioxidants such as ascorbic acid or glutathione, chelating agents, lower-molecular compounds or other stabilisers or adjuvants (see also Remington's Pharmaceutical Sciences, 18th Edition, Mack PubL, Easton.). The skilled person knows that the choice of a pharmaceutically acceptable carrier depends, for example, on the route of administration of the compound. The said pharmaceutical composition may also contain a vector according to the invention for gene therapy and may additionally contain, as adjuvant, a colloidal dispersion system or liposomes for targeted administration of the pharmaceutical composition. A host or a host cell which contains a vector according to the invention may also be used in a pharmaceutical composition within the scope of this invention, for example, for gene therapy.
Another important aspect of the present invention relates to the use of a pharmaceutical preparation according to the invention for treating or imaging tumours, wherein said tumours are associated with activated stromal fibroblasts.
This use according to the invention relates particularly to cases wherein said tumours can be categorised as one of the following types of cancer or form the basis thereof and are therefore selected from among colorectal cancer, non-small-cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer.
Yet another important aspect of the present invention relates to the use of an antibody protein according to the invention for preparing a pharmaceutical preparation for treating cancer.
Yet another important aspect of the present invention relates to the use of an antibody protein according to the invention for imaging activated stromal fibroblasts. An additional aspect of the present invention is a process for detecting activated stromal fibroblasts in wound healing, inflammatory processes or in a tumour which is characterised in that a probe, which might possibly contain activated fibroblasts, is contacted with an antibody protein according to the invention under conditions which are suitable for forming a complex from said antibody protein with its antigen and the formation of said complex and hence the presence of activated stromal fibroblasts in wound healing, inflammatory processes or in a tumour is detected. The process according to the invention described in the previous paragraph is particularly characterised in that said tumour is selected from among colorectal cancer, non-small-cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer.
The invention further includes a process for detecting tumour stroma wherein a suitable probe is contacted with an antibody protein according to the invention under suitable conditions for the formation of an antibody-antigen complex, the complex thus formed is detected and the presence of the complex thus formed is conelated with the presence of tumour stroma.
The process according to the invention described in the previous paragraph is particularly characterised in that said antibody is labelled with a detectable marker.
The following Examples are intended to aid the understanding of the invention and should in no way be regarded as limiting the scope of the invention.
Example 1
1 Cloning of a human VH repertoire for the guided selection method A) Development of anti-FAP antibodies with fully human V regions
Method of preparation: 1 Cloning of F19 VH 2. Preparation of human V-repertoire
• Reverse transcπption, PCR amplification of human VL (λ, K) repertoires from peripheral blood lymphocytes, an improved process according to Persson et al 1991, PNAS 88 2432.
• Cloning the VL repertoires in Phage display vector (pSEX81 , DKFZ, Heidelberg; Breithng et al., 1991, Gene 104:147) size of repertoire: VL 107 clones
• Reverse transcπption, PCR amplification of human VH repertoire (IgG, IgD, IgM) from peripheral blood lymphocytes, thymus gland, spleen, bone manow, tonsils, lymph nodes, foetal liver (improved according to Persson et al. 1991, PNAS 88: 2432)
• Improvement of process: Use of IgD and different lymphoid tissue
• Cloning the VH repertoire in Phage display vector (pSEX81 , DKFZ, Heidelberg; Breithng et al., 1991, Gene 104 147) size of repertoire: VH 3x10 clones 3. Selection of human VL regions which functionally replace VL F19:
• Phage display selection and Guided selection strategy with VH F19 as the guiding structure
(improved according to McCafferty et al., 1990, Nature 348- 552 and Jespers et al., 1994, Bio/Technology 12: 899) =>ιsolatιon of human FAP-specific VL regions (known as VL: III5, I IIO, IEE25, III43) 4. Selection of human VH regions which functionally replace VH F19 or impart FAP- specificity: • Phage display selection and Guided selection strategy with various VL as the guiding structures
(improved according to McCafferty et al., 1990, Nature 348: 552 and Jespers et al., 1994, Bio/Technology 12: 899) =>isolation of the following human FAP-specific scFv: scFv #13: VH #13, IgG; VL ITI25 scFv #46: VH #46, IgG; VL III25 scFv #50: VH #50, IgD, VL III25
Sequence of the selected VH and VL regions:
(see Figures)
Antigen binding properties
• ELESA: Detection of antigen specificity for human FAP
• Competition for antigen binding by cF19 (detected for scFv #13)
• Studies of binding to FAP+ cells: scFv #13 (as bivalent in minibody format) EC50: 8 - 12 nM (see below) scFv #50 (as bivalent in minibody format) EC50: 32 nM
• FAP-specific immunohistological staining of tumour biopsy material (detected for scFv #13 in the minibody format)
1) PCR amplification of the human VL- and VH repertoires: a) In order to prepare the VH and VL repertoires the various V-gene families are separately amplified from cDNA with the appropriate family-specific primers by PCR (see below). b) All Forward! 3 '-primers for VH- and VL-PCR amplification are complementary to the gene sequences of the constant immunoglobulin domains (IgG, IgD, IgM, K, λ). This allows efficient isotype-specific amplification of the V regions with very few 3 '-primers. By contrast, Marks et al., 1991 (J. Mol. Biol. 222: 581) use a plurality of different 3'- primers complementary to the J-sections of the V regions.
2) Preparation and cloning of a human VH repertoire: Preparation and cloning of a human VH repertoire consisting of a large number of clones (3 x 108) with high diversity (for method see below).
a) To ensure high diversity, commercially obtainable cDNA/RNA from different lymphoid s tissues from a very great number of donors was used as the starting material for the VH repertoires in addition to freshly isolated peripheral blood lymphocytes. By using bone manow and foetal liver naive V repertoires should be obtained and thus the prerequisites for isolating autoantibodies are created.
o Lymphoid tissues ( number of donors):
I) Commercial cDNA:
-Peripheral blood lymphocytes, PBL (550 donors) -spleen (5 donors) s -thymus gland (7 donors)
-bone manow (51 donors) -lymph nodes (59 donors) -tonsils (5 donors) -foetal livers (32 donors) o
ET) Commercial RNA which was subsequently circumscribed in cDNA in the laboratory (for method see Example 1 , A 1.2) -lymph nodes (25 donors)
- EH) PBL from fresh "buffy coats" (10 donors) (for method see below)
En the prior art only the following lymphoid tissues have hitherto been described as sources of V repertoires. (The combinations of the tissues and the numbers of donors are shown):
-PBL (15 donors), bone manow (4 donors), tonsils (4 donors) (Vaughan et al., 1996; o Nature Biotechnology 14: 309)
-spleen (3 donors) and PBL (2 donors) (Sheets et al., 1998; PNAS 95: 6157
-bone manow (Williamson et al., 1993; PNAS 90:4141) -lymph nodes (1 donors) (Clark et al., 1997; Clin. Exp. Immunol. 109: 166)
b) Moreover, the IgD repertoire was additionally amplified, as well as the IgM and IgG repertoires, to obtain a great repertoire diversity. For this, an IgD-specific PCR primer was developed (see below).
c) It proved to be very important to purify the PCR fragments of the human VH repertoire after the treatment with restriction enzymes, over an agarose gel. In subsequent cloning of this repertoire into a Phage display vector it was thus possible to achieve a very high proportion of clones with a functional scFv expression cassette. This is an essential prerequisite to obtaining a genetically stable Phage display repertoire (for method see Example 1, A 1.4).
3) Preparation of a combination repertoire consisting of a human VH repertoire and various human FAP-specific VL regions: Definition of combination repertoire:
Combination of a V repertoire with conespondingly complementary V-sequences by genetic engineering (complementary with regard to VH to VL and vice versa). The V- sequences used for the combination may consist of one V-sequence, a plurality of different sequences or a V repertoire.
a) Cloning strategy: En a Phage display vector the human VH repertoire was combined with a defined, non- FAP-specific VL region (dummy- L). This dummy-VL region could very efficiently be replaced by FAP-specific VL regions using restriction cutting sites. This created the conditions for effectively combining a previously tested human VH repertoire with specific human VL, in order to guarantee a diverse combination repertoire which contains a very high proportion (>95 %) of functional clones (in relation to the integrity of the scFv reading frame) (for method see below).
b) In order to increase the probability of selected a fully human scFv analogous to F19, the human VH repertoire was combined with the sequences of different human FAP-specific VL regions (VL: III10, EH25, EII5, EEI43). These human VL regions served as the guiding structures for selecting human FAP-specific VH. The FAP-specific human VL themselves had been isolated from a human VL repertoire in a previous Guided selection step with F19 VH.
c) DNA contamination of the combination repertoires with phagemid vectors which code for existing FAP-specific scFv (e.g. murine scFv from the hybridoma line F19 or the chimeric anti-FAP scFv with human VL and F19 VH), is a major technical problem. To overcome this, the following strategy proved necessary: After the Guided Selection step for the human anti-FAP VL-sequences with murine F19 VH as the guiding structure, this human VL-sequence without a VH-sequence was first sub-cloned in a plasmid (pUCBM21). Then this human VL region was excised using restriction enzymes and combined with the human VH repertoire which was already present in a Phage display vector. This prevented any FAP-specific V regions, apart from the VL-sequences of the relevant guide structure, from being introduced into the combination repertoire (for method see below).
4) Phage display selection:
The Phage display selection of the FAP-specific human V regions required the development of selective washing methods to prevent the accumulation of cross-reactive scFv (for method see below).
B) Development of human anti-FAP antibodies which contain the murine HCDR3 F19 (HCDR3 retaining guided selection):
Method of preparation:
1. Cloning of F19 VH
2. Preparation of human V-repertoire
• Reverse transcription, PCR amplification of human VL (λ, K) repertoires from peripheral blood lymphocytes (modified according to Persson et al. 1991, PNAS 88: 2432)
• Cloning of the VL repertoires in Phage display vector (pSEX81 , DKFZ, Heidelberg; Breitling et al., 1991, Gene 104:147), size of repertoire: VL 107 clones • Reverse transcription, PCR amplification of human VH repertoire from penpheral blood lymphocytes, (improved according to Persson et al. 1991, PNAS 88: 2432), PCR amplification of the VH segment consisting of
FR 1 +CDR 1 +FR2+CDR2+FR3
• Cloning of a repertoire consisting of the VH segment (FR1+CDR1+FR2+CDR2+FR3) in Phage display vector (pSEX81 , DKFZ, Heidelberg; Breithng et al., 1991, Gene 104- 147), size of repertoire- VH 4xl07 clones
3 Selection of human VL regions which functionally replace VL F19- (see A) 3)
4 Selection of a human VH region which contains HCDR3 from F19 and functionally replaces VH F19:
• HCDR3 retaining guided selection strategy with VL IEI43 or VL III5 and HCDR3 F19 + human FR4 as the guiding structure
(Our own process development improved according to McCafferty et al, 1990, Nature 348: 552; Jespers et al., 1994, Bio/Technology 12: 899 and Rader et al., 1998, PNAS 95: 8910)
=>isolatιon of the following human FAP-specific scFv, which contain muπne
HCDR3 F19: scFv #34- VH #34, IgG; VL EII43 scFv #18: VH #18, IgG; VL EEI43
Structure
(see Figures)
Antigen binding properties
• ELISA: detection of antigen specificity for human FAP
• competition for antigen binding by cF19 and mAb F19
• Studies of binding to FAP+ cells: scFv #34 and #18 (monovalent) EC5o: about 6 nM • FAP-specific immunohistological staining of tumour biopsy matenal (as an scFv #34-mmιbody)
1 1 RNA isolation
The mRNA source used was isolated total RNA from fresh lymphocytes from a total of 10 Buffy coats
In order to isolate the lymphocytes from Buffy Coat, 15 ml of Ficoll (LYMPHOPREP) were placed at ambient temperature in a 50 ml Falcon Tube and covered with 30 ml of Buffy Coat diluted 1 4 in RPMI medium After centπfuging for 30 mm at 700 g the interphase was removed and after the addition of 40 ml of RPMI medium centnfuged for 5 mm at 700 g The cell pellet was then washed once more with RPMI medium and once with PBS The cells were centnfuged after the last washing step and 200μl of RNA- Clean™ solution (AGS, Heidelberg) were added per 10° cells Immediately after the addition of the denatuπng solution the cells were homogenised by repeatedly passing up and down through a coarse cannula (size 1) and then through a finer cannula (size 18) The thin liquid lysate was mixed with 1/ 10 volume chloroform (p a ), shaken thoroughly and incubated on ice for 5 mm After centπfuging (15 min at 12000 g) the supernatant was roughly removed and mixed with an equal volume of isopropanol, incubated for 45 min at 4°C and then centnfuged at 12000 g for 45 min The supernatant was carefully poured off and the pellet was washed with ice-cold 70% ethanol The RNA pellet was then washed again with components of the RNA-Quick-Prep (Pharmacia) To do this, the pellet was taken up in a mixture of 113 μl of extraction buffer, 263μl of LiCl solution and 375μl of Cs-tπfluoroacetate, mixed thoroughly (Vortex) and centnfuged in an Eppendorf centπfuge tube (12000g) The RNA pellet was again washed with 70% ethanol, air-dned for 10 mm and adjusted with H20 to a concentration of lμg/ μl
Alternatively, the total RNA was isolated using an RNA isolation column made by QIAGEN (Midi) according to the manufacturer's instructions The mRNA was prepared from total RNA using the Ohgotex-Kit (Midi) made by QIAGEN The method used was in accordance with the manufacturer's instructions The isolated mRNA was mixed with 1/10 volume of 2 5 M RNAse-free K-acetate, pH 5 2, and precipitated by the addition of 2 5 volumes of ethanol p a at -20°C for 2 hours or overnight After centπfuging (45 min, 13000g , 4°C) the mRNA was washed twice with ice-cold 70 % ethanol (centnfugation for 5 mm at 12000g, 4°C) and after bπef air-drying dissolved in 10-20 μl of RNAse-free H20. In order to estimate the concentration the mRNA was compared with a total RNA standard dilution seπes. In order to do this, lμl of the sample to be measured was combined with 10 μl of ethidium bromide solution (l μg ml), dnpped onto a film and compared with the standardised concentration using a UV lamp The mRNA was used directly for the cDNA synthesis or frozen for storage at -80°C.
1.2 cDNA synthesis of the human VH regions
IgG, IgM and IgD specific VH-cDNA was prepared with mRNA using the cDNA Synthesis Kit produced by Boehπnger-Mannheim and Amersham. The first cDNA strand was synthesised with the Ig-specific pnmers HuIgGl-4 RT for the IgG library, HulgM-RT for the IgM library or HulgDelta for the IgD library Optionally, ohgo(dT) and ohgo-hexa- nucleotides were used. The cDNA synthesis was earned out with 100 ng of mRNA according to the manufacturer's instructions; to detach the secondary structures the mRNA had to be heated to 70 °C for 10 min immediately before use. The cDNA was synthesised in a 20 μl mixture with AMV-Reverse transcnptase in a Thermocycler for 60 min at 42°C. The quality of the cDNA was checked by PCR amplification using the pair of pnmers HulgGFOR and HuVHBl, by way of example. For this purpose 10" dilutions of the cDNA were used as template and the maximum dilution at which a specific band of the PCR product was still detectable in agarose gel after 36 cycles was determined.
1.3 PCR amplification of the human VH repertoire
The cDNA of each human lymphatic organ was used separately as a Template for the PCR amplification of the VH regions. Six separate PCR batches were set up from each lymphatic organ, one of the six VH-specific 5 'pnmers (HuVHBl to HuVHB6) being combined with one of the isotype-specific 3 'pnmers HulgGFOR, HulgMFOR or HulgDFOR. The amplification was earned out in a 50μl reaction mixture with 1 μl of Template cDNA (200pg), 25mM MgCl2 , 5μl of Goldstar reaction buffer, 200μM of each dNTP (Pharmacia) and 25pmol of each pnmer. After 10 min at 95°C , 0.6 U of Goldstar- polymerase was added and the preparation was coated with PCR wax. 36 amplification cycles were earned out, each with 15 s denatunng at 94°C, 30 s addition at 52-55°C and 30 s elongation at 72 °C. After the last amplification step had ended, an additional elongation was carried out for 15 min at 72°C.
In order to introduce the restriction cutting sites Nco I and Hind III onto the amplified VH regions a second PCR amplification was carried out with the primers extended by the restriction cutting sites (HuIgGFORHENDIII, HuIgMFORHENDIII, HuIgDHINDI I as the 3' primers and HuVHBlNCOI to HuVHBόNCOI as the 5' primers), lμl of the reaction solution of the first PCR mixtures were used as the template. The second PCR amplification was carried out over 15 cycles with in each case 15 s denaturing at 94°C, 30 s addition at 65°C and 30 s elongation at 72 °C. The final amplification step is followed once again by an additional elongation step for 5 min at 72 °C. The amplified materials which were based on the same isotype were combined and, in order to reduce the volume, precipitated by the addition of 1/10 volume of Na-acetate, pH 5.2, and 2.5 volumes of ethanol p. a. for 2 hours at -20°C and dissolved in TE buffer. En order to eliminate the primers the precipitated PCR fragments were separated on a 1.5% agarose gel and the 400 Bp fragment of the VH region was excised. The fragment was isolated according to the manufacturer's instructions using the QIA ExEI-Kit made by QIAGEN (Hilden). Elution was performed with preheated elution buffer for 5 min at 50 °C.
1.4 Digestion of the PCR-amplified VH regions with restriction enzymes
The gel-purified VH regions (of the three isotypes) were first digested in a lOOμl mixture with 70 U of Hind EH for 2 hours in buffer B and then incubated for a further 2 hours by the addition of 20 μl of buffer H, 60 U of Ncol and topping up to 200μl. Any digested overhangs were eliminated using the QIA-Quick PCR-Kit and the fragments were eluted with preheated EB buffer. The eluate was purified once more over a 1 % agarose gel and eluted with the QIA ExII Kit in 25 μl of EB buffer. Et was found that this additional gel purification step significantly increases the percentage of functional inserts after ligation into the vector. The digested PCR fragments were divided into aliquots and stored at -20°C.
1.5 Ligation of the human VH repertoire into a phagemid vector A Phage display vector pSEX81 which already contained the human VL-sequence of a hapten-specific Ab (Dummy PX-sequence) was used to clone the PCR-amplified VH repertoire
20 μg of vector pSEX81(VH&VLphox) were digested in a total volume of 125 μl with 40 U of Ncol (Boehnnger-Mannheim) and 60 μl of Hind III (Boehnnger-Mannheim) in buffer H for 2 hours at 37°C After the addition of 30μl of 6-tιmes concentrated Loading Buffer (30% glycerol, 30 mM EDTA) the digestion mixture was heated to 65°C for 10 min and slowly cooled at ambient temperature Vector DNA was separated from the insert in a 1 % agarose gel and isolated using the QIAGEN Gel elution kit The elution was done twice, each time with 50 μl of elution buffer (preheated to 50°C) for 5 min The elution fractions were pooled and the cut vector DNA was precipitated by the addition of 1/ 10 volume of sodium acetate, pH 5 2, and 2 5 volumes of ethanol p a at -20°C for 2 hours If necessary the vector DNA thus cut may also be stored at -20°C After 30 minutes' centπfugation (13000 g, 4°C ) and washing with -20°C cold 70% ethanol, the DNA was dπed and dissolved in 50 μl of 10 mM TRIS pH 7.9
In order to estimate the precise amount for the subsequent ligation, 2 μl of the vector DNA was compared with standardised DNA fragments (High-Mass Ladder, Gibco Life Technologies) For a direct compaπson, the VH-PCR fragments prepared in Example 1, A 1 4 were compared with standardised DNA fragments of lower molecular weight on the same gel (Low-Mass Ladder, Gibco Life Technologies)
A ligation mixture with an equimolar insert to vector ratio proved to be ideal In 40 μl of final volume, 500 ng of vector DNA and 50ng Insert DNA) were incubated with l μl of hgase and 4μl of ligation buffer The ligation was earned out overnight at 16°C using the T4 DNA-hgase made by Boehnnger Mannheim The ligation reaction was stopped by the addition of 60 μl of TE buffer The proteins were eliminated by the addition of 100 μl of chloroform/phenol mixture (1 1), bπef mixing (Vortex) and subsequent centnfuging at 13000 g The aqueous phase was removed and extracted again with chloroform to eliminate the phenol completely 90μl of vector DNA solution were precipitated by the addition of 9 μl of 3 M Na acetate (pH 5 2), 225 μl of ethanol p a. and lμl of glycogen (Boehnnger Mannheim) as earner (see above) for 2 hours at -20°C After centπfuging at 12000 g (4°C) and washing with ice-cold 70 % ethanol the DNA was air-dπed and taken up in 25 μl of water Inefficient restπction digestion duπng the vector preparation lead to vector DNA which is uncut or cut once, with the result that in the VH repertoire cloning the size of repertoire is falsified by rehgation of the incompletely cut vector For early momtoπng of the completeness of the restnction digestion, the prepared vector was ligated comparatively, with and without a VH insert, transformed in E coli and the number of clones was determined With efficient restπction digestion of the vector the number of clones in the vector sample without an insert was <1 %, compared with the mixture in which the vector with a VH insert had been used
2 Subcloning the human FAP-specific VL regions, combining the human VH- repertoires with various human FAP-specific VL
In order to avoid DNA contamination with existing FAP specific DNA-sequences in the construction of the scFv gene libraπes, the human VL-chains selected were first cloned in the expression vector pUCBM21 (Boehnnger-Mannheim) To do this, the FAP-specific VL-chains were each excised from the phagemid vector (pSEX 81 ) used for the selection with Mlul and Notl (Boehnnger-Mannheim ) and recloned into the conespondingly cut pUCBM21. After transformation in E coli a clone was picked for each VL-chain, amplified in LBAτ -medium and the vector DNA was isolated using the Nucleobond Kit (Macherey & Nagel). The human VL chains were excised from 15μg of pUC-plasmid in 150μl of restπction mixture with Mlul (60U) and Notl (60U) and isolated in a 1% agarose gel. These human FAP-specific VL were cloned into conespondingly cut Phage display vectors which contain the VH repertoires The method used to clone the VH regions was as descπbed above The combination banks with the different VL region were kept separate Ahquots of these combination banks were frozen and used for the selection of fully human FAP specific scFv
3 Phage display selection
Production of the phage-associated scFv
En order to avoid possible growth advantages for the vaπous VL-chains in the first round of panning, the phage-associated scFv of the vanous combination banks which contain the different human VL regions (see point 2) were produced independently of one another. To do this, 10ml of 2YTAτ medium in a chicane shaking flask were inoculated with one aliquot of the VL/VH combination banks with an OD of 0.4 and cultivated, with agitation (180 rpm) at 37°C until an OD of 0.8 was reached. After infection with 1012 helper phages (New England Biolabs) incubation was carried out, without agitation, for 15 min at 37°. After subsequent incubation with agitation at 37°C the bacteria were removed by centrifuging (4000g for 5 min) and the pellet was resuspended in 50 ml of glucose- free 2YTAT medium containing kanamycin (65 μg/ml). The phage-associated scFv was produced overnight with vigorous agitation (200rpm) at 30°C. In order to harvest the phages the bacteria were removed by centrifuging (9000 g) and the supernatant was mixed with PEG and incubated on ice for one hour in order to precipitate it. After subsequently centrifuging for 30 minutes at 9000 g at 4 °C the phages precipitated were resuspended in 45 ml of 4°C cold PBS and mixed with 5 ml of 5x PEG. After a further hour's incubation on ice, the mixture was again centrifuged at 9000 g and the phage pellet was resuspended in 5 ml PBS. The phages were filtered through a 0.45 μm filter and 500 μl of each phage preparation were combined and mixed with 2 ml of 4% milk powder suspension in PBS (MPBS) for 15 min. The phage suspension was clarified by centrifuging twice with 14000 g in a bench centrifuge. The phages thus preadsorbed had to be used the same day.
Selection of antigen-specific scFv:
Immunotubes (N nc-Maxi-Soτb-Immunotubes 3.5 ml ) immobilised with 5-30 μg CD8- FAP the day before were used for the selection. The immobilisation was carried out at 4°C overnight in PBS, then the tubes were washed twice with PBS and the unspecific binding sites were blocked for one hour with ROTI-Block (Roth). In order to investigate the specificity of the phage display selection, an immunotube without immobilised antigen was used for control purposes. After washing three times with PBS, the phage-associated scFv preadsorbed in MPBS were placed in the antigen-coated test tubes or the control test tubes and incubated on a roller for 2 hours.
To prepare the Plating bacteria, 20 ml of 2YTtet per mixture were inoculated with one aliquot of an XL- 1 -Blue overnight culture with an OD of 0.0125 and cultivated at 37°C with agitation (180 rpm). After three hours' incubation the Plating bacteria reached an OD of 0.8 and were then available for this time for infection with the eluted phages. One hour before infection, the phage suspensions were emptied out of the Immunotubes. Then the Immunotubes were washed to eliminate any unspecific and cross-reactive scFv. n the first round of panning the preparations were washed lOx with TPBS (0.1% Tween 20) and then lOx with PBS. The stringency was increased in the second and third rounds of panning by extending the washing steps to 15x TBBS (2nd round of panning) and 20x TPBS (3rd round of panning) as well as by increasing the concentration of Tween20 to 0.5%). To increase the stringency further, in the last two rounds of panning a vortex was briefly used during the washing with TPBS in order to mix the washing solution more thoroughly.
The final washing solution was discarded and 1 ml of 1 M TEA (triethylamine) was added to the immunotubes. After five minutes' incubation in a roll incubator, the eluted phages were neutralised with 0.5 ml of 1 M TRIS, pH 7.4, and added directly to the 20 ml of plating bacteria for infection.
After incubation for 15 min without agitation at 37°C the bacteria were agitated for 45 min and removed by centrifuging at 3000g for 10 min. The bacteria were resuspended in 500μl of 2YT medium and incubated on large SOBGAT plates (15cm) overnight at 37°C. For harvesting, the cells were scraped from the plate with LBAT medium, mixed with 25%> final concentration of glycerol and frozen in aliquots at -80°C or used for inoculation of another round of amplification.
The phage titre of each round of panning was determined by titration of 0.01-10 μl of the infected plating bacteria. In order to determine the specific concentration, in each selection round the number of eluted phages from CD8-FAP immobilised immunotubes was compared with that of the conesponding control immunotubes without an antigen. The ratio of quantities of the eluted phages from the antigen-coated immunotubes and the uncoated immunotubes yielded the concentration factor.
An increase in the concentration factor after successive amplification round indicated a concentration of specifically binding phages.
Example 2
Expression of the human FAP-specific scFv derivatives
Screening process on a microtitre scale for evaluating phage display-selected scFv The scFv-pIII- fusion proteins expressed using pSEX81 may be used both for Screening, i.e. sampling, and for analysis of scFv clones selected from phage display banks,.
Bacterial Production of scFv-pIH-fusion protein on a microtitre scale
300 μl aliquots of 2YTGAτwere inoculated with colonies set out individually on LBGAT plates and incubated overnight (o-n) in 96-well microtitre plates (Beckman) at 37°C and 300 rpm with agitation. If the colonies to be analysed were not to be stored frozen, this initial incubation was carried out in U-shaped 96-well tissue culture plates (Greiner). The next morning, 10 μl aliquots of these o-n cultures were transfened into a fresh 100 μl of 2YT and incubated again, with agitation, in U-shaped 96-vve// tissue culture plates in a damp chamber at 37°C. The residue of the cultures left in the Beckman microtitre plates was able to be mixed with glycerol at 20 %> and frozen at -80°C. The growth of the 100 μl of cultures could be checked if necessary with an ELISA Reader at a filter wavelength of 630 nm. After about 6-8 h the cultures were centrifuged at 1200 rpm (5 min, RT) and the supernatants were removed with a multichannel pipette. The pelleted bacteria were resuspended in 100 μl aliquots of 2YTAτ (without glucose) incl. 50 μM EPTG and incubated o-n with agitation in the damp chamber at 30°C and 300 rpm. After o-n incubation the cultures were each mixed with 25 μl of 0.5 % Tween and incubated with agitation for a further 3-4 h to achieve partial lysis. Finally, the cultures were centrifuged for 10 min at 1200 rpm and the supernatants were carefully removed. These were used directly for Western-Blot analysis or after preadsorption used in the ELISA.
Production of scFv-pHI-fusion protein on the ml scale
If only small numbers of clones were to be investigated for their expression and/or for the functionality of the scFv-pEH- fusion protein expressed, the overnight precultivation as well as the main cultivation of the bacteria were carried out in a volume of 3-10 ml in test tubes or in 50 ml PP-test tubes with agitation at about 200 rpm. If the bacterial growth had reached its logarithmic phase (O.D.όoonm about 0.7) the cultures were centrifuged (2500 rpm, 5 min, RT) and resuspended in an equal volume of fresh SBAτ or 2YTAτ incl. 50 μM- EPTG for induction. After o-n incubation at 25-30°C either the cultures were mixed with Tween 20 (ad 0.1 %) and the supernatants were removed after 3 h of further incubation. However, in order to increase the concentration of the fusion proteins, the bacteπal pellet could also be opened up (see below)
The scFv-glll-fusion proteins were used to demonstrate the integπty of the reading frames of the scFv-coding region (Western blot) and to investigate the FAP specificity of the scFv selected in the ELISA on immobilised FAP or in the cell analyser on FAP+ cells. An anti- glll-specific monoclonal antibody combined with a peroxidase- or alkaline phosphatase- conjugated detection antibody (Western-Blot and ELISA) was used to detect the scFv-glll- fusion proteins. In the case of cell binding studies with the scFv-glll proteins in the cell analyser an FITC-labelled detection antibody was used.
Prokaryotic Expression-
Media
All the data relate to a final volume of 1 L, the pH was adjusted to 7.0. The following additions of media were filtered stenle and optionally added to the autoclaved medium.
G:100 mM glucose (stock solution.: 2 M), A: ampicilhn 100 μg/ml, T- tetracychne 12,5 μg/ml, K: kanamycin 50 μg/ml
Liquid media for the bactenal culture:
BHI Brain Heart Enfusi on (DEFCO)
35 g yeast extract 5 g
dYT peptone 17 g yeast extract 10 g
NaCl 5 g
LB peptone 10 g yeast extract 10 g
NaCl 5 g SB peptone 30 g yeast extract 10 g
MOPS 10 g
SOC peptone 20 g yeast extract 5 g
NaCl 10 mM
KC1 2 5 mM
After autoclaving, stenle MgCl2 and MgSO4 are added ad 10 mM in each case, as well as stenle glucose ad 20 mM
Agar dishes
BHI (amounts per Petπ dish)
BHI (without yeast)30 ml agar agar 1 % saccharose (60 %) 0.5 ml horse serum 2.5 ml yeast extract (20 %) 1 ml glucose (20 %) 0 5 ml saccharose, serum, yeast extract, glucose are all added stenle
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, stenle MgCl is added ad 10 mM Other abbreviations- G: glucose, A: ampicilhn, T. tetracycline, K. kanamycin Bacteπal expression of scFv in E coli pOPE vectors and denvatives obtained therefrom were used to prepare a simple soluble scFv denvative with cmyc- and HIS6-Tag in E coli (Dubel et al , 1993, Gene 128 97-101) The scFv expression in E coli and the puπfication thereof are earned out according to the processes of Moosmayer et al , 1995 (Ther Immunol 2 31-40)
The scFv was produced in E coli XLl-Blue in volumes of 3-100 ml. The incubation took place wither in test tubes or in 50 ml PP-test tubes with agitation at about 200 rpm or in Erlenmeyer chicane flasks at 180 rpm in LB or 2YT medium The media were buffered with 1/10 vol MOPS (pH 7) and mixed with tetracycline (12 5μg/ml) for the strain XLl- Blue
2YTGAT OΓ LBGAI was inoculated with colonies separated out on LBGAT plates to form a preliminary culture and incubated o-n at 37°C with agitation The next day the main culture was inoculated 1 50 therewith and incubated at 37°C For induction the centnfuged bactena (2500 rpm, 1000 x g, 10 min, RT) were taken up in an equal volume of medium (without glucose) with 50 μM-EPTG and agitated for 2-3 h at 22-25°C and 220 rpm The bactenal pellet was harvested after centnfugation at 1000 x g (10 mm, RT) and broken up as follows The harvested pellets of the induced E coli cultures were taken up in 1/20 - 1/30 vol of ice-cold PBS and thoroughly resuspended, incubated for about 30 min on ice with occasional mixing and flash-frozen in liquid nitrogen or m a mixture of ethanol and dry ice The frozen sample could then be stored at -80°C To break it up the sample was slowly thawed and subjected to ultrasound treatment (25-30 cycles while cooling with ice water) until it was homogeneous and clear In order to obtain the entire soluble fraction of bacteπal protein, the sample was centnfuged for 20 min at 13000 rpm, the supernatant was carefully removed and the pellet was discarded For longer storage, if desired, the supernatants were mixed with BSA (ad 1%) , flash frozen and stored at -80°C In the preparation of scFv F19 in E coli, a drastic detenoration in the functionality of the recombinant proteins was observed if excessively rich (SB medium) or unbuffered culture media were used
Expression of scFv denvatives in Proteus mirabilis (L VI)
Monomenc scFv as well as dimeπc scFv (minibodies) were expressed in Proteus mirabilis
The expression and puπfication process was analogous to that which we have already published for soluble monovalent scFv (Rippmann et al., 1998, Applied and Environmental Microbiology 64: 4862-4869). Transformation of plasmid DNA in P. mirabilis LVI:
The incubation of P. mirabilis L VI was carried out in Erlenmeyer flasks (without chicanes) at >200 rpm. For transformation of the L VI bacteria they had to be in the stationary growth phase (OD550 ~ 6). To do this, 20 ml of a BHIκ culture were inoculated 1 :20 from a 4°C culture and incubated o-n at 37°C with agitation. Every 100 μl of the o-n culture were mixed with 20 μl of the prepared plasmid and 150 μl of PEG (incl. 0.4 M-saccharose) and stored on ice for 10 min. The temperature shock lasted for 5 min with occasional gentle agitation in a water bath at 37°C. The transformed LVI-bacteria were taken up in 1 ml of BYS medium ( 1 ml BHI, 0.5 % yeast extract, 1 % saccharose) and incubated for 3 h with vigorous agitation in a small steep-walled container at 37°C. 100 μl of each transformation mixture were plated out on a BHIK plate. After 24 - 48 h incubation (37°C) significantly large colonies were pricked out using a sterile spatula and transfened into 20 ml of BHIK medium. After o-n growth and microscopic monitoring for the presence of L-form bacteria, this culture was mixed with cryomedium and frozen at -80°C. Unfrozen transformed P. mirabilis cultures remained viable for at least 4 weeks if they were stored at 4°C. n order to induce expression in transformed P. mirabilis, two successive o-n or 11 - 12 h preliminary cultures were inoculated ( 20 ml each) and incubated at 30°C, the first of them from a 4°C culture. Depending on the density of the preliminary culture achieved and the length of incubation of the following culture, it was always overinoculated 1 :10 or 1:20. The BHIK induction cultures (incl. 0.5 mM-EPTG) had a volume of 20 - 50 ml and were also inoculated, then incubated at 30°C with agitation for at least 11 h. Before the harvesting of the bacteria, the OD550 (≥ 4), the pH (7.5 - 8.5) and the optical appearance of the L forms were examined under the microscope. The expression culture was centrifuged (5000 rpm, 3800 x g, 4°C) and the pellet was discarded. The supernatant could be used directly for ELISA or Western Blot analysis or it could be purified. En this study, the minibodies were purified by EMAC (immobilized metal affinity chromatography). 1 ml HiTrap columns made by Pharmacia Biotech were used for this. Gel chromatography was carried out as the second purification step.
Before the induction supernatant was applied, it was thoroughly dialysed against 5 1 of cold PBS (pH 8), then ultracentrifuged for at least 30 min (113000 x g, 4°C, rotor: Beckman 45 Ti). The column had to be charged with Zn2+ ions before each purification: The solutions used were filtered sterile beforehand to prevent clogging by the particles. Residues of metal ions were eliminated with 5 ml of 50 mM EDTA. After rinsing with 10 ml of H20b,d charging was carried out with 10 ml of 100 mM ZnSO . After rinsing again with 20 ml of H20b,d the column was equilibrated with 10 ml of PBS (pH 8). The supernatant was applied to the column using a peristaltic pump (1.5 ml/min), followed by a washing step (10 ml PBS incl. 5 - 20 mM imidazole). Elution was carried out in 1 ml fractions with 10 ml PBS incl. 300 mM imidazole. The elution fractions were stored on ice.
For the gel chromatography a Superdex 200 column (10/30) made by Pharmacia Biotech was used. In conjunction with an FPLC apparatus made by the same manufacturer. The IMAC-purified sample was centrifuged for 5 min (13000 rpm, 4°C) before the injection. After the equilibration of the pump system and column with the chosen elution buffer (PBS, pH 8), 500 μl (conesponding to 0.75 - 1 mg) of EMAC-purified MB #34 were injected into the system, pumped at a flow rate of 0.5 ml/min, detected with a UN-detector and automatically collected in 500 μl fractions.
Structure of the recombinant human antibodies
The pro- and eukaryotic expression of the human recombinant anti-FAP-antibodies took place as monovalent scFv and bivalent scFv (so-called minibodies). The structure of the minibodies produced and the expression cassettes used for this purpose is comparable with those described by Hu et al. 1996 (Cancer Res. 56: 3055-61). In addition, the minibodies we prepared have a c-myc domain at the C-terminus for immunological detection (with the monoclonal antibody 9E10) and a HIS6 domain for chromatographic purification. The cmyc- and HIS6-coding sequences conespond to those from pOPE 101 (S. Dubel, University of Heidelberg).
Structure of the minibodies:
Ν-signal sequence-scFv(VH-linker- VL)-hinge-linker-CH3 -cmyc-HISό-C
Prokaryotic expression of antibody proteins according to the invention: The expression vectors used and the processes for the expression and punfication of monovalent scFv denvatives in E coli (Moosmayer et al., 1995, Ther. Immunol. 2→ 31-40) and Proteus mirabilis LVI (Rippmann et al, 1998, Applied and Environmental Microbiology 64 4862-4869) are known from the pnor art The vector pACK02scKan and the processes from Rippmann et al , 1998 were also used to prepare and puπfy a minibody in Proteus mirabilis L VI
Eukaryotic expression of the antibody proteins according to the invention
The minibodies descπbed were also prepared in mammalian cells The expression vectors used for the minibody expression cassettes were p AD-CM V-l and a pgldl05 deπvative
Transient expression in COS cells
For transfecting COS 7 cells, the expression vector was first amplified in E coli (XLl- Blue) and then punfied The vector DNA was adjusted to a concentration of l μg/μl under steπle conditions and stored at -20°C
On the day before the transfection 5xl05 COS7 cells were seeded in a cell culture Petπ dish (8cm diameter, Greiner ) in DEMEM 10%>FCS and incubated for 16 h at 37°C in a CO2 heating cupboard On the day of the transfection, a suspension was prepared consisting, per Petπ dish, of 1 ml of OptiMEM (Gibco), 35 μl of hpofectamine (Gibco Life Science) and 10 μg of expression vector DNA After incubation at ambient temperature for 45 min a further 4 ml of OptMEM were added and the suspension was carefully pipetted over the cells which had previously been washed with PBS. The solution was distπbuted by gentle tilting and incubated for 5 hours at 37°C. The Petπ dish was filled with 5 ml of preheated DEMEM 20%FCS and incubated for 16 h at 37°C. Then the incubation medium is carefully suction filtered and replaced by 10 ml of OptiMEM After another 48 hours' incubation time at 37°C the supernatant was removed for harvesting and the cells were removed by centnfuging at 700 g A further centnfugation step at 12000g pelleted the remaining cell fragments The supernatant was either ultracentπfuged for 30 mm (60000 xg for 30 min) and then added to an EMAC column (Amersham-Pharmacia) or evaporated down to 1/ 40 to 1/80 volume in centπfugal concentrators with a 30 kDa separation threshold (Fugisept-Midi or MaxiRohrchen, Intersept). The centnfugation was earned out according to the manufacturer's instructions at 6000 g and usually took 6 hours The concentrated protein solution was mixed with 1% BSA, divided into lOOμl aliquots and after flash freezing in N2 stored at -80°C.
Stable expression in CHO cells:
Stable transfectants of CHO DG44 were prepared for the expression of FAP-specific minibodies.
Transfection:
1st day: 2xl05 cells were seeded in one well of a 6-well plate
2nd day: Careful suction filtering of the cell culture supernatant and subsequent addition of 800 μl CHO-SFM II medium plus HT supplement (Gibco BRL).
Preparation of the transfection suspension: 6 μl of lipofectamine + 200μl of CHO- SFM El with HT supplement + 3 μl (3μg) of expression vector. The suspension was mixed and carefully added to the cells. 3rd day: Change of medium: addition of CHO-SFM II without an HT supplement.
The change of medium was repeated regularly. For the gene amplification and for increasing the expression of foreign genes methotrexate was added to the medium from a period 10 - 14 days after the transfection. The methotrexate concentration was slowly increased; the concentrations were between 10 and 1000 nM.
The minibodies were produced in T-culture flasks or in a bioreactor.
Determining the apparent cell binding affinity of the recombinant anti-FAP antibodies
FAP+ cells were incubated in parallel batches with various concentrations of mono- or bivalent scFv derivatives. The binding of these recombinant antibodies was determined using an FETC-labelled detection antibody in a cell analyser (Coulter). The concentration of the scFv derivatives at which half the maximum saturation of the binding signal was achieved was chosen as a measurement of the apparent affinity.
Example 3 Sequences The sequences are shown here by way of example. Smaller mutations, e.g. the substitution of one or a few amino acids or the nucleotides coding therefor are also included in the invention.
- VH13 Protein sequence such as may be found in the minibody vector, for example. The first amino acid may also be an E (glutamate).
QVQLVESGGTLVQPGGSLRLSCAASGFTFSSYAMSWERQAPGKGLEWVSGISASG GYEDYADSVKGRVTESRDNSKNMAY .- LQMSSLRAEDTALYYCAKGGNYQMLLDHWGQGTLVTVSSASTKGPKL
Nucleotide sequence corresponding to VH13
CAGGTACAGCTGGTGGAGTCTGGGGGAACCTTGGTACAGCCTGGGGGGTCCCT is GAGACTCTCCTGTGCAGCCTCTGGATT
CACCTTTAGCAGCTATGCCATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGC
TGGAGTGGGTCTCAGGTATTAGTGCTA
GTGGTGGTTATATAGACTATGCCGATTCCGTGAAGGGCCGGGTCACCATCTCC
AGAGACAATTCCAAGAACATGGCATAT 20 CTACAAATGAGCAGCCTGAGAGCCGAGGACACGGCCCTTTATTACTGTGCGAA
AGGAGGCAACTACCAGATGCTATTGGA
CCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC
CAAAGCTT
-- VH 46 Protein sequence.
QVQLVQSGAEVKKDGASVKVSCKATGGTFSGHAISWVRQAPGQRLEWMGEISPM
FGTPNYAQSFQGRVTITADESTSYME
VSSLRSEDTATYYCARGANYRALLDYWGQGTLVTVSSASTKGPKL
Nucleotide sequence conesponding to VH46 such as may occur in the minibody, for example. The sixth nucleotide may also be an A instead of a G - a silent mutation, hence having no effect on the amino acid sequence.
« CAGGTACAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGGATGGGGCCTCAGT
GAAGGTCTCCTGCAAGGCTACTGGAGG
CACTTTCAGCGGTCACGCTATCAGTTGGGTGCGACAGGCCCCTGGGCAAAGAC
TTGAGTGGATGGGGGAGATCAGCCCTA
TGTTTGGAACACCAAACTACGCACAGAGCTTCCAGGGCAGAGTCACGATTACC 0 GCGGACGAATCTACGAGTTACATGGAG
GTGAGCAGCCTGAGATCTGAGGACACGGCCACTTATTACTGTGCGAGAGGTGC
GAACTACCGGGCCCTCCTTGATTACTG
GGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAAGC
TT VH50 Protein sequence as occurs in the minibody Again, the same applies as for VH13 The first amino acid may also be an E
QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQD GSEKYYVDSVKGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCARGSLCTDGSCPTIGPGPNWGQGTLVTVSSAPTKAPKL Nucleotide sequence conesponding to VH50 as occurs in the mmibody, for example
CAGGTACAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGCCTCTGGATT
CACCTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGC
TGGAGTGGGTGGCCAACATAAAGCAAG
ATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCC
AGAGACAACGCCAAGAACTCACTGTAT
CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAG
AGGTTCACTCTGTACTGATGGTAGCTG
CCCCACCATAGGGCCTGGGCCAAACTGGGGCCAGGGAACCCTGGTCACCGTCT
CCTCAGCACCCACCAAGGCTCCGAAGC
TT
VLIII25 Protein
DIQMTQSPSSLSASTGDRVTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQT
GVPSRFSGSGSGTDFTLTITSLQS
EDFATYYCQQYYEYPPTFGQGTRVEIKRTVAAPSVFAA
Nucleotide sequence conesponding to VLIII25
GACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAGGAGACAG
AGTCACCATCACTTGTCGGGCGAGTCA
AGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAAGCCCCTC
ATCTCCTGATGTCTGGAGCAACCACTT
TACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCACGTCCCTGCAGTCT
GAAGATTTTGCAACTTATTACTGTCAACAGTATTATATTTACCCTCCGACGTTC
GGCCAAGGGACCAGGGTGGAAATCAA
ACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC
Protein VH34 with the first 8 amino acids of CH I including
QVQLQQSGAEVKKPGSSVKVSCKASGGTFSTHTΓNWVRQAPGQGLEWMGGIAPM
FGTANYAQKFQGRVTITADKSTSTAY
MEMSSLRSDDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKL
Nucleic acid sequence conesponding to VH34 CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGT
GAAGGTCTCCTGCAAGGCTTCTGGAGG
CACCTTCAGCACCCATACTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGC
TTGAGTGGATGGGAGGGATCGCCCCTA
TGTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACAATTACC
GCGGACAAATCCACGAGCACAGCCTAC
ATGGAGATGAGCAGCCTGAGATCTGACGACACGGCTGTGTATTACTGTGCAAG
AAGAAGAATCGCGTACGGTTACGACGA
GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAG
CCTCCACCAAGGGGCCAAAGCTT
VH18 with some amino acids of CEI1 :
QVQLVQSGAELKKPGSSMKVSCKASGDTFSTYSINWVRQAPGQGLEWMGVΓNPS
GGSTSYAQKFQGRVTMTRDTSTSTVY
MELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKL
Nucleic acid sequence conesponding to VH 18 :
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGTTGAAGAAGCCTGGGTCCTCGAT
GAAGGTCTCCTGCAAGGCTTCTGGAGA
CACCTTCAGCACCTATTCTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGC
TTGAGTGGATGGGAGTAATCAACCCTA
GTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACC
AGGGACACGTCCACGAGCACAGTTTAC
ATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTGCGAG
AAGAAGAATCGCGTACGGTTACGACGA
GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAG
CCTCCACCAAGGGCCCAAAGCTT
VL chain III43
DIQMTQSPSSLSASTGDRVTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQT
GVPSRFSGSGSGTDFTLTISSLQA
EDVAVYYCQQYYRTPFTFGQGTKLEEKRTVAAPSVFAA
Nucleic acid sequence conesponding to III43:
GACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAGGAGACAG
AGTCACCATCACTTGTCGGGCGAGTCA
AGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAAGCCCCTC
ATCTCCTGATGTCTGGAGCAACCACTT
TACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGCCTGCAGGCT
GAAGATGTGGCAGTTTATTACTGTCAGCAATATTATCGTACTCCGTTTACTTTT
GGCCAGGGGACCAAGTTGGAGATCAA
ACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC VH 13 YOL VL III25 Protein sequence of the total antibody protein, as occurs in the minibody
QVQLVESGGTLVQPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWVSGISASG GYIDYADSVKGRVTISRDNSKNMAY
LQMSSLRAEDTALYYCAKGGNYQMLLDHWGQGTLVTVSSASTKGPKLEEGEFSE ARVDIQMTQSPSSLSASTGDRVTITC
RASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTITSLQS EDFATYYCQQYYEYPPTFGQGTR o VEIKRTVAAPSVFAA
Nucleotide sequence conesponding to VH 13 YOL VL III25
CAGGTACAGCTGGTGGAGTCTGGGGGAACCTTGGTACAGCCTGGGGGGTCCCT < GAGACTCTCCTGTGCAGCCTCTGGATT
CACCTTTAGCAGCTATGCCATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGC
TGGAGTGGGTCTCAGGTATTAGTGCTA
GTGGTGGTTATATAGACTATGCCGATTCCGTGAAGGGCCGGGTCACCATCTCC
AGAGACAATTCCAAGAACATGGCATAT o CTACAAATGAGCAGCCTGAGAGCCGAGGACACGGCCCTTTATTACTGTGCGAA
AGGAGGCAACTACCAGATGCTATTGGA
CCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC
CAAAGCTTGAAGAAGGTGAATTTTCAG
AAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTA 5 CAGGAGACAGAGTCACCATCACTTGT
CGGGCGAGTCAAGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGG
GAAAGCCCCTCATCTCCTGATGTCTGG
AGCAACCACTTTACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTG
GGACAGATTTCACTCTCACCATCACGT . CCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATATTTACC
CTCCGACGTTCGGCCAAGGGACCAGG
GTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC
VH46 YOL VL III25 Protein sequence of the total antibody protein as occurs in the minibody, for example
QVQLVQSGAEVKKDGASVKVSCKATGGTFSGHAISWVRQAPGQRLEWMGEISPM FGTPNYAQSFQGRVTITADESTSYME
VSSLRSEDTATYYCARGANYRALLDYWGQGTLVTVSSASTKGPKLEEGEFSEARV 0 DIQMTQSPSSLSASTGDRVTITCRA
SQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTITSLQSED
FATYYCQQYYEYPPTFGQGTRVE
EKRTVAAPSVFAA
5 Nucleotide sequence conesponding to VH46 YOL VL III25 CAGGTACAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGGATGGGGCCTCAGT
GAAGGTCTCCTGCAAGGCTACTGGAGG
CACTTTCAGCGGTCACGCTATCAGTTGGGTGCGACAGGCCCCTGGGCAAAGAC
TTGAGTGGATGGGGGAGATCAGCCCTA , TGTTTGGAACACCAAACTACGCACAGAGCTTCCAGGGCAGAGTCACGATTACC
GCGGACGAATCTACGAGTTACATGGAG
GTGAGCAGCCTGAGATCTGAGGACACGGCCACTTATTACTGTGCGAGAGGTGC
GAACTACCGGGCCCTCCTTGATTACTG
GGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAAAGC w TTGAAGAAGGTGAATTTTCAGAAGCAC
GCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTCTCTGCATCTACAGGAG
ACAGAGTCACCATCACTTGTCGGGCG
AGTCAAGATATTAGCAGTTATTTAGCCTGGTATCAACAGGCACCCGGGAAAGC
CCCTCATCTCCTGATGTCTGGAGCAAC CACTTTACAGACTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAG
ATTTCACTCTCACCATCACGTCCCTGC
AGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATATTTACCCTCCGA
CGTTCGGCCAAGGGACCAGGGTGGAA
ATCAAACGAACTGTGGCTGCACCATCTGTCTTCGCGGCCGC
VH 50 YOL VL III25 Protein sequence of the total antibody protein as occurs in the minibody, for example (for possible variation see VH50, above)
QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQD .< GSEKYYVDSVKGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCARGSLCTDGSCPTIGPGPNWGQGTLVTVSSAPTKAPKLE
EGEFSEARVDIQMTQSPSSLSASTG
DRVTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFT
LTITSLQSEDFATYYCQQYYEYPP so TFGQGTRVEEKRTVAAPSVFAA
Nucleotide sequence conesponding to VH 50 YOL VL III25
CAGGTACAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT -. GAGACTCTCCTGTGCAGCCTCTGGATT
CACCTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGC
TGGAGTGGGTGGCCAACATAAAGCAAG
ATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCC
AGAGACAACGCCAAGAACTCACTGTAT 40 CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAG
AGGTTCACTCTGTACTGATGGTAGCTG
CCCCACCATAGGGCCTGGGCCAAACTGGGGCCAGGGAACCCTGGTCACCGTCT
CCTCAGCACCCACCAAGGCTCCGAAGC
TTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCT 45 CCATCCTCACTCTCTGCATCTACAGGA
GACAGAGTCACCATCACTTGTCGGGCGAGTCAAGATATTAGCAGTTATTTAGC
CTGGTATCAACAGGCACCCGGGAAAGC CCCTCATCTCCTGATGTCTGGAGCAACCACTTTACAGACTGGAGTCCCATCAAG GTTCAGCGGCAGTGGATCTGGGACAG
ATTTCACTCTCACCATCACGTCCCTGCAGTCTGAAGATTTTGCAACTTATTACT GTCAACAGTATTATATTTACCCTCCG
ACGTTCGGCCAAGGGACCAGGGTGGAAATCAAACGAACTGTGGCTGCACCATC TGTCTTCGCGGCCGC
VH34YOLIII43 Protein sequence of the total antibody protein:
QVQLQQSGAEVKKPGSSVKVSCKASGGTFSTHTENWVRQAPGQGLEWMGGIAPM
FGTANYAQKFQGRVTITADKSTSTAY
MEMSSLRSDDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKLEE
GEFSEARVDIQMTQSPSSLSASTGDR
VTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTI
SSLQAEDVAVYYCQQYYRTPFTF
GQGTKLEEKRTVAAPSVFAA
Nucleotide sequence conesponding to VH34YOLIII43:
CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGT
GAAGGTCTCCTGCAAGGCTTCTGGAGG
CACCTTCAGCACCCATACTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGC
TTGAGTGGATGGGAGGGATCGCCCCTA
TGTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACAATTACC
GCGGACAAATCCACGAGCACAGCCTAC
ATGGAGATGAGCAGCCTGAGATCTGACGACACGGCTGTGTATTACTGTGCAAG
AAGAAGAATCGCGTACGGTTACGACGA
GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAG
CCTCCACCAAGGGGCCAAAGCTTGAAG
AAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCC
TCACTCTCTGCATCTACAGGAGACAGA
GTCACCATCACTTGTCGGGCGAGTCAAGATATTAGCAGTTATTTAGCCTGGTAT
CAACAGGCACCCGGGAAAGCCCCTCA
TCTCCTGATGTCTGGAGCAACCACTTTACAGACTGGAGTCCCATCAAGGTTCAG
CGGCAGTGGATCTGGGACAGATTTCA
CTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGC
AATATTATCGTACTCCGTTTACTTTT
GGCCAGGGGACCAAGTTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTT
CGCGGCCGC
VH18 YOL III43 Protein sequence of the total antibody protein:
QVQLVQSGAELKKPGSSMKVSCKASGDTFSTYSENWVRQAPGQGLEWMGVENPS GGSTSYAQKFQGRVTMTRDTSTSTVY
MELSSLRSEDTAVYYCA-RRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPKLEE GEFSEARVDIQMTQSPSSLSASTGDR VTITCRASQDISSYLAWYQQAPGKAPHLLMSGATTLQTGVPSRFSGSGSGTDFTLTI
SSLQAEDVAVYYCQQYYRTPFTF
GQGTKLEIKRTVAAPSVFAA
Nucleotide sequence conesponding to VH18 YOL III43
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGTTGAAGAAGCCTGGGTCCTCGAT
GAAGGTCTCCTGCAAGGCTTCTGGAGA
CACCTTCAGCACCTATTCTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGC
TTGAGTGGATGGGAGTAATCAACCCTA
GTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACC
AGGGACACGTCCACGAGCACAGTTTAC
ATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTGCGAG
AAGAAGAATCGCGTACGGTTACGACGA
GGGCCATGCTATGGACTACTGGGGTCAAGGAACCCTTGTCACCGTCTCCTCAG
CCTCCACCAAGGGCCCAAAGCTTGAAG
AAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCC
TCACTCTCTGCATCTACAGGAGACAGA
GTCACCATCACTTGTCGGGCGAGTCAAGATATTAGCAGTTATTTAGCCTGGTAT
CAACAGGCACCCGGGAAAGCCCCTCA
TCTCCTGATGTCTGGAGCAACCACTTTACAGACTGGAGTCCCATCAAGGTTCAG
CGGCAGTGGATCTGGGACAGATTTCA
CTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGC
AATATTATCGTACTCCGTTTACTTTT
GGCCAGGGGACCAAGTTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTT
CGCGGCCGC
Example 4
MATERIALS AND METHODS construction of a V- gene library
Total RNA was isolated from penpheral blood lymphocytes (buffy coats) of two naive donors mRNA was prepared with an mRNA isolation kit (Qiagen, Germany) cDNA was synthesized by ohgo dT-pπming
For the amplification of K and λ light chains a pπmary PCR was used applying the 5'- ohgonucleotides descnbed by Marks et al. (1991) as "human VK and Vλ back pnmers" and the 3' oligonucleotides descπbed as constant kappa and constant lambda pnmers by Welschof et al. (1995) 30 cycles with annealing at 56°C were chosen Secondary PCRs (max 14 cycles) served for adding the VL 5' cloning site Mlul and the 3' site Notl to the first amplificates Here, the 5' extension TAC AGG ATC CAC GCG TA served for adding the 5' cloning site Mlul to the back pnmers and the 5' extension TGA CAA GCT TGC GGC CGC added the Notl site to the constant VL pnmers The resulting 2nd PCR VL amplificates were run on an agarose gel and puπfied with a QiaEx kit (Qiagen, Germany) To clone the VL repertoire, the phagemid vector pSEX 81 (essentially as described in Breithng et al., 1991) was overdigested with Mlul and Notl. The restricted DΝA was purified using QiaQuick (Qiagen, Germany) and ligated overnight with VL PCR products, overdigested with the same endonucleases. The ethanol-precipitated ligations were used to transform E. coli XLl-Blue (Stratagene, California). Transformands were plated on 2YT plates containing 100 mM-glucose, 100 μg/ml ampicillin, 12.5 μg/ml tetracylin and grown overnight at 30 °C. Diversity of the cloned libraries was tested by ZfatΝI-digests of PCR- amplified V regions and analysis on polyacrylamid gels.
For the amplification of heavy chains a primary PCR was used applying the 5'- oligonucleotides already described by Marks et al. (1991) as "human VH back primers" for the Ν-terminus of VH and the following 3 '-oligonucleotides for the C-terminus of FR3 regions within the functionally reananged gene segment families: HU VG VH 1/3/4: TCT CGC AC A 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
With an annealing temperature of 55-58 °C 30 cycles were carried out. Secondary PCRs (max. 14 cycles) served for adding the VH 5' cloning site Ncol and the 3' site SpH, the latter facilitating the coupling of the FR1 to FR3 gene segments with the patental HCDR3. A few microliters of the 1 st PCR were used as a template for the above primers, with the following 5' sequences added: 5' primers: GAA TAG GCC ATG GCG. 3' primers: GGG GGC GGG CGT ACG CGA TTC TTC T. The new Spli site was inserted into the parental HCDR3 via PCR without changing the coding sense of it. This site enabled the cloning of all VH gene segment families known to be functionally reananged (fig 9).
Phage preparations and selection
To obtain phage associated antibodies (phabs), the overinfection of exponentially growing E. coli was carried out following Schier et al. (1996). After growth at 30 °C overnight bacteria were pelleted and phages were precipitated twice with 20 %> polyethylene glycol in 2.5 M-ΝaCl. For selection 1-20 μg FAP were coated in Maxisorb immunotubes (Νunc) rotating overnight at 4°C. After washing twice with PBS the coated tubes were blocked with 3 % non fat dry milk in PBS or with Roti-Block (Roth, Germany). Immediately before the panning, the tubes were washed twice with PBS. 1010 -1012 cfu were preadsorbed in 6 % non fat dry milk (working cone.) and used for selection tumbling at RT for 2 h. In round 1 and 2 of selection, 10 to 15 washing steps with PBS followed the same number of steps with PBS-Tween 20 (0.1 %). In later rounds the washing was increased to max. 25 times PBS-Tween and the same number of pure PBS. For a higher stingency during washing, the Tween concentration was raised to 0.5 % and considerable vortexing of the immunotubes was introduced. Elution of phages was done by either 100 mM-triethylamine or 0.1 M-HC1, pH 2.2. Eluted phages were immediately neutralized with Tris and used for infection of XL-1 Blue. After overnight growth at 30 °C, the bacteria were scraped from the agar plates and either used for a further round of selection or frozen with glycerol.
Screening for specific phabs
The screening of selected phabs was carried out as described elsewhere (Mersmann et al., 1998). Briefly, we induced the expression of scFv-pIII fusion proteins without producing complete phages. These fusion proteins were tested in ELISA on purified FAP and inelevant Ag. Binders that turned out to be FAP-specific were analyzed in competion ELISA where different amounts of a chimeric bivalent construct of the parental F19 served for synchronous competition. DNA-sequencing was done using fluorescent dideoxynucleotides and an ALFexpress (Amersham Pharmacia, Sweden) or by commercial service.
Affinity measurements
To estimate the functional affinity of Ab constructs, their half maximal saturation concentrations were determined on FAP overexpressing fibrosarcoma cells (HT1080). 105 FAP+ or control cells were incubated for 90 min with serial dilutions of the Ab construct. Detection was carried out by the anti-c-myc Ab 9E10 followed by an FITC labeled goat anti-mouse specific serum (in case of scFv) or by an FITC labeled goat anti-human specific serum (in the case of Mb). Incubations and washings were done on ice except for the labeled Abs which were applied at RT. Bound Ab contructs were detected in a FACStar (Becton Dickinson) or in an EPICS Flow Cytometer (Coulter). The mean fluorescence was measured for 10 cells in each dilution. The concentration of the applied Ab derivatives were determined in repeated estimations against a scFv or Mb standard used in SDS-PAGE and western blotting. Cloning, expression and purification of minibody (Mb)
The scFv cassettes of the selected clones 18 and 34 were excised from the scFv expression vector pOPElOl (Dubel et al , 1992) by restπction with Ncol! Notl and inserted into an equally prepared Mb-vector, pDl , a deπvative of the published vector pACK02scKan- (Pack et al , 1993) E coli XLl-Blue were transformed as usual, subsequently, the cell wall and outer membrane deficient strain LVI of Proteus mirabilis was transformed and induced to overnight expression according to Rippmann et al (1998) After dialysis against PBS the Mb was ultracentπfuged ( 1 13,000 xg, 4°C, 30 min) and purified by means of IMAC with a Zn2+loaded HiTrap column (Pharmacia, Sweden) Fiactions wered tested by SDS-PAGE and subsequent Coomassie staining
Stability assay for the Mb
The thermal stability of Mb #34 in RPMI medium containing 5 % FCS was by incubation of purified, freshly thawed Mb at 37 °C for up to 72 h After incubation the solution was centnfuged (20,000 xg, 4 °C, 10 min) and used on immobilized FAP in an ELISA. A preceding expenment was used to determine an appropπate dilution for each of the Mb preparations to reach distinct but non-saturated ELISA signals
Immunohistochemistry
Aceton-fixed fresh frozen sections of tumor tissues were used The tissue section were incubated (16 h) at 4 °C with the recombinant antibodies (10 μg/ml) followed by the anti-c myc Mab 9E10 for 1 h at room temperature Subsequently, a biotinylated horse anti-mouse serum was applied Detection of the Ag/Ab complexes was done by the avidin-biotin lmmunoperoxidase method As a negative control the section was only treated with biotinylated serum antibodies followed by the colonmetπc reaction Hams haematoxyhn was used for counterstaimng of the sections results
1 Selection of human VLs
A guided selection approach based on the scFv format was chosen for the substitution of the munne VL of the FAP specific antibody F19 first, followed by the humanization of the F19 Vli The vector pSEX81 was used, in which a VL repertoire deπved from naive human donors was combined with VH F19 to obtain a combinatoπal library of about 3 x 106 different clones
This library was phage display selected on immobilized FAP to isolate human VL F19 analogues After three rounds of selection, the screening for binders by ELISA yielded several FAP binding clones. To ensure the diversity of these isolated chimeπc scFv (murine VH/ human VI ) their phagemid DNA was analyzed by restπction enzymes and sequenced Various chimeπc scFv (now shortly named after their VL) could be identified (III5, III10, 11125, III43), consisting of the guiding VH of the paternal scFv F19 and the itemized human VLs. Table 1 shows the aa sequence homology of the selected light chains IEI5 and III43 compared to the replaced VL F19. Both listed VLs belong to the human VL subgroup kappa I according to Kabat (httpV/immuno.bme nwu edu/), and the germline gene with the closest homology is a member of the subgroup Vκl family (IH5: Ve; III43 Ve). Looking at the aa sequence, clone 1115 had as much as 64 % identity in FR positions compared to the parental F19, and 59 % identity in CDR positions. EII43 had 69 % identity in FR positions and, again, 59 % identity in CDR positions compared to F19. Additionally, Eϋ5 and IEI43 showed a high degree of mutations compared to their putative germline genes III5 differed in 14 aa positions from the sequence of the closest germline, IXI43 showed 17 differences (ImMunoGeneTics database : http://imgt.cnusc.fr.8104, and Cox et al., 1994)
Concerning binding charactenstics, the chimeπc scFv were highly specific for FAP (fig 7) Binding competition in ELISA with cF19, a chimenc, bivalent Ab compπsing the vaπable fragments of F19 and human constant domains, demonstrated a common epitope specificity of the selected chimeπc scFvs and the parental Ab (Fig. 8) To assess the functional affinities of the selected scFv, the concentrations leading to half maximal saturation of binding (SC50) were determined by sandwich ELISA using the c-myc tag for detection (Table 2) Using this assay, the parental scFv F19 had a functional affinity of 20 nM, scFv IEI5 of 45 nM, and scFv EIJ.43 of 20 nM. This indicates that the performed guided selection of VLs resulted in chimeric scFv of retained epitope specificity and with functional affinities in the nanomolar range.
2. Selection of humanized VHs:
In order to avoid an epitope shift during humanization of VH by guided selection as previously reported (Watzka et al. 1998), the parental HCDR3 of the murine mAb F19 was retained for subsequent selections. For this approach a phagemid vector was constructed containing HCDR3 F19, a human FR4 (found in Kabat subgroups I, II and III), and a new restriction site, which was introduced in HCDR3 without changing the aa sequence (fig. 9). In this vector, the selected VL III5 and VL EEE43 were inserted, respectively, to encode the specific guiding structures. In a subsequent step a cDNA derived VH segment library spanning heavy chain segments from FR1 to FR3, covering reananged sequences of all known VH germline families, was integrated into the phagemid. The resulting VH segment library (size: 4 x 107 clones) was combined with either VL III5 or VL EII43 and phage display selected on immobilized FAP.
As the selection of scFvs in phage associated form was frequently associated with strong unspecific binding, thus complicatind data analyses, various selection strategies were applied (data not shown). Only highly stringent washing conditions during the panning procedure led to the isolation of two highly antigen specific, FAP -binding clones after five successive rounds of selection. In table 3, the aa sequences of VH clone #18 and VH clone #34 are compared with the parental VH F19 and VH OS4 (a CDR grafted version of F19). Confining the comparison to the gene segment region from FR1 to 3, the selected clone #18 showed 66 % identity with the aa sequence of scFv F19 in the FRs, and 50 % identity in the CDRs 1 and 2. For the selected clone #34 the FR identity was 67 %>, and 55 % in CDR 1 plus 2. Both isolated VH chains use VL EEI43 as complement and belong to the human VH subgroup I, according to Kabat. For both VH, the closest germline gene segments were shown to belong to the VHl segment family, which represents about 12 % of all human VH gene segments (Guigou et al., 1990; Brezinschek et al., 1995). Compared to the VH 1 family (#18: DP-7, #34: DP-88), VH #18 and #34 showed 10 and 9 amino acids differences, respectively. Figure 10 shows the stnct FAP-specificity of the humanized scFv #18 and #34 in ELISA But in view of a potential clinical application of the selected human scFv, their binding characteπstics to natural cell membrane expressed FAP is of particular importance By flow cytometry we could demonstrate that scFv #18 and #34 bound to a FAP expressing human fibrosarcoma cell line, HT1080 in the same manner as the parental scFv F19 (fig 1 1) Saturation studies yielded in a functional cell binding afffinity (SC50) of 6 nM for scFv #18 and scFv #34, each In a parallel assay the SCso for the parental scFv F19 and its CDR grafted derivative, scFv OS4. respectively, were found to be 20 nM and 4 6 nM, indicating an even higher affinity of the selected scFv compared to the original Ab (table 2) Moreover, binding competition of scFv #18 and #34 with cF19 was dose dependent in ELISA (data not shown) and on FAP overexpressing cells as measured by flow cytometry, demonstrating the retained epitope specificity of the selected scFvs (fig 12) In view of potential clinical applications, the selected scFv were expressed as minibodies (Mb) using the L-form strain LVI of Proteus mirabilis (Gumpert and Taubeneck, 1983) This Ab format is advantageous for tumor targeting because of its bivalency, high tumor uptake and rapid blood clearance, resulting in a selective accumulation in the tumor (Hu et al., 1996) As expected, Mb #18 and Mb #34 exerted a high antigen specificity and retained F19 epitope specificity as demonstrated in antigen binding assays and by competition with cF19 (data not shown) Moreover, after affinity and size exclusion chromatography the functional affinity of Mb #34 on FAP-overexpressing cells was determined to be 2 nM (fig 13), exactly equaling the affinity assessed for the minibody version of the CDR grafted scFv OS4 (Mb OS4) Moreover, the Mb #34 turned out to have a high stability at 37°C in serum containing media, after 72 h of incubation the loss of binding activity was only 20 % (fig 14)
Lmmunohistological analyses with Mb #34 on cryo-sections of different human tumors led to a specific staining of the tumor stroma in breast, lung and colon carcinoma Furthermore, the malignant cells of a desmoid tumor and a malignant fibrous histiocytoma could be specifically detected by Mb #34 (fig 15) Hence, for both, tumors of epithelial and tumors of mesenchymal ongin, this human Mb exhibited an lmmunohistological staining pattern undistinguishable from that of F19 and Mb OS4 REFERENCES
Arap, W , Pasqualini, R., Ruoslahti E. (1998). Cancer Treatment by Targeted Drug Delivery to Tumor Vasculature in a Mouse Model. Science 279. 377-80
Brezinschck, H.-P., Brezinschek, R.I., Lipsky, P.E. ( 1995). Analysis of the Heavy Chain Repertoire of Human Peripheral B Cells Using Single-Cell Polymerase Chain Reaction, J Immunol 155: 190-202,
Bunows, F J , Thorpe, P E. (1993) Eradication of large solid tumors in mice with an lmmunotoxin directed against tumor vasculature Proc Natl Acad. Sci U S A 1993 90 8996-9000
Chothia, C , Lesk, A.M (1987) Canonical Strucures for the Hypervanable Regions of Immunoglobulins J Mol. Biol 196: 901-17
Chowdhury, P S , Viner, J.L , Beers, R , Pastan, I (1998) Isolation of a high-affinity stable single-chain Fv specific for mesothehn from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity Immunology 95: 669-
74
Colcher, D , Milenic, D.E., Fenoni, P , Canasquillo, J.A , Reynolds, J.C., Roselli, M., Larson, S.M. Schlom, J. (1990). In vivo fate of mononclonal antibody B72.3 in patients with colorectal cancer. J. Nucl. Med 31 : 1133-42
Coney, L.R., Daniel, P.T., Sanborn, D., Dhein, J., Debatin, K.M., Krammer, P.H., Zurawski, V.R. Jr (1994). Apoptotic cell death induced by a mouse-human antι-APO-1 chimenc antibody leads to tumor regression. Int. J. Cancer 58: 562-567
Cox, J.P.L., Tomhnson, I., Winter, G. (1994): A directory of human germ-line VK segments reveals a strong bias in their usage. Eur. J. Immunol. 24, 827-36
Figini, Marks, Winter, Griffiths (1994) In Vitro Assembly of Repertoires of Antibody Chains on the Surface of Phage by Renaturation; J. Mol. Biol. 239: 68-78
Folkman, J. (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med, 285 1 182-86
Foote, J , Winter, G (1992). Antibody Framework Residues Affecting the Conformation of the Hypervanable Loops. J. Mol. Biol. 224: 487-99
Francisco, J.A., Gilliland, L.K., Stebbins, M.R., Norπs, N.A., Ledbetter, J.A., Siegall, C.B (1995) Activity of a single-chain immunotoxin that selecively kills lymphoma and other B-hneage cells expressing the CD40 antigen. Cancer Res. 55. 3099-104
Gaπn-Chesa, P., Old, L J., Rettig, W J. (1990). Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers; Proc. Natl. Acad. Sci. USA 87: 7235-39
Guigou, V , Cuisinier, A.M., Tonnelle, C, Moinier, D., Fougereau, M., Fumoux, F. (1990). Human immunoglobulin VH and Vk repertoire revealed by in sity hybndization. Mol. Immunol 27 (9): 935-40
Griffiths AD, Williams SC, Hartley O, Tomhnson EM, Waterhouse P, Crosby WL, Kontermann RE, Jones PT, Low NM, Allison TJ, Prospero, TD, Hoogenboom, HR, Nissim, A, Cox, JPL, Harπson, JL, Zaccolo, M, Gherardi, E, Winter, G (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J 13 3245-60
Gumpert, J., Taubeneck, U. (1983) Characteπstic properties and biological significance of stable protoplast type L-forms. Exper Suppl 46 227-41
Hu, S , Shively, L , Raubitschek, A , Sherman, M , Williams, L E , Wong, J.Y , Shively, J.E , Wu, A.M (1996) Minibody. A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts Cancer Res 56. 3055-61
Huang, X , Molerna, G , King, S , Watkins, L , Edgington, T S , Thorpe, P.E ( 1997) Tumor Infarction in Mice by Antibody-Directed Targeting of Tissue Factor to Tumor Vasculature. Science 275: 547-50
Jespers, L.S , Roberts, A , Mahler, S.M., Winter, G , Hoogenboo , H R (1994) Guiding the Selection of Human Antibodies from Phage Display Repertoires to a Single Epitope of an Antigen Bio/Technology 12. 899-903,
Jones, P.T , Dear, P H , Foote, J , Neuberger, M.S , Winter, G (1986) Replacing the complementanty-determining regions in a human antibody with those from a mouse. Nature 321. 522-25
Kang, A.S., Jones, T.M., Burton, D.R (1991). Antibody redesign by chain shuffling from random combinatonal immunoglobulin libraπes. Proc. Natl. Acad. Sci. USA 88 1 1 120-23
Kettleborough, C A., Saldanha, J., Heath, V J., Mornson, C.J., Bendig, M.M (1991) Humanization of a mouse monoclonal antibody by CDR-graftmg- the importance of framework residues on loop conformation Protein Eng. 4 773-83
Khazaeh, M.B., Corny, R.M., LoBugho, A.F. (1994). Human Immune Response to Monoclonal Antibodies. J. Immunother. 15: 42-52
Knappik A , Ge L.; Honegger A., Pack P., Fischer M., Wellnhofer G , Hoess A , Wolle J., Pluckthun A , Virnekas B. (2000). Fully synthetic human combinatonal antibody hbranes (HuCAL) based on modular consensus frameworks and CDRs randomized with tπnucleotides . J Mol Biol 296- 57-86
Langer, R., Conn, H., Vacanti, J., Haudenschild, C, Folkman, J. (1980) Control of tumor growth in animals by infusion of an angiogenesis inhibitor. Proc Natl Acad Sci U S A 77 4331-5
Mack, M., Gruber, R , Schmidt, S , Riethmuller, G., Kufer, P. (1997) Biologic Properties of a Bispecific Single-Chain Antibody Directed Against 17-1A (EpCAM) and CD3. J Immunol. 158- 3965-70
Ohhn, M , Owman, H , Mach, M., Bonebaeck, C.A.K (1996) Light chain shuffling of a high affinity antibody results in a dnft in epitope recognition. Mol Immunol 33, 47-56
Ono, K., Ohtomo, T., Yoshida, K., Yoshimura, Y., Kawai, S., Koishihara, Y., Ozaki, S., Kosaka, M., Tsuchiya, M. (1999). The humanized antι-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity. Mol. Immunol.36 (6), 387-95 Park, J.E., Lenter, M.C., Zimmermann R.N., Gann-Chesa, P. Old, L.J. Rettig, W.J. (1999). Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts J. Biol. Chem. 274: 36505-12
Pegram, M.D., Lipton. A., Hayes, D.F., Weber, B.L., Baselga, J.M., Tripathy, D., Baly, D., Baughman, S.A., Twaddell, T., Glaspy, J.A., Slamon, D.J. (1998). Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-pl 85Her2/neu- overexpressing metastatic breast cancer refractory to chemotherapy treatment. J. Clin. Oncol. 16: 2659-71
Rader, C, Cheresh, D.A., Barbas, CF. 3rd (1998). A phage display approach for rapid antibody humanization: designed combinatorial V gene libraries. Proc. Natl. Acad. Sci. U S A 95: 8910-5
Rettig, W.J., Garin-Chesa, P., Beresford, H.R., Oettgen, H.F., Melamed, M.R., Old, L.J. (1988). Cell-surface glycoproteins of human sarcomas: Differential expression in normal and malignant tissues and cultured cells. Proc. Natl. Acad. Sci. USA 85: 31 10-14
Rettig, W.J., Gann-Chesa, P., Healey, J.H., Su, S.L., Ozer, H.L., Schwab, M., Albino, A.P., Old, L.J. (1993). Regulation and heteromeric structure of the fibroblast activation protein in normal and transformed cells of mesenchymal and neuroectodermal origin. Cancer Res.
53: 3327-35
Rippmann, J.F., Klein, M., Hoischen, C, Brocks, B., Rettig, W.J., Gumpert, J., Pfizenmaier, K., Mattes, R., Moosmayer, D. (1998). Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli. Appl. Environ. Microbiol. 64: 4862-9
Rippmann, J.F., Pfizenmaier, K., Mattes, R., Rettig, W.J., Moosmayer, D. (2000). Fusion of tissue factor extracellular domain to a tumor stroma specific scFv antibody results in an antigen-specific coagulation promoting molecule. Biochem. J., in press
Reynolds, J.C., Del Vecchio, S., Sakahara, H., Lora, M.E., Canasquillo, J.A., Neumann, R.D., Larson, S.M. ( 1989). Anti-murine antibody response to mouse monoclonal antibodies: clinical findings and implications. Int. J. Rad. Appl. Instrum 16: 121-5
Roselli, M., Guadagni, F., Buonomo, O., Belardi, A., Fenoni, P., Diodati, A., Anselmi, D., Cipriani, C, Casciani, C.U., Greiner, J., Schlom, J. (1996). Tumor markers as targets for selective diagnostic and therapeutic procedures. Anticancer Res. 16: 2187-92
Scanlan, M.J., Raj, B.K.M., Calvo, B., Garin-Chesa, P., Sanz-Moncasi, M.P., Healey, J.H., Old, L.J., Rettig, W.J. (1994). Molecular cloning of fibroblast activation protein a, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. Proc. Natl. Acad. Sci. USA 91 : 5657-61
Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302: 575-81
Vaughan, T.J., Williams, A.J., Pritchard, K., Osbourn, J.K., Pope, A.R., Earnshaw, J.C., McCafferty, J., Hodits, R.A., Wilton, J., Johnson, K.S (1996). Human Antibodies with Sub-nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library; Nat Biotechnol. 14: 309-314 Watzka, H., Pfizenmaier, K., Moosmayer, D. (1998). Guided selection of antibody fragments specific for human interferon gamma receptor 1 from a human VH- and VL- gene repertoire. Immunotechnology 3: 279-291
Weidner, N., Semple, J.P., Welch, W.R., Folkman, J. (1991). Tumor angiogenesis and metastasis -conelation in invasive breast carcinoma. N. Engl. J. Med. 324: 1-8
Welt, S., Divgi, C.R., Scott, A.M., Garin-Chesa, P., Finn, R.D., Graham, M., Carswell, E.A., Cohen, A., Larson, S.M., Old, L.J., Rettig, WJ.(1994). Antibody targeting in metastatic colon cancer: a phase I study of monoclonal antibody F19 against a cell-surface protein of reactive tumor stromal fibroblasts. J. Clin. Oncol. 12: 1193-203
Yanase, T., Tamura, M., Fujita, K., Kodama, S., Tanaka, K. (1993). Inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and metastasis of human cell lines in vitro and in vivo. Cancer Res. 53: 2566-70
Legend relating to the Figures
Fig. 1 : HCDR3-retaining guided selection
Fig. 2: Schematic representation of the HCDR3 sequence with the integrated Spll (Pfl23ll)
Fig. 3: Binding of scFv #13 (minibody format) to FAP+-cells (FACS analyses)
Fig. 4: Primers used for PCR amplification of the human V repertoire
Fig. 5: Primers for amplifying the human VH-gene segment repertoire for the HCDR3 retaining guided selection process
Fig. 6: Sequences of the selected human FAP-specific VL regions
Fig. 7: Ag specificity of selected chimeric scFv. ELISA wells were coated with FAP or inelevant Ag. TTX: tetanus toxoid; BSA: bovine serum albumin; HSA: human serum albumin; TF: transfemn; CHY: chymotrypsinogen; LYS: lysozyme; Detection was done with 9E10 and POD-labeled goat anti-mouse serum. Data are derived from triplicate values.
Fig. 8: Epitope specificity of selected chimeric scFv. Different concentrations of competitor were mixed with the respective scFv and added to FAP coated ELISA wells. The applied competitors were: cF19 (chimeric F19, with murine variable and constant human regions); hu IgG (unspecific human IgG serum). Detection was done as in figure 1. Data are from double values.
Fig. 9: Construction of the human VH gene segment library with retained HCDR3 F19. Schematic drawing of the final construct of VH, linker, VL and phage protein gpfll. By creation of a new restriction site the VH segment repertoire could be ligated to the preexisting HCDR3 F19, linked later to the selected human VLs. Fig. 10: Ag specificity of selected humanized scFv. Coating of ELISA wells and detection was earned out as in fig. 1. PLA' plastic
Fig. 11 : Binding of humanized scFv and Mb to cell surface-bound FAP analysed by flow cytometry. A) Binding of scFv #18 and #34 to FAP+cells Cells were incubated with 100-200 nM scFv from E. coli extracts. B) Binding of Mb #18 and #34 to FAP+cells. Supernatants of P mirabilis LVI containg 20 nM MB C Control binding of scFv F19 (punfied by IMAC) to FAP+cells Area for binding to FAP control cells is gray. scFv were detected by 9E10 and FITC-labeled Fc-specific anti-mouse serum, Mb by FITC-labeled Fc- specific anti-human serum. Each curve represents cytometer values of 5,000 predefined and measured events.
Fig. 12: Epitope specificity of humanized scFv for cellbound FAP. Different concentrations of competitor were mixed with the respective scFv and added to FAP+cells. cF19. chimeπc F19 (chimenc F19, with muπne vaπable and constant human regions); hu IgG: unspecific human IgG serum. Detection by 9E10 and FITC-labeled Fc-specific anti- mouse serum. Data represent cytometer values of 10,000 predefined and measured events.
Fig. 13: Assessment of apparent affinity for Mb #34 on FAP+cells. Mb #34 was purified by EMAC and size exclusion chromatography. Data are deπved from the cytometer with values of 10,000 events for each Ab concentration after detection with FITC-labeled Fc- specific anti-human serum.
Fig. 14: Long term stability of Mb #34 at 37°C. After incubation in a tenfold volume of RPMI (5% FCS) for 0 to 42 h, the IMAC punfied Mb was diluted and used in an anti-FAP ELISA. Detection was carried out with POD-labeled anti-human serum. Data are based on triplicate values. Fig. 15: lmmunohistological staining of biopsy material from FAP+ tumor sections with Mb #34. Cryo-sections of A) breast carcinoma B) colon carcinoma C) lung carcinoma D) desmoid tumor E) malignant fibrous histiocytoma were stained with Mb #34. Bound Mb was detected by subsequent treatment of the section with an anti-c-myc mAb (9E10), a biotinylated horse anti-mouse serum and the avidin-biotin immunoperoxidase complex. As a negative control F) a cryo-section was only treated with the detection antibodies and the avidin-biotin immunoperoxidase complex.

Claims

Patent Claims
1. Human or humanised antibody protein, which specifically binds to fibroblast activating protein alpha (FAPα), characterised in that either it is fully human or it contains not more than one murine complementarity-determining region (CDR region) of the monoclonal antibody F19 (ATCC accession number HB 8269).
2. Antibody protein according to claim 1, characterised in that it comprises a heavy chain (VH) of the class IgM.
3. Antibody protein according to claim 1 or 2, characterised in that it comprises a heavy chain (VH) of the class IgG.
4. Antibody protein according to one of claims 1 to 3, characterised in that it comprises a heavy chain (VH) of the class IgD.
5. Antibody protein according to one of claims 1 to 4, characterised in that it comprises a light chain (V ) of the lambda type (λ).
6. Antibody protein according to one of claims 1 to 5, characterised in that it comprises a light chain (VL) of the kappa type (K).
7. Antibody protein according to one of claims 1 to 6, characterised in that it is a Fab fragment.
8. Antibody protein according to one of claims 1 to 7, characterised in that it is an F(ab')2 fragment.
9. Antibody protein according to one of claims 1 to 8, characterised in that it is a single- chain-Fv protein (scFv).
10. Antibody protein according to one of claims 1 to 9, characterised in that it is a diabody antibody fragment.
11. Antibody protein according to one of claims 1 to 10, characterised in that it is a minibody antibody fragment.
12. Antibody protein according to one of claims 1 to 11, characterised in that it is a multimerised antibody fragment.
13. Antibody protein according to one of claims 1 to 12, characterised in that it is fully human.
14. Antibody protein according to one of claims 1 to 13, characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 1 (VH13).
15. Antibody protein according to one of claims 1 to 14, characteπsed in that the vaπable region of the heavy chain (VH) contains the ammo acid sequence ED No. 2 (VH46)
16 Antibody protein according to one of claims 1 to 15, characteπsed in that the vanable region of the heavy chain (VH) contains the amino acid sequence ID No 3 (VH50)
17 Antibody protein according to one of claims 1 to 16, characteπsed in that the vaπable region of the light chain (VL) contains the amino acid sequence ED No 4 (VLIII25)
18 Antibody protein according to one of claims 1 to 17, characteπsed in that the vanable region of the heavy chain (VH) IS coded by the nucleotide sequence ID No 5 (VH13) or by fragments or degenerate vanants thereof
19 Antibody protein according to one of claims 1 to 18, charactensed in that the vanable region of the heavy chain (VH) IS coded by the nucleotide sequence ID No 6 (VH46) or by fragments or degenerate vanants thereof
20 Antibody protein according to one of claims 1 to 19, charactensed in that the vanable region of the heavy chain (VH) is coded by the nucleotide sequence ED No. 7 (VH50) or by fragments or degenerate vanants thereof.
21. Antibody protein according to one of claims 1 to 20, charactensed in that the vaπable region of the light chain (V ) is coded by the nucleotide sequence ED No. 8 (VLIEI25) or by fragments or degenerate vaπants thereof.
22 Antibody protein, charactensed in that the vanable region of the heavy chain (VH) contains the ammo acid sequence ED No 1 (VH13) and the vanable region of the light chain (VH) contains the amino acid sequence ED No 4 (VLEEE25)
23 Antibody protein, characteπsed in that the coding sequence of the vaπable region of the heavy chain (VH) contains the nucleotide sequence ED No. 5 (VH13) and the coding sequence of the vaπable region of the light chain (VL) contains the nucleotide sequence ED No. 8 (VLIEI25)
24. Antibody protein, charactensed in that the vanable region of the heavy chain (VH) contains the ammo acid sequence ED No. 2 (VH46) and the vaπable region of the light chain (VL) contains the amino acid sequence ED No. 4 (VLHE25)
25. Antibody protein, characteπsed in that the coding sequence of the vanable region of the heavy chain (VH) contains the nucleotide sequence ED No. 6 (VH46) and the coding sequence of the vaπable region of the light chain (VL) contains the nucleotide sequence ED No. 8 (VLIIE25)
26. Antibody protein, characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 3 (VH50) and the variable region of the light chain (V ) contains the amino acid sequence ED No. 4 (VLEII25).
27. Antibody protein, characterised in that the coding sequence of the variable region of the heavy chain (VH) contains the nucleotide sequence ED No. 7 (VH50) and the coding sequence of the variable region of the light chain (VL) contains the nucleotide sequence ED No. 8 (VLEII25).
28. Antibody protein according to one of claims 1 to 12 and 14 to 21, characterised in that it is humanised.
29. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28, characterised in that it contains murine CDR 1 of the light chain (V ) of the monoclonal antibody F19.
30. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28 to 29, characterised in that it contains murine CDR 2 of the light chain (VL) of the monoclonal antibody F19.
31. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28 to 30, characterised in that it contains murine CDR 3 of the light chain (VL) of the monoclonal antibody F19.
32. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28 to 31, characterised in that it contains murine CDR 1 of the heavy chain (VH) of the monoclonal antibody F19.
33. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28 to 32, characterised in that it contains murine CDR 2 of the heavy chain (VH) of the monoclonal antibody F19.
34. Antibody protein according to one of claims 1 to 12, 14 to 21 or 28 to 33, characterised in that it contains murine CDR 3 of the heavy chain (VH) of the monoclonal antibody F19.
35. Antibody protein according to one of claims 1 to 34, characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 9 (VH34).
36. Antibody protein according to one of claims 1 to 35, characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 10 (VH18).
37. Antibody protein according to one of claims 1 to 36, characterised in that the variable region of the light chain (V ) contains the amino acid sequence ED No. 1 1 (VLEII43).
38. Antibody protein according to one of claims 1 to 37, characterised in that the variable region of the heavy chain (VH) is coded by the nucleotide sequence ED No. 12 (VH34) or by fragments or degenerate variants thereof.
39. Antibody protein according to one of claims 1 to 38, characterised in that the variable region of the heavy chain (VH) is coded by the nucleotide sequence ED No. 13 (VH18) or by fragments or degenerate variants thereof.
40. Antibody protein according to one of claims 1 to 39, characterised in that the variable region of the light chain (VL) is coded by the nucleotide sequence ED No. 14 (VLIII43) or by fragments or degenerate variants thereof.
41. Antibody protein, characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ED No. 9 (VH34) and the variable region of the light chain (VH) contains the amino acid sequence ED No. 1 1 (VLIII43).
42. Antibody protein, characterised in that the coding sequence of the variable region of the heavy chain (VH) contains the nucleotide sequence ID No. 12 (VH34) and the coding sequence of the variable region of the light chain (VL) contains the nucleotide sequence ID No. 14 (VLEU43).
43. Antibody protein, characterised in that the variable region of the heavy chain (VH) contains the amino acid sequence ID No. 10 (VH18) and the variable region of the light chain (V ) contains the amino acid sequence ED No. 11 (VLEII43).
44. Antibody protein, characterised in that the coding sequence of the variable region of the heavy chain (VH) contains the nucleotide sequence ED No. 13 (VH18) and the coding sequence of the variable region of the light chain (VL) contains the nucleotide sequence ED No. 14 (VLEπ43).
45. Nucleic acid, characterised in that it codes for an antibody protein according to one of claims 1 to 44.
46. Recombinant DNA vector, characterised in that it contains a nucleic acid according to claim 45.
47. Recombinant DNA vector according to claim 46, characterised in that it is an expression vector.
48. Host, characterised in that it contains a vector according to claim 46 or 47.
49. Host according to claim 48, characterised in that it is a eukaryotic host cell.
50. Host according to claim 48 or 49, characterised in that it is a mammalian cell.
51. Host according to one of claims 48 to 50, characterised in that it is a BHK, CHO or COS cell.
52. Host according to claim 48, characterised in that it is a bacteriophage.
53. Host according to claim 48, characterised in that it is a prokaryotic host cell.
54. Process for preparing antibody proteins according to one of claims 1 to 44, characterised in that it comprises the following steps: a host according to one of claims 48 to 51 is cultivated under conditions in which said antibody protein is expressed by said host cell and said antibody protein is isolated.
55. Process according to claim 54, characterised in that said host is a mammalian cell, preferably a CHO or COS cell.
56. Process according to claim 54 or 55, characterised in that said host cell is co-transfected with two plasmids which carry the expression units for the light or the heavy chain.
57. Antibody protein according to one of claims 1 to 44, characterised in that said antibody protein is coupled to a therapeutic agent.
58. Antibody protein according to claim 57, characterised in that said therapeutic agent is selected from among the radioisotopes, toxins, toxoids, boron, fusion proteins, inflammatory agents and chemotherapeutic agents.
59. Antibody protein according to claim 57 or 58, characterised in that said radioisotope is a β-emitting radioisotope.
60. Antibody protein according to claim 59, characterised in that said radioisotope is selected from among 186rhenium, 188rhenium, 131 iodine and 90yttrium.
61. Antibody protein according to one of claims 1 to 44, characterised in that it is labelled.
62. Antibody protein according to claim 61, characterised in that it is labelled with a detectable marker.
63. Antibody protein according to claim 61 or 62, characterised in that the detectable marker is selected from among the enzymes, dyes, radioisotopes, digoxygenine, streptavidine and biotin.
64. Antibody protein according to one of claims 1 to 44, characterised in that it is coupled to an imageable agent.
65. Antibody according to claim 64, characterised in that the imageable agent is a radioisotope.
66. Antibody according to claim 64 or 65, characterised in that said radioisotope is a γ- emitting radioisotope.
67. Antibody protein according to claim 66, characterised in that said radioisotope is 125iodine.
68. Pharmaceutical preparation, characterised in that it contains an antibody protein according to one of claims 1 to 44 and one or more pharmaceutically acceptable carrier substances.
69. Pharmaceutical preparation, characterised in that it contains an antibody protein according to one of claims 57 to 60 and one or more pharmaceutically acceptable carrier substances.
70. Pharmaceutical preparation, characterised in that it contains an antibody protein according to one of claims 64 to 67 and one or more pharmaceutically acceptable carrier substances.
71. Use of a pharmaceutical preparation according to one of claims 68 to 70, characterised in that it is used for the treatment or imaging of tumours wherein said tumours are associated with activated stromal fibroblasts.
72. Use according to claim 71 wherein said tumours are selected from among colorectal cancer, non-small-cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer.
73. Use of an antibody protein according to one of claims 1 to 44 for preparing a pharmaceutical preparation for treating cancer.
74. Use of an antibody protein according to one of claims 57 to 60 for preparing a pharmaceutical preparation for treating cancer.
75. Use of an antibody protein according to one of claims 64 to 67 for imaging activated stromal fibroblasts.
76. Process for detecting activated stromal fibroblasts in wound healing, inflammatory processes or in a tumour, characterised in that a probe, which might possibly contain activated fibroblasts, is contacted with an antibody protein according to one of claims 1 to 44 or 61 to 64 under conditions which are suitable for forming a complex from said antibody protein with its antigen and the formation of said complex and hence the presence of activated stromal fibroblasts in wound healing, inflammatory processes or in a tumour is detected.
77. Process according to claim 76, characterised in that said tumour is selected from among colorectal cancer, non-small-cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic brain cancer.
78. Process according to claim 76 or 77, characterised in that said antibody protein is a protein according to one of claims 61 to 63.
79. Process for detecting tumour stroma, characterised in that a suitable probe is contacted with an antibody protein according to one of claims 1 to 44 under suitable conditions for the formation of an antibody-antigen complex, the complex thus formed is detected and the presence of the complex thus formed is conelated with the presence of tumour stroma.
80. Process according to claim 79, characterised in that said antibody is labelled with a detectable marker.
81. Antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 15 or a part thereof or a functional variant thereof.
82. Antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 16 or a part thereof or a functional variant thereof.
83. Antibody protein, characterised in that it contains an amino acid sequence according to sequence ID No. 17 or a part thereof or a functional variant thereof.
84. Antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 18 or a part thereof or a functional variant thereof.
85. Antibody protein, characterised in that it contains an amino acid sequence according to sequence ED No. 19 or a part thereof or a functional variant thereof.
86. Antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 20 or a part thereof or a functional variant thereof.
87. Antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 21 or a part thereof or a functional variant thereof.
88. Antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 22 or a part thereof or a functional variant thereof.
89. Antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 23 or a part thereof or a functional variant thereof.
90. Antibody protein, characterised in that it is coded by a nucleotide sequence according to sequence ED No. 24 or a part thereof or a functional variant thereof.
91. Antibody protein, characterised in that it conesponds to the amino acid sequence according to sequence ED No. 15.
92. Antibody protein, characterised in that it conesponds to the amino acid sequence according to sequence ED No. 16.
93. Antibody protein, characterised in that it conesponds to the amino acid sequence according to sequence ED No. 17.
94. Antibody protein, characterised in that it conesponds to the amino acid sequence according to sequence ED No. 18.
95. Antibody protein, characterised in that it conesponds to the amino acid sequence according to sequence ED No. 19.
96. Antibody protein, characterised in that it is coded by the nucleotide sequence according to sequence ED No. 20.
97. Antibody protein, characterised in that it is coded by the nucleotide sequence according to sequence ED No. 21.
98. Antibody protein, characterised in that it is coded by the nucleotide sequence according to sequence ED No. 22.
99. Antibody protein, characterised in that it is coded by the nucleotide sequence according to sequence ED No. 23.
100. Antibody protein, characterised in that it is coded by the nucleotide sequence according to sequence ED No. 24.
EP01929604A 2000-03-17 2001-03-16 Human fap-alpha-specific antibodies Withdrawn EP1268550A2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10013286A DE10013286A1 (en) 2000-03-17 2000-03-17 New human humanized antibody that specifically binds to fibroblasts activating protein alpha, useful for treating cancer or tumor, and for imaging tumors associated with activated stromal fibroblasts, e.g. lung or breast cancer
DE10013286 2000-03-17
GB0022216 2000-09-11
GB0022216A GB0022216D0 (en) 2000-09-11 2000-09-11 Human FAP-a-specific antibodies for use in cancer therapy
PCT/EP2001/004716 WO2001068708A2 (en) 2000-03-17 2001-03-16 Human and humanized fap-alpha-specific antibodies

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EP1268550A2 true EP1268550A2 (en) 2003-01-02

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JP (1) JP2003530092A (en)
AU (1) AU2001256325A1 (en)
CA (1) CA2401252A1 (en)
WO (1) WO2001068708A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018129497A1 (en) 2017-01-09 2018-07-12 Bioxcel Therapeutics, Inc. Predictive and diagnostic methods for prostate cancer

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PE20021080A1 (en) * 2001-04-12 2003-02-12 Boehringer Ingelheim Int A SPECIFIC ANTIBODY FAPO BIBH1 IN THE TREATMENT OF CANCER
EP1806365A1 (en) * 2006-01-05 2007-07-11 Boehringer Ingelheim International GmbH Antibody molecules specific for fibroblast activation protein and immunoconjugates containing them
WO2011069019A2 (en) 2009-12-02 2011-06-09 David Ho J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (psma) and methods for their use
MA34519B1 (en) 2010-08-13 2013-09-02 Roche Glycart Ag ANTI-FAP ANTIBODIES AND METHODS OF USE
EP2673294B1 (en) 2011-02-10 2016-04-27 Roche Glycart AG Mutant interleukin-2 polypeptides
UA118028C2 (en) 2013-04-03 2018-11-12 Рош Глікарт Аг Bispecific antibodies specific for fap and dr5, antibodies specific for dr5 and methods of use
WO2014167083A1 (en) 2013-04-12 2014-10-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for predicting the risk of developing a colonic neoplasia
GB201402006D0 (en) * 2014-02-06 2014-03-26 Oncomatryx Biopharma S L Antibody-drug conjugates and immunotoxins
KR20180050321A (en) 2015-08-07 2018-05-14 이미지냅 인코포레이티드 An antigen binding construct for targeting a molecule
UA125962C2 (en) 2015-10-02 2022-07-20 Ф. Хоффманн-Ля Рош Аг Bispecific antibodies specific for a costimulatory tnf receptor
BR112019000015A2 (en) 2016-06-30 2019-04-24 Oncorus, Inc. pseudotyped oncolytic virus delivery of therapeutic polypeptides
JP2018035137A (en) 2016-07-13 2018-03-08 マブイミューン ダイアグノスティックス エイジーＭａｂｉｍｍｕｎｅ Ｄｉａｇｎｏｓｔｉｃｓ Ａｇ Novel anti-fibroblast activated protein (FAP) binding agent and use thereof
EP3555627B1 (en) 2016-12-14 2023-11-22 Purdue Research Foundation Fibroblast activation protein (fap)-targeted imaging and therapy
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies
US11865081B2 (en) 2017-12-29 2024-01-09 Virogin Biotech Canada Ltd. Oncolytic viral delivery of therapeutic polypeptides
CA3145872A1 (en) 2019-07-08 2021-01-14 3B Pharmaceuticals Gmbh Compounds comprising a fibroblast activation protein ligand and use thereof
EP3763726A1 (en) 2019-07-08 2021-01-13 3B Pharmaceuticals GmbH Compounds comprising a fibroblast activation protein ligand and use thereof
AU2020310538A1 (en) 2019-07-08 2022-01-27 3B Pharmaceuticals Gmbh Compounds comprising a fibroblast activation protein ligand and use thereof
KR102363980B1 (en) * 2020-04-13 2022-02-15 전남대학교산학협력단 Biomarker for diagnosis or prognosis analysis of brain metastasis and diagnosis method using same
AU2021276332A1 (en) 2020-05-19 2022-11-17 Boehringer Ingelheim International Gmbh Binding molecules for the treatment of cancer
WO2022148843A1 (en) 2021-01-07 2022-07-14 3B Pharmaceuticals Gmbh Compounds comprising a fibroblast activation protein ligand and use thereof
EP4050018A1 (en) 2021-01-07 2022-08-31 3B Pharmaceuticals GmbH Compounds comprising a fibroblast activation protein ligand and use thereof
WO2023125796A1 (en) * 2021-12-30 2023-07-06 Concept To Medicine Biotech Co., Ltd. Human antibodies against fap-alpha
WO2024074727A1 (en) 2022-10-07 2024-04-11 Genethon Immunotherapy of skeletal myopathies using anti-fap car-t cells

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587299A (en) * 1994-04-20 1996-12-24 Memorial Sloan-Kettering Cancer Center Isolated nucleic acid molecule coding for fibroblast activation protein alpha and uses thereof
EP0953639A1 (en) * 1998-04-30 1999-11-03 Boehringer Ingelheim International GmbH FAPalpha-specific antibody with improved producibility

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0168708A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018129497A1 (en) 2017-01-09 2018-07-12 Bioxcel Therapeutics, Inc. Predictive and diagnostic methods for prostate cancer

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JP2003530092A (en) 2003-10-14
AU2001256325A1 (en) 2001-09-24
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WO2001068708A2 (en) 2001-09-20
CA2401252A1 (en) 2001-09-20

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