EP1268550A2 - Humane und humanisierte fap-alpha-spezifische antikörper - Google Patents
Humane und humanisierte fap-alpha-spezifische antikörperInfo
- 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- Withdrawn
Links
Classifications
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- 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
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- 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)
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- 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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DE10013286A DE10013286A1 (de) | 2000-03-17 | 2000-03-17 | Humane FAP-alpha-spezifische Antikörper |
DE10013286 | 2000-03-17 | ||
GB0022216A GB0022216D0 (en) | 2000-09-11 | 2000-09-11 | Human FAP-a-specific antibodies for use in cancer therapy |
GB0022216 | 2000-09-11 | ||
PCT/EP2001/004716 WO2001068708A2 (en) | 2000-03-17 | 2001-03-16 | Human and humanized fap-alpha-specific antibodies |
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WO2018129497A1 (en) | 2017-01-09 | 2018-07-12 | Bioxcel Therapeutics, Inc. | Predictive and diagnostic methods for prostate cancer |
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PE20021080A1 (es) * | 2001-04-12 | 2003-02-12 | Boehringer Ingelheim Int | Un anticuerpo especifico fapo bibh1 en el tratamiento del cancer |
EP1806365A1 (de) * | 2006-01-05 | 2007-07-11 | Boehringer Ingelheim International GmbH | Antikörper spezifisch für Fibroblasten-Aktivierungsprotein und Immunokonjugaten, die diese Antikörper enthalten |
CA3040276A1 (en) | 2009-12-02 | 2011-06-09 | Imaginab, Inc. | J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (psma) and methods for their use |
EP3333194B1 (de) | 2010-08-13 | 2021-03-17 | Roche Glycart AG | Anti-fap-antikörper und verfahren zu ihrer verwendung |
DK3489255T3 (da) | 2011-02-10 | 2021-08-23 | Roche Glycart Ag | Muterede interleukin-2-polypeptider |
UA118028C2 (uk) | 2013-04-03 | 2018-11-12 | Рош Глікарт Аг | Біспецифічне антитіло, специфічне щодо fap і dr5, антитіло, специфічне щодо dr5, і спосіб їх застосування |
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 |
CN108136012B (zh) | 2015-08-07 | 2022-10-04 | 伊麦吉纳博公司 | 靶向分子的抗原结合构建体 |
MA43017A (fr) | 2015-10-02 | 2018-08-08 | Hoffmann La Roche | Anticorps bispécifiques spécifiques d'un récepteur de co-stimulation du tnf |
MX2019000252A (es) | 2016-06-30 | 2019-10-09 | Oncorus Inc | Administracion viral oncolitica pseudotipada de polipeptidos terapeuticos. |
JP2018035137A (ja) | 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 | 新規な抗線維芽細胞活性化タンパク質(fap)結合薬剤およびその使用 |
US20200237936A1 (en) | 2016-12-14 | 2020-07-30 | 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 |
WO2019133847A1 (en) | 2017-12-29 | 2019-07-04 | Oncorus, Inc. | Oncolytic viral delivery of therapeutic polypeptides |
MX2022000252A (es) | 2019-07-08 | 2022-02-21 | 3B Pharmaceuticals Gmbh | Compuestos que comprenden un ligando de proteina de activacion de fibroblasto y uso del mismo. |
BR112022000144A2 (pt) | 2019-07-08 | 2022-02-22 | 3B Pharmaceuticals Gmbh | Compostos que compreendem um ligante de proteína de ativação de fibroblastos e uso dos mesmos |
EP3763726A1 (de) | 2019-07-08 | 2021-01-13 | 3B Pharmaceuticals GmbH | Verbindungen mit fibroblasten-aktivierendem proteinligand und deren verwendung |
KR102363980B1 (ko) * | 2020-04-13 | 2022-02-15 | 전남대학교산학협력단 | 전이성 뇌종양의 진단 또는 예후 분석용 바이오마커 및 이를 이용한 진단방법 |
MX2022014538A (es) | 2020-05-19 | 2022-12-15 | Boehringer Ingelheim Int | Moleculas de union para el tratamiento de cancer. |
EP4050018A1 (de) | 2021-01-07 | 2022-08-31 | 3B Pharmaceuticals GmbH | Verbindungen mit fibroblasten-aktivierendem proteinligand und deren verwendung |
JP2024503637A (ja) | 2021-01-07 | 2024-01-26 | 3ベー ファーマシューティカルズ ゲーエムベーハー | 線維芽細胞活性化タンパク質リガンドを含む化合物およびその使用 |
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 |
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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 (de) * | 1998-04-30 | 1999-11-03 | Boehringer Ingelheim International GmbH | FAPalpha-spezifischer Antikörper mit verbesserter Produzierbarkeit |
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