EP1592792A1 - Proteomic screen to identify disease-related biological molecules and inhibitors thereto - Google Patents
Proteomic screen to identify disease-related biological molecules and inhibitors theretoInfo
- Publication number
- EP1592792A1 EP1592792A1 EP03763866A EP03763866A EP1592792A1 EP 1592792 A1 EP1592792 A1 EP 1592792A1 EP 03763866 A EP03763866 A EP 03763866A EP 03763866 A EP03763866 A EP 03763866A EP 1592792 A1 EP1592792 A1 EP 1592792A1
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- European Patent Office
- Prior art keywords
- cells
- ligand
- target
- assay
- cell
- 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.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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)
Definitions
- target validation At the moment, a drug target is considered validated only if a drug has been developed that is safe and efficacious.
- This retrospective definition of target validation implies the infancy of the field and the inefficiencies encountered in drug discovery. The average time so far required from identifying a target to identifying a lead is 3.4 years and costs approximately $350M. Increase in efficiency or predictability in defining which biological target molecule is the best target will significantly reduce the time and costs in developing a drug.
- Chemical genetics is a burgeoning field that utilizes the use of organic molecules instead of genetic mutations to modulate protein function (Schreiber, (2000), Science 287, 1964-1969). This field offers promise in simultaneously modulating protein function and the corresponding modulators can serve as potential therapeutic drug leads. Although this has been applied, chemical genetics is limited by the diversity of small organic molecules to modulate all the possible protein classes. Furthermore, the use of small organic molecules to isolate their corresponding protein target remains technically challenging. These limitations can be circumvented by the use of highly diverse combinatorial biological libraries (e.g. antibodies, aptamers, etc.). Unfortunately, the use of highly diverse combinatorial libraries for phenotypic screens analogous to the chemical genetics approach is technically challenging because ofthe high complexity of biological libraries.
- highly diverse combinatorial biological libraries e.g. antibodies, aptamers, etc.
- the present invention relies on a purely protein-based approach for identifying the function of a biological target molecule, circumventing the problems and technical challenges associated with the chemical genetics approach while being able to manage the complexity of biological libraries.
- Additional diversity such as random combination of heavy-chain and light-chain gene fragments, is introduced in the antibody gene cloning step.
- different antibodies are generated by the use of phage displayed antibody repertoires compared to methods relying on monoclonal antibody expressing stable cell lines.
- the antibody selection process using phage displayed antibody repertoires, is an in vitro process not limited by the same restrictions as the in vivo selection process.
- antibodies against different antigens will be generated by the use of phage displayed antibody repertoires compared to methods relying on monoclonal antibody expressing stable cell lines.
- phage display libraries allow the rapid search through large libraries of compounds to identify compounds that bind a particular target molecule with high affinity.
- a major weakness of this technology is the requirement of pure target molecules for phage selection. Only very few targets are available in this form and novel, unknown targets could not be identified using this approach.
- a few authors have recently described methods to screen cell surfaces for antibody binders in the absence of purified recombinant proteins. Nevertheless, selection of binding antibodies from large na ⁇ ve libraries by selection on cell surfaces is greatly limited by high background binding of non-specific phages and relatively low binding of specific phages. (See, e.g. Pereira et al. (1997) J. Immunol. Methods 203, 11- 24; Watters et al. (1997) Immunotechnology 3, 21-29; Becerril et al. (1999) Biochem. Biophys. Res. Commun. 255, 386-393).
- Heitner et al. (2001) J. Immunol. Meth., 248, 17-30 described the selection of EGFR antibodies from a na ' ive scFv-phage library via selection against cells that either over-express EGFR or were transfected with the EGFR gene. Heitner et. al. generated EGFR antibodies that recognized the over-expressed receptor.
- Poul et al. (2000) J. Mol. Biol., 301 , 1149-1161 selected internalizing antibody fragments on cells from a phage displayed repertoire and subsequently characterized a small subset of antibody fragments with respect to the corresponding epitope of the receptor and the internalizing efficiency ofthe antibody fragments.
- the method disclosed in the present invention provides a way to generate inhibitors of yet unknown disease-relevant targets and to simultaneously identify those unknown biological target molecules, if desired.
- the method disclosed in the present invention provides for example a way to generate inhibitors against cancer-relevant targets.
- the present invention demonstrates for the first time that for specific, naturally occurring tumor cells MCAM function is necessary for the process of invasion, proliferation, adhesion and/or metastasis.
- the invention further provides new inhibitors modulating the MCAM, integrin and ephrin function.
- the invention provides a new paradigm for a screening method by combining the biochemical approach of specific binding between a ligand and a target, e.g. an antibody and an antigen, with the genetic principle of a functional screen.
- a target e.g. an antibody and an antigen
- the present invention demonstrates its utility for identifying novel disease-relevant proteins and inhibitory ligands, e.g. antibodies or antibody fragments, that may serve as therapeutic leads for a wide variety of diseases, whether the cause ofthe disease is derivable from a virus, a bacterium, a lower eukaryote or from a misregulation ofthe cells ofthe subject to be treated.
- novel disease-relevant proteins and inhibitory ligands e.g. antibodies or antibody fragments
- the present invention has the advantage of significantly reducing the time and costs involved in drug discovery. This approach represents a new method to develop drugs more efficiently and makes it possible to identify a functional molecule against a disease in the complete absence of a candidate drug target.
- the present invention provides a method of identifying a ligand, for example an antibody or an antibody fragment, binding specifically to the extracellular region of an antigen associated with the surface of a target, for example a mammalian cell.
- the ligand for example an antibody or antibody fragment, is derived from a library of amplifiable ligand- displaying units (ALDU), for example a phage library displaying antibodies or antibody fragments.
- ALDU library for example a phage library displaying antibodies or antibody fragments, is brought in contact with a target, for example a cell.
- ALDUs for example phages
- bound to the target for example the mammalian cell
- the bound ALDUs are amplified.
- Those ALDUs or ligands derived from ALDUs, for example phages or antibodies or antibody fragments derived from phages, which are capable of affecting the biological ability of the target, for example said mammalian cell, are then identified in a functional screening assay.
- This method can also comprise the step of identifying the surface antigen, which the ALDU or the ligand derived from an ALDU affected or modified.
- the method of the invention has been applied in an unbiased screen for molecules that can inhibit invasiveness, adhesiveness and/or proliferation of naturally occurring tumor cells. Surprisingly, several antibody fragments binding to the extracellular domain of MCAM, integrin and/or ephrin had been identified as useful inhibitors according to the invention.
- the invention relates to a method for the identification of antibody fragments useful for inhibiting the invasiveness and the metastatic potential of sarcoma cells.
- the present invention provides in a prototypic embodiment a method of identifying a ligand binding specifically to the extracellular region of a target, by screening e.g. a phage display library of antibody fragments for a ligand being capable of affecting a biological function of said target, wherein these ligands are capable of affecting and, in particular, inhibiting the invasiveness, proliferation and/or adhesion of tumor cells.
- Said method comprises the steps of: a. contacting a library of amplifiable ligand-displaying units (ALDU) with a target; b. separating said target and the ALDUs bound thereto from ALDUs not bound to said target; c. optionally amplifying the ALDUs bound to said target; d. identifying an ALDU or a ligand derived from an ALDU after step c), which affects the biological function ofthe target in a functional screening assay; and optionally e. determining the chemical identity ofthe ligand.
- ALDU amplifi
- a target presenting an antigen, a tumor specific surface molecule, like e.g. MCAM, integrins or ephrins, or a recombinant antigen.
- a tumor specific surface molecule like e.g. MCAM, integrins or ephrins, or a recombinant antigen.
- This selection is not to be understood as limiting in any way the choice of possible targets and/or antigens or surface molecules, but is used as examples only.
- the ligand e.g. an antibody or an antibody fragment specific for the accessible part of the extracellular region of the antigen or surface molecule, e.g. MCAM, presented on the target is obtained, since the initial selection step is performed on intact cells, which present only the accessible part of the extracellular region of the surface molecule e.g. MCAM for binding with the ligand.
- the above method comprises instead of step d. the further steps of: d') contacting isolated ALDU's with recombinant antigen or surface molecule; e') washing said antigen or surface molecule with a buffered detergent and/or high salt solution; and f ) eluting ALDU's bound to antigen or surface molecule; and g') determining the identity ofthe ligand represented by said eluted ALDU's.
- Identifying means the identification of a biomolecule having desired properties from a mixture of biomolecules comprising related but non-identical biomolecules with slightly different properties.
- a "ligand” as used herein is a molecule displayable by an amplifiable ligand-displaying unit.
- a ligand is that part of an ALDU through which the ALDU can bind to a target.
- it is a polypeptide as defined above, an RNA-oligonucleotide or a DNA-oligonucleotide, whereby the oligonucleotide comprisies more than 20 base units but less than 10,000, preferably less than 1 ,000 base units.
- a ligand can bind to an extracellular region of an antigen. This binding may have specificity in the sense that the ligand binds to one antigen with high affinity but to a moderately related antigen with lower, for example 10- or 50- or 200-fold lower affinity. Moderately related antigens are antigens with up to 30 % amino acid identity in the extracellular regions.
- a “target” as used herein is an entity for which it is screened for, preferably a biological target that is a complex mixture of biomolecules.
- a target may be a eukaryotic cell, a virus, a bacterial cell, or parts thereof.
- a target is not an individual, purified biomolecule.
- the target may be a particle with a size of more than 10 nm, preferably more than 30, 100 or even 500 nm.
- the biological target as used herein is capable of presenting a particular biomolecule, surface molecule or antigen, which is associated with its surface.
- a ligand "binding specifically to a target” as mentioned herein can be a ligand which binds to the target or any of the presented surface molecules or antigens under the buffer conditions given in Examples 1, 2, 15 or 16.
- the target is an identified biomolecule the dissociation constant between the ligand and the identified biomolecule can be measured, e.g. by use of the so-called BIACORE System (see, for example, Fivash et al. Curr Opin Biotechnol.
- the dissociation constant between the ligand and the identified biomolecule is lower than 10 ⁇ M, preferably lower than 1 ⁇ M, more preferably lower than 500, 400, 300, 200, 100, 50, 20 nM, most preferably from 0,1 nM to 20 nM if measured under standard conditions, for example at 20°C, ambient pressure and in a suitable buffer, e.g. 20 mM Tris, 100 mM NaCl, 0, 1 mM EDTA at an overall pH of 7.0.
- a ligand "derived from an ALDU” as mentioned herein is a ligand, which had been displayed by such an ALDU, which had been selected by binding to the surface of a target.
- the ALDU is a phage of a phage library displaying scFvs
- the "ligand derived from an ALDU” in this case is a polypeptide, here an scFv.
- the ligand "derived from an ALDU” is the polypeptide, which was presented by the complex comprising the ribosome, the RNA and the polypeptide.
- extracellular region of a molecule as used herein is that part of the molecule which is located on the outside of a biological structure, that is, if the target is a eukaryotic cell located on the outside of the cell membrane; if the target is a gram-negative eubacterium located on the outside of the outer bacterial membrane; if the target is a gram-positive eubacterium located on the outside of the bacterial membrane.
- extracellular region should be understood as that part of the virus that is accessible to other biomolecules, e.g. a polypeptide with a molecular weight of 1000 Da, when the virus is intact. This includes the outside of a viral capsid and the outside of a for example outer viral coat.
- the term "associated with the surface of a target” means that a biomolecule is either part of the surface of a biological target, e.g. a constituent of the surface, or is binding to another biomolecule which is a constituent of the surface of a target.
- a transmembrane polypeptide like a transmembrane receptor of an eukaryotic cell can be seen as a constituent of the surface if at least one part of it transverses the lipid bilayer of the cell membrane and is in direct contact with the lipids of the cell membrane.
- An example for a polypeptide that is binding to another biomolecule, which is a constituent of the surface of a target, is, for example, the B2-microglobuline ofthe class 1 major histocompatibility complex.
- the B2-microglobuline binds non-covalently to the MHC - a transmembrane polypeptide -, and is therefore also regarded as being a molecule associated with the surface.
- a biological function of, e.g., a cancer cell can be their proliferation, invasiveness or adhesiveness; of, e.g., a virus can be its ability to enter and infect a cell; of, e.g., a bacterium can be, e.g., its ability to adhere to a cell, e.g., a mammalian cell, or also its ability to adhere to another bacterial cell.
- biological function refers to those activities that can take place in an intact mammalian organism or are indicative of a corresponding function in an intact mammalian organism. It does not include generalized toxic effects resulting from the presence of a molecule in large excess of its naturally- occurring concentration. Preferably it does also not include effects that do not rely on specific binding of the ligand.
- ligands of the invention will alter the "biological function" of the target, e.g. either enhance or inhibit or change the biological function of the target.
- the effect of a ligand on a biological function may be demonstrated by in vitro tests, which are indicative of the corresponding biological function in vivo.
- contacting of two components means allowing the two components to physically associate with one another and bind to one another.
- ALDUs amplifiable ligand-displaying units
- RNAs useful for the process of selection-amplification (SELEX), phages of phage display libraries, viruses of viral display libraries, ribosomes of ribosome-display techniques, but also individualized beads carrying an identifiable molecule or ligand, particularly chemical entities, e.g. small molecules bound to beads, which are recognizable by inert chemical tags.
- ALDUs which comprise a nucleic acid as the identifiable component.
- Such ALDUs can be either amplified in vitro, e.g., by nucleic-acid amplification methods like RT-PCR, PCR, LCR and the like, or they can be amplified in vivo, for example an individual phage can infect a bacterium and yield, after several cycles of infection, millions of virtually identical progeny.
- a library of ALDUs is a collection of similar, but in general non-identical ALDUs, for example phages of a phage library that display different scFvs in the context of an otherwise identical phage surface.
- Separating means the physical separation of two or more components, for example a cell with an ALDU bound thereto can be separated from a free ALDU by centrifugation, wherein the cell with the ALDU bound thereto is pelleted and the free ALDU is still in the supernatant.
- “Amplifying” as used herein is any process that increases the number of ALDUs or ligands derived from ALDUs by at least a factor of two, preferably by a factor of 10, 100, 1.000, 10.000, 100.000, 1.000.000, 10.000.000 or even a billion.
- a single phage can be amplified by infecting a bacterium and the infected bacterium then produces several mostly identical copies of the infecting phage, or a DNA-molecule can be amplified by the process of PCR to yield 10 4 or more mostly identical daughter DNA-molecules.
- Determining the chemical identity of a ligand means determining the structural composition of a ligand. For example, if the ALDU library is a phage library displaying scFvs, then "determining the chemical identity of a ligand” would mean to determine the sequence of the scFv polypeptide, for example by sequencing the region encoding the scFv ofthe phage from which it was derived.
- a screening assay as used herein is aimed at detecting an individual, e.g. an ALDU, with special properties among other similar individuals with slightly different properties.
- an individual e.g. an ALDU
- special properties among other similar individuals with slightly different properties In order to qualify for a screening assay at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or even 100 individuals must be tested in a certain functional assay to find an individual/individuals with the desired function detected among them.
- “Inhibiting" a biological function as used herein means the reduction of the biological function at a statistically significant level, preferably by more than 30 %, 40 %, 50 %, or even 60 %. Inhibition can be measured in a functional assay, e.g. by adding a ligand, e.g. a certain antibody fragment at a concentration of one nM to 50 ⁇ M, preferably around 20 ⁇ M to the functional assay under standard conditions.
- a ligand e.g. a certain antibody fragment at a concentration of one nM to 50 ⁇ M, preferably around 20 ⁇ M
- Activating biological function means an increase in biological function at a statistically significant level, preferably by more than 30 %, 40 %, 50 %, or even 60 %. Activation can be measured in a functional assay, e.g. by adding a ligand, e.g. a certain antibody fragment at a concentration of one nM to 50 ⁇ M, preferably around 20 ⁇ M to the functional assay under standard conditions.
- a ligand e.g. a certain antibody fragment at a concentration of one nM to 50 ⁇ M, preferably around 20 ⁇ M
- an "antigen derivable from a virus” is an antigen, which can be present in a virus particle, preferably a viral surface antigen like capsid- or coat-proteins.
- a "virus” as used herein comprises animal viruses, plant viruses and bacteriophages and can in particular be an animal virus, more particularly a virus capable of infecting a mammalian, e.g. a human cell.
- the virus is typically selected from the group comprising lentivirus, retrovirus, hepadnavirus, picornavirus, alphavirus, flavivirus, reovirus, paramyxovirus, orthomyxovirus, arbovirus, polyomavirus, papillomavirus, rhabdovirus, herpes virus, adenovirus, parvovirus, in particular selected from the group consisting of HIV-I, HIV-II, hepatitis B virus, hepatitis C virus, smallpox virus, pox virus, poliomyelitis virus, west nile virus, adeno virus, herpes simplex virus, Eppstein-Barr virus, coxsackie virus, influenza virus and rubella virus.
- an "antigen derivable from a bacterium” is an antigen, which can be part of a bacterium. It can be part of an archaebacterium or eubacterium, preferably gram positive or gram negative eubacteria such as particularly a bacterium selected from the group consisting of Bordella pertussis, Vibrio Cholerae, Bacillus anthracis, Yersinia pestis, Staphylococcus aureus, Clostridium botulinum, Streptococci, Chlamydia, Chromobacter, Helicobacter pylori. Particularly an antigen derivable from a bacterium is an antigen associated with the surface of a bacterium.
- an "antigen derivable from a lower eukaryote” as used herein is an antigen, which can be part of a lower eukaryote.
- a lower eukaryote as used herein is defined as a fungus, a protozoon, in particular a member of the Phylae sarcomastigophora (for example the genera Trypanosoma, Leishmania, Trichomonas and Giardia), Rhizopoda (for example the genera Entamoeba, Naegleria and Acanthamoeba), Sporozoa (for example the genera Poxoplasma, Isospora, Sarcosystis, Cryptosporidium and Plasmodium) and Ciliophora (for example the genus Balantidium) or as a member of the metazoan Phylae plathelminthes, Mesozoa, Nematoda, Nematomorpha or Acantocephala.
- an “antigen” as used herein can be a polypeptide, a glycoprotein, a peptide, a proteoglycane, a lipid or a glycolipid.
- polypeptide as used herein is a molecule comprising more than 10, preferably more than 20, most preferably more than 30, and less than 10000, more preferably less than 2500, most preferably less than 1000 amino acids. Also polypeptides with substantial amino acid sequence identity and polypeptides, which contain a low percentage of modified or non- natural amino acids are encompassed. Particularly antibodies and antibody fragments are considered as polypeptides.
- antibody or "immunoglobulin”, as used herein refer to any immunological binding agent, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, and the like.
- the heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed alpha, delta, epsilon, gamma and mu, respectively.
- the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
- Antibodies may be also selected from modified immunoglobulins, for example chemically or recombinantly produced antibodies, CDR grafted antibodies or humanized antibodies, site directed mutagenized antibodies that exhibit substantial amino acid sequence identity in their CDR regions, particularly in their CDR3 region, to the corresponding antibody fragments of the invention and retain substantially the same affinity for MCAM binding as the corresponding antibody fragments.
- modified immunoglobulins for example chemically or recombinantly produced antibodies, CDR grafted antibodies or humanized antibodies, site directed mutagenized antibodies that exhibit substantial amino acid sequence identity in their CDR regions, particularly in their CDR3 region, to the corresponding antibody fragments of the invention and retain substantially the same affinity for MCAM binding as the corresponding antibody fragments.
- the CDRs (complementary determining region) of an antibody are the parts of these molecules that determine their specificity and make contact with specific ligands.
- the CDRs are the most variable parts of the molecule and contribute to the diversity of these molecules. They are structurally defined in a human IgG as amino acids 24 to 41 (CDR-L1), 50 to 57 (CDR-L2) and 90 to 101 (CDR-L3) ofthe light chain and amino acids 26 to 38 (CDR-H1), 51 to 70 (CDR-L2) and 100 to 125 (CDR-H3) ofthe heavy chain (see Kabat et al. (1987) 4th edn US Department of Health and Human Services, Public Health Service, NIH, Bethesda).
- the CDR regions of an antibody fragment can easily be determined by somebody skilled in the art by aligning the antibody fragment with said human IgG, e.g. using a program of the NCBI that allows to "Blast", and thereby align, two sequences with one another, and identifying the amino acids ofthe antibody fragment corresponding to the CDRs of a human IgG.
- Substantial amino acid sequence identity means that at least 70%, preferably at least 85%, more preferably all but 5, still more preferably all but 3 and even more preferably all but 1 of the amino acids of two aligned amino acid sequences, particularly of aligned CDRs, are identical.
- antibody fragment is used to refer to any fragment of an antibody-like molecule that has an antigen binding region, and this term includes antibody fragments such as scFv, dsFv, Fab', Fab, F(ab') 2 , Fv, single domain antibodies (DABs), diabodies, and the like.
- DABs single domain antibodies
- scFv antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
- a “Fv” fragment is the smallest antibody fragment that retains an intact antigen binding site.
- a “dsFv” is a disulfide stabilized Fv.
- a “Fab” fragment is an antigen binding fragment, containing complete light chains paired with the VH and CHI domains of the heavy chain.
- a “Fab”' fragment is a reduced F(ab') 2 fragment.
- a “F(ab') 2 " fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
- a “single domain antibody (DAB)” is an antibody with only one (instead of two) protein chain derived from only one ofthe domains ofthe antibody structure. Dabs exploit the finding that, for some antibodies, half of the antibody molecule binds to its target antigen almost as well as the whole molecule (Davies et al. (1996) Protein Eng. 9: 531-537.
- a “proteoglycane” can be seen as an organic compound comprising long polysaccharide chains (glycanes) covalently bound to a polypeptide core.
- a “glycoprotein” as used herein can be seen as an organic compound comprising both a polypeptide and a carbohydrate moiety joint by a covalent chemical linkage.
- a carbohydrate moiety of a glycoprotein can be a small sugar of around 4 to 10 individual - optionally substituted - sugar units, selected from the list of glucose, glucosamine, galactose, galactosamine, mannose, fucose and sialic acid.
- a "glycolipid” as used herein can be seen e.g. as 1,2-Di-O-acylglycerole with a carbohydrate moiety covalently bound to oxygen 3 by a glycosidic linkage.
- the carbohydrate moiety can comprise a mono-, di-, or trisaccharide.
- Bacterial glycolipids which can have an isolated sugar moiety, wherein the acylation can be by one or more fatty acids and where the glycerol part may even be absent, are also "glycolipids" within the scope ofthe invention.
- the invention relates to a method of identifying a ligand binding specifically to the extracellular region of an antigen associated with the surface of a target, wherein said ligand is capable of affecting a biological function of said target, comprising the steps of a) contacting a library of amplifiable ligand-displaying units (ALDU) with said target; b) separating said target cell and the ALDUs bound thereto from ALDUs not bound to said target; c) amplifying the ALDUs bound to said target; d) identifying an ALDU or a ligand derived from an ALDU after step c), which affects said biological activity by a functional screening assay; and optionally e) determining the chemical identity of the ligand.
- the preferred order ofthe steps is a, b, c, d and optionally e.
- the method is capable of identifying a ligand, e.g. an antibody or antibody fragment, with the ability to bind strongly and specifically to the extracellular region of an antigen which is associated with the surface of a target, e.g. the extracellular loops of transmembrane receptors of a mammalian cell or e.g. the lipopolysaccharides ofthe outer membrane of a gram negative bacterium or e.g. a capside protein of an animal virus.
- the method of the invention identifies such ligands which are binding to the antigen and thereby affect a biological function of a target, e.g. the method can identify a ligand, e.g. an antibody or antibody fragment which binds to e.g.
- the method of the invention is capable to identify ligands, which bind to the alpha-2/beta 1 - integrin and inhibit adhesiveness of a cancer cell.
- the method can identify a ligand, which binds to the surface of pathogenic bacteria and inhibits the colonization of the mammalian body by said pathogenic bacteria or, e.g., the method can identify a ligand which can bind to a viral surface protein and thereby inhibit infection of a mammalian cell by the virus.
- the method relies on a new rationale for screening assays: advantageous combination of a screening step based on binding to the surface of a target with a screening step based on a functional assay.
- a library of amplifiable ligand-displaying units is brought in contact with a target in such a way that those ALDUs of the library with specificity for a surface antigen of the target can bind to the target.
- ALDU amplifiable ligand-displaying units
- phages of a phage library displaying antibodies or antibody fragments can be allowed to bind to cultured cancer cells or to a preparation of a virus.
- the target and the ALDUs bound thereto are separated from unbound ALDUs, in order to select for those ALDUs with specificity for the target.
- the separation can be achieved, e.g., by removing the solution, in which the contacting step has been performed from, e.g., cultured cells grown adhesively in a culture flask and together with the solution the unbound ALDUs; or, as another example, viruses with ligands of a library bound thereto can be pelleted by centrifugation, while the unbound ligand remain in the supernatant as part of the solution, in which the contacting step had been performed.
- the ALDUs bound to the target are amplified, in order to obtain high enough concentrations of the ALDUs so that they are useful for being applied in a functional screen.
- This amplifying step makes use of the ALDUs' intrinsic property to be amplifiable.
- phages displaying, e.g., an antibody or antibody fragment, which have been separated from the non-bound phages in step b), but are still bound to the target, e.g. a mammalian cancer cell can be amplified by first recovering the bound phages, for example by eluting the phages from the target cells and then using the recovered phage eluate to infect bacteria, e.g. Escherichia coli. The amplification of the phages in Escherichia coli then leads to a significant increase in total phage number, but in such a way, that the concentration of those individual phages, which were capable of binding to the target, is dramatically increased.
- the phages need not necessarily be removed from the target, as a phage still bound to a target can infect added bacteria and thus be amplified.
- the mRNA of a ribosomal-display library which is associated by its attachment to a ribosome displaying the polypeptide corresponding to the mRNA, with the surface of a target, can be directly used for RT-PCR, without the need of removing the intact ALDU from the target before amplifying the target.
- a step in which the ALDU bound to a target is recovered from the target before amplifying the ALDU is an optional step.
- Amplifying the ALDU can also consist in determining the chemical identity of the ligand of such ALDUs, which have bound to the target and then amplifying the ligands. That is to say, e.g., that in the case of a phage library, displaying an antibody or an antibody fragment, DNA encoding those ligands can be cloned from phages, which had bound to the target, e.g., by PCR, and the ligand, e.g., the antibody or antibody fragment, can then be produced recombinantly to yield sufficient amounts ofthe ligand to be useful for a functional screening assay. In such a way, not the ALDU itself is amplified, but the ligand displayed by the ALDU.
- the aim of the amplification step is the generation of a ligand concentration high enough to be useful for a functional screening assay and this can be achieved by either amplifying the ligand or the ALDU displaying it.
- an ALDU or a ligand derived from an ALDU, which had bound to a target is detected based on its effects on a biological function to be tested in a functional screening assay.
- the ALDUs or ligands derived from them are advantageously individualized, such that a signal or a pattern from the functional screening assay is relatable to an individual ALDU or an individual type of ligand. This can be done, e.g., by filling separate wells of a multi-well plate with individual ligands each and then performing an assay for a desired biological function in those individual wells.
- phages which had bound to a desired target, e.g. to a cancer cell, and had been separated from unbound phages, can be individualized by infecting bacteria and plating the infected bacteria in, e.g., soft agar, so that individual plaques, which represent clones of individual phages, form. Those individual plaques can then be tested for their effects in a functional biological screening assay. In such an assay, an individual plaque with an effect in the functional screening assay is identifiable. The ligand displayed by the phages of the plaque can then be identified, e.g.
- the identity of the ligand displayed by the ALDU can be determined, if desired.
- the ligand displayed or derived from an ALDU is capable of activating a biological function of the target, e.g. a ligand, like an antibody or an antibody fragment could bind to the surface receptor of a mammalian cell, e.g. the FAS-receptor, and could activate a biological function of a cell, in this case it could activate apoptosis of this cell.
- the ligand is capable of inhibiting a biological function of the target.
- the biological function of a target can be the ability of the target to interact with an antigen derivable from a virus.
- the target is a mammalian cell, or even a human cell
- a ligand that binds to a surface antigen of the mammalian cell that is necessary for mediating the interaction with a viral antigen important for viral entry into a mammalian cell can be identified by the method of identifying a ligand of the invention.
- a ligand could be useful for inhibiting infection of the mammalian cell with the virus.
- ligands displayed by the ALDUs bound to the surface of a mammalian cell - which is infectable by the virus - by screening those individualized ligands for their ability to inhibit infection ofthe target, in this case the mammalian cell, by the virus.
- ligands e.g.
- antibodies or antibody fragments can be identified by the method of the invention which inhibit the ability of the target, for example a mammalian cell, to interact with an antigen derived from a virus, for example selected from the group consisting of lentivirus, retrovirus, hepadnavirus, picornavirus, alphavirus, flavivirus, reovirus, paramyxovirus, orthomyxovirus, arbovirus, polyomavirus, papillomavirus, rhabdovirus, herpes virus, adenovirus, parvovirus, in particular selected from the group consisting of HIV- I, HIV-II, hepatitis B virus, hepatitis C virus, smallpox virus, pox virus, poliomyelitis virus, west nile virus, adeno virus, herpes simplex virus, Eppstein-Barr virus, coxsackie virus, influenza virus and rubella virus and thereby protect a mammalian cell from viral infection.
- a virus for example
- a ligand that binds to a surface antigen of the mammalian cell that is necessary for mediating the interaction with a bacterial antigen important for bacterial attachment to a mammalian cell can be identified by the method of the invention.
- a ligand could be useful for inhibiting bacterial infection of the mammal, in particular in those cases where infection is caused by adhesion of bacteria to mammalian cells or by entry of bacteria into mammalian cells.
- Such a ligand can be identified among the ligands displayed by the ALDUs bound to the surface of a mammalian cell by screening those individualized ligands for their ability to inhibit the bacterial adhesion to the target or bacterial infection of the target, e.g. in the case of a mammalian cell, bacterial adhesion to the mammalian cell or bacterial entry into the mammalian cell.
- ligands e.g. antibodies or antibody fragments, which inhibit the ability of the target, e.g. a mammalian cell, to interact with an antigen derived from a bacterium, e.g. derived from a gram positive or gram negative bacterium, e.g.
- bacterium selected from the group consisting of Bordella pertussis, Vibrio cholerae, Bacillus anthracis, Yersinia pestis, Staphylococcus aureus, Clostridium botulinum, Streptococci, Clamydia, Chromobacter and Helicobacter pylori, can be identified by the method ofthe invention.
- ligands e.g. antibodies or antibody fragments, which inhibit the ability of a bacterium to enter the target, e.g. a mammalian cell
- This relates particularly to intracellular or facultative intracellular bacteria, for example selected from the group consisting of Mycobacterium, Brucella, Chlamydiae and Rickettsiaceae, in particular selected from the group consisting of the pathogen species of intracellular Chlamydiae and Rickettsiaceae, for example the pathogen species of the genera Coxiella, Ehrlichia and Rickettsia.
- Such ligands can be potential therapeutics for, for example, the treatment of typhus, Q fever, Rocky Mountain spotted fever or scrub typhus or other diseases, which are known to be caused by Rickettsiaceae or Chlamydiae.
- the biological function of a target can be the ability of the target to interact with an antigen derivable from a lower eukaryote.
- a ligand which binds to a surface antigen of the mammalian cell that is necessary for mediating the interaction with an antigen from a lower eukaryote e.g. Plasmodium falciparum
- a mammalian cell e.g. Plasmodium falciparum
- important for mediating cell-cell interaction between a mammalian cell and a lower eukaryotic cell e.g. for entry of Plasmodium into erythrocytes
- Such a ligand could be useful for inhibiting infection of the mammal with the parasitic lower eukaryote. It can be identified among the ligands displayed by the ALDUs bound to the surface of a mammalian cell by screening those individualized ligands for their ability to inhibit interaction between the target, in this example the mammalian cell, with the lower eukaryotic cell, in the example of P. falciparum the entry of P. falciparum into erythrocytes.
- ligands e.g. antibodies or antibody fragments, can be identified by the method of the invention which inhibit the ability of the target, e.g.
- a mammalian cell to interact with an antigen derived from a lower eukaryote and particularly inhibit cell-entry of intracellular or facultative-intracellular lower eucaryotes such as Plasmodium vivax, Plasmodium ovalae, Plasmodium malariae, Plasmodium falciparum, Leishmania, Toxoplasma (for example T. gondii), Sarcocystis and Pneumocystis.
- the surface antigen is a polypeptide, a glycoprotein, a peptide, proteoglycane, a lipid or a glycolipid.
- the target is a eukaryotic cell, in particular a mammalian cell, more particularly a cell selected from the group consisting of cells of tissue sections, primary cells or cell lines, in particular selected from the group consisting of cancer cells, nerve cells, stem cells, blood cells, platelet cells, adipocytes, liver cells, kidney cells, lung cells, endothelial cell, keratinocytes, melanoma cells, erythrocytes, osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow cells, smooth muscle cells, skeleton muscle cells and heart muscle cells.
- a mammalian cell more particularly a cell selected from the group consisting of cells of tissue sections, primary cells or cell lines, in particular selected from the group consisting of cancer cells, nerve cells, stem cells, blood cells, platelet cells, adipocytes, liver cells, kidney cells, lung cells, endothelial cell, keratinocytes, melanoma cells, erythrocytes,
- the biological functions of those mammalian cells, which can be affected by the ligands identified by the method of the invention, can be many-fold, e.g. selected from the group consisting of invasion, adhesion, proliferation, angiogenesis and cytotoxicity.
- Invasiveness is the ability of a cell to migrate through a layer of other cells or to migrate through the extracellular matrix. Invasiveness can be assessed by the Matrigel assay described in Example 5. Invasion is measured as cells that reach the lower surface ofthe filter during a certain incubation period. When more than 40% of cells within 6h to 12h reach the other side of the filter and form colonies in an invasion assay like in Example 5, the naturally occurring cancer cell is defined as invasive. The control cells instead form only 5% colonies in the same time frame and are defined as non-invasive.
- Adhesiveness is the ability of a cell to reattach after they have been removed from the matrix on which it had been grown, resuspended as a solution of single cells (not in direct contact with other cells of the solution), and replated on a matrix to which adhesion is possible.
- a cell is defined as adhesive if in an assay as described in Example 7, more than 40% of the cells adhere within a time of 30-120 min. Instead, only 5% of the control cells adhere within the same time frame.
- Angiogenesis is the process where cells induce blood vessel formation in their proximity. Angiogenesis can accompany the growth of malignant tissue and can therefore be a property of malignant cells. That is to say that malignant cells can have the property of inducing angiogenesis.
- Metastatic potential as used herein is the ability of a tumor cell to form a new tumor at a site distant from the primary tumor of which the tumor cell was derived (a metastase). Metastatic potential can be measured by injecting, e.g. lxlO 6 , cells into the lateral tail vein of athymic nude mice and determining the number of tumor nodules in the lung, e.g. 2 months post injection, e.g. as described in the section "Tumor cell injections" on page 2346 of Huang et al (1996) Oncogene 13, 2339-2347, or the sections “Animals and production of tumors" and " Histochemical analysis for calcified matrix” on page 1882 of Radinsky et al. (1994) Oncogene 9, 1877-1883.
- a cell line to produce more than 3, preferably more than 8, more preferably more than 20 tumor nodules in the lung in this assay is considered metastatic.
- the biological function inhibitable by a ligand e.g. an antibody or antibody fragment, identified by the method of the invention is the adhesiveness, proliferation and invasiveness of a cancer cell.
- the ligand is a polypeptide, in particular an antibody or antibody fragment.
- the ligand is an antibody it can be selected from the group consisting of IgA, IgG, IgD, IgE and IgM, and particularly from the group consisting of human IgG and IgM.
- the ligand is an antibody fragment it can be selected from the group consisting of scFv, dsFv, Fab', Fab, F(ab') 2 , Fv, single domain antibodies and diabodies.
- the ligand is selected from the group consisting of protein scaffolds with a tertiary structure, random polypeptides, small molecules, RNA-aptamers and DNA-aptamers.
- Protein scaffolds with tertiary structure are ligands obtainable by using a gene encoding a polypeptide with a defined tertiary structure, wherein one or more regions of the gene encoding the polypeptide can be replaced by randomized oligonucleotides or randomized oligonucleotides can be inserted into the gene encoding the polypeptide without changing the tertiary structure of the encoded polypeptide. Examples for these ligands are e.g.
- Anticalin, Affibody, cystein knot or protein scaffolds with an immnoglobulin fold Anticalin is described in Beste et al (1999) Proc Natl Acad Sci USA. 96, 1898-903 and ALDUs displaying Anticalin are described in Beste et al, see supra.
- An Affibody is a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and ALDUs displaying Affibodies are described in Hansson et al (1999) Immunotechnology. 4, 237-52.
- a Cystein knot is a protein scaffold involving interlocking disulfide bonds as exemplified by the structure of conotoxins and nerve growth factor.
- a cystein knot is described in McDonald et al (1991) Nature 354, 411-4 and ALDUs displaying Cystein knots are described in Smith et al. J Mol Biol. 1998, 277, 317-32. ).
- Protein Scaffolds with the immunoglobulin fold are antibodies and antibody fragments and other polypeptides having the tertiary structure of the immunoglobulin superfamily and ALDUs displaying protein scaffolds with the immunoglobulin fold are disclosed in McCafferty et al. (1990) Nature 348, 552-4.
- ALDUs displaying random polypeptides are disclosed in Scott et al. (1990) Science, 249, 386-90.
- RNA-aptamers are structural mimics composed of ribonuncleic acid and are disclosed in Tuerk and Gold (1990) Science 249, 505- 10, and Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA 89, 6988-92.
- DNA-aptamers are structural mimics composed of deoxyribonucleic acid and ALDUs displaying DNA-aptamers are disclosed in Bock et al (1992) Nature 355, 564-6.
- amplification of the ALDU can be achieved by increasing the number of an individual ALDU, but also by increasing the number of a ligand displayed by an individual ALDU. Since this can in principle also be achieved by an individual bead displaying a small chemical in combinatorial chemical libraries (see e.g., Ohlmeyer et al. (1993) Proc. Natl. Acad. Sci USA 90, 10922-26; Liu et al. J. Am. Chem. Soc. (2002) 124, 7678-80; Liang et al Science (1996) 274, 1520-2) such bead-format libraries are also libraries of ALDUs in a way.
- Small molecules can be synthesized on microsphere beads, which can be indexed with inert chemical tags. During each step ofthe synthetic scheme a tag molecule that encodes the step number and the chemical reagent used in that step is attached to the bead. This array of tags can then be used as a binary code to record the reaction history of each bead. The identified compound can then easily be synthesized in a larger scale.
- the ALDU is a biological ALDU that is the information about its identity is stored as a nucleic acid, e.g. RNA or DNA.
- the ALDU is selected from the group consisting of viruses useful for viral display, phages useful for phage display, bacteria useful for bacterial display, ribosomes useful for ribosome display, yeasts useful for yeast display and oligonucleotides useful for selection and amplification.
- viruses useful for viral display can be, e.g., plant viruses, viruses of fungi or animal viruses, in particular viruses that can be obtained from cell culture, are amenable to genetic engineering techniques and can display a foreign ligand on their surface.
- Viruses useful for viral display on retroviruses are disclosed in Russell et al. (1993) Nucleic Acids Res. 21, 1081-5.
- Viruses useful for display by plant viruses e.g. the Cowpea mosaic virus are disclosed in Brendan et al. (1999) Microbiology 145, 211-20.
- Phages useful for phage display can be all phages that can be obtained from culture, are amenable to genetic engineering techniques and are capable of displaying a foreign ligand on their surface. Phage display has been disclosed in Smith et al. (1985) Science, 228, 1315-17, phage display of an antibody has been disclosed in WO 91/17271.
- Bacteria useful for bacterial display are bacteria, which can be kept in culture, are amenable to genetic engineering techniques and are able to display a ligand on their surface. Examples for bacteria useful for bacterial display are disclosed in Dougherty et al., (1999) Protein Eng. 12,613-21; and Westerlund-Wickstrom (2000) Int. J. Meth. Microbiol. 290, 223-30.
- Ribosomes useful for ribosomal display have been disclosed in Shaffizel et al., (1999) J. Immunol. Methods. 231, 119-35; and Willson et al., (2001) Proc. Natl. Acad. Sci. USA 98, 3750-5.
- Oligonucleotides useful for selection/amplification have been disclosed in Tuerk and Gold (1990) Science 249, 505-10, and Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA 89, 6988-92.
- ALDUs e.g. genetically modified yeast, as disclosed in Boder and Wittrup (2000) Methods Enzymol. 328, 430-44.
- the ALDU library is selected from the group consisting of immune or naive libraries, in particular wherein said naive library is a synthetic, semi- synthetic or natural library.
- naive library is a synthetic, semi- synthetic or natural library.
- the ligand displayed on the ALDU is an antibody or an antibody fragment.
- immune libraries the library members, e.g. antibody fragment genes, can be obtained from lymphocytes, e.g. B lymphocytes, of immunized animals.
- lymphocytes e.g. B lymphocytes
- the library members have not gone through the process of an active immune response.
- the naive library can be, e.g., a semi- synthetic library, in which library members are obtained by use of germline V-genes as starting material.
- the regions coding for one or several V-gene CDRs, encoded e.g. by CDR1 and CDR2 can be replaced by randomized oligonucleotides, or randomized oligonucleotides can be added to one or several CDRs, either at the beginning, as an insert into the CDR, or at the end of the CDR, and regions coding for one or several of the non V-gene encoded CDRs e.g. CDR3 can be added to the V-gene as randomized oligonucleotides.
- An antibody-based naive libraries can also be synthetic libraries, e.g. in which library members are obtained by use of non-germline V-genes as starting material.
- the regions coding for one or several V-gene CDRs encoded e.g. by CDR1 and CDR2 can be replaced by randomized oligonucleotides, or randomized oligonucleotides can be added to one or several CDRs, either at the beginning, as an insert into the CDR, or at the end of the CDR, and regions coding for one or several of the non V-gene encoded CDRs e.g. CDR3 can be added to the V-gene as randomized oligonucleotides (Griffiths et al. (1994) EMBO J. 13, 3245-60; Knappik et al. J Mol Biol. 296, 57-86).
- a naive library can be a natural library wherein the library members e.g. antibody fragment genes, can be obtained from lymphocytes, e.g. B-lymphocytes, of non-immunized donors.
- lymphocytes e.g. B-lymphocytes
- An example of a natural library is disclosed in Griffiths et al. see supra.
- the ALDUs are derived from viruses and phages, in particular from Cowpea mosaic virus, retroviruses, lambda phage, T7 phage, T4 phage and filamentous phages, more particularly derived from Ff phages.
- An ALDU library derived from Cowpea mosaic virus is disclosed in Brendan et al. (1999) Microbiology 145, 211-20.
- An example of an ALDU library derived from a retrovirus is disclosed in Russell et al. (1993) Nucleic Acids Res. 21, 1081-5.
- An example of an ALDU library derived from lambda phage is disclosed in Maruyama et al. Proc Natl Acad Sci U S A.
- the Ff phages of Escherichia coli have proven particularly useful as phages capable of displaying random and foreign ligands on their surface (Smith, Science (1985) 228, 1315-7).
- the ligand e.g. a random polypeptide
- the phage protein is derived from a phage surface protein, e.g. selected from the group consisting of pill, pVIII and pVI.
- Phage display based on fusion proteins with pill are disclosed in Smith, Science. (1985) 228, 1315-7.
- Phage display based on fusion proteins with pVIII are disclosed in Gram et al. Proc Natl Acad Sci U S A (1992) 89, 3576-80.
- Phage display based on fusion proteins with pVI are disclosed in Jespers et al. Biotechnology (N Y) (1995) 378-82.
- step d) ofthe method of identifying a ligand ofthe invention comprises detecting an ALDU or a ligand derived from an ALDU, which affects the biological function of a target by a functional screening assay, wherein the target can particularly be a mammalian cell, e.g.
- the functional screening assay applied for detecting the ALDU or a ligand derived from an ALDU is selected from the group consisting of an invasion assay, an adhesion assay, a proliferation assay, an apoptosis assay, a cytotoxicity assay, an angiogenesis assay, a protein translocation assay, a protein-protein interaction assay, a migration assay, an ion transport assay, a colorimetric assay, a gene activation assay and a phenotypic assay.
- An invasion assay measures the invasiveness of a cell in other words the capability of cells to spread out through a matrix.
- An invasion assay is, e.g., the assay performed in Example 5 of this application.
- An adhesion assay measures the adhesiveness of a target, e.g. a cell to something else, e.g. another cell, a virus, a matrix structure comprising e.g. collagen, fibronectin and laminin, or a complex biological mixture like, e.g., a population of vehicles derived from a cell or, e.g., the basal lamina.
- a target e.g. a cell to something else, e.g. another cell, a virus, a matrix structure comprising e.g. collagen, fibronectin and laminin, or a complex biological mixture like, e.g., a population of vehicles derived from a cell or, e.g., the basal lamina.
- a proliferation assay measures the ability of a target, e.g. a mammalian cell, to divide and give raise to daughter cells.
- a proliferation assay can comprise simply counting the cell number of a culture over time or it can also make use of the increase in total DNA of the cell culture, as does, e.g., a so called BrdU-Assay, which measures the amount of bromo- deoxyuridine incorporation into DNA during replication.
- a BrdU-ELISA commercially available from Roche Diagnostics, #1647229.
- Other proliferation assays are disclosed in Semlitsch et al. (2002) Anticancer. Res. 22, 1689-97 and Kaur et al. (2002) Gastroenterology 123, 60-7.
- An apoptosis assay measures the number of cells, particularly mammalian cells, which undergo cell-suicide (apoptosis) within a cell culture.
- Apoptosis can be measured by counting the number of cells, which undergo characteristic morphological changes during the process of apoptosis (Hoppner et al. (2001) Nature 412, 202-6), but apoptosis can also be measured by use of biochemical markers characteristic for the process of apoptosis.
- the cell death detection ELISA-Plus distributed by Roche Diagnostics #1774425, is an example of such an assay and measures apoptosis by quantifying the number of free nucleosomes in the cell lysate.
- An apoptosis assay can be useful in screens relating to biological function of cancer cells, cell types affected in cardiovascular diseases and inflammatory diseases, like, e.g., endothelial cells, heart muscle cells, smooth muscle cells or nerve cells.
- Other apoptosis assays are disclosed in Smolewski et al. (2002) J. Immunol. Methods 265, 111-21 and Chen et al. (2001) Mol. Cell. Biol. 21, 6322-31.
- a cytotoxicity assay measures the cell-killing activity of an agent or process that kills cells. Cytotoxicity assays are disclosed in Wang and Zheng (2002) J. Immunol. 268, 179 and Eriksson et al. (2002) Anticancer Res. 22, 1787-93.
- An angiogenesis assay measures the ability of a cell to induce blood vessel formation, a process, which usually accompanies the growth of malignant tissue.
- An angiogenesis assay is disclosed in Kanda et al (2002) J. Natl. Cancer Inst. 94, 1311-9.
- a protein translocation assay measures the qualitative or quantitative change of the steady state localization of a protein within a cell.
- a protein which at steady state is located in the cytoplasm may translocate as a response to a signal received by the cell, to, e.g., the nucleus.
- An example for such a behavior is the nuclear translocation of NFkappaB upon stimulation of an inflammatory response of the cell.
- a commercially available example of a protein translocation assay is the NFkappaB Nuclear Translocation Assay commercially available from Cellomics #K01-0001-1.
- Other protein translocation assays are disclosed in Schlegel et al. (1999) J. Biol. Chem. 274, 33551-6.
- a protein-protein interaction assay can measure the strength of the interaction between a first type of polypeptide and a second type of polypeptide. The result of the measurement can be a binding constant for this protein-protein interaction.
- Such an assay may be done in a BIACORE format (Fivash et al. Curr Opin Biotechnol. (1998) 9, 97-101) or also in the form of a so-called Pull-down (Phizicky at al. Microbiol Rev. (1995) 59, 94-123) or an ELISA.
- a protein-protein interaction assay also the quantity of a protein present in a biological sample can be determined. Examples for such ELISAs are cytokine ELISAs, disclosed in Foster (2001) Int. J. Ex.
- a cytokine ELISA measures the concentration of cytokines, e.g.
- interferon- ⁇ , interleukin-l ⁇ , -2, -4, -5, -6, -8, -10, tumor necrosis factor alpha, TRIAL or others, in a biological sample e.g. in culture supernatants and body fluids
- Matrix Metalloproteinase ELISAs measure the amount of active matrix metalloproteinase. This can be useful for functional screening assays of targets involved in invasiveness, e.g. invasiveness of cancer cells.
- a VEGF ELISA measures the amount of vascular endothelial growth factor.
- a FGF ELISA measures the amount of fibroblast growth factor.
- a FGF ELISA measures the amount of fibroblast growth factor.
- This can be useful for a functional screening assay in the case of targets involved in angiogenesis, wound healing or neural protection.
- the ability of cancer cells to induce angiogenesis can be assessed with such an assay, or e.g. an effect on a heart muscle cell or a smooth muscle cell or an endothelial cell can be assessed with such an assay or, e.g., an effect on nerve cells, e.g. a neural protective effect, can be assessed with such an assay.
- a migration assay measures the ability of a target, e.g.
- a migration assay can be useful for assessing, e.g. the chemotactic motility of cells.
- An ion transport assay measures the amount of ions translocated, or transported across the cell membrane.
- An example of an ion transport assay is disclosed in Lockwich et al. (2001) J. Biol. Chem. 276. 42401-8.
- An ion transport assay can be useful for assessing signaling events in immune cell activation, in cell migration, especially for targets involved in inflammatory diseases, allergy or cancer.
- a colorimetric assay measures a biological function by producing a detectable color change. For example some assays which measure enzymatic activity make use of a colorimetric assay system, e.g. as disclosed in Xu et al., (2000) J. Biol. Chem. 275, 38981-9.
- a gene activation assay measures a biological function, e.g. the strength of a signaling event, by quantification of induced gene expression, e.g. expression of a gene known to be induced by said signaling event.
- a gene activation assay can be performed by way of an ELISA, Chip assay, a Northern, an RT-PCR, a Western blot or another method capable of determining the quantity of a gene product. Examples of gene activation assays are disclosed in Tan et al., (2002) Proc. Natl. Acad. Sci. USA 99, 11387-92 and Zou et al., (2002) Oncogene 21, 4855-62.
- a phenotypic assay can make use of a morphologic change of a target, e.g. an eukaryotic cell, upon a stimulus.
- a target e.g. an eukaryotic cell
- An example of a phenotypic assay is the one described by H ⁇ ppner et al., above.
- the separation step b) of the method of identifying a ligand of the invention is achieved by centrifugation, filtration, density centrifugation, FACS, immobilization onto solid support, affinity chromatography or separation by magnetic beads.
- an ALDU bound to a target can be separated from unbound ALDUs making use of the fact that the complex of an ALDU bound to a target has different biochemical properties compared to an unbound ALDU. Those changed biochemical properties can be size, specific binding, charge density, density or the like. Filtration can separate the bound ALDUs from the unbound ALDUs, e.g.
- the size of the filter is such that ALDUs bound to a target are retained by the pores of the filter, while the unbound ALDUs can pass through the pores of the filter. Filtration is therefore useful if the target is large, e.g. a eukaryotic cell, and the ALDU is small, e.g. a phage, a virus, a nucleic acid or a ribosome displaying a ligand.
- Density centrifugation separates biomolecules according to their density. Density centrifugation can be applied if the density of an ALDU bound to a target is different from that of an unbound ALDU. For example, the density centrifugation can be applied if an ALDU is dense, e.g. a ribosome displaying a ligand, and the target is not as dense, e.g. an eukaryotic cell or vehicles derived from the membrane of a cell.
- FACS can separate the complex of ALDU bound to a target from unbound ALDUs based on fluorescence of the target.
- FACS can be performed by labeling the target with a fluorescent dye and then passing the labeled target in suspending medium through a narrow dropping nozzle so that each target is in a small, separate droplet.
- a laser-based detector system is often used to excite the fluorescent dye and droplets with e.g. fluorescence-positive targets are given an electric charge. Charged and uncharged droplets can then be separated as they pass charge plates and can be collected in two different tubes. In this way small droplets containing ALDUs that are bound to a target are separated from bulk solution containing unbound ALDUs.
- Immobilization of the target onto solid support can be performed in order to separate ALDUs bound to the immobilized target from unbound ALDUs. Immobilization of the target onto solid support is often performed using hydrophobic or hydrophilic interactions between the support and the target or covalent interactions formed by using surface activated solid support or specific binding of the surface of the solid support with the target using e.g. biomolecules with specific binding properties towards the target. In order to separate ALDUs that are bound to the immobilized target from unbound ALDUs, the immobilized target is separated from the bulk solution containing unbound ALDUs.
- Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by removing the bulk solution containing the unbound ALDUs from the immobilized target. For example, if the target is immobilized onto e.g. the well of a plastic micro titer plate via hydrophobic interactions then the bulk solution containing the unbound ALDUs can be removed by removing the solution and washing the wells with washing solution.
- Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by FACS whereby the solid support is labeled with a fluorescent dye and separated as described above.
- the solid support consists of e.g. plastic beads containing a fluorescent dye whereto the target can be immobilized via hydrophobic interactions and the ALDU does not contain a fluorescent dye.
- Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by filtration if the size of the filter is such that the solid support is retained by the pores of the filter, while the unbound ALDUs can pass the filter.
- filtration can be used if the solid support is e.g. surface activated pieces of glass with N-hydroxysuccinimid groups whereto the target can be immobilized via covalent interactions and the pieces of glass are large enough to be retained by the filter while the unbound ALDUs are not.
- Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can also be performed by density centrifugation if the solid support has a different density compared to the bulk solution.
- the solid support consists of e.g. sepharose beads whereto the target is covalently coupled then the sepharose beads containing the target and bound ALDU can be pelleted by centrifugation.
- Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can further be performed by affinity chromatography where the solid support is packed into a column and the bulk solution containing unbound ALDUs is removed by washing the column with washing solution.
- affinity chromatography where the solid support is packed into a column and the bulk solution containing unbound ALDUs is removed by washing the column with washing solution.
- the solid support consists of e.g. sepharose beads covalently coupled to an antibody specific for the target then the sepharose beads can be packed into a column and unbound ALDUs removed by washing the column with washing solution.
- Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by magnetic separation if the solid support has magnetic properties different from the unbound ALDUs.
- the solid support consists of e.g. a paramagnetic particle whereto streptavidin is immobilized via hydrophobic interactions and the target contains a biotin molecule then the target bound to the solid support can be separated from non-paramagnetic ALDUs by applying a magnetic field.
- the amplification step c) of the method of identifying a ligand of the invention, particularly wherein the target is a eukaryotic cell is achieved by either eluting the ALDU bound to a target, or by lysing a target, in particular eukaryotic cells, under conditions where the ALDU remains amplifiable, and subsequent amplification.
- ALDUs bound to a target can be eluted from the target first, e.g. by use of solutions with low pH and/or high salt, as demonstrated in Example 1 and Example 2, or Example 15 and Example 16, and subsequently amplified, e.g.
- the eluted phages of Example 1 or Example 2, or Example 15 and Example 16 are used to infect Escherichia coli in order to produce large numbers of progeny phages.
- the elution step from the target while it may be convenient, is not necessary, as, e.g., in the case of phage libraries even the phages still bound to a target, e.g. the eukaryotic cell, are capable of infecting Escherichia coli and thus capable of producing large numbers of identical progeny, and thus are amplifiable even when still bound to the target, e.g. a eukaryotic cell.
- phages that have entered the target can be amplified, e.g. by lysing the target, e.g. a eukaryotic cell, under conditions where the ALDUs remain amplifiable, and subsequent amplification of the ALDUs.
- the amplification of internalized phages is disclosed in Poul et al. (2000).
- the identity of phages representing the antibody or the antibody fragment obtained with step d) can be determined by, e.g., sequencing the DNA encoding the antibody or the antibody fragment, or, in the case of a commercial library with gridded or numbered phages, by determining the grid position or the number of the phage. The grid position or the number then can reveal the identity ofthe antibody or the antibody fragment represented by the phage.
- step c) the pool of phages is enriched in phages binding to a particular surface molecule. Those phages binding to the surface molecule or antigen can finally be identified by numerous methods known in the art.
- Phages can be separated to form individual clones and the clones of the phages can be contacted with recombinant antigen or can be probed with labeled antigen, or a labeled part of the antigen, e.g. an at least seven amino acid long peptide of the extracellular region of antigen. Clones binding to such a probe are identified as specific antigen-binders. Phages can also be affinity purified on purified antigen or on recombinant antigen.
- the open reading frame encoding the antibody or antibody fragment can be recloned from the whole enriched pool into an expression vector, the antibody or the antibody fragment can then be expressed in clones of another host cell, and the clone of the host cell carrying the expression vector comprising a nucleic acid encoding for the antibody or the antibody fragment binding specifically to the antigen can be identified, e.g. by the method described above for the identification of relevant phage clones, by the method of Examples 2 or 9, or by affinity purification on recombinant antigen.
- the ALDUs or the ligands derived from an ALDU, which are used for the functional screening assay of step d) of the method of identifying a ligand according to the invention are chemically modified to increase their biological activity or endow the ALDU or the ligand with biological activity.
- the ALDU or the ligand derived from an ALDU and used for the functional screening assay of step d) of the method of identifying a ligand of the invention are unmodified.
- the chemical modification of the ALDU or the ligand derived from an ALDU can be the addition of chromophores.
- a chromophore is that part of a molecule that possesses high optical activity due to mobile electrons that interact with light.
- Some examples of chromophores are, e.g., fluorescein derivatives, rhodamine derivatives, coumarin derivatives, porphyrin derivatives, phthalocyanine derivatives, naphthalocyanines derivatives eosin derivatives, triphenylmethane derivatives e.g. malachite green, or acridine derivatives.
- Chromophores useful for chemically modifying biomolecules are well-known in the art.
- a useful chromophore is a chromophore that is non-toxic in the concentration range used and has preferably an absorbance in the visible region of spectrum or can be excited with laser light. Another preferable feature is, that the chromophore is commercially available as a functional derivative or can easily be converted into a functional derivative that can be covalently bound to the ligands of the invention.
- Functional derivatives suitable for covalent binding are e.g. sucinimidyl esters, isocyanates, isothiocyanates or carboxylic acids.
- the method of identifying a ligand of the invention comprises as the identification step d) a spatial separation of the different ALDUs of a pool of ALDUs and then the screening of the spatially separated ALDUs for their effect on a biological function of a target in such a way that the result obtained can be assigned to an individual ALDU.
- Spacial separation of the different ALDUs of a pool of ALDUs can be done, e.g., by filling separate wells of a multi-well plate with individual ligands each and then use those multi-well plates in a functional screening assay.
- the method of identifying a ligand according to the invention can comprise the step of determining the identity of the ligand, wherein this determination step is achieved by sequencing the DNA of an identified ALDU, taking a PCR fingerprint of the nucleic acid of an identified ALDU or also, in the case of a ligand derived from an ALDU, by mass spectrometry of such a ligand.
- PCR fingerprinting of, e.g., phages is disclosed in (Marks et al. J Mol Biol. (1991) 222, 581-97). Mass spectrometrical analysis of a ligand, e.g. a polypeptide, is disclosed in Shevchenko et al. (1996) Anal Chem. 68, 850-58 or Spengler et al (1992) Rapid Commun Mass Spectrom. 6, 105-8.
- the method of identifying a ligand according to the invention may further comprise at least one additional step of screening ALDUs, e.g. wherein the screening is based on biochemical properties of the ALDUs.
- Such an additional screening step may comprise a method selected from the group consisting of flow cytometry, ELISA, Immunoprecipitation, binding assays, immunohistochemical experiments, affinity studies, immunoblots and protein arrays.
- Biochemical properties can be determined by the size, the shape, the density, the charge density, the hydrophobicity, or the binding specificity of an ALDU or the ligand derived from an ALDU.
- the biochemical properties of the ALDU or the ligand derived from an ALDU form the basis of the applicability of the above mentioned methods for screening the ALDUs.
- Flow cytometry is usually performed by labeling cells with a fluorescent dye and then passing those labeled cells in suspending medium through a narrow dropping nozzle so that each cell is in a small, separate droplet.
- a laser-based detector system is often used to excite the fluorescent dye and droplets with e.g. fluorescence-positive cells are registered.
- Immunoprecipitation is a procedure by which a ligand, e.g. an antibody or antibody fragment, that reacts specifically with an antigen, e.g. an antigen associated with the surface of a target, for example a eukaryotic cell, is used to remove said antigen from a solution, e.g. a cell lysate.
- the immunoprecipitate consisting of the ligand bound to the antigen can then be examined by utilizing biochemical methods like, e.g. 2D gel electrophoresis.
- the identity of the antigen can then be determined by use of, e.g., mass spectrometry.
- the method of identifying a ligand of the invention can further comprise a subtractive selection step.
- a subtractive selection step is a step, which removes ALDUs with an undesired property.
- a subtractive selection step can be affected by removing the ALDUs capable of binding to a control cell, if the property of binding to a control cell is undesired.
- one wants to select for ALDUs specific for cancer cells one could first select those ALDUs which bind to cancer cells, elute the bound ALDUs, e.g.
- phages and then remove those ALDUs which are capable of binding to non-cancer-cells, by for example contacting the pool of eluted ALDUs with non-cancer-cells and removing those ALDUs bound to the non-cancer-cells.
- the ALDUs remaining in the supernatant are then ALDUs specific for cancer cells and can then be used in functional screening assays according to the method of identifying a ligand ofthe invention.
- the method of identifying a ligand of the invention can further comprise the step of identifying the surface antigen to which the ligand ofthe invention binds, for example by immunoprecipitation followed by mass spectrometrical analysis, preferably excluding ligands such as small molecules.
- the identification of said surface antigen is not a necessary step of the method of identifying a ligand according to the invention, it is still particularly useful, as this step leads to the identification of both, a ligand which affects a biological function of the target, e.g. a eukaryotic cell, and the antigen associated with the surface of the target, which is involved in said biological function.
- ligands e.g.
- the potential drug is in such a way used to identify the corresponding drug target, and the ability of this embodiment of the invention to simultaneously identify a potential drug and the corresponding potential drug target is a great advantage.
- the present invention further provides a method to identify unknown antigen proteins via an immunoprecipitation protocol that uses a Streptag modified scFv and Streptactin coupled to a solid carrier material like Sepharose, magnetic beads etc.
- This method allows the use of single chain Fv to isolate their unknown, cognate (membrane) protein targets from a very complex mixture of proteins such as whole cell lysate.
- the precipitate is loaded on a gel and the isolated bands can conveniently be investigated by mass spectrometry analysis.
- the scFv have been selected against known, purified proteins, and the immunoprecipitation experiments were conducted in order to confirm the specificity of the binder.
- the advantage of the disclosed method is the simultaneous identification of scFv specificity and antigen identity.
- the method of identifying a ligand binding specifically to the extracellular region of an antigen associated with the surface of a target of the invention also comprises the step of incorporating the ligand identified into a therapeutic, prophylactic or diagnostic composition. This can, e.g., be done by mixing the identified ligand with a pharmaceutically acceptable carrier known in the art, wherein the ligand is present in an amount, which is therapeutically effective.
- the invention in another preferred embodiment relates to a method for the production of a pharmaceutical composition, comprising the method of identifying a ligand binding specifically to the extracellular region of an antigen associated with the surface of a target and further the step of mixing the identified ligand, or a modified or a labeled version thereof, with a pharmaceutical acceptable carrier known in the art.
- a modified ligand is a ligand with a covalent modification. Examples of modified ligands are labeled ligands.
- Figure 2 shows - presented as a table - results of the Invasion assay performed with HT1080 (human fibrosarcoma) cells.
- the scFv tested are identified by their number as shown in Fig. 6.
- the symbol "*" indicates that the respective scFv - as identified by its number as shown in Fig. 6 - was cloned into an IgG4 format before performing the Invasion assay.
- the invasion of cells through the matrix is measured and shown in % of inhibition of invasion.
- Figure 3 shows - presented as a table - results of FACS analysis performed on various cell types, such as HT1080 (human fibrosarcoma), KHOS-NP (human osteosarcoma), MCF-7 (human breast adenocarcinoma), BT-474 (human breast cancer, mammary gland), PC-3 (human adenocarcinoma, prostate), Jurkat (human T cell leukemia), HL-60 (human acute myeloid leukemia), HeLa (human cervix carcinoma), SW480 (human colon carcinoma), LS174T (human colon carcinoma), HT-29 (human colon carcinoma) in comparison to a control cell (Hs27 (human skin fibroblast)).
- HT1080 human fibrosarcoma
- KHOS-NP human osteosarcoma
- MCF-7 human breast adenocarcinoma
- BT-474 human breast cancer, mammary gland
- PC-3 human adenocarcinoma, prostate
- Jurkat human T cell leukemia
- Table 3a depicts scFv identified by its number as shown in Fig. 6.
- Table 3b depicts FACS results of IgG. The symbol * means that the respective scFv -as identified by its number as shown in Fig. 6 was cloned into an IgG4 format before performing the FACS experiment.
- FIG. 4 shows results of the immunoprecipitation experiments.
- the immuno-complexes were separated by SDS-PAGE and silver stained. While scFv2 recognized a protein with approximately 120 kDa, scFv7 and scFv9 recognized the 1 lOkDa MCAM protein.
- Figure 5 show the vector map of pXP14 (SEQ ID No. 39) as well as sequence of the scFv expression vector.
- Figure 6 shows in Table 1 the peptide sequences of the identified single chains: scFvl to scFcl9.
- the CDR3 region is underlined in the depicted peptide sequence. Relevant SEQ ID No.'s are indicated aside.
- Figure 7 shows in Table 2 the nucleotide sequences encoding for the polypeptides scFvl to scFcl9. Relevant SEQ ID No.'s are indicated.
- Figure 8 shows - presented as a table - results of the Adhesion assay performed on various matrixes, such as CI (collagen S type I), CIV (collagen IV), FN (fibronectin) and LN (laminin), with various cell types, such as HT1080 (human fibrosarcoma), PC-3 (human adenocarcinoma, prostate), HeLa (human cervix carcinoma) and HT-29 (human colon carcinoma).
- the scFv tested are identified by their number as shown in Fig. 6.
- the symbol "*" indicates that the respective scFv -as identified by its number as shown in Fig. 6- was cloned into an IgG4 format before performing the Adhesion assay.
- the adhesion is measured and shown in % of inhibition of adhesion.
- inhibition values “+” represents 1-10% inhibition, "++” represents an inhibition value of > 10-40% and "+++” represents an inhibition value of >40-100%.
- “n.d.” represents not determined.
- Figure 9 shows the vector map of scFv expression vector pXPIO (SEQ ID No. 40) and sequence ofthe pXPIO.
- Figure 10 shows the results of the immunohistochemistry using the MCAM specific antibodies (scFv7* and scFv9*) (scFv7 and scFv9 were cloned into IgGl format).
- Figure 11 shows a MALDI-MS spectrum (a) of the peptide mixture obtained from the band with an approximate size of 110 kDa.
- the matching peptides cover 23%) (151/646 residues) of the amino acid sequence SEQ ID No. 58 as shown in Figure l ib.
- Figure l ie shows a MALDI-MS spectrum of the peptide mixture obtained from the band with an approximate size of 145 kDa immunoprecipitated with scFvl4.
- Figure 12 shows the results of the Proliferation assay as the reduction of SW-480 and PC-3 proliferation by IgG's.
- scFv 7, scFv9 and scFvl ⁇ were cloned into an IgG4 format before performing the proliferation assay.
- Proliferation of SW-480 colon cancer cells (Fig. 12b) and of PC-3 prostate cancer cells (Fig. 12a) was measured with an MTS cell viability assay at the indicated time points after first antibody addition. Data are shown as means of % medium control, pooled from 3 independent experiments. Error bars: Tukey HSD 95% confidence intervals.
- Figure 13 shows - presented as a table - the anti-tumor effect of compounds in a subcutaneously grown human tumor xenograft in athymic mice.
- a lung adenocarcinoma (LXFA) was used as a xenograft.
- the table shows the tumor size at day 0 and day 7 after compound injection.
- scFv7* scFv7 cloned into IgGl format
- Docetaxel A result of the combination therapy of scFv* with Docetaxel is also shown.
- Figure 14 shows construction primers for a mouse library
- Example 1 Selection and screening of scFv (Selection on cells in suspension)
- Single chain Fv were selected from a large non-immune phage displayed repertoire of human origin containing 1011 independent clones, provided by Cambridge Antibody Technology Ltd., Cambridge, UK.
- HT1080 cells human fibrosarcoma cell line; ATCC, CCL-121
- ATCC human fibrosarcoma cell line
- FCS fetal calf serum
- Two times 1012 cfu of phage library/107 cells were pre-blocked for 1 hour at 25°C with DMEM + 10% FCS and subsequently incubated with end-over-end rotation for 1.5 hour at 25°C with the cells in Eppendorf tubes pre-blocked with DMEM + 10% FCS.
- Three times 107cells were used for the first round of selection and 1x107 cells were used for the 2nd round of selection, respectively.
- the cells were washed by centrifugation at 220xg for five minutes, followed by removal of the supernatant and re-suspension in wash buffer. Five washes with DMEM + 10% FCS + 0.05% Tween-20 as wash buffer and five washes with DMEM + 10% FCS as wash buffer were performed. Bound phages were eluted by the addition of 10 mM Glycine pH 2.2, neutralized with IM Tris/HCl pH 7.4. Typically, between 103 and 106 cfu were eluted in the 1st round of selection, thus the diversity of the enriched repertoire is decreased compared to the original repertoire. The eluate containing the enriched repertoire was amplified by infecting exponentially growing E.
- Phagemid containing E. coli were selected and propagated by overnight growth at 30°C on LB agar plates supplemented with lOO ⁇ g/ml ampicillin and 1% glucose.
- the enriched repertoire can either be amplified as a polyclonal pool and used for further rounds of selection in an iterative manner even until convergence to desired properties is achieved or be spatially separated and screened for a desired function on a single clone level.
- Phage particles for the next round of selection were produced by super-infecting exponentially growing cultures of the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin. Selection ready phage were precipitated with 0.5 M NaCl/4% PEG-6000 from the cleared bacterial supernatant and re-suspended in PBS. In this example two rounds of selection were performed followed by screening on a single clone level.
- Example 2 Selection and screening of scFv (Screening on adherent cells)
- the genes encoding the selected scFv, contained in the phage display vector were re-cloned to the expression vector pXP14.
- This vector directs the expression of a scFv in fusion with a Streptag and E-tag and does not contain a filamentous phage gene-3.
- Expression vector containing E. coli TGI from single colonies were grown in individual wells of a microtiter plate so that each well contains only one scFv clone. The bacteria were grown at 30°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 0.1% glucose in 96-well microtiter plates (#9297, TPP) until an OD600 of 0.7.
- Sequencing of scFv genes was performed by Sequiserve GmbH, Vaterstetten, Germany using the primer pXP2 Seq2 (5'-CCCCACGCGGTTCCAGC-3'; SEQ ID No. 41) and pXP2 Seql (5 ACCTATTGCCTACGGC-3 '; SEQ ID No. 42). Amino acid sequences are shown in Table 1 and nucleotide sequences are shown in Table 2.
- the cultures were grown at 25°C shaking until an OD 6 oo 0.5 - 0.6 was reached and then induced with IPTG 0.1 mM final concentration.
- Fresh Ampicillin was added to 50 ⁇ g/ml and incubation was proceeded at 22°C o/n shaking.
- the cultures were centrifuged at 5000 x g for 15 minutes at 4°C, supernatants discarded and the pellets resuspended carefully on ice with a pipette in 10 ml pre-cooled PBS-0.5 M Na buffer containing protease inhibitors complete (#1697498, Roche).
- bacterial suspensions were transferred to 20 ml oakridge centrifuge tubes and hen-egg lysozyme (#L-6876, Sigma) added to a final concentration of 50 ⁇ g/ml for 1 hour on ice.
- the lysed bacteria were centrifuged at 20000 x g for 15 minutes at 4°C and the supernatants (lysate) transferred to a 15 ml plastic tube.
- affinity purification the lysates were loaded with 1 ml/min onto 1ml StrepTactin (# 2-1505-010, IB A) columns equilibrated with 10 column volumes (CV) PBS-0.5 M Na buffer via a parallel protein purification system (self- made).
- the scFvs consist of the sequence of a variable light and heavy chain linked by a linker sequence.
- the variable light chain and the variable heavy chain were amplified by PCR separately with the usage of primer, which contain restriction sites. Those restriction sites are also present in the vectors, which contain the appropriate constant domains for the heavy and light chain.
- the amplified variable domains were cut with the restriction enzymes and cloned into the cut vectors. The correct sequence was confirmed via sequencing
- the vectors contained an Epstein Ban- virus origin of replication (oriP sequence) which enhances the level of transcription in 293- EBNA-HEK cells, because the EBNA protein leads to the replication ofthe episomal vector.
- a co -transfection was carried out with the vector for the heavy chain and the vector for the light chain leading to the expression of both chains in the cell and the assembly of the IgG in the Endoplasmic Reticulum. The assembled IgG was then secreted to the medium.
- Calcium-phosphate transfection was used, where a precipitate of Calcium-phosphate and the DNA is formed and incorporated into the cell. After the transfection the medium was changed to serum-free medium. Three harvests per IgG were done every 3 days. The supernatant (media) were sterile-filtrated and stored at 4°C.
- the supernatants were purified via Protein A Sepharose either by gravity flow or by HPLC depending on the volume. For up to 200 ml a gravity flow method was used. For both purification types the supernatant was loaded on the Protein A column, washed with 50 mM Tris pH 7 buffer and eluted with 0.1 M Citrate pH ⁇ 2. To the elution fraction 0.25 M Tris pH 9 was added leading to a pH of 5.5-6.0. Depending on the further use ofthe IgGs they were dialysed against PBS buffer and stored at -20°C.
- Example 4 FACS analysis for tumor cell specific binding
- FACS fluorescence-activated cell sorter
- ChemoTx® system Neuro Probe Inc.# 106-8, Gaithersburg was used as a disposable chemotaxis/cell migration chamber in a 96 well format with an 8 ⁇ m filter Track etched Polycarbonate pore size, 5.7 mm diameter/site.
- Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycan, entactin and nidogen.
- TGF- ⁇ fibroblast growth factor also contains TGF- ⁇ fibroblast growth factor, tissue plasminogen activator, and other growth factors which occur naturally in the EHS tumor
- Becton Dickenson, BD #356234 diluted in Dulbeccos PBS (Gibco #14040-091) was applied on the membrane filter ofthe 96-well plate on row B-H and on row A 1,2 ⁇ g/site of collagen S type I (Roche #10982929) diluted in 0,05 M HCl (Sigma #945-50) and incubated over night at 20°C in a desiccator for gelation.
- HT1080 cells were grown to 70-80% confluence in DMEM supplemented with Glutamaxl (862mg/l (Gibco #31966-021) with 10%) FCS (Gibco #10270106). The cells were then labeled in situ with Bisbenzimide H 33342 (Sigma #B-2261) diluted 1 :100 in DMEM/GlutamaxI/0.1 % BSA (Sigma #A-7030) for 15 min at 37°C, 7,5% CO 2 . Cells were washed 2x with DMEM/GlutamaxI/0.1 % BSA and loaded with DMEM/GlutamaxI/0.1 % BSA for 15 min at 37°C, 7,5% CO 2 for recovering.
- Bisbenzimide H 33342 Sigma #B-2261
- DMEM/GlutamaxI/0.1 % BSA Sigma #A-7030
- the cells were detached with 0.5mM EDTA (Sigma #E8008), collected with Dulbeccos PBS/0.1% BSA/lOmM Hepes (Gibco #15630-056), washed 2x with Dulbeccos PBS/0.1% BSA/lOmM Hepes, suspended in Dulbeccos PBS/0.1% BSA/lOmM Hepes and diluted to 6,7 x 10 6 cells/ml with Dulbeccos PBS/0.1% BSA/lOmM Hepes.
- DMEM/GlutamaxI with 5% FCS was used as a chemo attractant in the lower chamber.
- a standard curve from lxl 0 4 to 4xl0 4 cells/site is performed on collagen S type I coated row A of the chemotaxis chamber.
- DMEM/GlutamaxI/0.1%> BSA was used in the lower chamber (cells are not migrating). After scraping the non-migrating cells from the top of the membrane (except the Standard curve on row A) fluorescence of cells, which had migrated through the membrane (not migrated in case of the Standard curve), was measured on the Fluostar Galaxy (bMG) microplate reader using excitation/emission wavelengths of 370/460nm.
- Viable cells were detected by measuring the conversion of the tetrazolium dye MTS (MTS, Celltiter Aqueous one, Promega #G4000) to formazan.
- MTS tetrazolium dye
- HT1080 cells and HT1080 cell/scFv dilutions obtained from the dilutions prepared in the Invasion assay were pipetted in triplicate at a density of 3,4 x 10 4 cells/well were plated in a 96-well plate (black, ultra thin clear flat bottom, special optics, Costar #3615) 10 ⁇ l MTS was added to each well and incubated for 1 hour at 37°C, 7,5% CO 2 . Absorbance was measured at 492 nm with the Fluostar Galaxy (bMG) microplate reader. For all tested scFvs, no effect on viability of cells was seen.
- Example 6.2 Proliferation assay
- SW-480 and PC-3 cells were cultured in RPMI containing L-Glutamine and 10% FCS (Invitrogen #21875-034, Carlsbad, California).
- SW-480 and PC-3 cells were seeded in culture medium in a volume of lOO ⁇ l/well into a 96well plate (Corning Costar #3904, Acton, Massachusetts). Cells were incubated 24h at 37°C, 5% CO . Medium was aspirated and 15 ⁇ g/ml antibody or 50 mM NaN was added diluted in culture medium. Control cells were incubated in medium alone. 48h after the first addition, antibody was added a second time at the same concentration. MTS absorbance was measured before, 24h, 48h and 72h after the first treatment.
- TPP #9296 cell culture treated
- collagen S type I 1 ⁇ g/well (Roche #10982929)
- Dulbeccos PBS (Gibco #14040-091) at 4°C over night.
- the Adhesion assay was also performed using several matrix proteins, such as CIV (collagen IV), FN (fibronectin) and LN (laminin), and with various cell types, such as HT1080 (human fibrosarcoma), PC-3 (human adenocarcinoma, prostate), HeLa (human cervix carcinoma) and HT-29 (human colon carcinoma) cells. Additionally to the scFv also IgG cloned from the single chains according to Example 3.2 were tested for their capacity to inhibit adhesion of tumor cells. Results are shown in Figure 8.
- CIV collagen IV
- FN fibronectin
- LN laminin
- HT1080 human fibrosarcoma
- PC-3 human adenocarcinoma
- HeLa human cervix carcinoma
- HT-29 human colon carcinoma
- single cell suspensions of HT1080 were harvested with 0,5mM EDTA/PBS. Approximately 1 x 10 6 cells were incubated in CellWash (BD, #349524) with lO ⁇ g/ml scFv for one hour at 4°C. After washing with Cell Wash lO ⁇ g/ml FITC labeled scFv was added and incubated for 20 min at 4°C. Signals of bound FITC labeled scFvs with and without pre incubation of other binders were analyzed on a Becton Dickinson FACSscan.
- HT1080 specific single chain Fv were cloned to the expression vector pXP14, which directs the expression of a scFv in fusion with a Streptag and an E-tag at its c-terminal end.
- the Streptag-scFv were used for immunoprecipitation as follows.
- HT1080 and Hs-27 cells (10 8 ) were lysed in 3ml 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100 (v/v) containing protease inhibitor cocktail (1 pill in 50ml buffer) (Boehringer Mannheim, Cat. -No.
- HT1080 specific single chain Fv 50 ⁇ g/1 mg cell extract
- samples rotated for 2h at 4°C gently centrifuged at 700 x g to pellet the Streptactin Sepharose
- the immuno-complexes were separated by SDS-PAGE and silver stained for MS analysis.
- scFv 2 pulled down a protein, detected as a band on SDS-PAGE by silver staining at a molecular weight of approximately 120 kDa. This band was absent in the control samples with Hs-27 cells.
- Single chain Fv 1, 3, 5, 6, 7, 9, 15 and 19 pulled down a protein, detected as a band on SDS- PAGE by silver staining at a molecular weight of approximately 1 10 kDa.
- Immunoprecipitations were performed using cell extract from HT1080 cells and Hs-27 cells as control cells. It was observed that this particular band was also present in the control samples, but in all cases to a much lower extent indicating that this protein is likely to be over-expressed in the HT1080 cell line.
- Single chain Fv 4, 1 1 and 14 pulled down 2 proteins, detected as 2 bands on SDS-PAGE by silver staining at a molecular weight of approximately 150 kDa and approximately 130 kDa, respectively.
- the upper band was also present in the control samples, but a much lower extent, whereas the lower band was absent or extremely faint in the controls, indicating that the 2 proteins are likely to be overexpressed in the HT1080 cell line.
- Example 10 Protein identification via mass spectrometry
- the two fractions were pooled and one microliter of the obtained peptide mixtures was mixed in a 1 :1 ratio with a solution of ⁇ -cyano-4-hydroxycinnamic acid (3 mg/ml), co-crystallized on a Teflon-coated stainless steel target and analyzed on a MALDI-TOF instrument yielding peptide mass fingerprints (PMF) in a mass range of m/z 800-3000.
- the obtained PMF were used to search all entries for the species Homo sapiens in the NCBI and SwissProt databases. In all cases, only peptides matching a given protein with a mass deviation of less than 13 ppm were considered for identification.
- a MALDI spectrum that led to the identification of MCAM is shown in Figure 11a.
- the peptide coverage is shown in Figure l ib.
- the molecular weight of any of the melanoma adhesion proteins is around 72 kDa, but since the protein is expected to be glycosylated it is not surprising to find the band on the gel around 110 kDa.
- the molecular weight of this protein is around 119 kD, but it is expected to be glycosylated, thus explaining the higher observed molecular weight observed on SDS-PAGE.
- the band with an approximate size of 150 kDa which is the higher one of the two bands obtained by using scFvl l-scFvl4, yielded 17 to 21 peptides, which matched integrin alpha-2, with a maximum protein coverage of 19% (225/1181 residues).
- a MALDI spectra is shown in Fig. l ie.
- the molecular weight of integrin alpha-2 is around 129 kDa, and the molecular weight of integrin beta-1 is around 88 kDa.
- both proteins are expected to be highly glycosylated, which might explain the higher apparent molecular weight observed on SDS- PAGE.
- Example 11 Methods for epitope mapping
- membranes are equilibrated and unspecific binding sites are blocked. After incubation with the antibody of interest and several washing steps the detection is performed using an HRP- conjugated secondary antibody in combination with the ECL-System. Membranes can be stripped, regenerated, and re-used up to 10 times depending on the antibody. Small overlapping oligo-peptides that ideally cover the complete amino acid sequence ofthe antigen of interest are synthesized on a solid support. This method allows the identification of linear epitopes on the amino acid level. It also allows rapid mutational studies.
- Example 13 Anti-tumor effect of compounds in subcutaneously growing human tumor xenografts A lung adenocarcinoma (bronchial adenocarcinoma) was used as a xenograph.
- mice of NMRI background approximately 7 weeks old at the start of treatment with an average weight of 35g were used. Animal health was examined before study commencement to ensure that only animals of good health entered testing procedures. Identification of mice was achieved by individual ear tag number, each cage by labeling with a record card, indicating the number of the experiment, date of randomization, mouse strain, gender, individual mouse number, test compound, dosage, schedule, and route of administration. The animals were housed in Macrolon M type III cages with filter hoods in air-conditioned rooms at 24 ⁇ 1°C and relative humidity at 60+10% and fed Altromin Extrudat 1439 Rat/Mouse diet, demineralized sterilized water containing 0.9 g/1 potassium sorbate with the pH adjusted to 2 with IN HCl.
- the group size is 6 mice and in order to obtain 24 mice with similar-sized tumors, 32 mice were implanted with a total of 64 tumor fragments (bilateral implantation).
- Tumor fragments for implantation were obtained from xenografts in serial passage in nude mice. After removal of the tumor from donor mice, the tumor was cut into fragments (2-3 mm diameter) and placed in RPMI 1640 culture medium (maximally for 30-45 minutes) until subcutaneous implantation in the mice.
- the mice were anaesthetized by inhalation of vaporized isoflurane and two small incisions were made in the skin of the back (left and right), and 2 tumor fragments were transplanted per mouse. Treatment started if tumors had grown to mean diameters of between 6 and 8 mm.
- mice were divided into 3 categories according to tumor diameter: large: >8 mm; medium: between 6 and 8 mm; small: 5 mm.
- tumor categories were equally distributed among the different groups.
- Tumor size and mouse body weight were measured twice a week and mortalities and clinical signs recorded daily.
- Relative volume of individual tumors was calculated as the ratio between the tumor volume on Day x and the tumor volume on Day 0 (Tx/TO, Day 0 is the day of randomization and the first day of treatment).
- Relative body weights of individual mice were calculated correspondingly. Plots of the relative tumor volume and the relative body weight over time were generated.
- T/C% value the ratio of the median relative tumor volumes for the antibody-treated and the vehicle-treated groups on a particular day was calculated (T/C% value).
- Tumor-bearing mice were treated twice a week for 4 weeks. Tumors were collected at study termination for further analysis.
- FIG. 13 A comparison of the tumor size after different treatments at Day 7 is shown in Figure 13.
- blood samples of 300 ⁇ l were retrieved by sublingual bleeding of mice in the antibody-treated group at 6 different time points during the in vivo therapy study. Trough levels were determined on Day 4 just before administration of the second dose and on Day 22 before administration of the 7 th dose.
- Blood samples (three blood samples per time point) were obtained on Day 26, 27, 28 and 29. Samples on Days 4, 26 and 28 were obtained from mice 1-3, samples on Days 22, 27 and 29 from mice 4-6. The blood sample size was sufficient to prepare at least 100 ⁇ l plasma. Plasma samples were prepared using EDTA, stored at -80°C until analysis.
- mice Two BALB/c mice were each immunized intradermally with 2 x 10 7 paraformaldehyde fixed HT1080 cells (human fibrosarcoma cell line; ATCC, CCL-121). Following the first immunization, the injections were repeated twice in a period of 39 days, the mice sacrificed and the spleens isolated and frozen in liquid nitrogen. The immunizations were performed by Charles River, Germany GmbH, KiBlegg.
- VH genes were PCR amplified from l ⁇ l of cDNA using 36 individual combinations of 9 forward primers (M-VH1 (SEQ ID NO.: 81), M-VH2 (SEQ ID NO.: 82), M-VH3 (SEQ ID NO.: 83), M-VH4 (SEQ ID NO.: 84), M-VH5 (SEQ ID NO.: 85), M-VH6 (SEQ ID NO.: 86), M-VH7 (SEQ ID NO.: 87), M-VH8 (SEQ ID NO.: 88), M-VH9 (SEQ ID NO.: 89)) and 4 backward primers (M-JH1 (SEQ ID NO.: 90), M-JH2 (SEQ ID NO.: 91), M-JH3 (SEQ ID NO.: 92), M-JH4 (SEQ ID NO.: 93)) without restriction sites.
- M-VH1 SEQ ID NO.: 81
- VL genes were PCR amplified with one primer mix (M-VK1 (SEQ ID NO.: 94), M- VK2 (SEQ ID NO.: 95), M-VK3 (SEQ ID NO.: 96), M-VK4 (SEQ ID NO.: 97), M-VL1 (SEQ ID NO.: 98), M-JK1 (SEQ ID NO.: 99), M-JK2 (SEQ ID NO.: 100), M-JK3 (SEQ ID NO.: 101), M-JL1 (SEQ ID NO.: 102)) without restriction sites.
- M-VK1 SEQ ID NO.: 94
- M- VK2 SEQ ID NO.: 95
- M-VK3 SEQ ID NO.: 96
- M-VK4 SEQ ID NO.: 97
- M-VL1 SEQ ID NO.: 98
- M-JK1 SEQ ID NO.: 99
- M-JK2 SEQ ID NO.: 100
- PCR products were gel-purified (QIAquick Gel Extraction Kit, #28706) and reamplified using individual combinations of 9 forward primers (MVH1 Sfil (SEQ ID NO.: 59), MVH2 Sfil (SEQ ID NO.: 60), MVH3 Sfil (SEQ ID NO.: 61), MVH4 Sfil (SEQ ID NO.: 62), MVH5 Sfil (SEQ ID NO.: 63), MVH6 Sfil (SEQ ID NO.: 64), MVH7 Sfil (SEQ ID NO.: 65), MVH8 Sfil (SEQ ID NO.: 66), MVH9 Sfil (SEQ ID NO.: 67)) and 4 backward primers (M-JH1 Sail (SEQ ID NO.: 68), M-JH2 Sail (SEQ ID NO.: 69), M-JH3 Sail (SEQ ID NO.: 70), M-JH4 Sail (SEQ ID NO.: 71))
- PCR products were gel-purified (QIAquick Gel Extraction Kit, #28706) and cloned into the phage display vector pXPIO (SEQ ID No.: 40) using the restriction sites Sfil/Sall for VH and ApaLI/Notl for VL.
- the ligation mix was transfected into E.coli TG-1 by electroporation resulting in a library size of 10 7 independent clones (phages) expressing different single chain antibody fragments (scFv).
- Single chain Fv were selected from a phage display library generated from mice immunized with fixed HT 1080 cells.
- HT1080 cells were harvested with 0.05% EDTA, fixed with paraformaldehyde, diluted to lxl0 7 cells/ml in PBS and immobilized onto wells of a 96 well UV cross-link plate (Corning Costar). The wells of the UV cross-link plate were blocked with 5% Skim Milk Powder (#70166, Fluka) in PBS (MPBS). 10 12 cfu (colony forming units) of phage library/10 6 cells were pre-blocked for 1 hour at 25°C with MPBS and subsequently incubated for 1.5 hour at room temperature (RT) with the cells.
- RT room temperature
- coli were selected and propagated by overnight growth at 30°C on LB agar plates supplemented with 100 ⁇ g/ml ampicillin and 1% glucose. Following this step, the enriched repertoire can either be amplified as a polyclonal pool and used for further rounds of selection in an iterative manner until convergence to desired properties is achieved or be spatially separated and screened on a single clone level.
- Phage particles for the next round of selection were produced by super-infecting exponentially growing cultures of the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin. Selection ready phage were precipitated with 0.5 M NaCl/4% PEG- 6000 from the cleared bacterial supernatant and re-suspended in PBS. One round of selection was performed followed by screening on a single clone level.
- the genes encoding the selected scFv, contained in the phage display vector were re-cloned to the expression vector pXP14.
- This vector directs the expression of a scFv in fusion with a Streptag and E-tag and does not contain a filamentous phage gene-3.
- Expression vector containing E. coli TGI from single colonies were grown in individual wells of a microtiter plate so that each well contains only one scFv clone. The bacteria were grown at 30°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 0.1% glucose in 96-well microtiter plates (#9297, TPP) until an OD 60 o of 0.7.
- HT1080 cells were harvested with 0.05% EDTA, fixed with paraformaldehyde, diluted to lxl0 7 cells/ml in PBS and immobilized onto wells of a 96 well UV cross-link plate (Corning Costar). The wells of the UV cross-link plate were blocked with MPBS and the scFv containing blocked cleared lysates added for 1.5 hours at 25°C.
- the plates were washed 2x with PBS + 0.1% Tween-20 and lx with PBS, incubated with HRP conjugated ⁇ -E-tag (#27-9413-01, Pharmacia Biotech; diluted 1 :5000 in MPBS with 0.1 % Tween-20) for 1 hour, washed 3x with PBS + 0.1% Tween-20 and 3x with PBS, developed with POD (#1 484 281, Roche) and signals read at 370 nm. Positive clones were retested against HT1080 cells and control human fibroblasts Hs- 27 (ATCC CRL-1634) using the ELISA screening procedure described above.
- Cell proliferation is measured by quantifying the amount of bromo-deoxyuridine incorporated into DNA during replication.
- This assay can be used in a broad range of applications, e.g. in cancer (screen for proliferation inhibitors) or inflammatory diseases (screen for anti- inflammatory agents).
- cancer screen for proliferation inhibitors
- inflammatory diseases screen for anti- inflammatory agents.
- composition of buffers and reagents we refer to the manufacturer's instructions.
- Treated and non-treated cells are cultured in a 96-well plate in 100 ⁇ l of normal growth medium per well. After addition of 10 ⁇ l BrdU-labeling solution per well (final concentration: 10 ⁇ M), cells are incubated for 2-24 hours, depending on the doubling time of the cell line used. The labeling medium is removed after centrifugation for 10 minutes at 300 x g, and the cells are dried for 1 h at 60°C. 200 ⁇ l of FixDenat reagent and then 100 ⁇ l of anti-BrdU-POD working solution are added to each well and the plate is incubated for 90 minutes at 15-25°C.
- the wells are rinsed with 200-300 ⁇ l of washing solution, and 100 ⁇ l of substrate solution are added. Following an incubation of 5-30 minutes at room temperature and addition of 25 ⁇ l IM H 2 SO 4 , the absorbance ofthe samples is read in a plate reader at 450 nm (reference wavelength: 690 nm).
- Apoptosis is measured by quantifying the number of free nucleosomes in a cell lysate. This assay can be applied in screen for apoptosis inducing or -inhibiting agents in wide range of indications (cancer, cardiovascular diseases, inflammatory diseases).
- composition of buffers and reagent we refer to the manufacturer's instructions.
- the 96-well plate with the cells induced to undergo apoptosis and the control cells is spun at
- the medium is removed and 200 ⁇ l of lysis buffer are added.
- the plate is shaken for 30 minutes at room temperature, then centrifuged at
- the antibody mixture consisting of 72 ⁇ l of incubation buffer, 4 ⁇ l of anti- histone- and 4 ⁇ l of anti-DNA-POD-antibody, is added and the plate is shaken for 2 h at room temperature, covered with an adhesive foil. After removal ofthe solution and 3x washing with
- Activation of the transcription factor NFKB is measured by quantifying the portion of NFkB translocated from the cytoplasm to the nucleus using automated fluorescence microscopy;
- NFkB actvation mainly immune and inflammatory diseases, but also cardiovascular disease and cancer.
- composition of buffers and reagents we refer to the manufacturer's instructions.
- Example 20 Identification of virulence factors in alphaviruses and corresponding neutralizing scFvs
- Alphaviruses are a group of 26 icosahedral positive-sense RNA viruses transmitted by mosquitoes.
- Ross River (RR) virus is a member and causes polyarthritis in humans and is endemic in Australia.
- Viruses are produced as described (Vrati et al. (1988) Virology 162, 346-353; Smith et al. (1995) PNAS 92, 10648-52; Sharkey et al. (2001) J. Virol. 75, 2653-2659).
- the T48 strain of RR is rescued from full-length cDNA pRR64 (Kuhn et al. (1991) Virology 182, 430-441) and is the source of virus.
- Mosquito cell line C6/36 is used for propagation. Cells are grown at 30°C in roller bottles using Eagle minimal essential medium/10% fetal calf serum and are infected with virus at a multiplicity of infection (MOI) of 1. After a 36 hour incubation, cell supernate are harvested and viruses are concentrated by precipitation in 10% polyethylene glycol 600 (PEG)/0.5 M NaCl. The virus is purified by sucrose density centrifugation.
- single chain Fv are selected from a large non- immune phage displayed repertoire of human origin containing 10 11 independent clones, provided by Cambridge Antibody Technology Ltd., Cambridge, UK.
- Purified virions at different concentrations are tested for selection.
- Purified virions are coated on a 96 well UV cross-link plate (Coming Costar).
- the wells of the UV cross-link plate are blocked with 5% Skim Milk Powder (#70166, Fluka) in PBS (MPBS).
- MPBS 5% Skim Milk Powder
- Two times 10 12 cfu (colony forming units) of phage library/well are pre-blocked for 1 hour at 25°C with MPBS and subsequently incubated for 1.5 hour at room temperature (RT) with the wells.
- the wells of the UV cross-link plate are washed six times with PBS + 0.05% Tween-20 followed by six washes with PBS.
- Bound phage are eluted by the addition of 10 mM Glycine pH 2.2, and neutralized with IM Tris/HCl pH 7.4. Typically, between 10 3 and 10 6 cfu are eluted in the 1 st round of selection, thus the diversity of the enriched repertoire is decreased compared to the original repertoire.
- the eluate containing the enriched repertoire are amplified by infecting exponentially growing E. coli TGI. Phagemid containing E. coli are selected and propagated by overnight growth at 30°C on LB agar plates supplemented with lOO ⁇ g/ml ampicillin and 1%) glucose.
- the enriched repertoire can either be amplified as a polyclonal pool and used for further rounds of selection in an iterative manner until convergence to desired properties is achieved or be spatially separated and screened on a single clone level.
- Phage particles for the next round of selection are produced by super- infecting exponentially growing cultures of the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin. Selection ready phage are precipitated with 0.5 M NaCl/4% PEG-6000 from the cleared bacterial supernatant and resuspended in PBS. Two round of selection are performed followed by screening on a single clone level.
- the genes encoding the selected scFv, contained in the phage display vector are re-cloned to the expression vector pXP14.
- This vector directs the expression of a scFv in fusion with a Streptag and E-tag and does not contain a filamentous phage gene-3.
- Expression vector containing E. coli TGI from single colonies are grown in individual wells of a microtiter plate so that each well contains only one scFv clone.
- the bacteria are grown at 30°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 0.1 %> glucose in 96-well microtiter plates (#9297, TPP) until an OD 6 oo of 0.7. Expression is induced with IPTG at a final concentration of 0.5 mM and continued at 25°C overnight.
- Single chain Fv containing cleared lysates are prepared by addition of hen-egg lysozyme (#L-6876, Sigma) to a final concentration of 50 ⁇ g/ml for 1 hour at 25°C and centrifugation for 15 minutes at 3000 x g. Prior to the screening ELISA, the cleared lysates are blocked by the addition of an equal volume of 5% MPBS for 1 hour.
- purified virions are coated on a 96 well UV cross-link plate (Corning Costar).
- the wells of the UV cross-link plate are blocked with MPBS and the scFv containing blocked cleared lysates added for 1.5 hours at 25°C.
- the plates are washed 2x with PBS + 0.1% Tween-20 and lx with PBS, incubated with HRP conjugated ⁇ -E-tag (#27-9413-01, Pharmacia Biotech; diluted 1 :5000 in MPBS with 0.1 % Tween-20) for 1 hour, washed 3x with PBS + 0.1% Tween-20 and 3x with PBS, developed with POD (#1 484 281, Roche) and signals read at 370 nm.
- the specific scFvs are expressed and purified as described and tested in a functional neutralization assay.
- Plaque reduction neutralization assays is performed by incubating approximately 100-200 plaque forming units of virus (in 100 ⁇ l) with different dilutions of scFv (in 100 ⁇ l of Hanks' balanced salt solution - HBSS) at 37°C for 1 hour. Duplicate 100 ⁇ l aliquots of incubation mixtures are assayed for residual infective virus by plaquing on Vero cells. Percentage plaque reduction is calculated relative to that of virus controls incubated without any RR-specific scFv. The scFv dilution carrying 50% plaque reduction are taken as the neutralization titer.
- corresponding target of neutralizing scFv one milligram of RR- purified virions are solubilized with Triton-X.
- the soluble fractions are mixed with the neutralizing scFv as described in the immunoprecipitation from cell extracts.
- the immunoprecipitate is run in a SDS-PAGE and the corresponding protein band is cut out and is identified via mass spectrometry with database query.
- 20 micrograms of purified RR virions are solubilized in SDS-PAGE sample buffer, split into 2 and then loaded into an SDS-PAGE and the proteins are electrophoretically separated.
- the first half of the sample is processed for Western blot as described, using the neutralizing scFv as the detection reagent.
- the protein band recognized by the neutralizing scFv corresponds to the target protein.
- the identity of the target is derived from the second half of the sample which is run in SDS-PAGE but is stained by Coomassie blue or silver.
- the protein migrating to the same distance as identified from the Western blot is cut out and subsequently identified by mass spectrometry and database query.
- Example 21 Identification of virulence factors in Vibrio cholerae and its corresponding neutralizing scFvs.
- Vibrio cholerae is the aetiological agent responsible for the acute gastrointestinal disease cholera. In order to exert the pathogenic effects of cholera toxin, it is necessary for V. cholerae to colonize the small intestine. It has been shown that colonization of Vibrio of human intestine correlates with its ability to self-agglutinate. This process involves the interaction of surface proteins on V. cholerae with the human intestine. Single colonies of V.
- V. cholerae-specific phages V. cholerae cells (1 x 10E7) are coated on a well of a 96-well ELISA plate. The same protocol for blocking and washing is used as described in example 20. Phage selection and screening is performed as described in example 20.
- V. cholerae-specific scFvs are expressed and purified as described and tested in a functional Autoagglutination assay.
- the autoagglutination assay is performed as described in Chiang et al. (1995) Mol. Microbiol. 17,1133-1142. Cultures from single colonies are grown for 12-15 hours at 30°C in LB broth, pH 6.5 on a roller shaker. Agglutination is scored visually. To quantify the extent of autoagglutination, 1 ml aliquots of cultures are allowed to stand at room temperature for 30 minutes and the optical density at 550 nm of the culture is determined by spectrophotometry.
- cholerae cells are grown under conditions promoting virulence at a total amount of lxl 0E9. The cells are resuspended in 1 ml of lysis buffer lysed with lysozyme and EDTA under hypotonic conditions (same used for purification of scFvs) to release the outer membrane proteins. Triton X-100 is added to solubilize membrane proteins. The solubilized membrane proteins including the pilins of V. cholerae are used for subsequent immunoprecipitation with the neutralizing scFv. The immunoprecipitate is separated in an SDS-PAGE and the corresponding band is cut out and is analysed by mass spectrometry.
- Example 22 Identification of cellular receptors necessary for viral infection and corresponding neutralizing scFvs
- Poliovirus serotype 1, Mahoney strain is grown in HeLa cells in suspension and purified by differential centrifugation and CsCl density gradient fractionation as described in Yeates et al. (1991) EMBO J. 10, 2331 -2341.
- the infectivity of the virus preparation is determined by a plaque assay on HeLa cell monolayers as described in Chow and Baltimore (1982) Proc. Natl. Acad. Sci. 79(23), 7518-21.
- the functional screening assay is a plaque reduction assay, performed essentially as previously described (Chow and Baltimore (1982) Proc. Natl. Acad. Sci. 79, 7518-21) with the following modifications.
- HeLa cells grown in a 24 well format are incubated with different dilutions (lOnM to lOO ⁇ M final concentration in the well) of recombinantly expressed scFvs - derived from the spatially separated clones of Example 1 - in Dulbeccos PBS/0, 1%BSA/10 mM HEPES (Gibco 15630-056) on ice for 15 min.
- plaque forming units of poliovirus suspended in Phosphate-buffered saline containing 0,5mM MgCl 2 , 0,7 mM CaCl 2 , and 0,5%) (vol/vol) fetal calf serum are added and allowed to attach to the HeLa cell monolayer for 30 min at 37C.
- the cells are then overlaid with growth medium comprising 1% agarose and incubated at 37C for about 36 hours. Plaques are then visualized by staining with crystal violet, as described in Chow and Baltimore above. ScFvs effecting an inhibition of poliovirus infection, i.e.
- the receptor involved in viral infection is the identified according to the protocol for immunprecipitation of Example 9, with the modification that HeLa cells are lysed and used as starting material instead of Hs-27 cells.
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Abstract
The invention provides a new paradigm for a screening method by combining the biochemical approach of specific binding between a ligand and a target, e.g. an antibody and an antigen, with the genetic principle of a functional screen. The present invention demonstrates its utility for identifying novel disease-relevant proteins and inhibitory ligands, e.g. antibodies or antibody fragments, that may serve as therapeutic leads for a wide variety of diseases, whether the cause of the disease is derivable from a virus, a bacterium, a lower eukaryote or from a misregulation of the cells of the subject to be treated.
Description
PROTEOMIC SCREEN TO IDENTIFY DISEASE-RELATED BIOLOGICAL MOLECULES AND INHIBITORS THERETO
BACKGROUND OF THE INVENTION
The classical way of identifying a function of a biological target molecule is through genetics (Botstein and Maurer (1982) Ann. Rev. Genet. 16, 61-83). This process starts with a population of mutants or variants, naturally occurring or induced. Desired phenotypes representing the biological function of interest are then isolated and the genetic determinants responsible for the phenotype are identified by various genetic methods.
Despite the success of genomics, there is still a lack of understanding in the function of proteins, the major regulators of biological function. Better understanding of protein function is critical in the treatment of diseases in order to design drugs that attack the correct target molecules. Although there is an abundance of targets and combinatorial chemistry/biology methods can generate multitudes of lead molecules, there is still a major gulf in efficiently deciding which target to develop a drug against. This segment of drug discovery is called target validation. At the moment, a drug target is considered validated only if a drug has been developed that is safe and efficacious. This retrospective definition of target validation implies the infancy of the field and the inefficiencies encountered in drug discovery. The average time so far required from identifying a target to identifying a lead is 3.4 years and costs approximately $350M. Increase in efficiency or predictability in defining which biological target molecule is the best target will significantly reduce the time and costs in developing a drug.
Chemical genetics is a burgeoning field that utilizes the use of organic molecules instead of genetic mutations to modulate protein function (Schreiber, (2000), Science 287, 1964-1969). This field offers promise in simultaneously modulating protein function and the corresponding modulators can serve as potential therapeutic drug leads. Although this has been applied, chemical genetics is limited by the diversity of small organic molecules to modulate all the possible protein classes. Furthermore, the use of small organic molecules to isolate their corresponding protein target remains technically challenging. These limitations can be circumvented by the use of highly diverse combinatorial biological libraries (e.g. antibodies, aptamers, etc.). Unfortunately, the use of highly diverse combinatorial libraries for
phenotypic screens analogous to the chemical genetics approach is technically challenging because ofthe high complexity of biological libraries.
The present invention relies on a purely protein-based approach for identifying the function of a biological target molecule, circumventing the problems and technical challenges associated with the chemical genetics approach while being able to manage the complexity of biological libraries.
One approach in the antibody discovery field that several authors have described relates to methods to produce antibodies with hybridoma-technology using mice that were immunized with cell lines (see, e.g. Miyake et al. (1991) Biochem. 30, 3328-3334; Imhof et al. (1983) Cell 35, 667-675) or from auto-immunized human cancer patients (see, Vollmers et al. (1994) Cancer 74, 1525-1532). In these references, methods to select and characterize the obtained hybridomas in assays for cell perturbing functions are described. However, the use of hybridoma technology to generate antibodies is labor intensive and limits the number of different antibodies that can be screened due to the relative inefficiency of establishing monoclonal antibody expressing stable cell lines. In addition, the antibody selection process is purely in vivo, thus being biased against non-immunogenic antigens and making it impossible to direct the antibody selection process. Contrary to this, the use of phage displayed antibody repertoires makes it possible to screen a very large number of antibodies, due to the high efficiency of cloning of antibody genes in bacteria. Thus, more antibodies - at least three orders of magnitude - can be screened by utilizing phage displayed antibody repertoires compared to methods relying on monoclonal antibody expressing stable cell lines. Additional diversity, such as random combination of heavy-chain and light-chain gene fragments, is introduced in the antibody gene cloning step. Thus, different antibodies are generated by the use of phage displayed antibody repertoires compared to methods relying on monoclonal antibody expressing stable cell lines. The antibody selection process, using phage displayed antibody repertoires, is an in vitro process not limited by the same restrictions as the in vivo selection process. Thus, especially for na'ive phage displayed antibody repertoires, antibodies against different antigens will be generated by the use of phage displayed antibody repertoires compared to methods relying on monoclonal antibody expressing stable cell lines.
The utilization of phage display libraries allows the rapid search through large libraries of compounds to identify compounds that bind a particular target molecule with high affinity. However, a major weakness of this technology is the requirement of pure target molecules for
phage selection. Only very few targets are available in this form and novel, unknown targets could not be identified using this approach. A few authors have recently described methods to screen cell surfaces for antibody binders in the absence of purified recombinant proteins. Nevertheless, selection of binding antibodies from large naϊve libraries by selection on cell surfaces is greatly limited by high background binding of non-specific phages and relatively low binding of specific phages. (See, e.g. Pereira et al. (1997) J. Immunol. Methods 203, 11- 24; Watters et al. (1997) Immunotechnology 3, 21-29; Becerril et al. (1999) Biochem. Biophys. Res. Commun. 255, 386-393).
Heitner et al. (2001) J. Immunol. Meth., 248, 17-30 described the selection of EGFR antibodies from a na'ive scFv-phage library via selection against cells that either over-express EGFR or were transfected with the EGFR gene. Heitner et. al. generated EGFR antibodies that recognized the over-expressed receptor.
Noronha et al. (1998) J. Immunology, 161, 2968-2976 described a selection with a scFv phage library against two different melanoma cell lines to identify antibody fragments specific for human melanoma-associated antigens. Melanoma cell-specific markers were identified by this method.
Poul et al. (2000) J. Mol. Biol., 301 , 1149-1161 selected internalizing antibody fragments on cells from a phage displayed repertoire and subsequently characterized a small subset of antibody fragments with respect to the corresponding epitope of the receptor and the internalizing efficiency ofthe antibody fragments.
The selection techniques discussed above yielded cell surface binding antibody fragments. Cell surface selections were carried out with respect to over-expressed and/or known receptor molecules. None of these methods showed a screening for compounds that are able to inhibit the function of a disease-relevant, unknown and not pre-selected target nor showed a method to identify the unknown target molecule.
In contrast, the method disclosed in the present invention provides a way to generate inhibitors of yet unknown disease-relevant targets and to simultaneously identify those unknown biological target molecules, if desired. Specifically, the method disclosed in the present invention provides for example a way to generate inhibitors against cancer-relevant targets.
As one example the present invention demonstrates for the first time that for specific, naturally occurring tumor cells MCAM function is necessary for the process of invasion, proliferation, adhesion and/or metastasis. The invention further provides new inhibitors modulating the MCAM, integrin and ephrin function.
SUMMARY OF THE INVENTION
The invention provides a new paradigm for a screening method by combining the biochemical approach of specific binding between a ligand and a target, e.g. an antibody and an antigen, with the genetic principle of a functional screen.
The present invention demonstrates its utility for identifying novel disease-relevant proteins and inhibitory ligands, e.g. antibodies or antibody fragments, that may serve as therapeutic leads for a wide variety of diseases, whether the cause ofthe disease is derivable from a virus, a bacterium, a lower eukaryote or from a misregulation ofthe cells ofthe subject to be treated.
The present invention has the advantage of significantly reducing the time and costs involved in drug discovery. This approach represents a new method to develop drugs more efficiently and makes it possible to identify a functional molecule against a disease in the complete absence of a candidate drug target.
In one aspect, the present invention provides a method of identifying a ligand, for example an antibody or an antibody fragment, binding specifically to the extracellular region of an antigen associated with the surface of a target, for example a mammalian cell. The ligand, for example an antibody or antibody fragment, is derived from a library of amplifiable ligand- displaying units (ALDU), for example a phage library displaying antibodies or antibody fragments. The ALDU library, for example a phage library displaying antibodies or antibody fragments, is brought in contact with a target, for example a cell. ALDUs, for example phages, bound to the target, for example the mammalian cell, are separated from ALDUs not bound to the target and the bound ALDUs are amplified. Those ALDUs or ligands derived from ALDUs, for example phages or antibodies or antibody fragments derived from phages, which are capable of affecting the biological ability of the target, for example said mammalian cell, are then identified in a functional screening assay. This method can also
comprise the step of identifying the surface antigen, which the ALDU or the ligand derived from an ALDU affected or modified.
The method of the invention has been applied in an unbiased screen for molecules that can inhibit invasiveness, adhesiveness and/or proliferation of naturally occurring tumor cells. Surprisingly, several antibody fragments binding to the extracellular domain of MCAM, integrin and/or ephrin had been identified as useful inhibitors according to the invention.
Accordingly, in another embodiment the invention relates to a method for the identification of antibody fragments useful for inhibiting the invasiveness and the metastatic potential of sarcoma cells.
DETAILED INSCRIPTION OF THE INVENTION
The present invention provides in a prototypic embodiment a method of identifying a ligand binding specifically to the extracellular region of a target, by screening e.g. a phage display library of antibody fragments for a ligand being capable of affecting a biological function of said target, wherein these ligands are capable of affecting and, in particular, inhibiting the invasiveness, proliferation and/or adhesion of tumor cells. Said method comprises the steps of: a. contacting a library of amplifiable ligand-displaying units (ALDU) with a target; b. separating said target and the ALDUs bound thereto from ALDUs not bound to said target; c. optionally amplifying the ALDUs bound to said target; d. identifying an ALDU or a ligand derived from an ALDU after step c), which affects the biological function ofthe target in a functional screening assay; and optionally e. determining the chemical identity ofthe ligand.
For describing a preferred embodiment in the following several references are made to the method of the invention involving a target presenting an antigen, a tumor specific surface molecule, like e.g. MCAM, integrins or ephrins, or a recombinant antigen. This selection, however is not to be understood as limiting in any way the choice of possible targets and/or antigens or surface molecules, but is used as examples only.
A particular advantage of this method is that the ligand e.g. an antibody or an antibody fragment specific for the accessible part of the extracellular region of the antigen or surface molecule, e.g. MCAM, presented on the target is obtained, since the initial selection step is performed on intact cells, which present only the accessible part of the extracellular region of the surface molecule e.g. MCAM for binding with the ligand.
In a preferred embodiment of the invention, the above method comprises instead of step d. the further steps of: d') contacting isolated ALDU's with recombinant antigen or surface molecule; e') washing said antigen or surface molecule with a buffered detergent and/or high salt solution; and f ) eluting ALDU's bound to antigen or surface molecule; and g') determining the identity ofthe ligand represented by said eluted ALDU's.
In order that the invention described herein may be more fully understood, the following definitions are provided, and shall apply unless otherwise indicated.
"Identifying" as used herein means the identification of a biomolecule having desired properties from a mixture of biomolecules comprising related but non-identical biomolecules with slightly different properties.
A "ligand" as used herein is a molecule displayable by an amplifiable ligand-displaying unit. A ligand is that part of an ALDU through which the ALDU can bind to a target. Preferably it is a polypeptide as defined above, an RNA-oligonucleotide or a DNA-oligonucleotide, whereby the oligonucleotide comprisies more than 20 base units but less than 10,000, preferably less than 1 ,000 base units. A ligand can bind to an extracellular region of an antigen. This binding may have specificity in the sense that the ligand binds to one antigen with high affinity but to a moderately related antigen with lower, for example 10- or 50- or 200-fold lower affinity. Moderately related antigens are antigens with up to 30 % amino acid identity in the extracellular regions.
A "target" as used herein is an entity for which it is screened for, preferably a biological target that is a complex mixture of biomolecules. By way of example, a target may be a eukaryotic cell, a virus, a bacterial cell, or parts thereof. A target is not an individual, purified
biomolecule. The target may be a particle with a size of more than 10 nm, preferably more than 30, 100 or even 500 nm. The biological target as used herein is capable of presenting a particular biomolecule, surface molecule or antigen, which is associated with its surface.
A ligand "binding specifically to a target" as mentioned herein can be a ligand which binds to the target or any of the presented surface molecules or antigens under the buffer conditions given in Examples 1, 2, 15 or 16. When the target is an identified biomolecule the dissociation constant between the ligand and the identified biomolecule can be measured, e.g. by use of the so-called BIACORE System (see, for example, Fivash et al. Curr Opin Biotechnol. (1998) 9, 97-101 page 24) and "binding specifically" can then be understood to mean that the dissociation constant between the ligand and the identified biomolecule is lower than 10 μM, preferably lower than 1 μM, more preferably lower than 500, 400, 300, 200, 100, 50, 20 nM, most preferably from 0,1 nM to 20 nM if measured under standard conditions, for example at 20°C, ambient pressure and in a suitable buffer, e.g. 20 mM Tris, 100 mM NaCl, 0, 1 mM EDTA at an overall pH of 7.0.
A ligand "derived from an ALDU" as mentioned herein is a ligand, which had been displayed by such an ALDU, which had been selected by binding to the surface of a target. As an example, if the ALDU is a phage of a phage library displaying scFvs the "ligand derived from an ALDU" in this case is a polypeptide, here an scFv. As another example, in the case of ribosomal display the ligand "derived from an ALDU" is the polypeptide, which was presented by the complex comprising the ribosome, the RNA and the polypeptide.
The "extracellular region" of a molecule as used herein is that part of the molecule which is located on the outside of a biological structure, that is, if the target is a eukaryotic cell located on the outside of the cell membrane; if the target is a gram-negative eubacterium located on the outside of the outer bacterial membrane; if the target is a gram-positive eubacterium located on the outside of the bacterial membrane. Even though a virus is not a cell, in the case of a virus the term "extracellular region" should be understood as that part of the virus that is accessible to other biomolecules, e.g. a polypeptide with a molecular weight of 1000 Da, when the virus is intact. This includes the outside of a viral capsid and the outside of a for example outer viral coat.
As used herein, the term "associated with the surface of a target" means that a biomolecule is either part of the surface of a biological target, e.g. a constituent of the surface, or is binding
to another biomolecule which is a constituent of the surface of a target. For example, a transmembrane polypeptide, like a transmembrane receptor of an eukaryotic cell can be seen as a constituent of the surface if at least one part of it transverses the lipid bilayer of the cell membrane and is in direct contact with the lipids of the cell membrane. An example for a polypeptide that is binding to another biomolecule, which is a constituent of the surface of a target, is, for example, the B2-microglobuline ofthe class 1 major histocompatibility complex. In this case the B2-microglobuline binds non-covalently to the MHC - a transmembrane polypeptide -, and is therefore also regarded as being a molecule associated with the surface.
As used herein, "capable of affecting a biological function" means that the ligand is able at a reasonable concentration, e.g. in the micromolar range, for example 50 μM, to either inhibit or activate a biological activity that the target possesses in the context of alive mammals. By way of example, a biological function of, e.g., a cancer cell can be their proliferation, invasiveness or adhesiveness; of, e.g., a virus can be its ability to enter and infect a cell; of, e.g., a bacterium can be, e.g., its ability to adhere to a cell, e.g., a mammalian cell, or also its ability to adhere to another bacterial cell. Thus, as used herein, "biological function" refers to those activities that can take place in an intact mammalian organism or are indicative of a corresponding function in an intact mammalian organism. It does not include generalized toxic effects resulting from the presence of a molecule in large excess of its naturally- occurring concentration. Preferably it does also not include effects that do not rely on specific binding of the ligand. By "affecting" the biological function of a target, ligands of the invention, will alter the "biological function" of the target, e.g. either enhance or inhibit or change the biological function of the target. The effect of a ligand on a biological function may be demonstrated by in vitro tests, which are indicative of the corresponding biological function in vivo.
As used herein "contacting" of two components means allowing the two components to physically associate with one another and bind to one another.
As used herein "amplifiable ligand-displaying units" (ALDUs) are molecules or collections of molecules with dual function: they can bind to a target via a ligand; and they carry physically associated with the ligand an information about its identity, which allows individual units to be identified, and amplified. Examples of ALDUs are oligonucleotides, particularly RNAs, useful for the process of selection-amplification (SELEX), phages of phage display libraries, viruses of viral display libraries, ribosomes of ribosome-display techniques, but also
individualized beads carrying an identifiable molecule or ligand, particularly chemical entities, e.g. small molecules bound to beads, which are recognizable by inert chemical tags. Preferred are such ALDUs, which comprise a nucleic acid as the identifiable component. Such ALDUs can be either amplified in vitro, e.g., by nucleic-acid amplification methods like RT-PCR, PCR, LCR and the like, or they can be amplified in vivo, for example an individual phage can infect a bacterium and yield, after several cycles of infection, millions of virtually identical progeny. A library of ALDUs is a collection of similar, but in general non-identical ALDUs, for example phages of a phage library that display different scFvs in the context of an otherwise identical phage surface.
"Separating" as used herein means the physical separation of two or more components, for example a cell with an ALDU bound thereto can be separated from a free ALDU by centrifugation, wherein the cell with the ALDU bound thereto is pelleted and the free ALDU is still in the supernatant.
"Amplifying" as used herein is any process that increases the number of ALDUs or ligands derived from ALDUs by at least a factor of two, preferably by a factor of 10, 100, 1.000, 10.000, 100.000, 1.000.000, 10.000.000 or even a billion. For example a single phage can be amplified by infecting a bacterium and the infected bacterium then produces several mostly identical copies of the infecting phage, or a DNA-molecule can be amplified by the process of PCR to yield 104 or more mostly identical daughter DNA-molecules.
"Determining the chemical identity of a ligand" as used herein means determining the structural composition of a ligand. For example, if the ALDU library is a phage library displaying scFvs, then "determining the chemical identity of a ligand" would mean to determine the sequence of the scFv polypeptide, for example by sequencing the region encoding the scFv ofthe phage from which it was derived.
"A screening assay" as used herein is aimed at detecting an individual, e.g. an ALDU, with special properties among other similar individuals with slightly different properties. In order to qualify for a screening assay at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or even 100 individuals must be tested in a certain functional assay to find an individual/individuals with the desired function detected among them.
"Inhibiting" a biological function as used herein means the reduction of the biological function at a statistically significant level, preferably by more than 30 %, 40 %, 50 %, or even
60 %. Inhibition can be measured in a functional assay, e.g. by adding a ligand, e.g. a certain antibody fragment at a concentration of one nM to 50 μM, preferably around 20 μM to the functional assay under standard conditions.
"Activating" biological function as used herein means an increase in biological function at a statistically significant level, preferably by more than 30 %, 40 %, 50 %, or even 60 %. Activation can be measured in a functional assay, e.g. by adding a ligand, e.g. a certain antibody fragment at a concentration of one nM to 50 μM, preferably around 20 μM to the functional assay under standard conditions.
An "antigen derivable from a virus" is an antigen, which can be present in a virus particle, preferably a viral surface antigen like capsid- or coat-proteins.
A "virus" as used herein comprises animal viruses, plant viruses and bacteriophages and can in particular be an animal virus, more particularly a virus capable of infecting a mammalian, e.g. a human cell. The virus is typically selected from the group comprising lentivirus, retrovirus, hepadnavirus, picornavirus, alphavirus, flavivirus, reovirus, paramyxovirus, orthomyxovirus, arbovirus, polyomavirus, papillomavirus, rhabdovirus, herpes virus, adenovirus, parvovirus, in particular selected from the group consisting of HIV-I, HIV-II, hepatitis B virus, hepatitis C virus, smallpox virus, pox virus, poliomyelitis virus, west nile virus, adeno virus, herpes simplex virus, Eppstein-Barr virus, coxsackie virus, influenza virus and rubella virus.
An "antigen derivable from a bacterium" is an antigen, which can be part of a bacterium. It can be part of an archaebacterium or eubacterium, preferably gram positive or gram negative eubacteria such as particularly a bacterium selected from the group consisting of Bordella pertussis, Vibrio Cholerae, Bacillus anthracis, Yersinia pestis, Staphylococcus aureus, Clostridium botulinum, Streptococci, Chlamydia, Chromobacter, Helicobacter pylori. Particularly an antigen derivable from a bacterium is an antigen associated with the surface of a bacterium.
An "antigen derivable from a lower eukaryote" as used herein is an antigen, which can be part of a lower eukaryote. A lower eukaryote as used herein is defined as a fungus, a protozoon, in particular a member of the Phylae sarcomastigophora (for example the genera Trypanosoma, Leishmania, Trichomonas and Giardia), Rhizopoda (for example the genera Entamoeba,
Naegleria and Acanthamoeba), Sporozoa (for example the genera Poxoplasma, Isospora, Sarcosystis, Cryptosporidium and Plasmodium) and Ciliophora (for example the genus Balantidium) or as a member of the metazoan Phylae plathelminthes, Mesozoa, Nematoda, Nematomorpha or Acantocephala.
An "antigen" as used herein can be a polypeptide, a glycoprotein, a peptide, a proteoglycane, a lipid or a glycolipid.
A "polypeptide" as used herein is a molecule comprising more than 10, preferably more than 20, most preferably more than 30, and less than 10000, more preferably less than 2500, most preferably less than 1000 amino acids. Also polypeptides with substantial amino acid sequence identity and polypeptides, which contain a low percentage of modified or non- natural amino acids are encompassed. Particularly antibodies and antibody fragments are considered as polypeptides.
The terms "antibody" or "immunoglobulin", as used herein refer to any immunological binding agent, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, and the like. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antibodies may be also selected from modified immunoglobulins, for example chemically or recombinantly produced antibodies, CDR grafted antibodies or humanized antibodies, site directed mutagenized antibodies that exhibit substantial amino acid sequence identity in their CDR regions, particularly in their CDR3 region, to the corresponding antibody fragments of the invention and retain substantially the same affinity for MCAM binding as the corresponding antibody fragments.
The CDRs (complementary determining region) of an antibody are the parts of these molecules that determine their specificity and make contact with specific ligands. The CDRs are the most variable parts of the molecule and contribute to the diversity of these molecules. They are structurally defined in a human IgG as amino acids 24 to 41 (CDR-L1), 50 to 57 (CDR-L2) and 90 to 101 (CDR-L3) ofthe light chain and amino acids 26 to 38 (CDR-H1), 51 to 70 (CDR-L2) and 100 to 125 (CDR-H3) ofthe heavy chain (see Kabat et al. (1987) 4th edn
US Department of Health and Human Services, Public Health Service, NIH, Bethesda). The CDR regions of an antibody fragment can easily be determined by somebody skilled in the art by aligning the antibody fragment with said human IgG, e.g. using a program of the NCBI that allows to "Blast", and thereby align, two sequences with one another, and identifying the amino acids ofthe antibody fragment corresponding to the CDRs of a human IgG.
Substantial amino acid sequence identity as used herein means that at least 70%, preferably at least 85%, more preferably all but 5, still more preferably all but 3 and even more preferably all but 1 of the amino acids of two aligned amino acid sequences, particularly of aligned CDRs, are identical.
The term "antibody fragment" is used to refer to any fragment of an antibody-like molecule that has an antigen binding region, and this term includes antibody fragments such as scFv, dsFv, Fab', Fab, F(ab')2, Fv, single domain antibodies (DABs), diabodies, and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al. (1991) J. Immunol. 147, 1709-19), specifically incorporated herein by reference.
"scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. A "Fv" fragment is the smallest antibody fragment that retains an intact antigen binding site. A "dsFv" is a disulfide stabilized Fv. A "Fab" fragment, is an antigen binding fragment, containing complete light chains paired with the VH and CHI domains of the heavy chain. A "Fab"' fragment, is a reduced F(ab')2 fragment. A "F(ab')2" fragment, is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
A "single domain antibody (DAB)" is an antibody with only one (instead of two) protein chain derived from only one ofthe domains ofthe antibody structure. Dabs exploit the finding that, for some antibodies, half of the antibody molecule binds to its target antigen almost as well as the whole molecule (Davies et al. (1996) Protein Eng. 9: 531-537.
A "proteoglycane" can be seen as an organic compound comprising long polysaccharide chains (glycanes) covalently bound to a polypeptide core.
A "glycoprotein" as used herein can be seen as an organic compound comprising both a polypeptide and a carbohydrate moiety joint by a covalent chemical linkage. A carbohydrate moiety of a glycoprotein can be a small sugar of around 4 to 10 individual - optionally substituted - sugar units, selected from the list of glucose, glucosamine, galactose, galactosamine, mannose, fucose and sialic acid.
A "glycolipid" as used herein can be seen e.g. as 1,2-Di-O-acylglycerole with a carbohydrate moiety covalently bound to oxygen 3 by a glycosidic linkage. The carbohydrate moiety can comprise a mono-, di-, or trisaccharide. Bacterial glycolipids, which can have an isolated sugar moiety, wherein the acylation can be by one or more fatty acids and where the glycerol part may even be absent, are also "glycolipids" within the scope ofthe invention.
In the most general embodiment the invention relates to a method of identifying a ligand binding specifically to the extracellular region of an antigen associated with the surface of a target, wherein said ligand is capable of affecting a biological function of said target, comprising the steps of a) contacting a library of amplifiable ligand-displaying units (ALDU) with said target; b) separating said target cell and the ALDUs bound thereto from ALDUs not bound to said target; c) amplifying the ALDUs bound to said target; d) identifying an ALDU or a ligand derived from an ALDU after step c), which affects said biological activity by a functional screening assay; and optionally e) determining the chemical identity of the ligand. The preferred order ofthe steps is a, b, c, d and optionally e.
The method is capable of identifying a ligand, e.g. an antibody or antibody fragment, with the ability to bind strongly and specifically to the extracellular region of an antigen which is associated with the surface of a target, e.g. the extracellular loops of transmembrane receptors of a mammalian cell or e.g. the lipopolysaccharides ofthe outer membrane of a gram negative bacterium or e.g. a capside protein of an animal virus. The method of the invention identifies such ligands which are binding to the antigen and thereby affect a biological function of a target, e.g. the method can identify a ligand, e.g. an antibody or antibody fragment which binds to e.g. the MCAM protein or the Ephrin protein or the alpha-3 integrin protein and thereby inhibits invasiveness, proliferation or adhesiveness of a cancer cell. Furthermore, the method of the invention is capable to identify ligands, which bind to the alpha-2/beta 1 - integrin and inhibit adhesiveness of a cancer cell. Moreover the method can identify a ligand, which binds to the surface of pathogenic bacteria and inhibits the colonization of the mammalian body by said pathogenic bacteria or, e.g., the method can identify a ligand which
can bind to a viral surface protein and thereby inhibit infection of a mammalian cell by the virus.
The method relies on a new rationale for screening assays: advantageous combination of a screening step based on binding to the surface of a target with a screening step based on a functional assay. A library of amplifiable ligand-displaying units (ALDU) is brought in contact with a target in such a way that those ALDUs of the library with specificity for a surface antigen of the target can bind to the target. For example phages of a phage library displaying antibodies or antibody fragments can be allowed to bind to cultured cancer cells or to a preparation of a virus.
In another step, the target and the ALDUs bound thereto are separated from unbound ALDUs, in order to select for those ALDUs with specificity for the target. The separation can be achieved, e.g., by removing the solution, in which the contacting step has been performed from, e.g., cultured cells grown adhesively in a culture flask and together with the solution the unbound ALDUs; or, as another example, viruses with ligands of a library bound thereto can be pelleted by centrifugation, while the unbound ligand remain in the supernatant as part of the solution, in which the contacting step had been performed.
As another step the ALDUs bound to the target are amplified, in order to obtain high enough concentrations of the ALDUs so that they are useful for being applied in a functional screen. This amplifying step makes use of the ALDUs' intrinsic property to be amplifiable. For example phages displaying, e.g., an antibody or antibody fragment, which have been separated from the non-bound phages in step b), but are still bound to the target, e.g. a mammalian cancer cell, can be amplified by first recovering the bound phages, for example by eluting the phages from the target cells and then using the recovered phage eluate to infect bacteria, e.g. Escherichia coli. The amplification of the phages in Escherichia coli then leads to a significant increase in total phage number, but in such a way, that the concentration of those individual phages, which were capable of binding to the target, is dramatically increased.
However, the phages need not necessarily be removed from the target, as a phage still bound to a target can infect added bacteria and thus be amplified.
As another example, the mRNA of a ribosomal-display library, which is associated by its attachment to a ribosome displaying the polypeptide corresponding to the mRNA, with the
surface of a target, can be directly used for RT-PCR, without the need of removing the intact ALDU from the target before amplifying the target. Thus, a step in which the ALDU bound to a target is recovered from the target before amplifying the ALDU is an optional step.
Amplifying the ALDU can also consist in determining the chemical identity of the ligand of such ALDUs, which have bound to the target and then amplifying the ligands. That is to say, e.g., that in the case of a phage library, displaying an antibody or an antibody fragment, DNA encoding those ligands can be cloned from phages, which had bound to the target, e.g., by PCR, and the ligand, e.g., the antibody or antibody fragment, can then be produced recombinantly to yield sufficient amounts ofthe ligand to be useful for a functional screening assay. In such a way, not the ALDU itself is amplified, but the ligand displayed by the ALDU. This, however, is within the scope of the invention, as the aim of the amplification step is the generation of a ligand concentration high enough to be useful for a functional screening assay and this can be achieved by either amplifying the ligand or the ALDU displaying it.
In another step, an ALDU or a ligand derived from an ALDU, which had bound to a target is detected based on its effects on a biological function to be tested in a functional screening assay. For such a functional screening assay, the ALDUs or ligands derived from them are advantageously individualized, such that a signal or a pattern from the functional screening assay is relatable to an individual ALDU or an individual type of ligand. This can be done, e.g., by filling separate wells of a multi-well plate with individual ligands each and then performing an assay for a desired biological function in those individual wells.
In another example phages, which had bound to a desired target, e.g. to a cancer cell, and had been separated from unbound phages, can be individualized by infecting bacteria and plating the infected bacteria in, e.g., soft agar, so that individual plaques, which represent clones of individual phages, form. Those individual plaques can then be tested for their effects in a functional biological screening assay. In such an assay, an individual plaque with an effect in the functional screening assay is identifiable. The ligand displayed by the phages of the plaque can then be identified, e.g. by sequencing the DNA ofthe phages of the plaque, which represent a clone of a single phage, initially selected by its ability to bind to the target. Thus, in those cases where an ALDU itself is used in the functional screening assay, the identity of the ligand displayed by the ALDU can be determined, if desired.
In a preferred embodiment, the ligand displayed or derived from an ALDU is capable of activating a biological function of the target, e.g. a ligand, like an antibody or an antibody fragment could bind to the surface receptor of a mammalian cell, e.g. the FAS-receptor, and could activate a biological function of a cell, in this case it could activate apoptosis of this cell.
In another preferred embodiment the ligand is capable of inhibiting a biological function of the target. For example, the biological function of a target can be the ability of the target to interact with an antigen derivable from a virus. If, for example, the target is a mammalian cell, or even a human cell, a ligand that binds to a surface antigen of the mammalian cell that is necessary for mediating the interaction with a viral antigen important for viral entry into a mammalian cell can be identified by the method of identifying a ligand of the invention. Such a ligand could be useful for inhibiting infection of the mammalian cell with the virus. It can be identified among the ligands displayed by the ALDUs bound to the surface of a mammalian cell - which is infectable by the virus - by screening those individualized ligands for their ability to inhibit infection ofthe target, in this case the mammalian cell, by the virus. Thus, ligands, e.g. antibodies or antibody fragments, can be identified by the method of the invention which inhibit the ability of the target, for example a mammalian cell, to interact with an antigen derived from a virus, for example selected from the group consisting of lentivirus, retrovirus, hepadnavirus, picornavirus, alphavirus, flavivirus, reovirus, paramyxovirus, orthomyxovirus, arbovirus, polyomavirus, papillomavirus, rhabdovirus, herpes virus, adenovirus, parvovirus, in particular selected from the group consisting of HIV- I, HIV-II, hepatitis B virus, hepatitis C virus, smallpox virus, pox virus, poliomyelitis virus, west nile virus, adeno virus, herpes simplex virus, Eppstein-Barr virus, coxsackie virus, influenza virus and rubella virus and thereby protect a mammalian cell from viral infection.
As another example, if the target is a mammalian cell, and even a human cell, a ligand that binds to a surface antigen of the mammalian cell that is necessary for mediating the interaction with a bacterial antigen important for bacterial attachment to a mammalian cell can be identified by the method of the invention. Such a ligand could be useful for inhibiting bacterial infection of the mammal, in particular in those cases where infection is caused by adhesion of bacteria to mammalian cells or by entry of bacteria into mammalian cells. Such a ligand can be identified among the ligands displayed by the ALDUs bound to the surface of a mammalian cell by screening those individualized ligands for their ability to inhibit the
bacterial adhesion to the target or bacterial infection of the target, e.g. in the case of a mammalian cell, bacterial adhesion to the mammalian cell or bacterial entry into the mammalian cell. Thus, ligands, e.g. antibodies or antibody fragments, which inhibit the ability of the target, e.g. a mammalian cell, to interact with an antigen derived from a bacterium, e.g. derived from a gram positive or gram negative bacterium, e.g. from a bacterium selected from the group consisting of Bordella pertussis, Vibrio cholerae, Bacillus anthracis, Yersinia pestis, Staphylococcus aureus, Clostridium botulinum, Streptococci, Clamydia, Chromobacter and Helicobacter pylori, can be identified by the method ofthe invention.
Also ligands, e.g. antibodies or antibody fragments, which inhibit the ability of a bacterium to enter the target, e.g. a mammalian cell, can be identified by the method of the invention. This relates particularly to intracellular or facultative intracellular bacteria, for example selected from the group consisting of Mycobacterium, Brucella, Chlamydiae and Rickettsiaceae, in particular selected from the group consisting of the pathogen species of intracellular Chlamydiae and Rickettsiaceae, for example the pathogen species of the genera Coxiella, Ehrlichia and Rickettsia. Such ligands can be potential therapeutics for, for example, the treatment of typhus, Q fever, Rocky Mountain spotted fever or scrub typhus or other diseases, which are known to be caused by Rickettsiaceae or Chlamydiae.
As another example the biological function of a target can be the ability of the target to interact with an antigen derivable from a lower eukaryote. For example, if the target is a mammalian cell, and even a human cell, a ligand which binds to a surface antigen of the mammalian cell that is necessary for mediating the interaction with an antigen from a lower eukaryote, e.g. Plasmodium falciparum, important for mediating cell-cell interaction between a mammalian cell and a lower eukaryotic cell, e.g. for entry of Plasmodium into erythrocytes, can be identified by the method of the invention. Such a ligand could be useful for inhibiting infection of the mammal with the parasitic lower eukaryote. It can be identified among the ligands displayed by the ALDUs bound to the surface of a mammalian cell by screening those individualized ligands for their ability to inhibit interaction between the target, in this example the mammalian cell, with the lower eukaryotic cell, in the example of P. falciparum the entry of P. falciparum into erythrocytes. Thus, ligands, e.g. antibodies or antibody fragments, can be identified by the method of the invention which inhibit the ability of the target, e.g. a mammalian cell, to interact with an antigen derived from a lower eukaryote and particularly inhibit cell-entry of intracellular or facultative-intracellular lower eucaryotes such as
Plasmodium vivax, Plasmodium ovalae, Plasmodium malariae, Plasmodium falciparum, Leishmania, Toxoplasma (for example T. gondii), Sarcocystis and Pneumocystis.
In another preferred embodiment the surface antigen is a polypeptide, a glycoprotein, a peptide, proteoglycane, a lipid or a glycolipid.
In another preferred embodiment the target is a eukaryotic cell, in particular a mammalian cell, more particularly a cell selected from the group consisting of cells of tissue sections, primary cells or cell lines, in particular selected from the group consisting of cancer cells, nerve cells, stem cells, blood cells, platelet cells, adipocytes, liver cells, kidney cells, lung cells, endothelial cell, keratinocytes, melanoma cells, erythrocytes, osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow cells, smooth muscle cells, skeleton muscle cells and heart muscle cells.
The biological functions of those mammalian cells, which can be affected by the ligands identified by the method of the invention, can be many-fold, e.g. selected from the group consisting of invasion, adhesion, proliferation, angiogenesis and cytotoxicity.
"Invasiveness" as used herein is the ability of a cell to migrate through a layer of other cells or to migrate through the extracellular matrix. Invasiveness can be assessed by the Matrigel assay described in Example 5. Invasion is measured as cells that reach the lower surface ofthe filter during a certain incubation period. When more than 40% of cells within 6h to 12h reach the other side of the filter and form colonies in an invasion assay like in Example 5, the naturally occurring cancer cell is defined as invasive. The control cells instead form only 5% colonies in the same time frame and are defined as non-invasive.
"Adhesiveness" as used herein is the ability of a cell to reattach after they have been removed from the matrix on which it had been grown, resuspended as a solution of single cells (not in direct contact with other cells of the solution), and replated on a matrix to which adhesion is possible. A cell is defined as adhesive if in an assay as described in Example 7, more than 40% of the cells adhere within a time of 30-120 min. Instead, only 5% of the control cells adhere within the same time frame.
"Proliferation" of a eukaryotic or mammalian cell, can be seen as a division of a cell into daughter cells, which leads, e.g., in a cell culture, to an increase in overall cell number of the culture over time.
"Angiogenesis" is the process where cells induce blood vessel formation in their proximity. Angiogenesis can accompany the growth of malignant tissue and can therefore be a property of malignant cells. That is to say that malignant cells can have the property of inducing angiogenesis.
Metastatic potential as used herein is the ability of a tumor cell to form a new tumor at a site distant from the primary tumor of which the tumor cell was derived (a metastase). Metastatic potential can be measured by injecting, e.g. lxlO6, cells into the lateral tail vein of athymic nude mice and determining the number of tumor nodules in the lung, e.g. 2 months post injection, e.g. as described in the section "Tumor cell injections" on page 2346 of Huang et al (1996) Oncogene 13, 2339-2347, or the sections "Animals and production of tumors" and " Histochemical analysis for calcified matrix" on page 1882 of Radinsky et al. (1994) Oncogene 9, 1877-1883. A cell line to produce more than 3, preferably more than 8, more preferably more than 20 tumor nodules in the lung in this assay is considered metastatic.
Preferably, the biological function inhibitable by a ligand, e.g. an antibody or antibody fragment, identified by the method of the invention is the adhesiveness, proliferation and invasiveness of a cancer cell.
In a preferred embodiment of the invention, the ligand is a polypeptide, in particular an antibody or antibody fragment. For example, if the ligand is an antibody it can be selected from the group consisting of IgA, IgG, IgD, IgE and IgM, and particularly from the group consisting of human IgG and IgM. If the ligand is an antibody fragment it can be selected from the group consisting of scFv, dsFv, Fab', Fab, F(ab')2, Fv, single domain antibodies and diabodies.
In another preferred embodiment of the invention the ligand is selected from the group consisting of protein scaffolds with a tertiary structure, random polypeptides, small molecules, RNA-aptamers and DNA-aptamers. Protein scaffolds with tertiary structure are ligands obtainable by using a gene encoding a polypeptide with a defined tertiary structure, wherein one or more regions of the gene encoding the polypeptide can be replaced by randomized oligonucleotides or randomized oligonucleotides can be inserted into the gene encoding the polypeptide without changing the tertiary structure of the encoded polypeptide. Examples for these ligands are e.g. Anticalin, Affibody, cystein knot or protein scaffolds with an immnoglobulin fold. Anticalin is described in Beste et al (1999) Proc Natl Acad Sci USA.
96, 1898-903 and ALDUs displaying Anticalin are described in Beste et al, see supra. An Affibody is a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and ALDUs displaying Affibodies are described in Hansson et al (1999) Immunotechnology. 4, 237-52. A Cystein knot is a protein scaffold involving interlocking disulfide bonds as exemplified by the structure of conotoxins and nerve growth factor. A cystein knot is described in McDonald et al (1991) Nature 354, 411-4 and ALDUs displaying Cystein knots are described in Smith et al. J Mol Biol. 1998, 277, 317-32. ). Protein Scaffolds with the immunoglobulin fold are antibodies and antibody fragments and other polypeptides having the tertiary structure of the immunoglobulin superfamily and ALDUs displaying protein scaffolds with the immunoglobulin fold are disclosed in McCafferty et al. (1990) Nature 348, 552-4. ALDUs displaying random polypeptides are disclosed in Scott et al. (1990) Science, 249, 386-90. RNA-aptamers are structural mimics composed of ribonuncleic acid and are disclosed in Tuerk and Gold (1990) Science 249, 505- 10, and Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA 89, 6988-92. DNA-aptamers are structural mimics composed of deoxyribonucleic acid and ALDUs displaying DNA-aptamers are disclosed in Bock et al (1992) Nature 355, 564-6.
This makes clear that the nature of the ligand displayed on the ALDU is not critical, but the ability ofthe ALDU to be identifiable and amplifiable.
However, as explained before, amplification of the ALDU can be achieved by increasing the number of an individual ALDU, but also by increasing the number of a ligand displayed by an individual ALDU. Since this can in principle also be achieved by an individual bead displaying a small chemical in combinatorial chemical libraries (see e.g., Ohlmeyer et al. (1993) Proc. Natl. Acad. Sci USA 90, 10922-26; Liu et al. J. Am. Chem. Soc. (2002) 124, 7678-80; Liang et al Science (1996) 274, 1520-2) such bead-format libraries are also libraries of ALDUs in a way. Small molecules can be synthesized on microsphere beads, which can be indexed with inert chemical tags. During each step ofthe synthetic scheme a tag molecule that encodes the step number and the chemical reagent used in that step is attached to the bead. This array of tags can then be used as a binary code to record the reaction history of each bead. The identified compound can then easily be synthesized in a larger scale.
However, preferably the ALDU is a biological ALDU that is the information about its identity is stored as a nucleic acid, e.g. RNA or DNA. In a preferred embodiment the ALDU is selected from the group consisting of viruses useful for viral display, phages useful for phage
display, bacteria useful for bacterial display, ribosomes useful for ribosome display, yeasts useful for yeast display and oligonucleotides useful for selection and amplification. Viruses useful for viral display can be, e.g., plant viruses, viruses of fungi or animal viruses, in particular viruses that can be obtained from cell culture, are amenable to genetic engineering techniques and can display a foreign ligand on their surface. Viruses useful for viral display on retroviruses are disclosed in Russell et al. (1993) Nucleic Acids Res. 21, 1081-5.
Viruses useful for display by plant viruses, e.g. the Cowpea mosaic virus are disclosed in Brendan et al. (1999) Microbiology 145, 211-20.
Phages useful for phage display can be all phages that can be obtained from culture, are amenable to genetic engineering techniques and are capable of displaying a foreign ligand on their surface. Phage display has been disclosed in Smith et al. (1985) Science, 228, 1315-17, phage display of an antibody has been disclosed in WO 91/17271.
Bacteria useful for bacterial display are bacteria, which can be kept in culture, are amenable to genetic engineering techniques and are able to display a ligand on their surface. Examples for bacteria useful for bacterial display are disclosed in Dougherty et al., (1999) Protein Eng. 12,613-21; and Westerlund-Wickstrom (2000) Int. J. Meth. Microbiol. 290, 223-30.
Ribosomes useful for ribosomal display have been disclosed in Shaffizel et al., (1999) J. Immunol. Methods. 231, 119-35; and Willson et al., (2001) Proc. Natl. Acad. Sci. USA 98, 3750-5.
Oligonucleotides useful for selection/amplification (Selex) have been disclosed in Tuerk and Gold (1990) Science 249, 505-10, and Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA 89, 6988-92.
However, also lower eukaryotes which can be cultured and are amenable for genetic engineering techniques and able to display a foreign ligand on their surface are useful as ALDUs, e.g. genetically modified yeast, as disclosed in Boder and Wittrup (2000) Methods Enzymol. 328, 430-44.
In another preferred embodiment the ALDU library is selected from the group consisting of immune or naive libraries, in particular wherein said naive library is a synthetic, semi- synthetic or natural library. This relates particularly to the case wherein the ligand displayed on the ALDU is an antibody or an antibody fragment. In those cases, one can distinguish
between immune or naive ALDU libraries. In the case of immune libraries the library members, e.g. antibody fragment genes, can be obtained from lymphocytes, e.g. B lymphocytes, of immunized animals. In the case of naive libraries the library members have not gone through the process of an active immune response. Depending on the way of its production, the naive library can be, e.g., a semi- synthetic library, in which library members are obtained by use of germline V-genes as starting material. The regions coding for one or several V-gene CDRs, encoded e.g. by CDR1 and CDR2 can be replaced by randomized oligonucleotides, or randomized oligonucleotides can be added to one or several CDRs, either at the beginning, as an insert into the CDR, or at the end of the CDR, and regions coding for one or several of the non V-gene encoded CDRs e.g. CDR3 can be added to the V-gene as randomized oligonucleotides.
Semi-synthetic libraries are disclosed in Williamson et al (1993) Proc Natl Acad Sci U S A. 90, 4141-5.
An antibody-based naive libraries can also be synthetic libraries, e.g. in which library members are obtained by use of non-germline V-genes as starting material. The regions coding for one or several V-gene CDRs encoded e.g. by CDR1 and CDR2 can be replaced by randomized oligonucleotides, or randomized oligonucleotides can be added to one or several CDRs, either at the beginning, as an insert into the CDR, or at the end of the CDR, and regions coding for one or several of the non V-gene encoded CDRs e.g. CDR3 can be added to the V-gene as randomized oligonucleotides (Griffiths et al. (1994) EMBO J. 13, 3245-60; Knappik et al. J Mol Biol. 296, 57-86).
A naive library can be a natural library wherein the library members e.g. antibody fragment genes, can be obtained from lymphocytes, e.g. B-lymphocytes, of non-immunized donors. An example of a natural library is disclosed in Griffiths et al. see supra.
In another preferred embodiment of the invention, the ALDUs are derived from viruses and phages, in particular from Cowpea mosaic virus, retroviruses, lambda phage, T7 phage, T4 phage and filamentous phages, more particularly derived from Ff phages. An ALDU library derived from Cowpea mosaic virus is disclosed in Brendan et al. (1999) Microbiology 145, 211-20. An example of an ALDU library derived from a retrovirus is disclosed in Russell et al. (1993) Nucleic Acids Res. 21, 1081-5.
An example of an ALDU library derived from lambda phage is disclosed in Maruyama et al. Proc Natl Acad Sci U S A. (1994) 91, 8273-7. An example of an ALDU library derived from T7 phage is disclosed in Danner et al. Proc Natl Acad Sci U S A. (2001) 98, 12954-9. An example of an ALDU library derived from T4 phage is disclosed in Efimov et al. Virus Genes. (1995) 10, 173-7. Often filamentous phages have been used as the basis for ALDU libraries (Phage Display of Peptides and Proteins: A Laboratory Manual/ edited by Brian K. Kay, Jill Winter, John McCafferty, Academic Press 1996). The Ff phages of Escherichia coli have proven particularly useful as phages capable of displaying random and foreign ligands on their surface (Smith, Science (1985) 228, 1315-7). In those cases the ligand, e.g. a random polypeptide, is often displayed as a fusion protein with a phage protein, wherein the phage protein is derived from a phage surface protein, e.g. selected from the group consisting of pill, pVIII and pVI. Phage display based on fusion proteins with pill are disclosed in Smith, Science. (1985) 228, 1315-7. Phage display based on fusion proteins with pVIII are disclosed in Gram et al. Proc Natl Acad Sci U S A (1992) 89, 3576-80. Phage display based on fusion proteins with pVI are disclosed in Jespers et al. Biotechnology (N Y) (1995) 378-82.
In another preferred embodiment step d) ofthe method of identifying a ligand ofthe invention comprises detecting an ALDU or a ligand derived from an ALDU, which affects the biological function of a target by a functional screening assay, wherein the target can particularly be a mammalian cell, e.g. a human cell, and the functional screening assay applied for detecting the ALDU or a ligand derived from an ALDU is selected from the group consisting of an invasion assay, an adhesion assay, a proliferation assay, an apoptosis assay, a cytotoxicity assay, an angiogenesis assay, a protein translocation assay, a protein-protein interaction assay, a migration assay, an ion transport assay, a colorimetric assay, a gene activation assay and a phenotypic assay. Preferred are a proliferation assay, an invasion assay and an adhesion assay.
An invasion assay measures the invasiveness of a cell in other words the capability of cells to spread out through a matrix. An invasion assay is, e.g., the assay performed in Example 5 of this application.
An adhesion assay measures the adhesiveness of a target, e.g. a cell to something else, e.g. another cell, a virus, a matrix structure comprising e.g. collagen, fibronectin and laminin, or a complex biological mixture like, e.g., a population of vehicles derived from a cell or, e.g., the
basal lamina. An example of an adhesion assay is given in Example 7 of the present application.
A proliferation assay measures the ability of a target, e.g. a mammalian cell, to divide and give raise to daughter cells. A proliferation assay can comprise simply counting the cell number of a culture over time or it can also make use of the increase in total DNA of the cell culture, as does, e.g., a so called BrdU-Assay, which measures the amount of bromo- deoxyuridine incorporation into DNA during replication. An example for such an assay is a BrdU-ELISA, commercially available from Roche Diagnostics, #1647229. Other proliferation assays are disclosed in Semlitsch et al. (2002) Anticancer. Res. 22, 1689-97 and Kaur et al. (2002) Gastroenterology 123, 60-7.
An apoptosis assay measures the number of cells, particularly mammalian cells, which undergo cell-suicide (apoptosis) within a cell culture. Apoptosis can be measured by counting the number of cells, which undergo characteristic morphological changes during the process of apoptosis (Hoppner et al. (2001) Nature 412, 202-6), but apoptosis can also be measured by use of biochemical markers characteristic for the process of apoptosis. The cell death detection ELISA-Plus, distributed by Roche Diagnostics #1774425, is an example of such an assay and measures apoptosis by quantifying the number of free nucleosomes in the cell lysate. An apoptosis assay can be useful in screens relating to biological function of cancer cells, cell types affected in cardiovascular diseases and inflammatory diseases, like, e.g., endothelial cells, heart muscle cells, smooth muscle cells or nerve cells. Other apoptosis assays are disclosed in Smolewski et al. (2002) J. Immunol. Methods 265, 111-21 and Chen et al. (2001) Mol. Cell. Biol. 21, 6322-31.
A cytotoxicity assay measures the cell-killing activity of an agent or process that kills cells. Cytotoxicity assays are disclosed in Wang and Zheng (2002) J. Immunol. 268, 179 and Eriksson et al. (2002) Anticancer Res. 22, 1787-93.
An angiogenesis assay measures the ability of a cell to induce blood vessel formation, a process, which usually accompanies the growth of malignant tissue. An angiogenesis assay is disclosed in Kanda et al (2002) J. Natl. Cancer Inst. 94, 1311-9.
A protein translocation assay measures the qualitative or quantitative change of the steady state localization of a protein within a cell. For example a protein, which at steady state is located in the cytoplasm may translocate as a response to a signal received by the cell, to, e.g.,
the nucleus. An example for such a behavior is the nuclear translocation of NFkappaB upon stimulation of an inflammatory response of the cell. A commercially available example of a protein translocation assay is the NFkappaB Nuclear Translocation Assay commercially available from Cellomics #K01-0001-1. Other protein translocation assays are disclosed in Schlegel et al. (1999) J. Biol. Chem. 274, 33551-6.
A protein-protein interaction assay can measure the strength of the interaction between a first type of polypeptide and a second type of polypeptide. The result of the measurement can be a binding constant for this protein-protein interaction. Such an assay may be done in a BIACORE format (Fivash et al. Curr Opin Biotechnol. (1998) 9, 97-101) or also in the form of a so-called Pull-down (Phizicky at al. Microbiol Rev. (1995) 59, 94-123) or an ELISA. By a protein-protein interaction assay also the quantity of a protein present in a biological sample can be determined. Examples for such ELISAs are cytokine ELISAs, disclosed in Foster (2001) Int. J. Ex. Pathol. 82, 171-92; Matrix Metalloproteinase ELISAs, as, e.g., disclosed in Moran et al, (2002) Cancer Res. 62, 3855-60; VEGF ELISAs, as, e.g., disclosed in Kim et al., (2001) J. Biol. Chem. 276, 38781-6; FGF ELISAs, as, e.g., disclosed in Ribatti et al., (2001) Int. J. Cancer 94, 314-21. A cytokine ELISA measures the concentration of cytokines, e.g. interferon- α, interleukin-lβ, -2, -4, -5, -6, -8, -10, tumor necrosis factor alpha, TRIAL or others, in a biological sample, e.g. in culture supernatants and body fluids, and can be useful for functional screening assays in the case of targets involved in inflammation, allergy, carcinogenesis, invasiveness or cardiovascular disease. Matrix Metalloproteinase ELISAs measure the amount of active matrix metalloproteinase. This can be useful for functional screening assays of targets involved in invasiveness, e.g. invasiveness of cancer cells. A VEGF ELISA measures the amount of vascular endothelial growth factor. This can be useful for functional screening assays of targets with a function in angiogenesis, e.g. cancer cells with the ability to induce angiogenesis. A FGF ELISA measures the amount of fibroblast growth factor. This can be useful for a functional screening assay in the case of targets involved in angiogenesis, wound healing or neural protection. For example the ability of cancer cells to induce angiogenesis can be assessed with such an assay, or e.g. an effect on a heart muscle cell or a smooth muscle cell or an endothelial cell can be assessed with such an assay or, e.g., an effect on nerve cells, e.g. a neural protective effect, can be assessed with such an assay. A migration assay measures the ability of a target, e.g. a mammalian cell, to move on a substrate or through a substrate. An example for migration is disclosed in Rilla et
al., (2002) J. Cell Sci. 115, 3633-3643. A migration assay can be useful for assessing, e.g. the chemotactic motility of cells.
An ion transport assay measures the amount of ions translocated, or transported across the cell membrane. An example of an ion transport assay is disclosed in Lockwich et al. (2001) J. Biol. Chem. 276. 42401-8. An ion transport assay can be useful for assessing signaling events in immune cell activation, in cell migration, especially for targets involved in inflammatory diseases, allergy or cancer.
A colorimetric assay measures a biological function by producing a detectable color change. For example some assays which measure enzymatic activity make use of a colorimetric assay system, e.g. as disclosed in Xu et al., (2000) J. Biol. Chem. 275, 38981-9. A gene activation assay measures a biological function, e.g. the strength of a signaling event, by quantification of induced gene expression, e.g. expression of a gene known to be induced by said signaling event. A gene activation assay can be performed by way of an ELISA, Chip assay, a Northern, an RT-PCR, a Western blot or another method capable of determining the quantity of a gene product. Examples of gene activation assays are disclosed in Tan et al., (2002) Proc. Natl. Acad. Sci. USA 99, 11387-92 and Zou et al., (2002) Oncogene 21, 4855-62.
A phenotypic assay can make use of a morphologic change of a target, e.g. an eukaryotic cell, upon a stimulus. An example of a phenotypic assay is the one described by Hδppner et al., above.
In a preferred embodiment of the invention, the separation step b) of the method of identifying a ligand of the invention is achieved by centrifugation, filtration, density centrifugation, FACS, immobilization onto solid support, affinity chromatography or separation by magnetic beads. For example an ALDU bound to a target can be separated from unbound ALDUs making use of the fact that the complex of an ALDU bound to a target has different biochemical properties compared to an unbound ALDU. Those changed biochemical properties can be size, specific binding, charge density, density or the like. Filtration can separate the bound ALDUs from the unbound ALDUs, e.g. if the size of the filter is such that ALDUs bound to a target are retained by the pores of the filter, while the unbound ALDUs can pass through the pores of the filter. Filtration is therefore useful if the target is large, e.g. a eukaryotic cell, and the ALDU is small, e.g. a phage, a virus, a nucleic acid or a ribosome displaying a ligand.
Density centrifugation separates biomolecules according to their density. Density centrifugation can be applied if the density of an ALDU bound to a target is different from that of an unbound ALDU. For example, the density centrifugation can be applied if an ALDU is dense, e.g. a ribosome displaying a ligand, and the target is not as dense, e.g. an eukaryotic cell or vehicles derived from the membrane of a cell.
FACS can separate the complex of ALDU bound to a target from unbound ALDUs based on fluorescence of the target. FACS can be performed by labeling the target with a fluorescent dye and then passing the labeled target in suspending medium through a narrow dropping nozzle so that each target is in a small, separate droplet. A laser-based detector system is often used to excite the fluorescent dye and droplets with e.g. fluorescence-positive targets are given an electric charge. Charged and uncharged droplets can then be separated as they pass charge plates and can be collected in two different tubes. In this way small droplets containing ALDUs that are bound to a target are separated from bulk solution containing unbound ALDUs.
Immobilization of the target onto solid support can be performed in order to separate ALDUs bound to the immobilized target from unbound ALDUs. Immobilization of the target onto solid support is often performed using hydrophobic or hydrophilic interactions between the support and the target or covalent interactions formed by using surface activated solid support or specific binding of the surface of the solid support with the target using e.g. biomolecules with specific binding properties towards the target. In order to separate ALDUs that are bound to the immobilized target from unbound ALDUs, the immobilized target is separated from the bulk solution containing unbound ALDUs.
Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by removing the bulk solution containing the unbound ALDUs from the immobilized target. For example, if the target is immobilized onto e.g. the well of a plastic micro titer plate via hydrophobic interactions then the bulk solution containing the unbound ALDUs can be removed by removing the solution and washing the wells with washing solution.
Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by FACS whereby the solid support is labeled with a fluorescent dye and separated as described above. For example, if the solid support consists of
e.g. plastic beads containing a fluorescent dye whereto the target can be immobilized via hydrophobic interactions and the ALDU does not contain a fluorescent dye.
Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by filtration if the size of the filter is such that the solid support is retained by the pores of the filter, while the unbound ALDUs can pass the filter. For example, filtration can be used if the solid support is e.g. surface activated pieces of glass with N-hydroxysuccinimid groups whereto the target can be immobilized via covalent interactions and the pieces of glass are large enough to be retained by the filter while the unbound ALDUs are not.
Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can also be performed by density centrifugation if the solid support has a different density compared to the bulk solution. For example, if the solid support consists of e.g. sepharose beads whereto the target is covalently coupled then the sepharose beads containing the target and bound ALDU can be pelleted by centrifugation.
Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can further be performed by affinity chromatography where the solid support is packed into a column and the bulk solution containing unbound ALDUs is removed by washing the column with washing solution. For example, if the solid support consists of e.g. sepharose beads covalently coupled to an antibody specific for the target then the sepharose beads can be packed into a column and unbound ALDUs removed by washing the column with washing solution.
Separation of ALDUs bound to the immobilized target from the bulk solution containing unbound ALDUs can be performed by magnetic separation if the solid support has magnetic properties different from the unbound ALDUs. For example, if the solid support consists of e.g. a paramagnetic particle whereto streptavidin is immobilized via hydrophobic interactions and the target contains a biotin molecule then the target bound to the solid support can be separated from non-paramagnetic ALDUs by applying a magnetic field.
In another preferred embodiment the amplification step c) of the method of identifying a ligand of the invention, particularly wherein the target is a eukaryotic cell, is achieved by either eluting the ALDU bound to a target, or by lysing a target, in particular eukaryotic cells, under conditions where the ALDU remains amplifiable, and subsequent amplification.
ALDUs bound to a target can be eluted from the target first, e.g. by use of solutions with low pH and/or high salt, as demonstrated in Example 1 and Example 2, or Example 15 and Example 16, and subsequently amplified, e.g. such that the eluted phages of Example 1 or Example 2, or Example 15 and Example 16, are used to infect Escherichia coli in order to produce large numbers of progeny phages. However, the elution step from the target, while it may be convenient, is not necessary, as, e.g., in the case of phage libraries even the phages still bound to a target, e.g. the eukaryotic cell, are capable of infecting Escherichia coli and thus capable of producing large numbers of identical progeny, and thus are amplifiable even when still bound to the target, e.g. a eukaryotic cell.
Also even phages that have entered the target, e.g. a eukaryotic cell, can be amplified, e.g. by lysing the target, e.g. a eukaryotic cell, under conditions where the ALDUs remain amplifiable, and subsequent amplification of the ALDUs. For example in the case of a phage library, the amplification of internalized phages is disclosed in Poul et al. (2000).
The identity of phages representing the antibody or the antibody fragment obtained with step d) can be determined by, e.g., sequencing the DNA encoding the antibody or the antibody fragment, or, in the case of a commercial library with gridded or numbered phages, by determining the grid position or the number of the phage. The grid position or the number then can reveal the identity ofthe antibody or the antibody fragment represented by the phage.
After step c) the pool of phages is enriched in phages binding to a particular surface molecule. Those phages binding to the surface molecule or antigen can finally be identified by numerous methods known in the art.
Phages can be separated to form individual clones and the clones of the phages can be contacted with recombinant antigen or can be probed with labeled antigen, or a labeled part of the antigen, e.g. an at least seven amino acid long peptide of the extracellular region of antigen. Clones binding to such a probe are identified as specific antigen-binders. Phages can also be affinity purified on purified antigen or on recombinant antigen.
Alternatively, the open reading frame encoding the antibody or antibody fragment can be recloned from the whole enriched pool into an expression vector, the antibody or the antibody fragment can then be expressed in clones of another host cell, and the clone of the host cell carrying the expression vector comprising a nucleic acid encoding for the antibody or the antibody fragment binding specifically to the antigen can be identified, e.g. by the method
described above for the identification of relevant phage clones, by the method of Examples 2 or 9, or by affinity purification on recombinant antigen.
In one embodiment ofthe invention the ALDUs or the ligands derived from an ALDU, which are used for the functional screening assay of step d) of the method of identifying a ligand according to the invention are chemically modified to increase their biological activity or endow the ALDU or the ligand with biological activity. In another embodiment of the invention, the ALDU or the ligand derived from an ALDU and used for the functional screening assay of step d) of the method of identifying a ligand of the invention are unmodified.
The chemical modification of the ALDU or the ligand derived from an ALDU can be the addition of chromophores. A chromophore is that part of a molecule that possesses high optical activity due to mobile electrons that interact with light. Some examples of chromophores are, e.g., fluorescein derivatives, rhodamine derivatives, coumarin derivatives, porphyrin derivatives, phthalocyanine derivatives, naphthalocyanines derivatives eosin derivatives, triphenylmethane derivatives e.g. malachite green, or acridine derivatives. Chromophores useful for chemically modifying biomolecules are well-known in the art. A useful chromophore is a chromophore that is non-toxic in the concentration range used and has preferably an absorbance in the visible region of spectrum or can be excited with laser light. Another preferable feature is, that the chromophore is commercially available as a functional derivative or can easily be converted into a functional derivative that can be covalently bound to the ligands of the invention. Functional derivatives suitable for covalent binding are e.g. sucinimidyl esters, isocyanates, isothiocyanates or carboxylic acids.
In another preferred embodiment of the invention, the method of identifying a ligand of the invention comprises as the identification step d) a spatial separation of the different ALDUs of a pool of ALDUs and then the screening of the spatially separated ALDUs for their effect on a biological function of a target in such a way that the result obtained can be assigned to an individual ALDU. Spacial separation of the different ALDUs of a pool of ALDUs can be done, e.g., by filling separate wells of a multi-well plate with individual ligands each and then use those multi-well plates in a functional screening assay. If, e.g., in one well the biological function of the target assessed by the functional screening assay is, e.g., inhibited, then a ligand inhibiting said biological function of the target is identified, as the experimentator knows the identity of each ligand he put into each individual well.
In another preferred embodiment the method of identifying a ligand according to the invention can comprise the step of determining the identity of the ligand, wherein this determination step is achieved by sequencing the DNA of an identified ALDU, taking a PCR fingerprint of the nucleic acid of an identified ALDU or also, in the case of a ligand derived from an ALDU, by mass spectrometry of such a ligand. PCR fingerprinting of, e.g., phages is disclosed in (Marks et al. J Mol Biol. (1991) 222, 581-97). Mass spectrometrical analysis of a ligand, e.g. a polypeptide, is disclosed in Shevchenko et al. (1996) Anal Chem. 68, 850-58 or Spengler et al (1992) Rapid Commun Mass Spectrom. 6, 105-8.
The method of identifying a ligand according to the invention may further comprise at least one additional step of screening ALDUs, e.g. wherein the screening is based on biochemical properties of the ALDUs. Such an additional screening step may comprise a method selected from the group consisting of flow cytometry, ELISA, Immunoprecipitation, binding assays, immunohistochemical experiments, affinity studies, immunoblots and protein arrays. Biochemical properties can be determined by the size, the shape, the density, the charge density, the hydrophobicity, or the binding specificity of an ALDU or the ligand derived from an ALDU. The biochemical properties of the ALDU or the ligand derived from an ALDU form the basis of the applicability of the above mentioned methods for screening the ALDUs. Flow cytometry is usually performed by labeling cells with a fluorescent dye and then passing those labeled cells in suspending medium through a narrow dropping nozzle so that each cell is in a small, separate droplet. A laser-based detector system is often used to excite the fluorescent dye and droplets with e.g. fluorescence-positive cells are registered.
Immunoprecipitation is a procedure by which a ligand, e.g. an antibody or antibody fragment, that reacts specifically with an antigen, e.g. an antigen associated with the surface of a target, for example a eukaryotic cell, is used to remove said antigen from a solution, e.g. a cell lysate. The immunoprecipitate consisting of the ligand bound to the antigen can then be examined by utilizing biochemical methods like, e.g. 2D gel electrophoresis. In particular the identity of the antigen can then be determined by use of, e.g., mass spectrometry.
In another preferred embodiment of the invention, the method of identifying a ligand of the invention can further comprise a subtractive selection step. A subtractive selection step is a step, which removes ALDUs with an undesired property. For example a subtractive selection step can be affected by removing the ALDUs capable of binding to a control cell, if the property of binding to a control cell is undesired. By way of example, if one wants to select
for ALDUs specific for cancer cells one could first select those ALDUs which bind to cancer cells, elute the bound ALDUs, e.g. phages, and then remove those ALDUs which are capable of binding to non-cancer-cells, by for example contacting the pool of eluted ALDUs with non-cancer-cells and removing those ALDUs bound to the non-cancer-cells. The ALDUs remaining in the supernatant are then ALDUs specific for cancer cells and can then be used in functional screening assays according to the method of identifying a ligand ofthe invention.
In a further preferred embodiment of the invention, the method of identifying a ligand of the invention can further comprise the step of identifying the surface antigen to which the ligand ofthe invention binds, for example by immunoprecipitation followed by mass spectrometrical analysis, preferably excluding ligands such as small molecules. Although the identification of said surface antigen is not a necessary step of the method of identifying a ligand according to the invention, it is still particularly useful, as this step leads to the identification of both, a ligand which affects a biological function of the target, e.g. a eukaryotic cell, and the antigen associated with the surface of the target, which is involved in said biological function. For example ligands, e.g. antibody fragments, which inhibit the invasiveness of naturally occurring, invasive cancer cells, can be identified. Those ligands have a great value in themselves, because they might be useful as therapeutics. If they are also used, e.g., in an immunoprecipitation step they can help to identify the antigen responsible for mediating the function of cancer cell under regular conditions. Thus, the potential drug is in such a way used to identify the corresponding drug target, and the ability of this embodiment of the invention to simultaneously identify a potential drug and the corresponding potential drug target is a great advantage.
Accordingly, the present invention further provides a method to identify unknown antigen proteins via an immunoprecipitation protocol that uses a Streptag modified scFv and Streptactin coupled to a solid carrier material like Sepharose, magnetic beads etc. This method allows the use of single chain Fv to isolate their unknown, cognate (membrane) protein targets from a very complex mixture of proteins such as whole cell lysate. The precipitate is loaded on a gel and the isolated bands can conveniently be investigated by mass spectrometry analysis.
So far, either biotinylated scFv or scFv fused to an affinity tag such as HSV (Herpes simplex virus) (R. W. Siegel et al, J. Mol. Biol. (2000) 302, 285-293), c-myc (C. Peneff et al, Journal of Immunological Methods (2000) 263, 105-115, S. Dauvillier, The Journal of Immunology (2002), 2274-2283) or Fc (B. Wang et al, Proc. Natl. Acad. Sci. USA (1999) 96, 1627-1632),
have been used in immunoprecipitation experiments. In most cases, the scFv have been selected against known, purified proteins, and the immunoprecipitation experiments were conducted in order to confirm the specificity of the binder. The advantage of the disclosed method is the simultaneous identification of scFv specificity and antigen identity.
In another preferred embodiment the method of identifying a ligand binding specifically to the extracellular region of an antigen associated with the surface of a target of the invention also comprises the step of incorporating the ligand identified into a therapeutic, prophylactic or diagnostic composition. This can, e.g., be done by mixing the identified ligand with a pharmaceutically acceptable carrier known in the art, wherein the ligand is present in an amount, which is therapeutically effective.
In another preferred embodiment the invention relates to a method for the production of a pharmaceutical composition, comprising the method of identifying a ligand binding specifically to the extracellular region of an antigen associated with the surface of a target and further the step of mixing the identified ligand, or a modified or a labeled version thereof, with a pharmaceutical acceptable carrier known in the art. A modified ligand is a ligand with a covalent modification. Examples of modified ligands are labeled ligands.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows invasion of HT1080 stained cells through an 8μm filter. Fluorescence was quantified after six hours incubation at 37°C. Data presented are the mean of n=3 wells +/- SD.
Figure 2 shows - presented as a table - results of the Invasion assay performed with HT1080 (human fibrosarcoma) cells. The scFv tested are identified by their number as shown in Fig. 6. The symbol "*" indicates that the respective scFv - as identified by its number as shown in Fig. 6 - was cloned into an IgG4 format before performing the Invasion assay. The invasion of cells through the matrix is measured and shown in % of inhibition of invasion.
Figure 3 shows - presented as a table - results of FACS analysis performed on various cell types, such as HT1080 (human fibrosarcoma), KHOS-NP (human osteosarcoma), MCF-7 (human breast adenocarcinoma), BT-474 (human breast cancer, mammary gland), PC-3 (human adenocarcinoma, prostate), Jurkat (human T cell leukemia), HL-60 (human acute myeloid leukemia), HeLa (human cervix carcinoma), SW480 (human colon carcinoma),
LS174T (human colon carcinoma), HT-29 (human colon carcinoma) in comparison to a control cell (Hs27 (human skin fibroblast)). The results are indicated by mean fluorescence intensity, whereby a mean fluorescence intensity of 0-12 is indicated by (+), a mean fluorescence intensity of 13-40 is indicated by (++) and a mean fluorescence intensity of above 40 is indicated by (+++). Table 3a depicts scFv identified by its number as shown in Fig. 6. Table 3b depicts FACS results of IgG. The symbol * means that the respective scFv -as identified by its number as shown in Fig. 6 was cloned into an IgG4 format before performing the FACS experiment.
Figure 4 shows results of the immunoprecipitation experiments. The immuno-complexes were separated by SDS-PAGE and silver stained. While scFv2 recognized a protein with approximately 120 kDa, scFv7 and scFv9 recognized the 1 lOkDa MCAM protein.
Figure 5 show the vector map of pXP14 (SEQ ID No. 39) as well as sequence of the scFv expression vector.
Figure 6 shows in Table 1 the peptide sequences of the identified single chains: scFvl to scFcl9. The CDR3 region is underlined in the depicted peptide sequence. Relevant SEQ ID No.'s are indicated aside.
Figure 7 shows in Table 2 the nucleotide sequences encoding for the polypeptides scFvl to scFcl9. Relevant SEQ ID No.'s are indicated.
Figure 8 shows - presented as a table - results of the Adhesion assay performed on various matrixes, such as CI (collagen S type I), CIV (collagen IV), FN (fibronectin) and LN (laminin), with various cell types, such as HT1080 (human fibrosarcoma), PC-3 (human adenocarcinoma, prostate), HeLa (human cervix carcinoma) and HT-29 (human colon carcinoma). The scFv tested are identified by their number as shown in Fig. 6. The symbol "*" indicates that the respective scFv -as identified by its number as shown in Fig. 6- was cloned into an IgG4 format before performing the Adhesion assay. The adhesion is measured and shown in % of inhibition of adhesion. For inhibition values: "+" represents 1-10% inhibition, "++" represents an inhibition value of > 10-40% and "+++" represents an inhibition value of >40-100%. "n.d." represents not determined.
Figure 9 shows the vector map of scFv expression vector pXPIO (SEQ ID No. 40) and sequence ofthe pXPIO.
Figure 10 shows the results of the immunohistochemistry using the MCAM specific antibodies (scFv7* and scFv9*) (scFv7 and scFv9 were cloned into IgGl format).
Figure 11 shows a MALDI-MS spectrum (a) of the peptide mixture obtained from the band with an approximate size of 110 kDa. Two trypsin auto digestion peaks, indicated as T, were used for internal calibration. A total of 15 peaks, marked with asterisks, matched an exemplary MCAM amino acid sequence (SwissProt, P43121), with a mass deviation of less than 13 ppm. The matching peptides cover 23%) (151/646 residues) of the amino acid sequence SEQ ID No. 58 as shown in Figure l ib. Figure l ie shows a MALDI-MS spectrum of the peptide mixture obtained from the band with an approximate size of 145 kDa immunoprecipitated with scFvl4. Two trypsin auto digestion peaks, indicated as T, were used for internal calibration. A total of 21 peaks, marked with asterisks, matched Integrin alpha-2 (SwissProt, P17301), with a mass deviation of less than 10 ppm. The matched peptides cover 19% (225/1181 residues) of the protein.
Figure 12 shows the results of the Proliferation assay as the reduction of SW-480 and PC-3 proliferation by IgG's. (scFv 7, scFv9 and scFvlδ were cloned into an IgG4 format before performing the proliferation assay). Proliferation of SW-480 colon cancer cells (Fig. 12b) and of PC-3 prostate cancer cells (Fig. 12a) was measured with an MTS cell viability assay at the indicated time points after first antibody addition. Data are shown as means of % medium control, pooled from 3 independent experiments. Error bars: Tukey HSD 95% confidence intervals.
Figure 13 shows - presented as a table - the anti-tumor effect of compounds in a subcutaneously grown human tumor xenograft in athymic mice. A lung adenocarcinoma (LXFA) was used as a xenograft. The table shows the tumor size at day 0 and day 7 after compound injection. scFv7* (scFv7 cloned into IgGl format) was used in comparison to Docetaxel. A result of the combination therapy of scFv* with Docetaxel is also shown. On the 7th day after scFv7* injection the tumor size is reduced by 12 %.
Figure 14 shows construction primers for a mouse library
The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting upon the present invention.
EXAMPLES
Example 1: Selection and screening of scFv (Selection on cells in suspension)
Single chain Fv were selected from a large non-immune phage displayed repertoire of human origin containing 1011 independent clones, provided by Cambridge Antibody Technology Ltd., Cambridge, UK.
For selection, HT1080 cells (human fibrosarcoma cell line; ATCC, CCL-121) were harvested with 0.05% EDTA and diluted to lxl07cells/ml in DMEM + 10% FCS. Two times 1012 cfu of phage library/107 cells were pre-blocked for 1 hour at 25°C with DMEM + 10% FCS and subsequently incubated with end-over-end rotation for 1.5 hour at 25°C with the cells in Eppendorf tubes pre-blocked with DMEM + 10% FCS. Three times 107cells were used for the first round of selection and 1x107 cells were used for the 2nd round of selection, respectively. The cells were washed by centrifugation at 220xg for five minutes, followed by removal of the supernatant and re-suspension in wash buffer. Five washes with DMEM + 10% FCS + 0.05% Tween-20 as wash buffer and five washes with DMEM + 10% FCS as wash buffer were performed. Bound phages were eluted by the addition of 10 mM Glycine pH 2.2, neutralized with IM Tris/HCl pH 7.4. Typically, between 103 and 106 cfu were eluted in the 1st round of selection, thus the diversity of the enriched repertoire is decreased compared to the original repertoire. The eluate containing the enriched repertoire was amplified by infecting exponentially growing E. coli TGI. Phagemid containing E. coli were selected and propagated by overnight growth at 30°C on LB agar plates supplemented with lOOμg/ml ampicillin and 1% glucose. Following this step, the enriched repertoire can either be amplified as a polyclonal pool and used for further rounds of selection in an iterative manner even until convergence to desired properties is achieved or be spatially separated and screened for a desired function on a single clone level. Phage particles for the next round of selection were produced by super-infecting exponentially growing cultures of the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20°C in 2xTY supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin. Selection ready phage were precipitated with 0.5 M NaCl/4% PEG-6000 from the cleared bacterial supernatant and re-suspended in PBS. In this example two rounds of selection were performed followed by screening on a single clone level.
Example 2: Selection and screening of scFv (Screening on adherent cells)
For screening, the genes encoding the selected scFv, contained in the phage display vector, were re-cloned to the expression vector pXP14. This vector directs the expression of a scFv in fusion with a Streptag and E-tag and does not contain a filamentous phage gene-3. Expression vector containing E. coli TGI from single colonies were grown in individual wells of a microtiter plate so that each well contains only one scFv clone. The bacteria were grown at 30°C in 2xTY supplemented with 100 μg/ml ampicillin and 0.1% glucose in 96-well microtiter plates (#9297, TPP) until an OD600 of 0.7. Expression was induced with IPTG at a final concentration of 0.5 mM and continued at 25°C overnight. Single chain Fv containing cleared lysates were prepared by addition of hen-egg lysozyme (#L-6876, Sigma) to a final concentration of 50 μg/ml for 1 hour at 25°C and centrifugation for 15 minutes at 3000xg. Prior to the screening ELISA, the cleared lysates were blocked by the addition of an equal volume of DMEM + 10% FCS for 1 hour. For the screening ELISA, HT1080 cells were seeded in a 96-well microtiter plate (#9296, TTP) at a density of 3x104 cells/well in DMEM + 10% FCS overnight at 37°C. The wells were blocked with DMEM + 10% FCS for 1 hour at 37°C and the scFv containing blocked cleared lysates added for 1.5 hours at 25°C. The plates were washed 2x with PBS + 0.1% Tween-20 and lx with PBS, incubated with HRP conjugated α-E-tag (#27-9413-01, Pharmacia Biotech; diluted 1 :5000 in 5% Skim Milk Powder (#70166, Fluka) in PBS with 0.1 % Tween-20) for 1 hour, washed 3x with PBS + 0.1% Tween-20 and 3x with PBS, developed with POD (#1 484 281, Roche) and signals read at 370 nm. Positive clones were retested against HT1080 cells and control human fibroblasts Hs-27 (ATCC CRL-1634) using the ELISA screening procedure described above.
In a typical screen, 1472 (16x92) clones were screened for binding to HT1080 cells with 16 % positives defined as clones giving a background subtracted signal > 0.1. 238 positive clones were retested for specific binding to HT1080 cells compared to the Hs-27 control cells with 28 % positives defined as clones giving a background subtracted signal on HT1080 of twice the value ofthe signal on Hs-27 control cells.
Example 3a: Sequencing and large scale expression
Sequencing of scFv genes was performed by Sequiserve GmbH, Vaterstetten, Germany using the primer pXP2 Seq2 (5'-CCCCACGCGGTTCCAGC-3'; SEQ ID No. 41) and pXP2 Seql (5 ACCTATTGCCTACGGC-3 '; SEQ ID No. 42). Amino acid sequences are shown in Table 1 and nucleotide sequences are shown in Table 2.
Unique clones identified by sequencing were streaked out from glycerol stocks onto LB/Amp(100μg/ml)/l% Glucose Agar plates and incubated o/n at 30°C. 10 ml LB/Amp/Glu (1%) media were inoculated with a single colony and grown o/n at 30°C and 200 rpm shaking. The next morning the overnight cultures were placed on ice until inoculation of IL 2xTY media supplemented with lOOμg/ml Ampicillin and 0.1% Glucose in 2L Erlenmeyer- flasks. The cultures were grown at 25°C shaking until an OD6oo 0.5 - 0.6 was reached and then induced with IPTG 0.1 mM final concentration. Fresh Ampicillin was added to 50 μg/ml and incubation was proceeded at 22°C o/n shaking. In the morning the cultures were centrifuged at 5000 x g for 15 minutes at 4°C, supernatants discarded and the pellets resuspended carefully on ice with a pipette in 10 ml pre-cooled PBS-0.5 M Na buffer containing protease inhibitors complete (#1697498, Roche). After re-suspension was completed, bacterial suspensions were transferred to 20 ml oakridge centrifuge tubes and hen-egg lysozyme (#L-6876, Sigma) added to a final concentration of 50 μg/ml for 1 hour on ice. The lysed bacteria were centrifuged at 20000 x g for 15 minutes at 4°C and the supernatants (lysate) transferred to a 15 ml plastic tube. For affinity purification the lysates were loaded with 1 ml/min onto 1ml StrepTactin (# 2-1505-010, IB A) columns equilibrated with 10 column volumes (CV) PBS-0.5 M Na buffer via a parallel protein purification system (self- made). After a 10 CV wash with PBS the elution was done with 5 CV PBS/5mM Desthiobiotin (#D-1411, Sigma) and 1 ml fractions collected. The fractions were measured at UV280, protein containing fractions were pooled and concentrated with Amicon Ultra Centrifugal Filter Devices 10.000 MWCO (#UFC801024, Millipore) at 4700 x g. The concentrated scFv were checked on 12% Bis-Tris SDS-PAGE gels stained with Coomassie Blue for purity and frozen in aliquots with 20 % glycerol at -80°C.
Example 3b: Cloning of scFv into IgG format and expression
The scFvs consist of the sequence of a variable light and heavy chain linked by a linker sequence. The variable light chain and the variable heavy chain were amplified by PCR separately with the usage of primer, which contain restriction sites. Those restriction sites are also present in the vectors, which contain the appropriate constant domains for the heavy and light chain. The amplified variable domains were cut with the restriction enzymes and cloned into the cut vectors. The correct sequence was confirmed via sequencing
Four vectors were used, one contained the constant domain of the heavy chain for IgGl format. The second contained the constant domain of the heavy chain for IgG4 format and two contained the constant domain of lambda and kappa light chains, respectively. Different restriction sites enabled to cut the vectors and to ligate the variable domains in the vectors.
For expression of the IgGs in mammalian cell lines the vectors contained an Epstein Ban- virus origin of replication (oriP sequence) which enhances the level of transcription in 293- EBNA-HEK cells, because the EBNA protein leads to the replication ofthe episomal vector. A co -transfection was carried out with the vector for the heavy chain and the vector for the light chain leading to the expression of both chains in the cell and the assembly of the IgG in the Endoplasmic Reticulum. The assembled IgG was then secreted to the medium. As transfection method Calcium-phosphate transfection was used, where a precipitate of Calcium-phosphate and the DNA is formed and incorporated into the cell. After the transfection the medium was changed to serum-free medium. Three harvests per IgG were done every 3 days. The supernatant (media) were sterile-filtrated and stored at 4°C.
For the purification of the IgGs the supernatants were purified via Protein A Sepharose either by gravity flow or by HPLC depending on the volume. For up to 200 ml a gravity flow method was used. For both purification types the supernatant was loaded on the Protein A column, washed with 50 mM Tris pH 7 buffer and eluted with 0.1 M Citrate pH ~ 2. To the elution fraction 0.25 M Tris pH 9 was added leading to a pH of 5.5-6.0. Depending on the further use ofthe IgGs they were dialysed against PBS buffer and stored at -20°C.
Example 4: FACS analysis for tumor cell specific binding
To test the ability of single chain Fv or IgG to bind specifically to the target cells, we performed a fluorescence-activated cell sorter (FACS) analysis using HT1080 cells, KHOS cells, PC-3 cells, BT 474 cells, MCF cells, Hela cells, Jurkat cells, HL60 cells, LS174T cells, and SW480 cells (106 cells/ml) and Hs-27 cells (106 cells/ml) as control cells. Cells were incubated with lOμg/ml of pure scFv in CellWash (BD (Becton, Dickinson and Company) #349524) for 20 min at 4°C, washed, and bound scFvs were detected with a secondary FITC labeled anti E-tag mab (Amersham #27-9412-01). Samples were washed and analyzed on a Becton Dickinson FACSscan. Results of selected scFv are shown in Figure 3 Table 3a. Results obtained with IgG's are shown in Table 3b.
Example 5: Invasion assay
The ChemoTx® system (Neuro Probe Inc.# 106-8, Gaithersburg) was used as a disposable chemotaxis/cell migration chamber in a 96 well format with an 8μm filter Track etched Polycarbonate pore size, 5.7 mm diameter/site.
13,3μl of 0.3mg/ml Matrigel (Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycan, entactin and nidogen. It also contains TGF-α fibroblast growth factor, tissue plasminogen activator, and other growth factors which occur naturally in the EHS tumor) (Becton Dickenson, BD #356234) diluted in Dulbeccos PBS (Gibco #14040-091) was applied on the membrane filter ofthe 96-well plate on row B-H and on row A 1,2 μg/site of collagen S type I (Roche #10982929) diluted in 0,05 M HCl (Sigma #945-50) and incubated over night at 20°C in a desiccator for gelation. HT1080 cells were grown to 70-80% confluence in DMEM supplemented with Glutamaxl (862mg/l (Gibco #31966-021) with 10%) FCS (Gibco #10270106). The cells were then labeled in situ with Bisbenzimide H 33342 (Sigma #B-2261) diluted 1 :100 in DMEM/GlutamaxI/0.1 % BSA (Sigma #A-7030) for 15 min at 37°C, 7,5% CO2. Cells were washed 2x with DMEM/GlutamaxI/0.1 % BSA and loaded with DMEM/GlutamaxI/0.1 % BSA for 15 min at 37°C, 7,5% CO2 for recovering. After washing 2x with PBS w/o Ca**, Mg++ (Gibco, 10010-015), the cells were detached with 0.5mM EDTA (Sigma #E8008), collected with Dulbeccos PBS/0.1% BSA/lOmM Hepes
(Gibco #15630-056), washed 2x with Dulbeccos PBS/0.1% BSA/lOmM Hepes, suspended in Dulbeccos PBS/0.1% BSA/lOmM Hepes and diluted to 6,7 x 106 cells/ml with Dulbeccos PBS/0.1% BSA/lOmM Hepes. 6,7 x 106 cells/ml were incubated 1 :1 with 40μg/ml HT1080 specific scFvl as a negative control for inhibition of invasion and with HT1080 specific scFv (scFv2-scFvlO) for lh on ice. After dilution to 6,7 x 105 cells/ml with DMEM/GlutamaxI/0.1 % BSA, HT1080 cells and HT1080 cell/scFv dilutions were pipetted in triplicate onto the chemotaxis chamber (row B-H) at a density of 3,4 x 10 cells/well and incubated for 6 h at 37°C, 7,5% CO2. DMEM/GlutamaxI with 5% FCS was used as a chemo attractant in the lower chamber. A standard curve from lxl 04 to 4xl04 cells/site is performed on collagen S type I coated row A of the chemotaxis chamber. DMEM/GlutamaxI/0.1%> BSA was used in the lower chamber (cells are not migrating). After scraping the non-migrating cells from the top of the membrane (except the Standard curve on row A) fluorescence of cells, which had migrated through the membrane (not migrated in case of the Standard curve), was measured on the Fluostar Galaxy (bMG) microplate reader using excitation/emission wavelengths of 370/460nm. In our experiments, a value of 45000 corresponded to 100% migrated cells. Average inhibition results of triplicate samples are shown in Figure 2. Each experiment was repeated three times with essentially similar results. The invasion phenotype of tumor cells was assessed by comparing their relative ability to invade tumor extracellular matrix (Matrigel) using the Transwell culture system described above.
Example 6.1: MTS viability assay
Viable cells were detected by measuring the conversion of the tetrazolium dye MTS (MTS, Celltiter Aqueous one, Promega #G4000) to formazan. HT1080 cells and HT1080 cell/scFv dilutions (obtained from the dilutions prepared in the Invasion assay) were pipetted in triplicate at a density of 3,4 x 104 cells/well were plated in a 96-well plate (black, ultra thin clear flat bottom, special optics, Costar #3615) 10 μl MTS was added to each well and incubated for 1 hour at 37°C, 7,5% CO2. Absorbance was measured at 492 nm with the Fluostar Galaxy (bMG) microplate reader. For all tested scFvs, no effect on viability of cells was seen.
Example 6.2: Proliferation assay
SW-480 and PC-3 cells were cultured in RPMI containing L-Glutamine and 10% FCS (Invitrogen #21875-034, Carlsbad, California).
SW-480 and PC-3 cells were seeded in culture medium in a volume of lOOμl/well into a 96well plate (Corning Costar #3904, Acton, Massachusetts). Cells were incubated 24h at 37°C, 5% CO . Medium was aspirated and 15 μg/ml antibody or 50 mM NaN was added diluted in culture medium. Control cells were incubated in medium alone. 48h after the first addition, antibody was added a second time at the same concentration. MTS absorbance was measured before, 24h, 48h and 72h after the first treatment. For this, lOμl CellTiter96 Aqueous One solution (Promega #G3581, Madison, Wisconsin) was added to lOOμl of cell suspension and the mixture incubated for 3h at 37°C, 5% CO2. Absorbance was measured at 492 nm in a FluoStar plate-reader (BMG LabTechnologies, Offenburg, Germany). Results are shown in Figure 12.
Example 7: Cell-matrix adhesion assay
96-well plates (TPP #9296) (cell culture treated) were coated with collagen S type I 1 μg/well (Roche #10982929) in Dulbeccos PBS (Gibco #14040-091) at 4°C over night. After washing with Dulbeccos PBS, blocking with 2% BSA (Sigma #A-7030)/Dulbeccos PBS for lh at 37°C and washing with Dulbeccos PBS, HT1080 cells and HT1080 cell/scFv dilutions (obtained from the dilutions prepared in the Invasion assay) were pipetted in triplicate at a density of 3,4 x 104 cells/well and incubated for one hour at 37°C, 7.5% CO2. After two additional washing steps with Dulbeccos PBS, where non-adherent cells were washed away, a Standard curve from lxlO4 to 4xl04 stained cells/well (obtained from the dilutions prepared in the Invasion assay) diluted in Dulbeccos PBS/0.1% BSA/lOmM Hepes was performed in row A. Washed wells were filled with 50μl Dulbeccos PBS and the absorbance of attached cells and of the Standard curve was measured on the Fluostar Galaxy (bMG) microplate reader using excitation emission wavelengths of 370/460nm. In our experiment, a value of 25000 corresponded to 100% adherent cells. The Adhesion assay was also performed using several matrix proteins, such as CIV (collagen IV), FN (fibronectin) and LN (laminin), and with various cell types, such as HT1080 (human fibrosarcoma), PC-3 (human adenocarcinoma, prostate), HeLa (human cervix carcinoma) and HT-29 (human colon
carcinoma) cells. Additionally to the scFv also IgG cloned from the single chains according to Example 3.2 were tested for their capacity to inhibit adhesion of tumor cells. Results are shown in Figure 8.
Example 8: Competition analysis by FACS
To test the ability of the certain inhibitory scFv to block common antigen epitopes on the target cells, single cell suspensions of HT1080 were harvested with 0,5mM EDTA/PBS. Approximately 1 x 106 cells were incubated in CellWash (BD, #349524) with lOμg/ml scFv for one hour at 4°C. After washing with Cell Wash lOμg/ml FITC labeled scFv was added and incubated for 20 min at 4°C. Signals of bound FITC labeled scFvs with and without pre incubation of other binders were analyzed on a Becton Dickinson FACSscan.
Example 9: Immunoprecipitation
HT1080 specific single chain Fv were cloned to the expression vector pXP14, which directs the expression of a scFv in fusion with a Streptag and an E-tag at its c-terminal end. The Streptag-scFv were used for immunoprecipitation as follows. HT1080 and Hs-27 cells (108) were lysed in 3ml 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100 (v/v) containing protease inhibitor cocktail (1 pill in 50ml buffer) (Boehringer Mannheim, Cat. -No. 1697498) and 100 μM Pefablock (Roth, Cat.-No. A154.1). Lysates were pre-incubated for 2h at 4°C with Streptactin Sepharose (IB A, # 2-1201-010) and the supernatants used for the immunoprecipitation reactions. HT1080 specific single chain Fv (50 μg/1 mg cell extract) were added to the cleared lysates, samples rotated for 2h at 4°C, gently centrifuged at 700 x g to pellet the Streptactin Sepharose, the pellet was washed 4 times with 1ml volume of PBS + 0.1% Tween buffer per wash, before the complexes were isolated by elution from the Streptactin Sepharose pellet with 50 μl 10 mM D-desthiobiotin in PBS 0.1% Tween 20. The immuno-complexes were separated by SDS-PAGE and silver stained for MS analysis. scFv 2 pulled down a protein, detected as a band on SDS-PAGE by silver staining at a molecular weight of approximately 120 kDa. This band was absent in the control samples with Hs-27 cells.
Single chain Fv 1, 3, 5, 6, 7, 9, 15 and 19 pulled down a protein, detected as a band on SDS- PAGE by silver staining at a molecular weight of approximately 1 10 kDa. Immunoprecipitations were performed using cell extract from HT1080 cells and Hs-27 cells as control cells. It was observed that this particular band was also present in the control samples, but in all cases to a much lower extent indicating that this protein is likely to be over-expressed in the HT1080 cell line. In the case of the scFv3 this band was only detected when HT1080 cell extract was used and not with the Hs-27 cells. scFv8 and 10 pulled down a protein, detected as a band on SDS-PAGE by silver staining at a molecular weight of approximately 130 kDa. This band was absent in the control samples with Hs-27 cells.
Single chain Fv 4, 1 1 and 14 pulled down 2 proteins, detected as 2 bands on SDS-PAGE by silver staining at a molecular weight of approximately 150 kDa and approximately 130 kDa, respectively. The upper band was also present in the control samples, but a much lower extent, whereas the lower band was absent or extremely faint in the controls, indicating that the 2 proteins are likely to be overexpressed in the HT1080 cell line.
Example 10: Protein identification via mass spectrometry
The gel bands obtained from immunoprecipitations followed by SDS PAGE were subjected to a tryptic in-gel digest over night at 37 °C. Peptides were extracted using 5% formic acid and the resulting peptide mixture was desalted using ZipTip μC18 (Millipore) and eluted first with 2 μl of 30% ACN/0.1% TFA, then with 2 μl of 70% ACN/0.1% TFA. The two fractions were pooled and one microliter of the obtained peptide mixtures was mixed in a 1 :1 ratio with a solution of α-cyano-4-hydroxycinnamic acid (3 mg/ml), co-crystallized on a Teflon-coated stainless steel target and analyzed on a MALDI-TOF instrument yielding peptide mass fingerprints (PMF) in a mass range of m/z 800-3000. The obtained PMF were used to search all entries for the species Homo sapiens in the NCBI and SwissProt databases. In all cases, only peptides matching a given protein with a mass deviation of less than 13 ppm were considered for identification.
The band with an approximate size of 120 kDa, obtained by using scFv2, yielded 9 to 10 peptide peaks, which matched Ephrin type-A receptor 2, with a maximum protein coverage of 14% (134/976 residues).
The band with an approximate size of 110 kDa yielded 6 to 15 peptides in different MALDI- TOF-MS experiments, which matched cell surface glycoprotein MUC18 precursor, with a maximum protein coverage of 23% (151/646 residues). Multiple entries of the same protein are available in the database, which contain slight deviations in the amino acid sequences. A MALDI spectrum that led to the identification of MCAM is shown in Figure 11a. The peptide coverage is shown in Figure l ib.
The molecular weight of any of the melanoma adhesion proteins is around 72 kDa, but since the protein is expected to be glycosylated it is not surprising to find the band on the gel around 110 kDa.
The band with an approximate size of 130 kDa, obtained by using scFvlO, yielded 7 peptides, which matched integrin-alpha 3, with a protein coverage of 6% (71/1019 residues).
The molecular weight of this protein is around 119 kD, but it is expected to be glycosylated, thus explaining the higher observed molecular weight observed on SDS-PAGE.
The band with an approximate size of 150 kDa, which is the higher one of the two bands obtained by using scFvl l-scFvl4, yielded 17 to 21 peptides, which matched integrin alpha-2, with a maximum protein coverage of 19% (225/1181 residues). A MALDI spectra is shown in Fig. l ie.
The lower one of the two bands with an approximate size of 130 kDa, yielded 9 to 13 peptides, which matched integrin beta-1, with a maximum protein coverage of 13% (106/798 residues).
The molecular weight of integrin alpha-2 is around 129 kDa, and the molecular weight of integrin beta-1 is around 88 kDa. However, both proteins are expected to be highly glycosylated, which might explain the higher apparent molecular weight observed on SDS- PAGE.
Example 11: Methods for epitope mapping
Example 11.1: "Classical" epitope mapping
Defined fragments of the cDNA for the antigen of interest are expressed as recombinant (fusion)proteins and probed in various assays such as Westernblot or ELISA.
Example 11.2: Phage display technology
The technique of epitope mapping using random peptide phage display libraries was developed to clone small random fragments of the cDNA for the antigen of interest into the phage protein pill of filamentous phages and display them on the surface of the phage (Fack et al., (1997) J. Immunol. Methods 7, 43-52). Epitope-displaying phages can be captured with antibodies in a procedure called "bio-panning". Sequencing ofthe inserts ofthe corresponding phages gives some information on the epitopes. This procedure is in principle capable to identify conformational epitopes.
Example 11.3: Peptide scan technology
It is based on the synthesis of immobilized peptides on activated membranes using the Fmoc chemistry. Amino acid solutions are applied to the activated membrane leading to a peptide bond between the amino-group on the membrane (the membrane is activated with PEG) and the activated carboxy-group of the applied amino acid. After each cycle a specific washing procedure, acetylation, deprotection and monotoring of free amino-groups is performed. In contrast to the in vivo protein-synthesis membrane bound oligo-peptide chains are stepwise synthesized from C- to the N-terminus. Oligo-peptides containing natural as well as modified amino acids can be synthesized up to a length of 20 amino acids. Following synthesis the membranes are equilibrated and unspecific binding sites are blocked. After incubation with the antibody of interest and several washing steps the detection is performed using an HRP- conjugated secondary antibody in combination with the ECL-System. Membranes can be stripped, regenerated, and re-used up to 10 times depending on the antibody. Small overlapping oligo-peptides that ideally cover the complete amino acid sequence ofthe antigen of interest are synthesized on a solid support. This method allows the identification of linear epitopes on the amino acid level. It also allows rapid mutational studies.
Example 12: Tissue profiling by immunohistochemistry
Two antibodies against MCAM (scFv7* and scFv9* - scFv7 and scFv9 cloned into IgGl format), were tested for their binding to the respective target in the tissue micro arrays containing 112 tumor specimens and 2 controls. The specimens were paraffin sections derived from human xenografts grown in nude mice and stained with biotin-streptavidin
/peroxidase/diaminobenzidine. Results of the binding to various tumor tissues are shown in Figure 10.
Example 13: Anti-tumor effect of compounds in subcutaneously growing human tumor xenografts A lung adenocarcinoma (bronchial adenocarcinoma) was used as a xenograph.
Nude mice of NMRI background approximately 7 weeks old at the start of treatment with an average weight of 35g were used. Animal health was examined before study commencement to ensure that only animals of good health entered testing procedures. Identification of mice was achieved by individual ear tag number, each cage by labeling with a record card, indicating the number of the experiment, date of randomization, mouse strain, gender, individual mouse number, test compound, dosage, schedule, and route of administration. The animals were housed in MacrolonM type III cages with filter hoods in air-conditioned rooms at 24±1°C and relative humidity at 60+10% and fed Altromin Extrudat 1439 Rat/Mouse diet, demineralized sterilized water containing 0.9 g/1 potassium sorbate with the pH adjusted to 2 with IN HCl. Water consumption was monitored visually daily, food and water provided ad libitum. The animal bedding SAWI produced by Altromin GmbH was analyzed and certified by Jelu Werk and autoclaved and distributed by Charles River and renewed twice a week. Antibodies were administered at a single high dose level (50 mg/kg twice a week for 4 weeks). One group received the vehicle only, the second scFv7* (scFv7 sequence cloned into IgGl format), the third the positive control with Docetaxel (20 mg/kg) for reference, and the forth with the combination of scFv7* and Docetaxel (50/20 mg/kg).
The group size is 6 mice and in order to obtain 24 mice with similar-sized tumors, 32 mice were implanted with a total of 64 tumor fragments (bilateral implantation). Tumor fragments for implantation were obtained from xenografts in serial passage in nude mice. After removal of the tumor from donor mice, the tumor was cut into fragments (2-3 mm diameter) and placed in RPMI 1640 culture medium (maximally for 30-45 minutes) until subcutaneous implantation in the mice. The mice were anaesthetized by inhalation of vaporized isoflurane and two small incisions were made in the skin of the back (left and right), and 2 tumor fragments were transplanted per mouse. Treatment started if tumors had grown to mean diameters of between 6 and 8 mm. For randomization, animals were divided into 3 categories according to tumor diameter: large: >8 mm; medium: between 6 and 8 mm; small: 5 mm. At randomization, tumor categories were equally distributed among the different groups.
Tumor size and mouse body weight were measured twice a week and mortalities and clinical signs recorded daily. Relative volume of individual tumors was calculated as the ratio between the tumor volume on Day x and the tumor volume on Day 0 (Tx/TO, Day 0 is the day of randomization and the first day of treatment). Relative body weights of individual mice were calculated correspondingly. Plots of the relative tumor volume and the relative body weight over time were generated. In order to evaluate anti-tumor activity, the ratio of the median relative tumor volumes for the antibody-treated and the vehicle-treated groups on a particular day was calculated (T/C% value). A minimum requirement for rating an antibody as active was a minimum T/C% value of <50%. Tumor-bearing mice were treated twice a week for 4 weeks. Tumors were collected at study termination for further analysis.
A comparison of the tumor size after different treatments at Day 7 is shown in Figure 13. To assess antibody plasma levels in NMRI nu nu mice, blood samples of 300 μl were retrieved by sublingual bleeding of mice in the antibody-treated group at 6 different time points during the in vivo therapy study. Trough levels were determined on Day 4 just before administration of the second dose and on Day 22 before administration of the 7th dose. Blood samples (three blood samples per time point) were obtained on Day 26, 27, 28 and 29. Samples on Days 4, 26 and 28 were obtained from mice 1-3, samples on Days 22, 27 and 29 from mice 4-6. The blood sample size was sufficient to prepare at least 100 μl plasma. Plasma samples were prepared using EDTA, stored at -80°C until analysis.
Example 14: Construction of immune library
Two BALB/c mice were each immunized intradermally with 2 x 107 paraformaldehyde fixed HT1080 cells (human fibrosarcoma cell line; ATCC, CCL-121). Following the first immunization, the injections were repeated twice in a period of 39 days, the mice sacrificed and the spleens isolated and frozen in liquid nitrogen. The immunizations were performed by Charles River, Germany GmbH, KiBlegg.
Total RNA was isolated using the RNeasy Midi Kit (QIAGEN #75142) as described by the manufacturer using half of each spleen preparation. The RNA concentration and purity was determined by a denaturing formaldehyde gel and photometric measurement. cDNA was synthesized using 8.9μg of freshly prepared RNA and lOpmol of a primer mix (IgGl-c (SEQ ID NO.: 103), IgG2a-c (SEQ ID NO.: 104), IgG2b-c (SEQ ID NO.: 105),
IgG3-c (SEQ ID NO.: 106), VLL-c (SEQ ID NO.: 107), VLK-c (SEQ ID NO.: 108)) using the Superscript™ II Kit (GibcoBRL Life Technologies #18064-014) These primers anneal to the RNA encoding the IgG heavy-chain genes and the light chain genes of the kappa and lambda family. VH genes were PCR amplified from lμl of cDNA using 36 individual combinations of 9 forward primers (M-VH1 (SEQ ID NO.: 81), M-VH2 (SEQ ID NO.: 82), M-VH3 (SEQ ID NO.: 83), M-VH4 (SEQ ID NO.: 84), M-VH5 (SEQ ID NO.: 85), M-VH6 (SEQ ID NO.: 86), M-VH7 (SEQ ID NO.: 87), M-VH8 (SEQ ID NO.: 88), M-VH9 (SEQ ID NO.: 89)) and 4 backward primers (M-JH1 (SEQ ID NO.: 90), M-JH2 (SEQ ID NO.: 91), M-JH3 (SEQ ID NO.: 92), M-JH4 (SEQ ID NO.: 93)) without restriction sites. VL genes were PCR amplified with one primer mix (M-VK1 (SEQ ID NO.: 94), M- VK2 (SEQ ID NO.: 95), M-VK3 (SEQ ID NO.: 96), M-VK4 (SEQ ID NO.: 97), M-VL1 (SEQ ID NO.: 98), M-JK1 (SEQ ID NO.: 99), M-JK2 (SEQ ID NO.: 100), M-JK3 (SEQ ID NO.: 101), M-JL1 (SEQ ID NO.: 102)) without restriction sites. PCR products were gel-purified (QIAquick Gel Extraction Kit, #28706) and reamplified using individual combinations of 9 forward primers (MVH1 Sfil (SEQ ID NO.: 59), MVH2 Sfil (SEQ ID NO.: 60), MVH3 Sfil (SEQ ID NO.: 61), MVH4 Sfil (SEQ ID NO.: 62), MVH5 Sfil (SEQ ID NO.: 63), MVH6 Sfil (SEQ ID NO.: 64), MVH7 Sfil (SEQ ID NO.: 65), MVH8 Sfil (SEQ ID NO.: 66), MVH9 Sfil (SEQ ID NO.: 67)) and 4 backward primers (M-JH1 Sail (SEQ ID NO.: 68), M-JH2 Sail (SEQ ID NO.: 69), M-JH3 Sail (SEQ ID NO.: 70), M-JH4 Sail (SEQ ID NO.: 71)) with restriction sites for VH and one primer mix (M-VK1 ApaLI (SEQ ID NO.: 72), M-VK2 ApaLI (SEQ ID NO.: 73), M-VK3 ApaLI (SEQ ID NO.: 74), M- VK4 ApaLI (SEQ ID NO.: 75), M-VL1 ApaLI (SEQ ID NO.: 76), M-JK1 Notl (SEQ ID NO.: 77), M-JK2 Notl (SEQ ID NO.: 78), M-JK3 Notl (SEQ ID NO.: 79), M-JL1 Notl (SEQ ID NO.: 80)) with restriction sites for VL. PCR products were gel-purified (QIAquick Gel Extraction Kit, #28706) and cloned into the phage display vector pXPIO (SEQ ID No.: 40) using the restriction sites Sfil/Sall for VH and ApaLI/Notl for VL. The ligation mix was transfected into E.coli TG-1 by electroporation resulting in a library size of 107 independent clones (phages) expressing different single chain antibody fragments (scFv).
Example 15: Selection and Screening of scFv (Selection on fixed cells)
Single chain Fv were selected from a phage display library generated from mice immunized with fixed HT 1080 cells.
HT1080 cells were harvested with 0.05% EDTA, fixed with paraformaldehyde, diluted to lxl07cells/ml in PBS and immobilized onto wells of a 96 well UV cross-link plate (Corning Costar). The wells of the UV cross-link plate were blocked with 5% Skim Milk Powder (#70166, Fluka) in PBS (MPBS). 1012 cfu (colony forming units) of phage library/106 cells were pre-blocked for 1 hour at 25°C with MPBS and subsequently incubated for 1.5 hour at room temperature (RT) with the cells. The wells of the UV cross-link plate were washed six times with PBS + 0.05% Tween-20 followed by six washes with PBS. Bound phage were eluted by the addition of 10 mM Glycine pH 2.2, and neutralized with IM Tris/HCl pH 7.4. Typically, between 103 and 106 cfu were eluted in the 1st round of selection, thus the diversity of the enriched repertoire is decreased compared to the original repertoire. The eluate containing the enriched repertoire was amplified by infecting exponentially growing E. coli TGI . Phagemid containing E. coli were selected and propagated by overnight growth at 30°C on LB agar plates supplemented with 100 μg/ml ampicillin and 1% glucose. Following this step, the enriched repertoire can either be amplified as a polyclonal pool and used for further rounds of selection in an iterative manner until convergence to desired properties is achieved or be spatially separated and screened on a single clone level. Phage particles for the next round of selection were produced by super-infecting exponentially growing cultures of the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20°C in 2xTY supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin. Selection ready phage were precipitated with 0.5 M NaCl/4% PEG- 6000 from the cleared bacterial supernatant and re-suspended in PBS. One round of selection was performed followed by screening on a single clone level.
Example 16: Selection and Screening of scFv (Screening on fixed cells)
For screening, the genes encoding the selected scFv, contained in the phage display vector, were re-cloned to the expression vector pXP14. This vector directs the expression of a scFv in fusion with a Streptag and E-tag and does not contain a filamentous phage gene-3. Expression vector containing E. coli TGI from single colonies were grown in individual wells of a microtiter plate so that each well contains only one scFv clone. The bacteria were grown at 30°C in 2xTY supplemented with 100 μg/ml ampicillin and 0.1% glucose in 96-well microtiter plates (#9297, TPP) until an OD60o of 0.7. Expression was induced with IPTG at a
final concentration of 0.5 mM and continued at 25°C overnight. Single chain Fv containing cleared lysates were prepared by addition of hen-egg lysozyme (#L-6876, Sigma) to a final concentration of 50 μg/ml for 1 hour at 25°C and centrifugation for 15 minutes at 3000 x g. Prior to the screening ELISA, the cleared lysates were blocked by the addition of an equal volume of DMEM + 10% FCS for 1 hour. For the screening ELISA, HT1080 cells were harvested with 0.05% EDTA, fixed with paraformaldehyde, diluted to lxl07cells/ml in PBS and immobilized onto wells of a 96 well UV cross-link plate (Corning Costar). The wells of the UV cross-link plate were blocked with MPBS and the scFv containing blocked cleared lysates added for 1.5 hours at 25°C. The plates were washed 2x with PBS + 0.1% Tween-20 and lx with PBS, incubated with HRP conjugated α-E-tag (#27-9413-01, Pharmacia Biotech; diluted 1 :5000 in MPBS with 0.1 % Tween-20) for 1 hour, washed 3x with PBS + 0.1% Tween-20 and 3x with PBS, developed with POD (#1 484 281, Roche) and signals read at 370 nm. Positive clones were retested against HT1080 cells and control human fibroblasts Hs- 27 (ATCC CRL-1634) using the ELISA screening procedure described above.
In a typical screen, 2760 (30x92) clones were screened for binding to HT1080 cells with 5 % positives defined as clones giving a background subtracted signal > 0.1. 155 positive clones were retested for specific binding to HT1080 cells compared to the Hs-27 control cells with 28 % positives defined as clones giving a background subtracted signal on HT1080 of twice the value ofthe signal on Hs-27 control cells.
Example 17: BrdU-ELISA (Roche Diagnostics, #1647229)
Cell proliferation is measured by quantifying the amount of bromo-deoxyuridine incorporated into DNA during replication. This assay can be used in a broad range of applications, e.g. in cancer (screen for proliferation inhibitors) or inflammatory diseases (screen for anti- inflammatory agents). For composition of buffers and reagents we refer to the manufacturer's instructions.
Treated and non-treated cells are cultured in a 96-well plate in 100 μl of normal growth medium per well. After addition of 10 μl BrdU-labeling solution per well (final concentration: 10 μM), cells are incubated for 2-24 hours, depending on the doubling time of the cell line used. The labeling medium is removed after centrifugation for 10 minutes at 300 x g, and the cells are dried for 1 h at 60°C. 200 μl of FixDenat reagent and then 100 μl of anti-BrdU-POD
working solution are added to each well and the plate is incubated for 90 minutes at 15-25°C. After removal of the antibody conjugate, the wells are rinsed with 200-300 μl of washing solution, and 100 μl of substrate solution are added. Following an incubation of 5-30 minutes at room temperature and addition of 25 μl IM H2SO4, the absorbance ofthe samples is read in a plate reader at 450 nm (reference wavelength: 690 nm).
Example 18: Cell Death detection ELISA-plus (Roche Diagnostics, #1774425)
Apoptosis is measured by quantifying the number of free nucleosomes in a cell lysate. This assay can be applied in screen for apoptosis inducing or -inhibiting agents in wide range of indications (cancer, cardiovascular diseases, inflammatory diseases).
For composition of buffers and reagent we refer to the manufacturer's instructions.
The 96-well plate with the cells induced to undergo apoptosis and the control cells is spun at
200 x g for 10 minutes, the medium is removed and 200 μl of lysis buffer are added. To induce lysis, the plate is shaken for 30 minutes at room temperature, then centrifuged at
200 x g for 10 minutes. 20 μl of lysate are pipetted to each well of the streptavidin-coated
ELISA plate, then the antibody mixture, consisting of 72 μl of incubation buffer, 4 μl of anti- histone- and 4 μl of anti-DNA-POD-antibody, is added and the plate is shaken for 2 h at room temperature, covered with an adhesive foil. After removal ofthe solution and 3x washing with
250 μl incubation buffer, 100 μl ABTS solution is added to each well. Absorbance is read out in a plate reader at 405 nm (reference wavelength 490 nm).
Example 19: NFKB nuclear translocation assay (Cellomics, #K01-0001-1)
Activation of the transcription factor NFKB is measured by quantifying the portion of NFkB translocated from the cytoplasm to the nucleus using automated fluorescence microscopy;
NFkB actvation; application: mainly immune and inflammatory diseases, but also cardiovascular disease and cancer.
For composition of buffers and reagents we refer to the manufacturer's instructions.
After stimulation cells cultured in a 96-well plate are washed with 200 μl pre-warmed PBS, and 200 μl of 3.7% formaldehyde in wash buffer is added. The plate is incubated at room temperature for 10 minutes, washed once with 200 μl of wash buffer per well, and 200 μl of permeabilization buffer are added. After incubation for 90 seconds, each well is washed with
200 μl of wash buffer, and 50 μl of diluted primary antibody (anti-NFκB p65, 1 :250 in wash
buffer) are added. The plate is incubated for 1 h at room temperature, then the wells are washed 3 x with detergent wash buffer and 3 x with wash buffer at room temperature. 50 μl of diluted secondary antibody (Alexa-488 -conjugated anti-rabbit IgG, 1 :80), containing Hoechst dye (1 :2000) are added and the plate incubated for 1 h at room temperature. After washing as above, the intensity difference between nucleus and cytoplasm in 20 fields per well is measured with the ArrayScan II automated microscope (Cellomics).
Example 20: Identification of virulence factors in alphaviruses and corresponding neutralizing scFvs
Alphaviruses are a group of 26 icosahedral positive-sense RNA viruses transmitted by mosquitoes. Ross River (RR) virus is a member and causes polyarthritis in humans and is endemic in Australia.
Viruses are produced as described (Vrati et al. (1988) Virology 162, 346-353; Smith et al. (1995) PNAS 92, 10648-52; Sharkey et al. (2001) J. Virol. 75, 2653-2659). The T48 strain of RR is rescued from full-length cDNA pRR64 (Kuhn et al. (1991) Virology 182, 430-441) and is the source of virus. Mosquito cell line C6/36 is used for propagation. Cells are grown at 30°C in roller bottles using Eagle minimal essential medium/10% fetal calf serum and are infected with virus at a multiplicity of infection (MOI) of 1. After a 36 hour incubation, cell supernate are harvested and viruses are concentrated by precipitation in 10% polyethylene glycol 600 (PEG)/0.5 M NaCl. The virus is purified by sucrose density centrifugation.
For the selection of RR virus-specific phages single chain Fv are selected from a large non- immune phage displayed repertoire of human origin containing 1011 independent clones, provided by Cambridge Antibody Technology Ltd., Cambridge, UK.
Purified virions at different concentrations are tested for selection. Purified virions are coated on a 96 well UV cross-link plate (Coming Costar). The wells of the UV cross-link plate are blocked with 5% Skim Milk Powder (#70166, Fluka) in PBS (MPBS). Two times 1012 cfu (colony forming units) of phage library/well are pre-blocked for 1 hour at 25°C with MPBS and subsequently incubated for 1.5 hour at room temperature (RT) with the wells. The wells of the UV cross-link plate are washed six times with PBS + 0.05% Tween-20 followed by six washes with PBS. Bound phage are eluted by the addition of 10 mM Glycine pH 2.2, and neutralized with IM Tris/HCl pH 7.4. Typically, between 103 and 106 cfu are eluted in the 1st
round of selection, thus the diversity of the enriched repertoire is decreased compared to the original repertoire. The eluate containing the enriched repertoire are amplified by infecting exponentially growing E. coli TGI. Phagemid containing E. coli are selected and propagated by overnight growth at 30°C on LB agar plates supplemented with lOOμg/ml ampicillin and 1%) glucose. Following this step, the enriched repertoire can either be amplified as a polyclonal pool and used for further rounds of selection in an iterative manner until convergence to desired properties is achieved or be spatially separated and screened on a single clone level. Phage particles for the next round of selection are produced by super- infecting exponentially growing cultures of the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20°C in 2xTY supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin. Selection ready phage are precipitated with 0.5 M NaCl/4% PEG-6000 from the cleared bacterial supernatant and resuspended in PBS. Two round of selection are performed followed by screening on a single clone level.
For selection and screening, the genes encoding the selected scFv, contained in the phage display vector, are re-cloned to the expression vector pXP14. This vector directs the expression of a scFv in fusion with a Streptag and E-tag and does not contain a filamentous phage gene-3. Expression vector containing E. coli TGI from single colonies are grown in individual wells of a microtiter plate so that each well contains only one scFv clone. The bacteria are grown at 30°C in 2xTY supplemented with 100 μg/ml ampicillin and 0.1 %> glucose in 96-well microtiter plates (#9297, TPP) until an OD6oo of 0.7. Expression is induced with IPTG at a final concentration of 0.5 mM and continued at 25°C overnight. Single chain Fv containing cleared lysates are prepared by addition of hen-egg lysozyme (#L-6876, Sigma) to a final concentration of 50 μg/ml for 1 hour at 25°C and centrifugation for 15 minutes at 3000 x g. Prior to the screening ELISA, the cleared lysates are blocked by the addition of an equal volume of 5% MPBS for 1 hour. For the screening ELISA, purified virions are coated on a 96 well UV cross-link plate (Corning Costar). The wells of the UV cross-link plate are blocked with MPBS and the scFv containing blocked cleared lysates added for 1.5 hours at 25°C. The plates are washed 2x with PBS + 0.1% Tween-20 and lx with PBS, incubated with HRP conjugated α-E-tag (#27-9413-01, Pharmacia Biotech; diluted 1 :5000 in MPBS with 0.1 % Tween-20) for 1 hour, washed 3x with PBS + 0.1% Tween-20 and 3x with PBS, developed
with POD (#1 484 281, Roche) and signals read at 370 nm. The specific scFvs are expressed and purified as described and tested in a functional neutralization assay.
Plaque reduction neutralization assays is performed by incubating approximately 100-200 plaque forming units of virus (in 100 μl) with different dilutions of scFv (in 100 μl of Hanks' balanced salt solution - HBSS) at 37°C for 1 hour. Duplicate 100 μl aliquots of incubation mixtures are assayed for residual infective virus by plaquing on Vero cells. Percentage plaque reduction is calculated relative to that of virus controls incubated without any RR-specific scFv. The scFv dilution carrying 50% plaque reduction are taken as the neutralization titer.
For the identification of corresponding target of neutralizing scFv one milligram of RR- purified virions are solubilized with Triton-X. The soluble fractions are mixed with the neutralizing scFv as described in the immunoprecipitation from cell extracts. The immunoprecipitate is run in a SDS-PAGE and the corresponding protein band is cut out and is identified via mass spectrometry with database query.
Alternatively, 20 micrograms of purified RR virions are solubilized in SDS-PAGE sample buffer, split into 2 and then loaded into an SDS-PAGE and the proteins are electrophoretically separated. The first half of the sample is processed for Western blot as described, using the neutralizing scFv as the detection reagent. The protein band recognized by the neutralizing scFv corresponds to the target protein. The identity of the target is derived from the second half of the sample which is run in SDS-PAGE but is stained by Coomassie blue or silver. The protein migrating to the same distance as identified from the Western blot is cut out and subsequently identified by mass spectrometry and database query.
Example 21: Identification of virulence factors in Vibrio cholerae and its corresponding neutralizing scFvs.
Vibrio cholerae is the aetiological agent responsible for the acute gastrointestinal disease cholera. In order to exert the pathogenic effects of cholera toxin, it is necessary for V. cholerae to colonize the small intestine. It has been shown that colonization of Vibrio of human intestine correlates with its ability to self-agglutinate. This process involves the interaction of surface proteins on V. cholerae with the human intestine.
Single colonies of V. cholerae 0393 of which each are re-suspended in 1 ml of LB broth and 10 μl of the suspension are inoculated into 5 ml LB, pH 6.5 at 30°C for 12-18 hours (conditions that promote formation of virulence factors) as described in (Kaufmann and Taylor (1994) Methods Enzymology 235, 426-448).
For the selection of V. cholerae-specific phages V. cholerae cells (1 x 10E7) are coated on a well of a 96-well ELISA plate. The same protocol for blocking and washing is used as described in example 20. Phage selection and screening is performed as described in example 20.
V. cholerae-specific scFvs are expressed and purified as described and tested in a functional Autoagglutination assay. The autoagglutination assay is performed as described in Chiang et al. (1995) Mol. Microbiol. 17,1133-1142. Cultures from single colonies are grown for 12-15 hours at 30°C in LB broth, pH 6.5 on a roller shaker. Agglutination is scored visually. To quantify the extent of autoagglutination, 1 ml aliquots of cultures are allowed to stand at room temperature for 30 minutes and the optical density at 550 nm of the culture is determined by spectrophotometry. For the inhibition of autoagglutination, different concentrations of specific scFvs are tested and the degree of autoagglutination is quantified as described. The results are quantified as the ratio of optical density at 550 nm with scFvs to the optical density at 550 nm without any scFv.
For the identification of corresponding target of neutralizing scFvs V. cholerae cells are grown under conditions promoting virulence at a total amount of lxl 0E9. The cells are resuspended in 1 ml of lysis buffer lysed with lysozyme and EDTA under hypotonic conditions (same used for purification of scFvs) to release the outer membrane proteins. Triton X-100 is added to solubilize membrane proteins. The solubilized membrane proteins including the pilins of V. cholerae are used for subsequent immunoprecipitation with the neutralizing scFv. The immunoprecipitate is separated in an SDS-PAGE and the corresponding band is cut out and is analysed by mass spectrometry.
Example 22: Identification of cellular receptors necessary for viral infection and corresponding neutralizing scFvs
Poliovirus serotype 1, Mahoney strain is grown in HeLa cells in suspension and purified by differential centrifugation and CsCl density gradient fractionation as described in Yeates et al.
(1991) EMBO J. 10, 2331 -2341. The infectivity of the virus preparation is determined by a plaque assay on HeLa cell monolayers as described in Chow and Baltimore (1982) Proc. Natl. Acad. Sci. 79(23), 7518-21.
The functional screening assay is a plaque reduction assay, performed essentially as previously described (Chow and Baltimore (1982) Proc. Natl. Acad. Sci. 79, 7518-21) with the following modifications. HeLa cells grown in a 24 well format are incubated with different dilutions (lOnM to lOOμM final concentration in the well) of recombinantly expressed scFvs - derived from the spatially separated clones of Example 1 - in Dulbeccos PBS/0, 1%BSA/10 mM HEPES (Gibco 15630-056) on ice for 15 min. After removal of the scFv-containing solution by suction approximately 50 plaque forming units of poliovirus suspended in Phosphate-buffered saline containing 0,5mM MgCl2, 0,7 mM CaCl2, and 0,5%) (vol/vol) fetal calf serum are added and allowed to attach to the HeLa cell monolayer for 30 min at 37C. The cells are then overlaid with growth medium comprising 1% agarose and incubated at 37C for about 36 hours. Plaques are then visualized by staining with crystal violet, as described in Chow and Baltimore above. ScFvs effecting an inhibition of poliovirus infection, i.e. a reduction in the number of plaques as compared to a control sample in which no scFv was added to Dulbeccos PBS/0,1%BSA/10 mM HEPES (Gibco 15630-056) during the incubation step on ice for 15 min, are identified.
The receptor involved in viral infection is the identified according to the protocol for immunprecipitation of Example 9, with the modification that HeLa cells are lysed and used as starting material instead of Hs-27 cells.
Claims
1. A method of identifying a ligand binding specifically to the extracellular region of an antigen associated with the surface of a target, wherein said ligand is capable of affecting a biological function of said target, comprising the steps of a) contacting a library of amplifiable ligand-displaying units (ALDU) with said target; b) separating said target and the ALDUs bound thereto from ALDUs not bound to said target; c) amplifying the ALDUs bound to said target; d) identifying an ALDU or a ligand derived from an ALDU after step c), which affects said biological function by a functional screening assay; and optionally e) determining the chemical identity ofthe ligand.
2. The method according to claim 1 further comprising at least one additional step of screening ALDUs, wherein the screening is based on biochemical properties ofthe ALDUs.
3. The method according to claim 1 or 2 additionally comprising after step c) a subtractive selection step effected by removing the ALDUs capable of binding to a control cell.
4. The method according to any ofthe claims 1 to 3 further comprising the step of identifying said surface antigen by immunoprecipitation with the ligand followed by mass-spectrometrical analysis.
5. The method according to claim 4 wherein for the immunoprecipitation the ligand is fused to streptac.
6. The method according to any ofthe claims 1 to 5 further comprising the step of incorporating the ligand into a therapeutic, prophylactic and/or diagnostic composition.
7. The method according to claim 6 for the production of a composition comprising the step of mixing a therapeutically effective amount of a ligand identified according to step d) of claim 1 and/or modified with a pharmaceutical acceptable carrier.
8. The method according to any ofthe claims 1 to 7, wherein said ligand is an antibody or antibody fragment or a small chemical molecule selected from
- the group consisting of IgA, IgG, IgD, IgE and IgM, particularly human IgG and IgM;
- the group consisting of scFv, dsFv, Fab', Fab, F(ab')2, Fv, single domain antibodies and diabodies; or, respectively,
- the group consisting of anticalin, affibody, cysteine-knot, protein scaffolds with an immunoglobulin fold, polypeptides, RNA aptamers and DNA aptamers.
9. The method according to any ofthe claims 1 to 7, wherein the ligand is displayed as a fusion protein with a phage protein derived from a protein selected from the group consisting of pill, pVIII and pVI.
10. The method according to any ofthe claims 1 to 7, wherein said target is a eukaryotic cell, in particular a mammalian cell, more particularly a cell selected from the group consisting of tissue sections, primary cells or cell-lines, in particular selected from the group consisting of cancer cells, nerve cells, stem cells, blood cells, platelet cells, adipocytes, liver cells, kidney cell, lung cell, endothelial cells, keratinocytes, melamoma cells, erythrocytes, osteoblasts, osteoclasts, chondrocytes, bone marrow cells smooth muscle cells, skeleton muscle cells and heart muscle cells.
11. The method according to any ofthe claims 1 to 7, wherein said ligand is capable of modulating, preferably inhibiting or activating, the biological function of said target.
12. The method according to any ofthe claims 1 to 7, wherein said biological function of said target is the ability to interact with an antigen derivable from a virus, a bacterium and/or a lower eukaryote.
13. The method according to any ofthe claims 1 to 7, wherein said biological function is a biological function selected from the group consisting of invasion, adhesion, proliferation, angiogenesis and cytotoxicity.
14. The method according to any ofthe claims 1 to 7, wherein said functional assay is selected from the group consisting of an invasion assay, an adhesion assay, a proliferation assay, an apoptosis assay, a cytotoxicity assay, angiogenesis assay, protein translocation assay, protein-protein interaction assay, migration assay, ion transport assay, colorimetric assay, gene activation assay and phenotypic assay.
15. The method according to any ofthe claims 1 to 7, wherein said surface antigen is a polypeptide, glycoprotein, peptide, proteoglycan, lipid or a glycolipid.
16. The method according to any ofthe claims 1 to 7, wherein the ALDU is selected from the group consisting of a) viruses useful for viral display, phages useful for phage display, bacteria useful for bacterial display, ribosomes useful for ribosome display and oligonucleotides useful for SELEX b) immune or naϊve libraries, in particular wherein said naϊ've library is a synthetic, semi-synthetic or natural library.
17. The method according to any ofthe claims 1 to 7, wherein the ALDUs are derived from viruses and phages, in particular from Cowpea Mosaic virus, retrovirus, lambda phage, T7 phage, T4 phage, and filamentous phages, more particularly derived from Ff phages.
18. The method according to any ofthe claims 1 to 7, wherein the ALDU or the ligand is chemically modified to increase its biological activity or endow the ALDU or the ligand with biological activity, particularly by addition of a chromophore.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP03763866A EP1592792A1 (en) | 2002-07-15 | 2003-07-15 | Proteomic screen to identify disease-related biological molecules and inhibitors thereto |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
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EP02015591A EP1382615A1 (en) | 2002-07-15 | 2002-07-15 | MCAM inhibitors |
EP02015591 | 2002-07-15 | ||
US41033102P | 2002-09-11 | 2002-09-11 | |
US410331P | 2002-09-11 | ||
EP03763866A EP1592792A1 (en) | 2002-07-15 | 2003-07-15 | Proteomic screen to identify disease-related biological molecules and inhibitors thereto |
PCT/EP2003/007660 WO2004007717A1 (en) | 2002-07-15 | 2003-07-15 | Proteomic screen to identify disease-related biological molecules and inhibitors thereto |
Publications (1)
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EP1592792A1 true EP1592792A1 (en) | 2005-11-09 |
Family
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Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
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EP02015591A Withdrawn EP1382615A1 (en) | 2002-07-15 | 2002-07-15 | MCAM inhibitors |
EP03763866A Withdrawn EP1592792A1 (en) | 2002-07-15 | 2003-07-15 | Proteomic screen to identify disease-related biological molecules and inhibitors thereto |
EP03763867A Withdrawn EP1601695A1 (en) | 2002-07-15 | 2003-07-15 | Mcam inhibitors |
Family Applications Before (1)
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EP02015591A Withdrawn EP1382615A1 (en) | 2002-07-15 | 2002-07-15 | MCAM inhibitors |
Family Applications After (1)
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EP03763867A Withdrawn EP1601695A1 (en) | 2002-07-15 | 2003-07-15 | Mcam inhibitors |
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EP (3) | EP1382615A1 (en) |
JP (1) | JP2006516085A (en) |
CN (1) | CN1675244A (en) |
AU (2) | AU2003250956A1 (en) |
CA (1) | CA2492559A1 (en) |
WO (2) | WO2004007717A1 (en) |
Families Citing this family (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7601351B1 (en) | 2002-06-26 | 2009-10-13 | Human Genome Sciences, Inc. | Antibodies against protective antigen |
WO2004003144A2 (en) | 2002-07-01 | 2004-01-08 | Human Genome Sciences, Inc. | Antibodies that specifically bind to reg iv |
GB0404936D0 (en) * | 2004-03-04 | 2004-04-07 | Univ London | Screen |
US20090053300A1 (en) * | 2006-02-23 | 2009-02-26 | Mitsubishi Tanabe Pharma Corporation | Monoclonal antibody, gene encoding the antibody, hybridoma, pharmaceutical composition, and diagnostic reagent |
USD564883S1 (en) * | 2006-03-21 | 2008-03-25 | Saverglass, Inc. | Bottle |
WO2009068630A1 (en) | 2007-11-27 | 2009-06-04 | Ablynx N.V. | Immunoglobulin constructs |
US8450278B2 (en) | 2008-10-15 | 2013-05-28 | Board Of Regents, The University Of Texas System | MUC18 targeting peptides |
EP2216399A1 (en) * | 2009-01-30 | 2010-08-11 | Université de la Méditerranée | Human soluble CD146, preparation and uses thereof |
US20130096070A1 (en) * | 2009-04-07 | 2013-04-18 | University Of Kansas | Peptide targeting of inner ear cells |
US8293468B2 (en) | 2009-06-10 | 2012-10-23 | Centre Hospitalier De L'université De Montréal | MCAM modulation and uses thereof |
US8628979B2 (en) * | 2009-10-21 | 2014-01-14 | Pronota N.V. | MCAM as a biomarker for fluid homeostasis |
US9220733B2 (en) | 2012-03-19 | 2015-12-29 | The Regents Of The University Of California | Solubilization of antigen components for removal from tissues |
TWI595007B (en) | 2012-09-10 | 2017-08-11 | Neotope Biosciences Ltd | Anti-mcam antibodies and associated methods of use |
JP2016519081A (en) | 2013-03-14 | 2016-06-30 | バイエル・ヘルスケア・エルエルシーBayer HealthCare LLC | Monoclonal antibody against antithrombin β conjugated with heparin |
WO2015136472A1 (en) | 2014-03-12 | 2015-09-17 | Prothena Biosciences Limited | Anti-laminin4 antibodies specific for lg4-5 |
KR20160127825A (en) * | 2014-03-12 | 2016-11-04 | 프로테나 바이오사이언시즈 리미티드 | Anti-mcam antibodies and associated methods of use |
TW201623331A (en) * | 2014-03-12 | 2016-07-01 | 普羅帝納生物科學公司 | Anti-MCAM antibodies and associated methods of use |
CN104710513A (en) * | 2015-04-06 | 2015-06-17 | 苏州普罗达生物科技有限公司 | Melanoma cell adhesion molecule antagonist polypeptide and application thereof |
CN104725489A (en) * | 2015-04-06 | 2015-06-24 | 苏州普罗达生物科技有限公司 | Melanoma cell adhesion molecule antagonist polypeptide and application thereof |
CN104725483A (en) * | 2015-04-06 | 2015-06-24 | 苏州普罗达生物科技有限公司 | Melanoma cell adhesion molecule antagonist polypeptide and application thereof |
US11230591B2 (en) * | 2016-04-20 | 2022-01-25 | Merck Sharp & Dohme Corp. | CMV neutralizing antigen binding proteins |
WO2018183747A1 (en) * | 2017-03-31 | 2018-10-04 | The Regents Of The University Of California | Shotgun proteomic antigen identification |
WO2018220467A1 (en) | 2017-05-30 | 2018-12-06 | Foroogh Nejatollahi | Anti-muc18 human immunotoxin and applications thereof |
Family Cites Families (2)
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WO2000058501A2 (en) * | 1999-03-26 | 2000-10-05 | The Wistar Institute | Novel genetic suppressor elements and methods of making the same |
WO2001077153A2 (en) * | 2000-04-10 | 2001-10-18 | The Curators Of The University Of Missouri | Phage display selection of anti fungal peptides |
-
2002
- 2002-07-15 EP EP02015591A patent/EP1382615A1/en not_active Withdrawn
-
2003
- 2003-07-15 JP JP2004520644A patent/JP2006516085A/en active Pending
- 2003-07-15 EP EP03763866A patent/EP1592792A1/en not_active Withdrawn
- 2003-07-15 EP EP03763867A patent/EP1601695A1/en not_active Withdrawn
- 2003-07-15 CN CNA038191571A patent/CN1675244A/en active Pending
- 2003-07-15 WO PCT/EP2003/007660 patent/WO2004007717A1/en not_active Application Discontinuation
- 2003-07-15 WO PCT/EP2003/007661 patent/WO2004007550A1/en not_active Application Discontinuation
- 2003-07-15 AU AU2003250956A patent/AU2003250956A1/en not_active Abandoned
- 2003-07-15 AU AU2003250065A patent/AU2003250065A1/en not_active Abandoned
- 2003-07-15 CA CA002492559A patent/CA2492559A1/en not_active Abandoned
Non-Patent Citations (2)
Title |
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See also references of WO2004007717A1 * |
SIEGEL ROBERT ET AL: "Mass spectral analysis of a protein complex using single-chain antibodies selected on a peptide target: Applications to functional genomics", JOURNAL OF MOLECULAR BIOLOGY, vol. 302, no. 2, 15 September 2000 (2000-09-15), pages 285 - 293, XP004465905, DOI: doi:10.1006/jmbi.2000.4070 * |
Also Published As
Publication number | Publication date |
---|---|
CN1675244A (en) | 2005-09-28 |
WO2004007717A8 (en) | 2005-05-12 |
WO2004007550A1 (en) | 2004-01-22 |
JP2006516085A (en) | 2006-06-22 |
EP1382615A1 (en) | 2004-01-21 |
AU2003250956A1 (en) | 2004-02-02 |
WO2004007717A1 (en) | 2004-01-22 |
CA2492559A1 (en) | 2004-01-22 |
EP1601695A1 (en) | 2005-12-07 |
AU2003250065A1 (en) | 2004-02-02 |
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