WO2012175201A1 - Procédés pour la caractérisation d'anticorps - Google Patents

Procédés pour la caractérisation d'anticorps Download PDF

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Publication number
WO2012175201A1
WO2012175201A1 PCT/EP2012/002606 EP2012002606W WO2012175201A1 WO 2012175201 A1 WO2012175201 A1 WO 2012175201A1 EP 2012002606 W EP2012002606 W EP 2012002606W WO 2012175201 A1 WO2012175201 A1 WO 2012175201A1
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antibody
antigen
immobilized
complexes
complex
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PCT/EP2012/002606
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English (en)
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Gerard Drewes
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Cellzome Ag
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to methods for the characterization of antibodies.
  • Antibody-antigen interactions can be described thermodynamically and kinetically by the affinity or the equilibrium dissociation constant (KD), the association rate constant (ko n ), or dissociation rate constant (kotr). This information is critical for lead selection and evaluation of an appropriate animal species for the conduct of efficacy and safety studies. Additionally, biophysical techniques are used to evaluate the cross-reactivity to orthologous antigens from species other than human. Conventionally, ELISA assays or biophysical methods are used to generate this information. Typical biophysical methods comprise the BiacoreTM technology, kinetic exclusion assays (KinExATM) and fluorescence activated cell-sorting (FACS) (Tabrizi et al., 2009. Drug Discov. Today 14(5-6): 298-305).
  • KD equilibrium dissociation constant
  • ko n association rate constant
  • kotr dissociation rate constant
  • the in vitro characterization of antibodies is typically performed using enzyme-linked immunosorbent (ELISA) assays.
  • ELISA enzyme-linked immunosorbent
  • recombinant protein antigens are usually expressed in E. coli bacteria and tested in ELISA assays.
  • endogenous antigens from mammalian cells or tissues.
  • BiacoreTM surface plasmon resonance
  • SPR surface plasmon resonance
  • a titration experiment allows measurements of the KD of a monoclonal antibody to a cell surface receptor where a constant number of cells in solution is titrated with increasing concentrations of a given antibody and the solutions are allowed to reach equilibrium. A fluorescently labeled polyclonal antibody is then used for detection of cell-bound antibody.
  • FACS flow cytometry instrumentation measures the fluorescence of cells moving individually through an excitation laser, hence, FACS is a method to detect cell-bound antibody (Drake and Klakamp, 2007. J. Immunol. Methods 318(1 -2): 147- 152).
  • a particular antibody of interest For the characterization of a particular antibody of interest, methods would be advantageous which allow the determination of the antigen specificity of this antibody.
  • it can be intended to provide an antibody that binds to a particular antigen (such as a drug target) but which does not interact with a closely related antigen, which could lead to unwanted side effects.
  • a particular antigen such as a drug target
  • the specificity of antibodies is studied by a variety of methods including Western blotting, immunofluorescence, immunohistochemistry (IHC) on tissue microarrays (TMAs), reverse phase protein arrays (RPAs) and antibody arrays (Brennan et al., 2010. Nat. Rev. Cancer 10(9): 605-617).
  • TMAs Tissue microarrays
  • biomarkers e.g. antigens
  • TMAs are assembled by acquiring cylindrical cores (0.6-2.0 mm in diameter) from donor paraffin-embedded tissues and re- embedding them in a single recipient block. The resultant TMA block is then sectioned, and immunohistochemistry and other assays such as immunoblotting are carried out on individual sections (Kononen et al., 1998. Nat. Med. 4(7): 844-847).
  • RPPAs Reverse phase protein microarrays
  • Antibody arrays are produced by printing antibodies onto a solid surface that is analogous to a DNA microarray. Two categories of antibody microarray formats have been described, namely direct labeling single-capture antibody arrays and dual antibody (capture and readout antibody) sandwich arrays (Brennan et al., 2010. Nat. Rev. Cancer 10(9): 605-617).
  • Biosimilars also referred to as the follow-on protein products in the U.S.
  • biotech drugs that have been shown to have comparable quality, safety and efficacy to the original product.
  • biosimilars are larger and more complex molecules with associated structural heterogeneities as compared to their small molecule counterparts. The exact manner in which the numerous product quality attributes of a biosimilar impact the safety and efficacy of the product in the clinic is generally not known completely.
  • the present invention relates to a method for the characterization of an antibody, comprising the steps of: a) providing two aliquots of a cell preparation comprising each at least one cell containing at least one antigen, b) incubating one aliquot with a given second antibody, c) harvesting the cells of each aliquot, d) lysing the cells in order to obtain protein preparations, e) contacting the protein preparations with an immobilized first antibody under conditions allowing the formation of one or more different complexes between one of the antigens and the immobilized first antibody, and f) determining the amount of the complex or the complexes formed in each
  • the present invention relates to a method for the characterization of an antibody, comprising the steps of: a) providing a protein preparation containing at least one antigen, b) contacting the protein preparation with an immobilized first antibody and with a given second antibody under conditions allowing the formation of one or more different complexes between one of the antigens and the immobilized first antibody, and c) detecting the complex or the complexes formed in step b).
  • the present invention relates to a method for the characterization of an antibody, comprising the steps of: a) providing two aliquots of a protein preparation containing at least one antigen, b) contacting one aliquot with an immobilized first antibody under conditions allowing the formation of one or more different complexes between one of the antigens and the immobilized first antibody, c) contacting the other aliquot with an immobilized first antibody and with a given second antibody under conditions allowing the formation of one or more different complexes between one of the antigens and the immobilized first antibody, and d) determining the amount of the complex or the complexes formed in steps b) and c).
  • the methods of the present invention are suitable for the characterization of the binding of antibodies to their targets, i.e. antigens, and, therefore, enable the characterization of the antibodies.
  • the method according to the first aspect of the invention is considered as especially suitable in cases where for the specific binding of some antibodies the native three-dimensional conformation of the antigen may be critical. In this case it is envisaged that the incubation of intact cells with the antibody may give more reliable results compared to adding the antibody to cell lysates. This is also expected in cases where the antigen is located on a protein that forms a protein complex (e.g. a dimer of receptors such as formed by members of the EGF receptor family).
  • bispecific antibodies are characterized, e.g engineered antibodies which recognize two different antigens which may be located on different proteins but occur in close vicinity, e.g. in a protein complex.
  • the term "antibody” generally refers to any kind of immunoglobulin-derived structure with binding specificity to an antigen, including, but not limited to, a full length antibody, an antibody fragment (a fragment derived, physically or conceptually, from an antibody structure), a derivative of any of the foregoing, a chimeric molecule, a fusion of any of the foregoing with another polypeptide, or any alternative structure/composition.
  • the antibody of the invention may be any polypeptide which comprises at least one antigen binding fragment.
  • Antigen binding fragments consist of at least the variable domain of the heavy chain and the variable domain of the light chain, arranged in a manner that both domains together are able to bind to the specific antigen.
  • An antibody fragment contains at least one antigen binding fragment as defined above, and exhibits essentially the same function and specificity as the complete antibody of which the fragment is derived from.
  • the antibody of the present invention may be a monoclonal antibody, a polyclonal antibody (e.g. an antibody derived from a patient suffering from an autoimmune disease), a recombinant antibody, a chimeric antibody, or an artificial antibody comprising one or more protein domain(s) that can interact with a target antigen of interest.
  • Monoclonal antibodies are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell.
  • a chimeric antibody is an antibody in which at least one region of an immunoglobulin of one species is fused to another region of an immunoglobulin of another species by genetic engineering in order to reduce its immunogenecity.
  • murine V L and VH regions may be fused to the remaining part of a human immunoglobulin.
  • the antibody of the present invention may be an antibody domain (Fab etc.), a single chain antibody, or a biological receptor or receptor fusion protein.
  • artificial antibodies with antibody-like binding activities are adnectins and lipocalins (Chames et al., 2009. Br. J. Pharmacol. 157(2): 220-233).
  • the first antibody and/or the second antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, chimeric antibodies, and antibody fragments.
  • FDA (U.S. Food and Drug Administration)-approved therapeutic antibody fragments are Abciximab (ReoPro®, Centocor, Ranibizumab (Lucentis®, Genentech) and Certolizumab pegol (Cimzia®, UCB).
  • the term "antigen" refers to any biomolecule to which an antibody or a ligand can bind, including, but not limited to peptides, proteins, lipids, glycans, metabolites and nucleic acids.
  • the antigen of the invention can further be any kind of a compound.
  • the antigen is a mutated form of the antigen.
  • the term “immobilized” means that the antibody is bound, preferably covalenty bound to a solid or nanostructured support.
  • solid support relates to every undissolved support being able to immobilize the antibody on its surface.
  • the solid support may be selected from the group consisting of agarose, modified agarose, sepharose beads (e.g. NHS-activated sepharose), latex, synthetic polymer, cellulose, and ferro- or ferrimagnetic particles.
  • the antibody may be coupled to the solid support either covalently or non-covalently.
  • the antibody is covalently coupled to the solid support.
  • the matrixes can contain active groups such as NHS, Carbodiimide etc. to enable the coupling reaction with the antibody.
  • the antibody can be coupled to the solid support by direct coupling (e.g. using functional groups such as amino-, sulfhydryl-, carboxyl-, hydroxyl-, aldehyde-, and ketone groups) and by indirect coupling (e.g.
  • the linkage to the solid support material may involve cleavable and non- cleavable linkers.
  • the cleavage may be achieved by enzymatic cleavage or treatment with suitable chemical methods.
  • the linker may be a CMO alkylene group, which is optionally interrupted or terminated by one or more atoms or functional groups selected from the group consisting of S, O, NH, C(0)0, C(O), and C(0)NH and wherein the linker is optionally substituted with one or more substituents independently selected from the group consisting of halogen, OH, NH 2 , C(0)H, C(0)NH 2 , S0 3 H, N0 2 , and CN.
  • the term “drawC 1-10 alkylene” means an alkylene chain having 1 - 10 carbon atoms, e.g.
  • Kits for covalently immobilizing antibodies to a solid support are commercially available including, for example, the AminoLink® Immobilization Kit from Thermo Scientific Inc., IL, USA. Equally preferred, the antibody is non-covalently coupled to the solid support.
  • the antibody can also be immobilized to protein A or protein G.
  • Protein G and protein A have been immobilized to several different matrices resulting in an excellent means of isolating IgG and IgG subclasses from ascites, cell culture supernatants, and serum.
  • the antibody of the invention is immobilized to protein A or protein G beads, preferably to protein A or protein G sepharose beads (commercially available from, e.g., GE Healthcare UK Ltd.). Subsequently, the bead bound antibodies may be covalently crosslinked to the beads (Whiteaker et al., 2007. Anal. Biochem. 362(1): 44-54).
  • the immobilized antibody is coupled to a solid support with a coupling density which results in non- depleting conditions for the antigen ("non-depleting coupling density").
  • non-depleting coupling density defines the concentration of the immobilized antibody (with respect to the volume of the hydrated resin) which is insufficient for the depletion of the antigen from the cell lysate. That is, as exemplified in the experiments shown in Figures 5 and 6, the antibody of the invention is coupled to a solid support at a concentration which allows that only a fraction of the antigen that the immobilized antibody is exposed to (for example, the antigen which is present in the given protein preparation) is bound by the immobilized antibody, and is also present in non- bound form in the supernatant including, e.g., the non-bound fraction(s). (Bantscheff et al., 2007. Nat. Biotechnol. 25(9): 1035-1044 especially the online supplement; Sharma et al., 2009. Nat. Methods 6(10): 741-744).
  • Non-depleting conditions are generally all conditions in which the antigen is not completely bound by the immobilized antibody resulting in only a partial depletion from the supernatant (for example, from the cell or protein preparation which contains the given antigen).
  • non-depleting conditions of the invention are conditions in which the antigen is bound to less than 50 % to the immobilized antibody and is non-bound to at least 50 %, i.e. present in the supernatant including, for example, the protein preparation which contains the antigen.
  • Non-depleting conditions according to the present invention which have to be experimentally verified and established for each antibody independently (see Figures 5 and 6), are favorable for elucidating the binding affinity and/or the dissociation constant KD of a particular antibody of interest for its antigen (Yamamoto et al., 2006. Anal. Biochem. 352(1): 15-23; Bantscheff et al., 2007. Nat. Biotechnol. 25(9): 1035-1044 especially the online supplement; Sharma et al., 2009. Nat. Methods 6(10): 741-744).
  • the protein preparation is contacted with the immobilized antibody under conditions allowing the formation of one or more different complexes between one of the antigens and the immobilized antibody.
  • the term "a complex between one of the antigens and the immobilized antibody” denotes a complex where the immobilized antibody interacts with at least one of the antigens which are present in the protein preparation by non-covalent binding.
  • the binding between the immobilized antibody and the antigen is, e.g., via salt bridges, hydrogen bonds, hydrophobic interactions or a combination thereof.
  • the term "under conditions allowing the formation of one or more different complexes” includes all conditions under which such formation, preferably such binding is possible. This includes the possibility of having the solid support on an immobilized phase and pouring the lysate onto it. In another preferred embodiment, it is also included that the solid support is in a particulate form and mixed with the cell lysate. Such conditions are known to the person skilled in the art.
  • the antigen containing protein preparation is first incubated with the second antibody and then contacted with the immobilized first antibody.
  • the simultaneous incubation of the second antibody and the immobilized first antibody with the antigen containing protein preparation is equally preferred (coincubation).
  • the antigen is preferably first incubated with the second antibody for 10 to 120 minutes, more preferred for 30 to 45 minutes at a temperature of 4 °C to 37 °C, even more preferred at 4 °C to 25 °C, and most preferred at 4 °C.
  • antibodies are used at concentrations ranging from 1 pM to 10 ⁇ , preferably from 100 pM to 1 ⁇ , preferably from 1 nM to 100 nM.
  • the second step, contacting with the immobilized first antibody, is preferably performed for 10 to 120 minutes at 4 °C, preferably for 60 minutes.
  • the antigen is preferably simultaneously incubated with the second antibody and the immobilized first antibody of the invention for 30 to 120 minutes, more preferred for 60 to 120 minutes at a temperature of 4 °C to 37 °C, even more preferred at 4 °C to 25 °C, and most preferred at 4 °C.
  • antibodies are used at concentrations ranging from 1 pM to 10 ⁇ , preferably from 100 pM to 1 ⁇ , preferably from 1 nM to 100 nM.
  • steps a) to c) of the third aspect of the invention may be performed with several protein preparations in order to test different antibodies. This embodiment is especially interesting in the context of medium or high throughput screenings.
  • the methods of the invention are performed as a medium or high throughput screening.
  • control samples may be needed to control for nonspecific binding, for example by using isotype-matched antibodies that do not bind to the antigen of interest.
  • Isotype controls need to be matched to the antibody of interest (species and isotype, including heavy and light chains) in order to accurately determine the level of specific binding by the free antibody.
  • the most common monoclonal antibody isotypes are IgGl , IgG2a, IgG2b, IgG3, IgM, and IgA.
  • the methods of the present invention can be performed with any protein preparation as a starting material, as long as the respective antigen is solubilized in the preparation.
  • Examples include a liquid mixture of several proteins, such as a tissue lysate, a cell lysate or a partial cell lysate which contains not all proteins present in the original tissue or cell, or a combination of several tissue or cell lysates.
  • protein preparation also includes dissolved purified protein.
  • Cell lysates or partial cell lysates can e.g. be obtained by isolating cell organelles (e.g. nucleus, mitochondria, ribosomes, golgi etc.) first and then preparing protein preparations derived from these organelles. Methods for the isolation of cell organelles are known in the art (Chapter 4.2 Purification of Organelles from Mammalian Cells in "Current Protocols in Protein Science", Editors: John.E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, ISBN: 0-471 -14098-8).
  • cell organelles e.g. nucleus, mitochondria, ribosomes, golgi etc.
  • Lysis of different cell types and tissues can be achieved by homogenizers (e.g. Potter-homogenizer), ultrasonic desintegrators, enzymatic lysis, detergents (e.g. NP-40, Triton® X-100, CHAPS, SDS), osmotic shock, repeated freezing and thawing, or a combination of these methods.
  • homogenizers e.g. Potter-homogenizer
  • ultrasonic desintegrators e.g. Potter-homogenizer
  • enzymatic lysis e.g. NP-40, Triton® X-100, CHAPS, SDS
  • detergents e.g. NP-40, Triton® X-100, CHAPS, SDS
  • osmotic shock repeated freezing and thawing, or a combination of these methods.
  • the lysis is performed simultaneously.
  • the cell is first harvested and then separately lysed.
  • protein preparations can be prepared by fractionation of cell extracts thereby enriching specific types of proteins such as cytoplasmic or membrane proteins (Chapter 4.3 Subcellular Fractionation of Tissue Culture Cells in "Current Protocols in Protein Science", Editors: John.E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, ISBN: 0-471-14098-8).
  • the steps of the formation of said complex or complexes are performed under essentially physiological conditions.
  • the physical state of proteins within cells is described in Petty, 1998 (Howard R. Petty, Chapter 1, Unit 1.5 in: Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada (eds.) Current Protocols in Cell Biology Copyright ⁇ 2003 John Wiley & Sons, Inc. All rights reserved. DOI: 10.1002/0471 143030.cb0101s00Online Posting Date: May, 2001 , Print Publication Date: October, 1998).
  • Essentially physiological conditions are inter alia those conditions which are present in the original, unprocessed sample material. They include the physiological protein concentration, pH, salt concentration, buffer capacity and post-translational modifications of the proteins involved.
  • the term "essentially physiological conditions” does not require conditions identical to those in the original living organism, wherefrom the sample is derived, but essentially cell-like conditions or conditions close to cellular conditions.
  • the person skilled in the art will, of course, realize that certain constraints may arise due to the experimental set-up which will eventually lead to less cell-like conditions. For example, the eventually necessary disruption of cell walls or cell membranes when taking and processing a sample from a living organism may require conditions which are not identical to the physiological conditions found in the organism.
  • essentially physiological conditions relates to conditions close to physiological conditions, as e. g. found in natural cells, but does not necessarily require that these conditions are identical.
  • "essentially physiological conditions” may comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20-37°C, and 0.001-10 mM divalent cation (e.g. Mg++, Ca++,); more preferably about 150 m NaCl or KC1, pH7.2 to 7.6, 5 mM divalent cation and often include 0.01-1.0 percent non-specific protein (e.g. BSA).
  • a non-ionic detergent can often be present, usually at about 0.001 to 2 %, typically 0.05-0.2 % (volume/volume).
  • buffered aequous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non-ionic detergents.
  • "essentially physiological conditions" mean a pH of from 6.5 to 7.5, preferably from 7.0 to 7.5, and / or a buffer concentration of from 10 to 50 mM, preferably from 25 to 50 mM, and / or a concentration of monovalent salts (e.g. Na or K) of from 120 to 170 mM, preferably 150 mM.
  • Divalent salts e.g. Mg or Ca
  • the buffer is selected from the group consisting of Tris-HCl or HEPES.
  • washing steps may be necessary. Such washing is part of the knowledge of the person skilled in the art.
  • the washing serves to remove non-bound components of the cell lysate from the solid support. Nonspecific (e.g. simple ionic) binding interactions can be minimized by adding low levels of detergent or by moderate adjustments to salt concentrations in the wash buffer.
  • the amount of the complex is determined by separating the antigen from the immobilized antibody and subsequent detection of the separated antigen or subsequent determination of the amount of the separated antigen, in particular wherein the antigen is detected or the amount of the antigen is determined by mass spectrometry or immunodetection methods, preferably with an antibody directed against the antigen.
  • separating means every action which destroys the interactions between the antigen and the immobilized antibody. This includes in a preferred embodiment the elution of antigen from the immobilized antibody.
  • the elution can be achieved by using non-specific reagents as described in detail below (ionic strength, pH value, detergents).
  • ionic strength, pH value, detergents it can be tested whether a compound of interest can specifically elute the antigen from the immobilized antibody.
  • Such nonspecific methods for destroying the interaction are principally known in the art and depend on the nature of the antibody-antigen interaction. Principally, change of ionic strength, the pH value, the temperature or incubation with detergents are suitable methods to dissociate the target antigen from the immobilized antibody.
  • the application of an elution buffer can dissociate binding partners by extremes of pH value (high or low pH; e.g. lowering pH by using 0.1 M citrate, pH 2-3), change of ionic strength (e.g.
  • the solid support has preferably to be separated from the released material.
  • the individual methods for this depend on the nature of the solid support and are known in the art. If the support material is contained within a column the released material can be collected as column flowthrough. In case the support material is mixed with the lysate components (so called batch procedure) an additional separation step such as gentle centrifugation may be necessary and the released material is collected as supernatant.
  • magnetic beads can be used as solid support so that the beads can be eliminated from the sample by using a magnetic device.
  • the amount of the complex or complexes formed in step c) is compared to the amount formed in step b).
  • a reduced amount of the complex formed in the aliquot incubated with the second antibody in comparison to the aliquot not incubated with the second antibody indicates that said antigen is a target of the second antibody.
  • the mass spectrometry analysis is performed in a quantitative manner, for example by stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification.
  • mass tags can be introduced into proteins or peptides metabolically, by chemical means, enzymatically, or provided by spiked synthetic peptide standards (Bantscheff et al., 2007; Anal. Bioanal. Chem. 389(4): 1017-1031).
  • the stable isotope is introduced into proteins by metabolic labeling during cell growth and division, for example by the stable isotope labeling by amino acids in cell culture (SILAC) approach (Ong et al., 2002; Mol. Cell. Proteomics. 1(5): 376-386).
  • SILAC amino acids in cell culture
  • the mass spectrometry analysis is performed in a quantitative manner, for example by using iTRAQ technology (isobaric tags for relative and absolute quatification) or cICAT (cleavable isotope-coded affinity tags) (Wu et al., 2006. J. Proteome Res. 5: 651- 658).
  • iTRAQ technology isobaric tags for relative and absolute quatification
  • cICAT cleavable isotope-coded affinity tags
  • TMT isobaric tagging reagent can be used.
  • the TMT reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides in up to six different biological samples enabling simultaneous identification and quantitation of peptides.
  • the samples are analyzed with a nano-flow liquid chromatography system coupled online to a tandem mass spectrometer (LC-MS/MS) experiment followed by reporter ion quantitation in the MS/MS spectra (Ross et al., 2004. Mol. Cell. Proteomics 3(12): 1 154-1 169; Dayon et al., 2008.
  • the characterization by mass spectrometry is performed by the identification of proteotypic peptides of the antigen.
  • the idea is that the antigen is digested with proteases and the resulting peptides are determined by MS.
  • proteotypic peptide As a result, peptide frequencies for peptides from the same source protein differ by a great degree, the most frequently observed peptides that "typically" contribute to the identification of this protein being termed "proteotypic peptide”. Therefore, a proteotypic peptide as used in the present invention is an experimentally well observable peptide that uniquely identifies a specific protein or protein isoform.
  • the characterization is performed by comparing the proteotypic peptides obtained in the course of practicing the methods of the invention with known proteotypic peptides. Since, when using fragments prepared by protease digestion for the identification of a protein in MS, usually the same proteotypic peptides are observed for a given target, it is possible to compare the proteotypic peptides obtained for a given sample with the proteotypic peptides already known for said target and thereby identifying the target being present in the sample.
  • the eluted antigen can be detected or its amount can be determined by using a specific antibody directed against said antigen.
  • Suitable antibody-based assays include but are not limited to Western blots, ELISA assays, sandwich ELISA assays and antibody arrays or any combination thereof.
  • the establishment of such assays is known in the art (Chapter 1 1, Immunology, pages 1 1-1 to 1 1-30 in: Short Protocols in Molecular Biology. Fourth Edition, Edited by F.M. Ausubel et al., Wiley, New York, 1999).
  • a “free antibody” according to the invention generally means an antibody which is not immobilized to a solid support, i.e. a non-immobilized antibody.
  • the detection of the formed complex or complexes, or the determination of the amount of the formed complex or complexes is used for determining the binding affinity, preferably the dissociation constant KD of the second non-immobilized antibody for its antigen.
  • the determination of the affinity of the antibody requires that the amount of the second, non-immobilized antibody is varied, resulting in the generation of a concentration- response curve from which the affinity of the antibody can be derived.
  • Such methods are known in the art (Cheng and Prusoff, 1973. Biochem. Pharmacol. 22(23): 3099-4108; Bantscheff et al., 2007. Nat. Biotechnol. 25(9): 1035-1044 especially the online supplement; Sharma et al., 2009. Nat. Methods 6(10): 741-744).
  • the detection of the formed complex or complexes, or the determination of the amount of the formed complex or complexes is used for analyzing the binding specificity of the second antibody for its antigen.
  • the specifity of two anti-EGFR antibodies was assessed by the methods of the invention.
  • the detection of the formed complex or complexes, or the determination of the amount of the formed complex or complexes is used for analyzing the species selectivity or cross-reactivity of the second antibody.
  • cross-reactivity also referred to as cross-immunity or cross-protective immunity generally means the reaction between an antibody and an antigen that differs from the immunogen.
  • Cross-reactivity is also a commonly evaluated parameter for the validation of immune and protein binding based assays such as, for example, ELISA. In this case it is normally quantified by comparing the assays response to a range of similar analytes and expressed as a percentage.
  • calibration curves are produced using fixed concentration ranges for a selection of related compounds and the mid-points (IC50) of the calibration curves are calculated and compared.
  • the first antibody and the second antibody are the same.
  • “the same” means that both antibodies have the same complementary determining regions (CDRs).
  • the first antibody and the second antibody are different, i.e. have different CDRs.
  • the methods of the invention are repeated with the requirement that the first and second antibody is exchanged. This enables the cross-profiling of the antibodies. Accordingly, in a preferred embodiment of the methods of the present invention, the first antibody and the second antibody are raised against the same antigen, preferably the same epitope. According to another preferred embodiment of the present invention, the antigen is identified.
  • This embodiment of the intention is particularly relevant if the antigen is unknown.
  • cancer cells are used for the screening of antibodies or antibody conjugates based on functional properties without knowing the antigen (such as, e.g., the Fusogenics approach; see Cizeau et al., 201 1.
  • Fusogenics a recombinant immunotoxin- based screening platform to select internalizing tumor-specific antibody fragments. J. Biomol. Screen. 16(1): 90-100). Fusogenics can potentially isolate antibodies against any target, known or novel, as long it is recognized by the patients ' immune system. Not knowing the identity of the target antigen could represent a challenge in drug development, for example for the design of toxicology studies.
  • immunoprecipitation with the antibody and subsequent identification of the antigen by mass spectrometry is used (Chahal et al., 2006. Biochem. Biophys. Res. Commun. 348(3): 1055-1062).
  • the antigen is identified by mass spectrometry.
  • peptide sequences of the captured protein can be identified by mass spectrometry which leads to the identification of the antigen.
  • the epitope is identified.
  • epitopope identification also referred to as “epitope mapping” means the systematic identification and characterization of the minimum recognition domain for antibodies (Brennan et al., 2010. Nat. Rev. Cancer 10(9): 605-617).
  • epitope generally refers to any distinct molecular surface features of an antigen capable of being bound by an antibody.
  • Antigenic molecules normally being “large” biological polymers, usually present several surface features that can act as points of interaction for specific antibodies. Any such, distinct molecular feature may constitute an epitope. Therefore, most antigens have the potential to be bound by several distinct antibodies, each of which is specific to a particular epitope.
  • the methods of the present invention as described above require the use of a first and a second antibody. In the context of the present invention, it is equally possible that only one antibody is used in combination with a compound capable or potentially capable of interacting with the antigen. For example, the antibody may be immobilized and the other compound is used as a soluble competitor.
  • this enables to determine whether and to which extend the compound is capable of interacting with the antigen.
  • the compound may be immobilized and the antibody is not immobilized and is used for competition with the immobilized compound for the binding to the antigen.
  • said compound is selected from the group consisting of synthetic or naturally occurring chemical compounds or organic synthetic drugs, more preferably small molecules, organic drugs or natural small molecule compounds.
  • said compound is identified starting from a library containing such compounds. Then, in the course of the present invention, such a library is screened.
  • a "library” according to the present invention relates to a (mostly large) collection of (numerous) different chemical entities that are provided in a sorted manner that enables both a fast functional analysis (screening) of the different individual entities, and at the same time provide for a rapid identification of the individual entities that form the library. Examples are collections of tubes or wells or spots on surfaces that contain chemical compounds that can be added into reactions with one or more defined potentially interacting partners in a high-throughput fashion.
  • the method of the invention according to the first aspect encompasses as initial steps a provision of two aliquots of a cell preparation comprising each at least one cell containing the antigen and incubating one aliquot with a given second antibody.
  • the cell preparation used in the context of this first aspect of the invention may be any cell preparation containing intact cells.
  • said cell preparation is derived from a cell culture system.
  • One aliquot of the cells of the cell culture system is then incubated with the given second antibody.
  • Methods for the incubation of cell culture system with antibodies are known in the art.
  • the cell preparation is obtained from an organism as e.g. isolated blood cells or a tissue.
  • the cell preparation may be obtained from an organism by biopsy, e.g. a tumour biopsy.
  • cell or tissue will be influenced by the purpose of the study. If the mode of action for a given antibody needs to be identified, then cells or tissues will be selected in which the desired therapeutic effect occurs (e.g. breast cancer tissue for antibodies to be used for cancer treatment). By contrast, for the elucidation of antigens mediating unwanted side effects the cell or tissue will be analysed in which the side effect is observed (e.g. skin tissue).
  • Figure 1 Amino acid sequence of human EGF receptor (IPI00018274.1 ; SEQ ID NO: 1). Peptides identified by mass spectrometry are underlined (Panitumumab experiment X013380).
  • Figure 2 Amino acid sequence of human EGF receptor (IPI00018274.1 ; SEQ ID NO: 2). Peptides identified by mass spectrometry are underlined (Cetuximab experiment X013381).
  • Figure 5 Optimization of coupling density for the anti-EGFR monoclonal antibody Panitumumab (Vectibix®, Amgen, USA).
  • Lane 1 Molecular weight marker. Lane 2: Cell lysate control (50 ⁇ g human placenta protein) without beads. Lanes 3-5: Eluate from beads with various antibody coupling densities. Lanes 6-8: Non-bound fraction from beads with various antibody coupling densities (0.2 mg/ml, 1 mg/ml, and 5 mg/ml, respectively).
  • Lane 1 Molecular weight marker. Lane 2: Cell lysate control (50 ⁇ g human placenta protein) without beads. Lanes 3-5: Eluate from beads with various antibody coupling densities. Lanes 6-8: Non-bound fraction from beads with various antibody coupling densities (0.04 mg/ml, 0.008 mg/ml, and 0.0016 mg/ml, respectively).
  • Proteins from the eluate and from the non-bound fractions were separated on a 4-12 % SDS-polyacrylamide gel (SDS-PAGE), blotted onto a PVDF membrane and subsequently visualized by Western blot analysis using an anti-EGFR detection antibody.
  • SDS-PAGE SDS-polyacrylamide gel
  • Lane 1 Molecular weight marker. Lane 2: Cell lysate control (50 ⁇ g human placenta protein) without beads. Lanes 3-5: Eluate from beads with various antibody coupling densities. Lanes 6-8: Non-bound fraction from beads with various antibody coupling densities (0.2 mg/ml, 1 mg ml, and 5 mg/ml, respectively).
  • Lane 1 Molecular weight marker. Lane 2: Cell lysate control (50 ⁇ g human placenta protein) without beads. Lanes 3-5: Eluate from beads with various antibody coupling densities. Lanes 6-8: Non-bound fraction from beads with various antibody coupling densities (0.04 mg/ml, 0.008 mg/ml, and 0.0016 mg/ml, respectively).
  • Proteins from the eluate and from the non-bound fractions were separated on a 12 % SDS- polyacrylamide gel (SDS-PAGE), blotted onto a PVDF membrane and subsequently visualized by Western blot analysis using an anti-EGFR detection antibody.
  • SDS-PAGE SDS- polyacrylamide gel
  • Figure 7 Binding specificity of Panitumumab (Vectibix®) and Cetuximab (Erbitux®).
  • Figure 8 Cross-competition analysis of Panitumumab (Vectibix®) and Cetuximab (Erbitux®).
  • Lane 1 Molecular weight marker.
  • Lane 2 Cell lysate control (25 ⁇ g protein of human placenta cell lysate) without beads.
  • Lanes 3-7 Eluate from beads where samples had been treated with different concentrations of the free antibody (30 nM, 15 nM, 7.5 nM, 3.75 nM, and 1.875 nM, respectively).
  • Lane 8 Eluate from control beads where the sample had been treated with buffer (PBS/10 % glycerol).
  • Lane 1 Molecular weight marker.
  • Lane 2 Cell lysate control (25 ⁇ g protein of human placenta cell lysate) without beads.
  • Lanes 3-7 Eluate from beads where samples had been treated with different concentrations of the free antibody (30 nM, 15 nM, 7.5 nM, 3.75 nM, and 1.875 nM, respectively).
  • Lane 8 Eluate from control beads where the sample had been treated with buffer (PBS/10 % glycerol).
  • Example 1 Selectivity profiling of anti-EGF receptor antibodies in cell lysate Principle of the assay
  • This example illustrates a competition binding assay in cell lysate to establish concentration-response curves for anti-EGF receptor (anti-EGFR) antibodies.
  • the free anti-EGFR antibody was added at defined concentrations (30 nM, 15 nM, 7.5 nM, 3.75 nM, 1.875 nM) to placenta cell lysate samples, thereby allowing the antibody to bind to the proteins in the lysate.
  • the lysate was subsequently contacted with the same immobilized anti-EGFR antibody (affinity matrix) to capture remaining free EGFR and other target proteins.
  • the beads with captured proteins were separated from the lysate and then bead-bound proteins were eluted in SDS sample buffer and subsequently separated by SDS- polyacrylamide gel electrophoresis.
  • the gel was stained with colloidal Coomassie and stained areas of each gel lane were cut out and subjected to in-gel proteolytic digestion with trypsin.
  • the peptide extracts corresponding to samples treated with different concentrations of the free anti-EGFR antibody (30 nM, 15 nM, 7.5 nM, 3.75 nM, 1.875 nM) and the solvent control (PBS, 10 % glycerol) were coupled with different variants of the isobaric TMT tagging reagents.
  • the TMT reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides in up to six different biological samples enabling simultaneous identification and quantitation of peptides.
  • the combined samples were fractionated using reversed- phase chromatography at pH 1 1 , and fractions were subsequently analyzed with a nano- flow liquid chromatography system coupled online to a tandem mass spectrometer (LC- MS/MS) experiment followed by reporter ion quantification in the MS/MS spectra (Ross et al., 2004. Mol. Cell. Proteomics 3(12): 1 154-1 169; Dayon et al., 2008. Anal. Chem.
  • the residual binding was plotted against the concentration of free anti-EGFR antibody and curve fitting was performed using the Xlfit program (ID Business Solutions Ltd.) as previously described (Bantscheff et al., 2007. Nature Biotechnology 25: 1035-1044).
  • the IC 50 value corresponds to the concentration of the free anti-EGFR antibody at which the residual binding is 50 % compared to the solvent control (PBS, 10 % glycerol).
  • the EGF receptor was quantified by mass spectrometry and the identified peptides are shown in Figures 1 and 2 for illustration. As a measure for binding affinity, concentration- response curves and IC 50 values for Panitumumab and Cetuximab are shown in Figures 3 and 4, respectively.
  • Figures 5 and 6 illustrate how the coupling density for the antibodies was optimized to establish non-depleting conditions.
  • the binding specificity of the two antibodies for the EGF receptor and other proteins is displayed in Figure 7.
  • Figure 7B shows the proteins captured from human placenta lysate by immobilized Panitumumab and competed ( ⁇ 50 % residual binding) by 30 nM free Panitumumab. The identity of these proteins is documented in Table 1.
  • Figure 7C displays the proteins captured from human placenta lysate by immobilized Cetuximab and competed ( ⁇ 50 % residual binding) by 30 nM free Cetuximab. The identity of these proteins is revealed in Table 2.
  • Table 1 Proteins captured from human placenta lysate by immobilized Panitumumab and competed ( ⁇ 50 % residual binding) by 30 nM free Panitumumab.
  • the antibodies Prior to immobilization the antibodies were dialyzed against phosphate-buffered-saline PBS/10% glycerol at 4°C using 3,500 MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, 66330). After 2 hours, the dialysis buffer was changed and the antibodies were dialyzed overnight. Dialyzed antibodies were transferred into fresh siliconized microfuge tubes and stored at 4°C. The protein concentration was determined by a Bradford assay (BioRad, 500-0006).
  • Antibodies were covalently coupled to activated beaded agarose through primary amines.
  • the AminoLink® Plus Coupling Reaction (Thermo Scientific Inc., Rockford, IL 61 105, USA) involves spontaneous formation of Schiff base bonds between aldehydes (on the support) and amines (on the antibody) and their subsequent stabilization by incubation with a mild reductant (sodium cyanoborohydride).
  • the AminoLink® resin was washed three times with 10 bead volumes of PBS and subsequently the dialyzed antibody solution was added to the resin at a ratio of 1 : 1 in a 1.5 ml siliconized microfuge tube.
  • 1M NaCNBH3 (Thermo Scientific Inc., 44892) was prepared freshly in 0.01M NaOH (prepared from 1M NaOH, Merck, 109137) and 25 ⁇ were added per 1 ml reaction volume. The mixture was incubated overnight at 4°C rotating (NeoLab Rotator, 2-1 175). A small amount of the supernatant was kept to determine the coupling efficiency by a Bradford Assay, the rest was discarded.
  • the beads were washed two times with 10 bead volumes of 1 M Tris pH 7.4 (Sigma-Aldrich, S5150). 1 M Tris pH 7.4 was added to the beads at a ratio of 1 : 1 , 25 ⁇ freshly prepared NaCNBH3 per 1 ml reaction volume was added and incubated rotating for 30 minutes at room temperature. The supernatant was discarded and the beads were washed three times with 10 bead volumes of 1 M NaCl (prepared from 5 M NaCl, Sigma-Aldrich, S5150). For storage, the beads with the immobilized antibodies were washed three times with 10 bead volumes of PBS and stored in PBS/0.05% sodium azide (Sigma-Aldrich, SA71289) at 4°C.
  • the tissue was cut into small pieces on ice and homogenized using a Polytron PT1200 in 5 volumes lysis buffer (50 mM Tris-HCl, 5 % glycerol, 150 mM NaCl, 1.5 mM MgCl 2 , 25 mM NaF, 1 mM sodium vanadate, 1 mM DTT, pH 7.5).
  • lysis buffer 50 mM Tris-HCl, 5 % glycerol, 150 mM NaCl, 1.5 mM MgCl 2 , 25 mM NaF, 1 mM sodium vanadate, 1 mM DTT, pH 7.5.
  • One complete EDTA-free tablet prote inhibitor cocktail, Roche Diagnostics, 1 873 580
  • Polytron-homogenisation NP40 detergent was added to a final concentration of 0.8 % and for lysis the suspension was transferred into a precooled Wheaton Dounce tissue grinder.
  • the material was dounced 10 times using a mechanized POTTER S, transferred to 50 ml falcon tubes, incubated for 30 minutes rotating at 4 °C and spun down for 10 minutes at 20,000 x g at 4°C (10,000 rpm in Sorvall RC5C plus, pre-cooled). The supernatant was transferred to an ultracentrifuge (UZ)-polycarbonate tube (Beckmann, 355654) and spun for 1 hour at 100.000 x g at 4 °C (33.500 rpm in Beckmann LE80K, precooled).
  • UZ ultracentrifuge
  • the supernatant was transferred again to a fresh 50 ml falcon tube, the protein concentration was determined by a Bradford assay (BioRad) and samples containing 50 mg of protein per aliquot were prepared. The samples were immediately used for experiments or frozen in liquid nitrogen and stored frozen at -80 °C.
  • the cell lysate was thawn, 1 : 1 diluted with with lysis buffer not containing DTT and NP40 and further diluted to 5 mg/ml protein concentration with lysis buffer containing 0.4 % NP40 (no DTT).
  • the lysate was transferred to an ultracentrifuge tube (Beckmann, 355654) and spun for 20 minutes at 121.400 x g at 4 °C (33.500 rpm in ⁇ 50.2, pre-cooled). The supernatant was transferred into a fresh falcon tube. Meanwhile a 200 x fold stock solution of the 5 desired antibody concentrations were prepared by diluting the dialyzed antibody in PBS/10 % glycerol.
  • Beads were collected by centrifugation at 2000 rpm for 2 minutes, a small amount of the non-bound fraction was kept and the remaining supernatant was discarded.
  • the beads were transferred to Mobicol-columns (MoBiTech, 10055) with 600 ⁇ 1 lysis buffer (no DTT) and washed with 10 ml lysis buffer containing 0.4 % NP40 detergent, followed by 10 ml lysis buffer containing 0.2 % detergent.
  • 50 ⁇ 2 x SDS sample buffer was added to the column. The column was incubated for 10 minutes at -95 °C and the eluate was transferred to a siliconized microfuge tube by centrifugation.
  • Proteins were then reduced with 50 mM DTT and afterwards alkylated with 108 mM iodoacetamide. Proteins were then separated by SDS- Polyacrylamide electrophoresis (SDS-PAGE). 5. Protein identification and quantitation by mass spectrometry 5.1 Protein digestion prior to mass spectrometric analysis
  • Gel-separated proteins were digested in-gel essentially following a previously described procedure (Shevchenko et al., 1996, Anal. Chem. 68: 850-858). In brief, gel-separated proteins were excised from the gel using a clean scalpel, destained twice using 100 ⁇ of 5 mM triethylammonium bicarbonate buffer (TEAB; Sigma T7408) and 40 % ethanol in water and dehydrated with absolute ethanol. Proteins were subsequently digested in-gel with porcine trypsin (Promega) at a protease concentration of 10 ng/ ⁇ in 5 mM TEAB.
  • porcine trypsin Promega
  • the peptide extracts corresponding to the different aliquots treated with different concentrations of the anti-EGFR antibody were labeled with variants of the isobaric tagging reagent as shown in Table 4 (TMT sixplex Label Reagent Set, part number 90066, Thermo Fisher Scientific Inc., Rockford, IL 61 105 USA).
  • the TMT reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides on amino groups in up to six different biological samples enabling simultaneous identification and quantification of peptides.
  • the TMT reagents were used according to instructions provided by the manufacturer.
  • the samples were resuspended in 10 ⁇ of 50 mM TEAB solution, pH 8.5 and 10 ⁇ acetonitrile were added.
  • the TMT reagent was dissolved in acetonitrile to a final concentration of 24 mM and 10 ⁇ of reagent solution were added to the sample.
  • the labeling reaction was performed at room temperature for one hour on a horizontal shaker and stopped by adding 5 ⁇ of 100 mM TEAB and 100 mM glycine in water.
  • the labeled samples were then combined, dried in a vacuum centrifuge and resuspended in 60 % 200 mM TEAB / 40 % acetonitrile.
  • Peptide samples were injected into a capillary LC system (CapLC, Waters) and separated using a reversed phase CI 8 column (X-Bridge 1 mm x 150 mm, Waters, USA). Gradient elution was performed at a flow-rate of 50 ⁇ 7 ⁇ .
  • Solvent A 20 mM ammoniumformiate, pH 1 1
  • solvent B 20 mM ammoniumformiate, pH 1 1, 60 % acetonitrile and 1 min fractions were automatically collected throughout the separation range (Micro-fraction collector, Sunchrom, Germany) and pooled to yield a total of 16 peptide fractions.
  • LTQ-Orbitrap XL and Orbitrap Velos instruments were operated with XCalibur 2.0/2.1 software. Intact peptides were detected in the Orbitrap at 30.000 resolution. Internal calibration was performed using the ion signal from (Si(CH 3 ) 2 0) 6 H + at m/z 445.120025 (Olsen et al., 2005. Mol. Cell Proteomics 4:2010-2021). Data dependent tandem mass spectra were generated for up to six peptide precursors using a combined CID/HCD approach (Kocher et al., 2009. J. Proteome Res. 8: 4743-4752). For CID up to 5000 ions (Orbitrap XL) or up to 3000 ions (Orbitrap Velos) were accumulated in the ion trap within a maximum ion accumulation time of 200 msec.
  • MascotTM 2.0 (Matrix Science) was used for protein identification using 10 ppm mass tolerance for peptide precursors and 0.8 Da (CID) tolerance for fragment ions. Carbamidomethylation of cysteine residues and iTRAQ TMT modification of lysine residues were set as fixed modifications and S,T,Y phosphorylation, methionine oxidation, N-terminal acetylation of proteins and iTRAQ/TMT modification of peptide N-termini were set as variable modifications.
  • the search data base consisted of a customized version of the IPI protein sequence database combined with a decoy version of this database created using a script supplied by Matrix Science (Elias et al., 2005. Nat.
  • Centroided iTRAQ/TMT reporter ion signals were computed by the XCalibur software operating and extracted from MS data files using customized scripts. Only peptides unique for identified proteins were used for relative protein quantification. Further spectra used for quantification were filtered according to the following criteria: Mascot ion score > 15, signal to background ratio of the precursor ion > 4, s2i > 0.5 (Savitski et al., 2010. J. Am. Soc. Mass Spectrom. 21(10): 1668-79). Reporter ion intensities were multiplied with the ion accumulation time yielding an area value proportional to the number of reporter ions present in the mass analyzer.
  • Panitumumab (Vectibix®, Amgen, USA) was coupled at 5.0 mg/ml, 1.0 mg/ml and 0.2 mg/ml density ( Figure 5A), and at 0.04 mg/ml, 0.008 mg/ml and 0.0016 mg/ml density ( Figure 5B). Coupling densities of 5.0 mg/ml, 1.0 mg/ml, 0.2 mg/ml and 0.04 mg/ml resulted in target depletion (little or no EGFR detected in the non-bound fraction). By contrast, low coupling densities of 0.008 mg/ml and 0.0016 mg/ml did not capture enough EGFR for reliable quantification in the eluate.
  • a coupling density of 0.02 mg/ml was selected as suitable for in lysate competition experiments to determine IC 50 values as shown in Figure 3.
  • Cetuximab Erbitux®, Merck KGaA, Darmstadt, Germany
  • Figure 6A was coupled at 5.0 mg/ml, 1.0 mg/ml and 0.2 mg/ml density
  • Figure 6B Coupling densities of 5.0 mg/ml, 1.0 mg/ml, and 0.2 mg/ml resulted in target depletion (little or no EGFR detected in the non-bound fraction).
  • proteins were captured from 1 mg cell lysate using 40 ⁇ AminoLink® resin with immobilized antibody.
  • the AminoLink® coupling was performed as described in section 2, but the dialyzed antibody solution was added to the resin at the ratio of 1 :2 in a 0.5 ml microfuge tube.
  • the lysate was diluted as described in section 4, but neither ultracentrifuged nor preincubated with free antibody.
  • the lysate was incubated with the immobilized antibody for 2.5 hours on an end-over-end shaker at 4 °C.
  • the beads were then washed two times with 3 ml lysis buffer (no DTT) and once with 3 ml lysis buffer without DTT and NP40.
  • the elution and reduction of proteins were performed as described in section 4.
  • Western Blot analysis at least half of the eluate, 25 ⁇ g to 50 ⁇ g lysate and an equal amount of the non-bound fraction were separated by SDS-PAGE.
  • the proteins were blotted onto an Immobilon FL PVDF membrane (Millipore, IPFL, 00010) using the semi dry method.
  • the membrane was dried on filter paper, rehydrated in methanol (Merck, 1.06018), rinsed with PBS and incubated with Odyssey blocking buffer (Licor, 927-40000) for 1 hour at room temperature.
  • the blocking buffer was discarded and the membrane was incubated with 15 ml primary antibody solution (15 ml odyssey blocking buffer, 0.1 % Tween® 20 (Sigma, Aldrich, PI 379) and appropriate amount of the primary anti-EGFR antibody) either 1 hour at room temperature or overnight at 4 °C.
  • the antibody solution was discarded and the membrane was washed three times for 10 minutes with PBS/0.1 % Tween® 20.
  • the membrane was then incubated with 15 ml secondary antibody solution (15 ml odyssey blocking buffer, 0.1 % Tween20, 0.02 % SDS (BioRad, 161-0418) and 1.5 ⁇ secondary antibody) for 1 hour at room temperature. Finally, the membrane was washed three times for 10 minutes with PBS/0.1 % Tween® 20 and rinsed with PBS prior to scanning.
  • Table 7 Sources and dilutions of antibodies used for detection
  • the 5x-DP buffer was filtered through a 0.22 ⁇ filter and stored in 40 ml-aliquots at -80 °C.
  • Stock solutions were obtained from the following suppliers: 1.0 M Tris/HCl pH 7.5 (Sigma, T-2663), 87 % Glycerol (Merck, catalogue number 04091.2500); 1.0 M MgCl 2 (Sigma, M-1028); 5.0 M NaCl (Sigma, S-5150).
  • This example illustrates a cross-competition binding assay of two different anti-EGF receptor (anti-EGFR) antibodies, Panitumumab and Cetuximab.
  • Anti-EGFR anti-EGF receptor
  • Cetuximab was added at defined concentrations as free antibody to human placentacell lysate samples and subsequently contacted with immobilized Panitumumab.
  • Panitumumab was added as free antibody to the cell lysate samples and subsequently contacted with immobilized Cetuximab.
  • the free anti-EGFR antibody was added at defined concentrations (30 nM, 15 nM, 7.5 nM, 3.75 nM, 1.875 nM) to placenta cell lysate samples thereby allowing the antibody to bind to the proteins in the lysate.
  • buffer PBS, 10 % glycerol
  • the lysate samples were contacted with the other immobilized anti-EGFR antibody (affinity matrix) to capture remaining free EGFR and other target proteins.
  • the beads with captured proteins were separated from the lysate and then bead-bound proteins were eluted in SDS sample buffer and subsequently separated by SDS-Polyacrylamide gel electrophoresis and analysed by Western blot analysis as described in Example 1.
  • Figure 8 A shows that added free Cetuximab at a concentration of 15 nM and 30 nM effectively prevented capturing of the EGF receptor by immobilized Panitumumab.
  • Figure 8B shows that added free Panitumumab at a concentration of 15 nM and 30 nM effectively prevented capturing of the EGF receptor by immobilized Cetuximab.
  • Example 3 Profiling of an anti-CD20 antibody in cell lysate Principle of the assay
  • This example illustrates a competition binding assay in cell lysate to establish a concentration-response curve for the anti-CD20 antibody Rituximab (MabThera®, Roche, Switzerland).
  • CD20 is a B lymphocyte specific integral membrane protein.
  • the free Rituximab was added at defined concentrations (100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM) to Ramos cell lysate samples, thereby allowing the antibody to bind to the proteins in the lysate.
  • the lysate was subsequently contacted with the immobilized Rituximab antibody (affinity matrix) to capture remaining free CD20 and other target proteins.
  • a suitable coupling density for Rituximab was determined at 2.5 mg/ml according to the methods as described in example 1.
  • Ramos cells (a human B cell line, ATCC number CRL-1596) were either obtained from an external supplier (CIL SA, Mons, Belgium) or grown in one litre Spinner flasks (Integra Biosciences, #182101) in suspension in RPMI 1640 medium (Invitrogen, #21875-034) supplemented with 10% Fetal Bovine Serum (Invitrogen, #10270-106) at a density between 0.2 x 10 6 and 1.0 x 10 6 cells/ml. Cells were harvested by centrifugation, washed once with 1 x PBS buffer (Invitrogen, #14190-094) and cell pellets were frozen in liquid nitrogen and subsequently stored at -80°C.
  • Ramos cells were lysed in buffer containing 1% of the detergent CHAPSO without DTT. Cells were homogenized in a Potter S homogenizer in lysis buffer: 50 mM Tris-HCl, 1% CHAPSO, 5% glycerol, 150 mM NaCl, 1.5 mM MgCl 2 , 25 mM NaF, 1 mM sodium vanadate, pH 7.5.
  • lysis buffer 50 mM Tris-HCl, 1% CHAPSO, 5% glycerol, 150 mM NaCl, 1.5 mM MgCl 2 , 25 mM NaF, 1 mM sodium vanadate, pH 7.5.
  • One complete EDTA-free tablet prote inhibitor cocktail, Roche Diagnostics, 1 873 580
  • the material was dounced 20 times using a mechanized POTTER S, transferred to 50 ml falcon tubes, incubated for 30 minutes rotating at 4° C and spun down for 10 minutes at 20,000 x g at 4°C (10,000 rpm in Sorvall SLA600, precooled). The supernatant was transferred to an ultracentrifuge (UZ)- polycarbonate tube (Beckmann, 355654) and spun for 1 hour at 145.000 x g at 4°C (40.000 rpm in ⁇ 50.2, precooled). The supernatant was transferred again to a fresh 50 ml falcon tube, the protein concentration was determined by a Bradford assay (BioRad) and samples containing 50 mg of protein per aliquot were prepared. The samples were immediately used for experiments or frozen in liquid nitrogen and stored frozen at -80°C. Competition binding assays and protein identification and quantification by mass spectrometry were performed as described in Example 1.
  • the CD20 antigen was quantified by mass spectrometry and as a measure for the binding affinity of free Rituximab to CD20, a concentration-response curve and IC50 value is shown in Figure 9.

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Abstract

Cette invention concerne des procédés pour la caractérisation d'anticorps faisant appel à un anticorps immobilisé.
PCT/EP2012/002606 2011-06-20 2012-06-20 Procédés pour la caractérisation d'anticorps WO2012175201A1 (fr)

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