WO2023149801A1 - Method for preparing a conformation-specific antibody. - Google Patents

Abstract

The invention provides a method for preparing a conformation-specific antibody against an oligomeric protein in a specific oligomeric state, the method comprising the steps of a) providing an oligomeric protein and its protein subunits under an in vitro condition that causes further oligomerization of said protein oligomer in the presence of an oligomerization-modulating compound that alters the oligomerization or depolymerization rate of said protein oligomer or its oligomeric state relative to said in vitro condition wherein said oligomerization-modulating compound is absent, wherein said oligomerization-modulating compound is selected from a protein interacting with said oligomeric protein or its protein subunits, a nucleoside phosphate or an hydrolysis-resistant analogue thereof, or a (de)stabilizing small molecule; b) arresting the oligomerization of said protein oligomer by addition of a cross-linking agent to thereby provide said protein oligomer in a stabilized quaternary conformation and specific oligomeric state; c) using the protein oligomer in said stabilized quaternary conformation and specific oligomeric state of step (b) as a conformation-specific antigen of said protein oligomer to immunize a non-human mammal, optionally in combination with an adjuvant, to thereby obtain antibodies against said conformation-specific antigen; d) selecting an antibody obtained in step (c) that binds to the said conformation-specific antigen but not to said protein subunits, to thereby obtain a conformation-specific antibody against said oligomeric protein in a specific oligomeric state.

Description

Title: Method for preparing a conformation-specific antibody.

FIELD OF THE INVENTION

The invention is in the field of antibody generation, more particularly the generation of an antibody that specifically binds to oligomeric proteins. The antibody produced by a method of the invention is capable of distinguishing between protein monomers and oligomers composed of those monomers. The antibody obtained by the method of the invention can be used for research towards fundamental mechanisms of the cell, as well as for understanding clinically relevant processes.

BACKGROUND OF THE INVENTION

Antibodies are proteins made by cells of the immune system (B cells) that recognize unique amino acid sequence epitopes on target proteins. Antibodies can be produced in vast quantities and labelled directly (for example with fluorescent or radioactive labels), or they can be recognized by labelled secondary antibodies. Antibodies/secondary antibodies are widely used in research, diagnostics and therapeutics to detect specific proteins in cells. For example, the intracellular localization and tissue distribution of proteins can be examined in fixed tissue sections using antibodies against the protein of interest and a fluorescence microscope-based assay.

Virtually all antibodies to date are made against a single protein, or a protein domain. In oligomers of proteins such as microtubules (MTs), specific protein conformations can occur. The microtubule (MT) network is part of the cytoskeleton of cells, and is important for the maintenance of cell shape and structure, as well as for the correct execution of many dynamic processes, including mitosis. Many antibodies have been made against tubulin, the building block of MTs, and these have been used to describe the MT network in fixed cells and tissues. However, these antibodies are not able to distinguish between tubulin and MTs, leading to noisy signals in microscopy images.

Perturbation of dynamic MT behaviour underlies the action of a number of successful anti-cancer drugs, including paclitaxel, or taxol, a compound that binds to, and stabilizes MTs, and which alters MT conformation. Classic anti-tubulin antibodies recognize MTs because the same epitope is present on soluble tubulin and on MTs. But MT-specific conformations, also present in normal MTs, or specific conformations of taxane-MTs, are not recognized. Hence, classic anti-tubulin antibodies cannot distinguish between taxol-decorated and normal MTs and they cannot be used to examine taxane penetrance in a body or tissue. Thus, the prior art anti-tubulin antibodies are not able to recognize specific conformations present in MTs but not in tubulin, e.g. those of taxol- stabilized MTs. In fact, there is an almost complete absence of antibodies recognizing specific protein conformations, hampering basic and applied research into cellular structures, such as the MT network.

Despite decades of research and use of paclitaxel in the clinic, it is still unknown exactly how this highly successful anti-cancer drug exerts its effect in vivo because it is unknown which tissues/cells of the (human) body (and hence of a particular cancer) are targeted, and to what extent they are targeted. The possibility to generate antibodies against taxane-bound MTs would allow the generation of reagents that can reliably detect taxol- decorated MTs in (formalin-fixed) tissue samples. This would allow researchers to describe taxane distribution and penetration in human tissues at high resolution. Such tools are essential in better understanding the (side) effects of taxol and other related compounds (i.e. taxanes).

Only one monoclonal antibody (MB 11) has been described to date that recognizes a specific conformation of tubulin/MTs. This MB 11 antibody was prepared by screening a phage display library of recombinant scFv (single-chain fragment variable) proteins. A drawback of this antibody is that it does not recognize taxol-decorated MTs, and it does not work in formalin -fixed tissue samples.

There is a need in the art for a method for producing conformationspecific antibodies that specifically recognize conformations present in oligomeric structures. The resulting antibodies would allow for the development of therapies that relate to protein oligomer interactions and the advancement of research towards protein oligomers and their role in disease.

SUMMARY OF THE INVENTION

The present inventors have discovered a method by which conformation-specific antibodies can reliably be generated.

Therefore, the invention provides in a first aspect a method for preparing a conformation-specific antibody against a microtubule in a specific oligomeric state, the method comprising the steps of: a) providing tubulin protein subunits of a microtubule under an in vitro condition and effecting oligomerization of said tubulin protein subunits into a microtubule in the presence of an oligomerization-modulating compound that alters the oligomerization rate of microtubule or its oligomeric state relative to said in vitro condition wherein said oligomerization-modulating compound is absent, wherein said oligomerization-modulating compound is selected from a protein interacting with microtubule or tubulin, a nucleoside phosphate or an hydrolysis-resistant analogue thereof, or a (de)stabilizing small molecule; b) arresting the oligomerization of said microtubule in a specific oligomeric state by addition of a cross-linking agent to thereby provide said microtubule in a stabilized quaternary conformation and specific oligomeric state; c) using the microtubule in said stabilized quaternary conformation and specific oligomeric state of step (b) as a conformation-specific antigen of said microtubule to immunize a non-human mammal, optionally in combination with an adjuvant, to thereby obtain antibodies against said microtubule conformation-specific antigen; d) selecting an antibody obtained in step (c) that binds to said microtubule conformation-specific antigen but not to said tubulin protein subunits, to thereby obtain a conformation-specific antibody against said microtubule in a specific oligomeric state.

In a preferred embodiment of a method of the invention said oligomerization-modulating compound is selected from the group comprising GTP, dGTP, GDP, dGDP, ATP, dATP, ADP, dADP, OTP, dCTP, GDP, dCDP, TTP, dTTP, TDP, dTDP, UTP, dUTP, m⁵UTP, UDP, dUTP, m⁵UDP, GMPCPP, GTPyS, GTPaS, GpCpp, GppCp, GppNHp, paclitaxel, paclitaxel derivatives, docetaxel, cabazitaxel, tesetaxel, paclitaxel poliglumex, nab- paclitaxel, larotaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, ixabepilone, laulimalide, peloruside A, discodermolide, taccalonolide AF, taccalonolide AJ, taccalonolide A, taccalonolide E, taccalonolide Al-epoxide, zampanolide, sagopilone, dictyostatin, cyclostreptin, eleutherobin, sarcodictyin A, sarcodictyin B, patupilone, rhazinilam, XMAP215, CLASP-1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP170, doublecourtin, and other proteins that stabilize microtubules, or a protein comprising one or more mutation with respect to stabilizing proteins XMAP215, CLASP-1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP170 or doublecourtin, wherein said protein affects the oligomerization rate or conformational state of said microtubules. In a preferred embodiment, said oligomerization-modulating compound is GMPCPP.

The present invention provides in a broader aspect a method for preparing a conformation-specific antibody against an oligomeric protein in a specific oligomeric state, the method comprising the steps of: a) providing protein subunits of an oligomeric protein under an in vitro condition and effecting oligomerization of said protein subunits into a protein oligomer in the presence of an oligomerization-modulating compound that alters the oligomerization rate of said protein oligomer or its oligomeric state relative to said in vitro condition wherein said oligomerization-modulating compound is absent, wherein said oligomerization-modulating compound is selected from a protein interacting with said oligomeric protein or its protein subunits, a nucleoside phosphate or an hydrolysis-resistant analogue thereof, or a (de)stabilizing small molecule; b) arresting the oligomerization of said protein oligomer in a specific oligomeric state by addition of a cross-linking agent to thereby provide said protein oligomer in a stabilized quaternary conformation and specific oligomeric state; c) using the protein oligomer in said stabilized quaternary conformation and specific oligomeric state of step (b) as a conformation-specific antigen of said protein oligomer to immunize a non-human mammal, optionally in combination with an adjuvant, to thereby obtain antibodies against said conformation-specific antigen; d) selecting an antibody obtained in step (c) that binds to said conformationspecific antigen but not to said protein subunits, to thereby obtain a conformation-specific antibody against said oligomeric protein in a specific oligomeric state.

Preferably, in a method for preparing a conformation -specific antibody against an oligomeric protein in a specific oligomeric state, said oligomeric protein is a constituent of the cytoskeleton, most preferably a microtubule.

In preferred embodiments of methods for preparing conformationspecific antibodies as described above, the step c) comprises the steps of: i) injecting a non-human mammal with the conformation-specific antigen obtained in step b); ii) selecting a B cell clone from the serum of said non-human mammal that produces an antibody against said conformation-specific antigen; iii) optionally converting said B cell clone into a hybridoma; and iv) having said B cell clone or said hybridoma produce the conformationspecific antibody.

Preferably, step ii) further comprises the step of selecting a B cell clone that produces an antibody with a binding affinity indicated by a KD value of less than IO^-6 M.

In further preferred embodiments of the above methods for preparing conformation-specific antibodies according to the invention as described above, said conformation-specific antibody binds to an epitope of the protein oligomer with a binding affinity indicated by a KD value of less than IO ⁶ M, and does not bind to the protein subunits or binds to said subunits with a binding affinity indicated by a KD value of more than IO ⁶ M.

In other preferred embodiments of the above methods for preparing conformation-specific antibodies according to the invention as described above, the cross-linking agent is an aldehyde, preferably glutaraldehyde.

In still other preferred embodiments of the above methods for preparing conformation-specific antibodies according to the invention as described above, at least one protein subunit of the oligomeric protein (e.g. tubulin) comprises at least one amino acid sequence mutation relative to the native protein subunit that affects the quaternary conformation and specific oligomeric state of the oligomer.

In another aspect, the present invention provides an antibody obtained by a method for preparing a conformation-specific antibody according to the invention as described above. In yet another aspect, the present invention provides a pharmaceutical composition comprising the antibody obtained by a method for preparing a conformation-specific antibody according to the invention and at least one pharmaceutically acceptable adjuvant.

In yet another aspect, the present invention provides a diagnostic composition comprising the antibody obtained by a method for preparing a conformation-specific antibody according to the invention, preferably for use in the detection of a microtubule in cancer cells.

In yet another aspect, the present invention provides the use of a method for preparing a conformation-specific antibody according to the invention or an antibody obtained by such a method, for measuring taxane loading of microtubules in a tissue sample or body fluid sample of a subject, preferably a subject suffering from cancer and treated with said taxane.

In yet another aspect, the present invention provides a method of detecting or monitoring an ohgomerization-modulating event of a protein oligomer, preferably in vitro in a cell, the method comprising staining the protein oligomer by an antibody obtainable by the method of of the present invention as described above, and comparing the staining result with a control staining not subjected to or affected by the oligomerization- modulating event, preferably wherein the ohgomerization-modulating event is a mutation in a gene, or regulatory element thereof, affecting the expression or expression product, more preferably wherein said gene is the gene for proteins that stabilize microtubules, most preferably XMAP215, CLASP-1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP170 or doublecourtin.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows the result of ELISA-based detection of a conformation-specific antibody produced by methods according to the invention as described in Examples 1 and 2. Biotylinlated GmpCpp- stabilized MT seeds (generated in vitro) on Streptavidin-coated ELISA plates served as antigen. Mouse monoclonal beta tubulin antibody (Sigma, T8328, 1:500) was used as primary antibody and Rabbit Polyclonal antiMouse IgG-HRP (Dako, 1:5000) was used as secondary antibody allowing HRP based colorimetric readout. X-axis shows raw absorbance at 450 nm averaged from triplicate wells.

Figure 2 shows the result of ELISA screening of antisera from G- TUB-injected mice (#4 and #5) after 6 rounds of immunizations using serially diluted antisera as described in Example 3.

Figure 3 shows representative confocal laser scanning microscopy (CLSM) images of paraformaldehyde (PF A) fixed HELA cells stained with three hybridoma supernatants from G-TUB mice as described in Example 4. Clone 27G7: top left and top right; Clone 50C12: bottom left; Clone 15B8: bottom right. DAPI (blue) was used to stain nuclei. The appearance of plusend like structures at 1:10 dilution of clone 27G7 was apparent (zoomed inset in top right image). Images are Maximum Intensity Projections of z- stacks acquired at 500nm step -size on a Leica SP5 laser scanning confocal microscope. Scale bar = 10 micron.

Figure 4 shows the results of immunostaining of MTs in HeLa cells using conformation -specific antibody 27G7 and control anti-beta-tubilin antibody as described in Example 5. Figure 4A shows staining of PFA-fixed HeLa cells with 27G7 (panels A2-A4) or regular anti -beta-tubulin antibodies (panel Al; beta-tub). 27G7 was used undiluted, except in panel A4, where the antibodies were used 1:10 diluted. Asterisks indicate centrosomal MTs. Arrows indicate midzone MTs at the end of mitosis. Figure 4B shows staining of PF A- or MetOH-fixed HeLa cells with 27G7 (green fluorescence; panels Bl, B2, B4) or regular anti-tubulin antibodies (red fluorescence; panel B3; beta-tub). 27G7 was used undiluted. Cells were counter stained with DAPI to mark nuclei (blue fluorescence). Asterisk indicates centrosomal MTs. Figure 5 shows the results of experiments as described in Example 6. Figure 5A: Immunostaining of MTs in Wild type (WT) and Clasp2 knockout (KO) embryonic stem (ES) cell colonies with antibody 27G7. Each colony comprises about 6 ES cells. Positive MT staining is shown in black against a white background. Figure 5B: Fluorescence intensity of 27G7 MT staining in WT and Clasp2 KO ES cell colonies as quantified by image analysis as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “antibody”, as used herein, includes reference to an intact immunoglobulin, or to an antigen-binding fragment of an immunoglobulin. An antibody specifically binds to an antigen. The term “antibody”, as used herein, includes all classes and subclasses of antibodies. Preferably, the antibody is monoclonal - displaying a single binding specificity and affinity within a population of antibodies. However, it is possible that variants of monoclonal antibodies are present in minor amounts due to naturally occurring mutations. The term “antibody” applies to antibodies of animal origin, including for example human, murine, lama, cow, rat, rabbit, goat and horse, as well as to artificial antibodies, chimeric antibodies and recombinant antibodies.

The term “conformation-specific antibody”, as used herein, includes reference to an antibody that specifically binds to a conformational epitope of an oligomeric protein, while it lacks the ability to bind the individual monomers (or individual repeating subunits) of which the oligomeric protein is composed. Hence, preferably, the quaternary structure of an oligomeric protein is recognized by the conformation-specific antibody, but preferably not the tertiary structure of the subunit constituents of the oligomeric protein. Said specific binding may include binding to an oligomeric conformation wherein additional molecules are present that are not part of the oligomeric protein sequence or structure. The term “conformation-specific antibody” includes reference to an oligomer-specific antibody, i.e., and antibody (or fragment thereof) that binds to the oligomer with higher affinity than to the monomers (or oligomer subunits) of which the oligomer is composed.

The term “oligomeric protein”, also referred to as a “protein oligomer”, as used herein, includes reference to a group of two or more monomeric forms (or subunits) that together form a quaternary structure. The monomeric forms are grouped as at least one, preferably at least two, repeating unit(s). Homo-oligomeric proteins are protein complexes wherein all polypeptide chains are identical (the protein assembly comprises a single repeating unit); hetero-oligomeric proteins comprise at least two distinct polypeptide chains or repeating units. Oligomeric proteins may further comprise other molecules, such as coenzymes and/or associating proteins.

The term “monomer”, as used herein, includes reference to a single polypeptide chain that is able to assemble into (or that is the building block of) a protein oligomer, optionally and dependent on the monomer together with monomers of a different group.

The term “oligomerization”, as used herein, includes reference to the process of assembly of monomers, or repeating subunits, or monomeric forms of an oligomeric protein, into a protein oligomer of higher polymerization degree containing a higher number of monomers, repeating subunits, or monomeric forms. Oligomerization of monomers, repeating subunits, or monomeric forms of an oligomeric protein may require energy. A term that may be used as an equivalent to oligomerization in the context of oligomeric proteins is “polymerization”, whereas the opposed term of depolymerization is used herein.

The term “oligomerization-modulating compound”, as used herein, includes reference to a molecule that promotes and/or facilitates, or, by contrast, reduces or inhibits, the oligomerization of a protein oligomer. Oligomerization-modulating compounds may for example be small molecules or proteins. The term “ohgomerization-modulating compound” also includes reference to molecules that to a certain extent prevent or hamper the depolymerization or degradation of an oligomeric protein into smaller structures and/or monomers. Presence of an oligomerizationmodulating compound during protein oligomer formation preferably results in a protein oligomer having a conformation that differs from the protein oligomer when formed in the absence of the oligomerization-modulating compound.

Examples of oligomerization-modulating compounds in the context of protein oligomers as components of the cytoskeleton include nucleoside triphosphates and nucleoside diphosphates, e.g. guanosine triphosphate (GTP), deoxyguanosine triphosphate (dGTP), guanosine diphosphate (GDP), deoxy guanosine diphosphate (dGDP), adenosine triphosphate (ATP), deoxyadenosine triphosphate (dATP), adenosine diphosphate (ADP), deoxyadenosine diphosphate (dADP), cytidine triphosphate (OTP), deoxycytidine triphosphate (dCTP), cytidine diphosphate (GDP), deoxycytidine diphosphate (dCDP), thymidine triphosphate (TTP), deoxythymidine triphosphate (dTTP), thymidine diphosphate (TDP), deoxythymidine diphosphate (dTDP), uridine triphosphate (UTP), deoxyuridine triphosphate (dUTP), 5 -methyluridine triphosphate (m⁵UTP), uridine diphosphate (UDP), deoxyuridine diphosphate (dUDP) and 5- methyluridine diphosphate (m⁵UDP); non-hydrolyzable and slowly hydrolysable nucleotides, e.g. guanylyl-(alpha, beta)-methylene- diphosphonate (GMPCPP), guanosine 5'-O-[gamma-thio]triphosphate (GTPyS), guanosine 5'-O-[alpha-thio]triphosphate (GTPaS), GpCpp, GppCp and GppNHp; small molecule microtubule stabilizers, e.g. taxanes, including paclitaxel, paclitaxel derivatives (such as nab-paclitaxel and paclitaxel poliglumex), docetaxel, cabazitaxel, tesetaxel and larotaxel; or epothilones, including epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, or other small molecule microtubule stabilizers, including ixabepilone, laulimalide, peloruside A, discodermolide, taccalonolide AF, taccalonolide AJ, taccalonolide A, taccalonolide E, taccalonolide Al-epoxide, zampanolide, sagopilone, dictyostatin, cyclostreptin, eleutherobin, sarcodictyin A, sarcodictyin B, patupilone and rhazinilam; and protein microtubule stabilizers, e.g. XMAP215, CLASP- 1 and CLASP-2, MAP2, tau, MAP4, STOP, CLIP170 and doublecourtin.

The term “cross-linking agent”, as used herein, includes reference to a chemical that is able to covalently link two or more biomolecules. Examples of cross-linking agents are aldehyde fixatives, such as formaldehyde, glutaraldehyde and acrolein (also referred to as propenal), and oxidizing fixatives such as osmium tetroxide. In general, cross-linking agents can react with side chains of biomolecules such as DNA, proteins and lipids, thereby creating a covalent chemical bond between that biomolecule and another biomolecule that is in close vicinity of the former biomolecule at the time of the reaction. For example, proteins that are non-covalently bound to each other may be cross-linked when subjected to a cross-linking agent. During the process of cross-linking, the cross-linked molecules may form a matrix in which other molecules are trapped. In this manner, molecules that do not directly react with the cross-linking agent may still be fixated by addition of the cross-linking agent. Formaldehyde is mainly used for cross-linking proteins, as it is able to covalently link most prominently lysine residues, but also tyrosine, asparagine, tryptophan, histidine, arginine, cysteine and glutamine residues of a protein to the same or another protein via the formation of a methylene bridge. The terms “formalin” and “paraformaldehyde” may be used interchangeably with the term “formaldehyde”, although strictly speaking formalin refers to a 37 % w/v or 40% v/v solution of the water-soluble gas formaldehyde in water together with up to 15% v/v methanol, and paraformaldehyde refers to higher polymers of formaldehyde that are poorly soluble in water. Glutaraldehyde can cross-link molecules that possess at least one free amino group, such as proteins and some lipids. Glutaraldehyde may occur as a polymer and may cross-link biomolecules as such. Osmium tetroxide is a cross-linking agent that is soluble in both polar and non-polar media. It can cross-link unsaturated carbon bonds, such as may be present in phospholipids and lipoproteins. A cross-linking agent may also comprise multiple fixatives; the most widely used cross-linking agent mixture is a formaldehyde-glutaraldehyde mixture.

The term “binding”, as used herein, includes reference to the non- covalent interaction between two proteins; in particular the non-covalent specific interaction between an antigen and an antibody.

The terms “B cell” and “B lymphocyte”, as used herein, includes reference to a type of blood cell that is part of the mammalian immune system. B cells are derived from the bone marrow and/or spleen. B cells produce antibodies, and may secrete those antibodies in the form of plasma cells. The term “B cell clone”, as used herein, includes reference to a copy of a B cell that produces and/or secretes the same antibody and/or antibodies. B cell clones are derived from B cells during expansion.

The term “serum”, as used herein, includes reference to the fluid and solute component of mammalian blood. Of all components of blood, only cells and clotting factors are not included in serum. Serum may inter alia comprise proteins, such as antibodies, as well as nutrients, gases and hormones.

The term “hybridoma”, as used herein, includes reference to a cell from a cell line that results from the fusion of an antibody-producing B cell and an immortal B cell (a myeloma). Hybridomas are used for the production of antibodies at large scale, as they are able to be grown in culture. B cells are mortal and can therefore only produce a finite number of antibodies. Myeloma cells are immortal, but to not produce the antibody of interest. The fusion of both cell types results in a cell line of which the cells are able to produce while staying alive. Hybridomas can be cultured from a single hybridoma. The hybridomas that are produced, are genetically identical to the first hybridoma and will produce identical antibodies, which makes hybridoma technology suitable for the large-scale production of monoclonal antibodies. Hybridomas are produced by chemical fusion or electrofusion of myeloma cells that lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene with B cells that produce an antibody of interest. Suitable hybridomas are then selected by culturing the cells that remain after the fusion procedure in hypoxanthine-aminopterin- thymidine medium (HAT-medium). Unfused myelomas die in HAT-medium, and unfused B cells have a short life span and will therefore also die over the course of culturing. Only hybridomas will survive selective culturing. Suitable hybridomas, which produce the desired antibodies, may subsequently be selected by screening.

The term “epitope”, as used herein, includes reference to a part of an antigen that is recognized by antibodies and to which antibodies bind. The amino acid residues that make up an epitope may be contiguous or discontiguous. An epitope may be formed by a single polypeptide chain, but may also exist of multiple proteins, optionally together with other molecules such as sugars and/or coenzymes. A change in the surface structure of the epitope may alter binding affinity of the antibody that binds the epitope.

The term “monomeric form”, as used herein, refers to a repeating polypeptide subunit of an protein oligomer and includes reference to a polypeptide chain as a constituent of a protein oligomer, that is either incorporated or that is not incorporated into said protein oligomer (e.g. nonpolymerized, free, or isolated therefrom). Proteins in monomeric form may oligomerize into protein oligomers, together with protein monomers with the same and/or with a different amino acid sequence; other molecules may also be incorporated during oligomerization. Tubulin, a heterodimer of alpha and beta tubulin chains, is considered a monomeric form herein. Tubulin may assemble into microtubules, thereby establishing the oligomeric protein.

The term “cytoskeleton”, as used herein, includes reference to a complex comprising filament-forming polypeptide chains, which are responsible for the cellular architecture. The cytoskeleton gives a cell its shape and mechanical resistance, and may (partially) deform the cell. Intracellular transport, vesicle formation, cell division and motility are examples of cellular processes that are dependent on components of the cytoskeleton. Components of the cytoskeleton are present in bacterial, archaeal and eukaryotic cells. In eukaryotic cells, the three main kinds of filaments of the cytoskeleton are microtubules, microfilaments and intermediate filaments. These filaments are oligomeric proteins, generally with a fixed width and variable and dynamic length. Other proteins, such as motor proteins, associate with said filaments (except intermediate filaments) and can facilitate contraction of and transport along filaments. A number of coenzymes interact with components of the cytoskeleton; most notably, ATP, ADP, GTP and GDP are involved in oligomerization and stabilization of the microfilament (ATP /ADP) and microtubule cytoskeletal filaments (GTP/GDP). Other small molecules may interact with the cytoskeleton and may function as cytoskeletal drugs. Those drugs may for example stabilize or destabilize filaments, and/or stimulate or prevent filament oligomerization.

The term “microtubule”, as used herein, includes reference to one of the filaments of the eukaryotic cytoskeleton, which is a protein oligomer composed of a- and B-tubulin heterodimers that form hollow cylinders. Microtubules nucleate from microtubule organizing centers (MTOCs) by addition of a- and B-tubulin heterodimers. Tubulins can bind to GTP and oligomerize in a GTP-bound state. The GTP bound to a-tubulin is stable, but the GTP bound to B-tubulin is prone to hydrolyzation. Tubulin is added to an end of the microtubule in GTP-bound state, which functions as a stabilizing cap at the tip of the microtubule. Upon hydrolyzation of the 6- tubulin-bound GTP, leading to the formation of GDP and inorganic phosphate, the tubulin inside the microtubule becomes more prone to depolymerization. Said depolymerization will occur when the stabilizing GTP-cap disappears, for example in a situation wherein hydrolysis occurs at a faster rate than oligomerization, leading to rapid depolymerization of the microtubule, via an event known as catastrophe. The inverse process is called rescue and refers to the switch of microtubule depolymerization into microtubule polymerization. As this is an energetically unfavourable process, it not only requires GTP-bound tubulin to be present but also other conditions, for example the presence of microtubule-stabilizing factors. Several proteins and small molecules may contribute to the polymerization, stability, or depolymerization of microtubule. Stabilizing small molecules, including taxanes, are for example paclitaxel, paclitaxel derivatives, docetaxel, cabazitaxel, tesetaxel, paclitaxel poliglumex, nab-paclitaxel, larotaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, ixabepilone, laulimalide, peloruside A, discodermolide, taccalonolide AF, taccalonolide AJ, taccalonolide A, taccalonolide E, taccalonolide Al-epoxide, zampanolide, sagopilone, dictyostatin, cyclostreptin, eleutherobin, sarcodictyin A, sarcodictyin B, patupilone and rhazinilam; examples of stabilizing proteins are XMAP215, CLASP- 1 and -2, MAP2, tau, MAP4, STOP, CLIP 170, and doublecourtin. Destabilizing small molecules are for example nocodazole, colchicine, vinblastine, vincristine, vinorelbine, CA-4, phenstatin, isocombrestatin A, CC-5079, 3,3- diarylacrylonitriles, alkenyl diarylmethanes, lavendustin A, 2- methoxyestradiol, 2-(l’-propynyl)estradiol, ABT-751, BNC105, verubulin, hemiasterlin, HTL286, tubulysin D, PM050489, ABL274, ABI-231, BZML, N-(methylindodyl)aminoquinazoline 10, lb, 4a, indanocine, millepachine, DJ101, SSE 15206, IMB5046, D4-9-31; examples of destabilizing proteins are kinesin-13, stathmins and katanin. Examples of other microtubule- associated proteins are MAPla, MAPlb, ensconsin, EB2, EB3, pl50Glued, Dynamitin, Lisi, CLIP115, catastrophin and patronin. Besides GTP and GDP, GTP analogues may also bind to tubulin and be incorporated into microtubules. These GTP analogues include non-hydrolyzable GTP analogues, such as GMPCPP, GTPyS, GTPaS, GpCpp, GppCp and GppNHp. These molecules cannot be hydrolyzed or are hydrolyzed at a much slower rate than GTP, and will thus prevent or slow down the formation of tubulin- bound GDP. This results in a more stable and less catastrophe-prone microtubule. Any molecule associated with tubulin and/or microtubules may alter the conformation of microtubules. Several isotypes of tubulin exist, and these isotypes may have physiological relevance; for example, some isotypes are known to be involved in developmental stages of the brain. Examples of isotypes of a-tubulin are TUBA1A and TUBA8. Examples of isotypes of 6- tubulin are TUBB2B, TUBB3 and TUBB5. Mutations in tubulin and/or one of its isotypes may result in disease. Preferably, the microtubule as disclosed herein is a hollow tube that comprises tubulin dimer protofilaments. Preferably, such a microtubule comprises 10-17, preferably 12-15 (such as 13 or 14), protofilaments, whereby each individual protofilament has a length (longitudinally) of at least 2, preferably at least 3, tubulin dimers.

The term “aldehyde”, as used herein, includes reference to molecules comprising the functional group -OHO. Aldehydes may react with amino acid residues, most prominently lysine, to form a covalent chemical bond. Aldehydes possessing two or more functional groups may be able to covalently link two or more previously non-covalently linked polypeptide chains that are in close proximity of each other, thereby cross-linking the polypeptide chains.

The term “glutaraldehyde”, as used herein, includes reference to a chemical that can be used as a cross-linking agent. Glutaraldehyde comprises two aldehyde groups. The term “mutation”, as used herein, includes reference to an alteration in the DNA and/or RNA sequence, which may result in an alteration (mutation) in the amino acid sequence of a polypeptide chain with respect to an amino acid sequence that is considered wild type’ or native polypeptide or protein (subunit). Mutations may result in a different variants of a protein or peptide. Variants may be the result of one or more mutations. In some instances, protein variants may underlie one or more diseases.

The term “aqueous solution”, as used herein, includes reference to a solution wherein the primary solvent is water. An aqueous solution may further comprise other components such as salts, sugars, proteins, amino acids, nucleic acids, lipids, fatty acids and carbohydrates.

The term “adjuvant”, as used herein, includes reference to substances that may be combined with an antigenic substance in order to elicit a more enhanced immune response when administered conjointly with said antigenic substance. One or more adjuvants may be constituents of a vaccine or immunogenic composition. Examples of adjuvants include incomplete Freund’s adjuvant, aluminum hydroxide, modified muramyldipeptide, dimethyl dioctadecyl ammonium bromide, MF59, SAF, aluminum salts, calcium salts and liposomes.

The term “cancer cell”, as used herein, includes reference to cells of the animal body, including human, that display abnormal cell growth, which often is not controlled by regular cell growth signals. The term comprises benign and malignant cancer cells. Cancer cells may form solid tumors or non-solid tumors, which may be invasive or non-invasive. The term comprises all types of cancer cells, including cells belonging to germ cell tumors, blastomas, lymphomas, carcinomas, and sarcomas. The term may also be used to refer to cultured cancer cells or cells derived thereof that exhibit abnormal cell growth compared to equivalent cells in the healthy body. The term “taxane”, as used herein, includes reference to molecules belonging to a class of diterpenes, which were discovered in plants of the genus Taxus. Examples of taxanes are paclitaxel, docetaxel, cabazitaxel, tesetaxel and larotaxel. The term also includes reference to derivatives of taxanes, such as the paclitaxel derivatives nab -paclitaxel (nanoparticle albumin-bound paclitaxel), DHA-paclitaxel (paclitaxel linked to docosahexaenoic acid), and paclitaxel poliglumex (also known as PPX, CT- 2103 and Xyotax; a macromolecular conjugate of paclitaxel and poly-L- glutamic acid).

The present invention allows for the generation of antibodies against specific protein conformations, for example conformations in oligomers of a single protein, such as conformations present in the MT lattice, which are not present in the tubulin subunit. This possibility now opens a new field of research with basic and clinical importance, as it is now possible to distinguish the oligomer (e.g. MTs) from the subunit (i.e. tubulin). For instance, applications of the present invention include the possibility to generate antibodies against specific conformations of normal MTs, but also against taxane-bound MTs, which are known to differ from normal MT lattices, and against the end of MTs, which are conformation ally different from the MT normal lattice. The method of the present invention provides a significant improvement over the "MB 11" method described above because it does not rely on the presence of an antibody in a preexisting phage library. Instead, the expression of the new antibodies is induced in a living animal, allowing one to create, in principle, antibodies against any conformation. Using the methods of the present invention, conformation-specific antibodies may be generated to visualize the ends of microtubules in fixed tissue samples. Also, using the methods of the present invention, conformation-specific antibodies may be generated to visualize taxanes bound to microtubules as their binding changes conformation of the microtubules. The invention thus provides methods to generate "conformation-specific antibodies", that is, antibodies against specific protein conformations. These are not normally found in single proteins or in protein domains, which are at the base of normal antibodies.

Specific MT conformations may be produced by first polymerizing short MT segments in the presence of GMPCPP (a GTP analogue), followed by stabilizing (fixating) the conformation by the addition of a fixative, resulting in a GMPCPP -MT antigen. Conformation-specific anti-MT antibodies may then be made by injecting GMPCPP-MT antigen into mice. B cells (e.g. hybridomas) expressing conformation-specific anti-MT antibodies may then be isolated by a suitable ELISA assay and antibodyspecificity may be confirmed by immunofluorescence experiments, for instance in HeLa cells. The invention, as exemplified herein by the generation of antibodies against GMPCPP -bound MTs, is described herein as a general method, which method can be used generate a library of antibodies against specific MT conformations useful in studying dynamic MT behavior, for instance in response to many different drugs, and to generate antibodies against other oligomeric structures in cells. The inventors envisage that this general method is more widely applicable to other oligomeric proteins.

One embodiment of the invention relates to the use of mutant proteins. For instance, a mutation in tubulin is known that prevents GTP- hydrolysis of the MT lattice. By preparing tubulin subunits of this hydrolysis-resistant mutant tubulin and allowing the oligomerization of these tubulin subunits into MTs, an MT is generated with a conformation that resembles the MT end. Thus, in addition to using compounds such as GMPCPP to prepare conformation-specific antibodies, the use of mutations in proteins that alter oligomeric conformations are contemplated herein as a means for making conformation-specific antibodies. Methods of the invention may comprise the use of commercial tubulin preparations to generate MTs in vitro in the presence of, for instance GMPCPP, a slowly hydrolyzable GTP analogue. This will result in the generation of MTs with a GTP-like conformation, which differs from that of normal MTs (which are GDP-bound and have a GDP-conformation). Following the fixation of the GMPCPP-MTs (using e.g. glutaraldehyde) in order to preserve the specific GMPCPP-MT conformation, and the injection of the fixed GMPCPP-MTs into mice, B cells expressing promising antisera may be developed into hybridomas, which may in turn be examined for the expression of antibodies against GMPCPP-MTs using a suitable ELISA assay for screening of B cell clones, each producing a unique antibody. Positive clones may subsequently be validated using a immunofluorescence microscopy-based assay. Using this method, the present inventors were able to isolate several clones expressing antibodies that reveal novel MT conformations in paraformaldehyde-fixed HeLa cells.

Methods for preparing a conformation-specific antibody

A method for preparing a conformation-specific antibody against an oligomeric protein in a specific oligomeric state in accordance with the present invention comprises a first step of providing protein subunits of an oligomeric protein under an in vitro condition that promotes oligomerization of said protein subunits in the presence of an oligomerization-modulating compound that alters the oligomerization rate of said protein oligomer or its oligomeric state relative to said in vitro condition wherein said oligomerization-modulating compound is absent, wherein said oligomerization-modulating compound is selected from a protein interacting with said oligomeric protein or an hydrolysis-resistant analogue thereof, or a (de)stabilizing small molecule. Alteration of the oligomerization rate of said protein oligomer or alteration of the oligomeric state of said protein oligomer is defined as an alteration relative to said in vitro condition wherein said oligomerization-modulating compound is absent.

In an embodiment, said protein subunits of an oligomeric protein as used in a method of the invention may be any subunits of any oligomeric protein, for example of hetero-oligomeric proteins or of homo-oligomeric proteins. Preferably, all different subunits that are required for the polymerization of said oligomeric protein are provided.

In a preferred embodiment, said oligomeric protein of a method of the invention is a constituent of the cytoskeleton. This may be the cytoskeleton of any organism, including eukaryotes, bacteria and archaea. The cytoskeleton of eukaryotes may include microfilaments, intermediate filaments and microtubules. Microfilaments are oligomeric proteins that are mainly composed of actin subunits. Intermediate filaments are oligomeric proteins that can for example be composed of vimentins, acidic keratins, basic keratins, neurofilaments, lamin, desmin, glial fibrillary acidic protein, peripherin, alpha-internexin, synemin, syncoilin, filensin, phakinin and nestin. Microtubules are oligomeric proteins that are mainly composed of alpha-tubulin and beta-tubulin, together forming the tubulin dimer, the repeating subunit of microtubules. Additional proteins may be associated with said microfilaments, intermediate filaments and microtubules. The method of the invention may be used to prepare conformation-specific antibodies against conformations of the main components of cytoskeletal oligomers, as well as against conformations of main components and associated proteins or other molecules. The cytoskeleton of prokaryotes, such as bacteria and archaea, may comprise oligomeric proteins mainly composed of subunits such as FtsZ, MreB, ParM, SopA, archaeal actin, crescentin, MinCDE-system proteins, bactofilin and CfpA.

Preferably, the oligomeric protein of a method of the invention is a microtubule. Microtubules are mainly composed of tubulin heterodimers, that in turn consist of the monomers alpha-tubulin and beta-tubulin. Tubulins may be post-translationally modified, for example by detyrosination, acetylation, polyglutamylation, polyglycylation, phosphorylation, ubiquitinylation, sumoylation, palmitoylation and by the removal of the last two residues from the C-terminus of alpha-tubulin. Microtubules of any organism may be used; preferably mammalian microtubules; more preferably human, pig, or bovine microtubules.

In a method of the invention, subunits of an oligomeric protein are provided under an in vitro condition that promotes oligomerization. This condition depends on the oligomeric protein. An exemplary condition that promotes polymerization of bovine tubulin is a temperature of 37°C. In some embodiments, a temperature between 5°C and 75°C is selected as a condition that promotes oligomerization; more preferably, a temperature between 20°C and 60°C is selected; even more preferably, a temperature between 32°C and 42°C is selected; most preferably, a temperature around 37°C is selected. Another exemplary condition that promotes oligomerization of bovine tubulin is a pH of 6.8. In some embodiments, a pH between 4 and 10 is selected as a condition that promotes oligomerization; more preferably, a pH between 5 and 9 is selected; even more preferably, a pH between 6 and 8 is selected; most preferably, a pH around 6.8 is selected. Another exemplary condition that promotes oligomerization of tubulin is the presence of a chelating agent. In some embodiments, a chelating agent is selected from the group comprising EGTA, EDTA, BAPTA and BINAP as a condition that promotes oligomerization; preferably, EGTA is selected as a chelating agent. Another exemplary condition that promotes oligomerization of tubulin is the presence of a source of cations. In some embodiments, a source of cations is provided as a condition that promotes oligomerization; preferably, a source of divalent cations is provided; more preferably, a source of Mg²⁺ is provided. An exemplary buffer that can be used for providing a condition that promotes oligomerization of bovine tubulin is MRB80 buffer, comprising 80 mM PIPES, pH 6.8; 1 mM EGTA; and 4 mM MgCh. In some embodiments, a buffer comprising 80 mM PIPES, pH 6.8; 1 mM EGTA; and 4 mM MgCh is used to provide a condition that promotes oligomerization of protein subunits.

In a method of the invention, the step of providing protein subunits of an oligomeric protein under an in vitro condition that promotes oligomerization of said protein in the presence of an oligomerizationmodulating compound may include incubation of said protein subunits with said oligomerization-modulating compound. The incubation time is dependent on the combination of protein subunits and oligomerizationmodulating compound. An exemplary incubation time that promotes polymerization of bovine tubulin in the presence of GmpCpp is 40 minutes. In some embodiments, an incubation time of at least 10 seconds is selected; preferably, the incubation time is at least 1 minute; more preferably, the incubation time is at least 10 minutes; even more preferably, the incubation time is at least 30 minutes; most preferably, the incubation time is around 40 minutes.

In a method of the invention, the step of providing protein subunits of an oligomeric protein under an in vitro condition that promotes oligomerization of said protein requires the presence of an oligomerizationmodulating compound that alters the oligomerization rate of said protein oligomer or its oligomeric state. Said oligomerization-modulating compound is selected from: i) a protein interacting with said oligomeric protein or its protein subunits; ii) a nucleoside phosphate or a hydrolysis-resistant analogue thereof; or iii) a (de)stabilizing small molecule. In some embodiments, an oligomerization-modulating compound is selected from the group comprising GTP, dGTP, GDP, dGDP, ATP, dATP, ADP, dADP, OTP, dCTP, GDP, dCDP, TTP, dTTP, TDP, dTDP, UTP, dUTP, m⁵UTP, UDP, dUTP, m⁵UDP, GMPCPP, GTPyS, GTPaS, GpCpp, GppCp, GppNHp, paclitaxel, paclitaxel derivatives, docetaxel, cabazitaxel, tesetaxel, paclitaxel poliglumex, nab-paclitaxel, larotaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, ixabepilone, laulimalide, peloruside A, discodermolide, taccalonolide AF, taccalonolide AJ, taccalonolide A, taccalonolide E, taccalonolide Al-epoxide, zampanolide, sagopilone, dictyostatin, cyclostreptin, eleutherobin, sarcodictyin A, sarcodictyin B, patupilone, rhazinilam, XMAP215, CLASP- 1, CLASP-2, MAP2, tan, MAP4, STOP, CLIP 170 and doublecourtin. In some preferred embodiments, an oligomerization-modulating compound is selected from the group comprising GTP, dGTP, GDP, dGDP, ATP, dATP, ADP, dADP, CTP, dCTP, CDP, dCDP, TTP, dTTP, TDP, dTDP, UTP, dUTP, m5UTP, UDP, dUTP and m⁵UDP. In preferred embodiments, an oligomerizationmodulating compound is selected from the group comprising GMPCPP, GTPyS, GTPaS, GpCpp, GppCp and GppNHp. In preferred embodiments, an oligomerization-modulating compound is a taxane or taxane derivative; more preferably, an oligomerization-modulating compound selected from the group comprising paclitaxel, paclitaxel derivatives, docetaxel, cabazitaxel, tesetaxel, paclitaxel poliglumex, nab -paclitaxel and larotaxel. In other preferred embodiments, an oligomerization-modulating compound is an epothilone; more preferably, an oligomerization-modulating compound selected from the group comprising epothilone A, epothilone B, epothilone C, epothilone D, epothilone E and epothilone F. In other preferred embodiments, an oligomerization-modulating compound is selected from the group comprising ixabepilone, laulimalide, peloruside A, discodermolide, taccalonolide AF, taccalonolide AJ, taccalonolide A, taccalonolide E, taccalonolide Al-epoxide, zampanolide, sagopilone, dictyostatin, cyclostreptin, eleutherobin, sarcodictyin A, sarcodictyin B, patupilone and rhazinilam. In still other preferred embodiments, an oligomerizationmodulating compound is selected from the group comprising XMAP215, CLASP-1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP170 and doublecourtin. In other embodiments, any other protein that stabilizes microtubules may be selected as ohgomerization-modulating compound. In other embodiments, said oligomerization-modulating compound is a protein comprising one or more mutation with respect to stabilizing proteins XMAP215, CLASP- 1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP170 or doublecourtin, wherein said protein affects the oligomerization state in a similar manner as said stabilizing proteins. The term “in a similar manner” includes reference to “similarly”, and preferably refers to “in the same manner”.

In a preferred embodiment, said oligomerization-modulating compound as used in a method of the invention is GMPCPP.

In some embodiments, a combination of oligomerizationmodulating compounds is used. This combination may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 oligomerization-modulating compounds.

In a method of the invention, the step of providing protein subunits of an oligomeric protein under an in vitro condition that promotes oligomerization of said protein in the presence of an oligomerizationmodulating compound may include centrifugation, and/or may be followed by centrifugation. In a step of centrifugation, polymerized oligomeric proteins are forced to the bottom of the container wherein centrifugation takes place. Exemplary conditions for centrifugation of polymerized microtubules include centrifugation in a Beckman Airfuge for 10 minutes at 90,000 rpm. For further handling of the microtubules, the microtubules are typically resuspended in buffer, for example MRB80 buffer.

In some embodiments, the method of the invention comprises at least one subunit of the oligomeric protein comprising at least one amino acid sequence mutation relative to the native protein subunit that affects the quaternary conformation and specific oligomeric state of the oligomer. Non-limiting examples of common mutations in human tubulin are T56M, P72S, L92V, N101S, V137D, S158L, I188L, R214H, D218Y, I238V, R263T, R264H, A270T, L286F, V303G, R320H, K326N, N329S, V371E, M377V, R402C, R402H, V409A, R422H, E429Q (all in TUBA1A), G58R, S172P, P173L, I210T, L228P, C239F, A248T, F265L, D294H, T312M (ah in TUBB2B), R62Q, G82R, T178M, E205K, R262C, R262H, A302V, M323V and M388V (all in TUBB3).

Oligomerization rates and depolymerization rates of protein oligomers can be affected by numerous (disease) conditions and (interacting) compounds, such as therapeutic drugs. Essentially all such conditions and compounds result in a protein oligomer that differs in its quarternary structure from the protein oligomer that has formed or depolymerized under normal, control or reference conditions in the absence of such (disease) conditions or (interacting) compounds. For one, such rates can be affected by mutations in the amino acid sequence of (one of) the oligomeric protein subunits. Alternatively, such rates can be affected by the interaction of proteins or small molecules with the protein oligomer or with its protein subunits. Also, such rates can be affected by nucleoside phosphates, in particular nucleoside phosphate derivatives or analogues with reduced rates of hydrolysis, e.g., hydrolysis resistant analogues. In addition to, or apart from, changes in the oligomerization or depolymerization rate, oligomerization-modulating compounds may also affect the oligomeric state of the oligomeric protein. With the term “oligomeric state”, reference is made to proteins self-assembling into specific quaternary structure whereby they adopt a particular oligomeric form. For instance, wild-type proteins can be mutated to generate variants of lower oligomeric order, which directly impacts the resulting proteins, in terms of structure, function and generic stability. A specific oligomeric state as used herein preferably includes the presence of specific epitopes in the oligomer, not present in the monomer or repeating subunit.

A method for preparing a conformation-specific antibody against an oligomeric protein in a specific oligomeric state in accordance with the present invention comprises a further step of arresting the oligomerization of said protein oligomer by addition of a cross-linking agent to thereby provide said protein oligomer in a stabilized quaternary conformation and specific oligomeric state. Arresting the oligomerization in aspects of this invention is preferably complete, i.e. the oligomerization has rate zero. The present inventors have found that arresting the oligomerization of the oligomeric protein by the addition of a cross-linking agent may result in the production of antibodies that can remarkably well differentiate between the protein oligomer and its subunits, and thereby distinguish oligomeric proteins in a specific oligomeric state, in particular, and preferably, a state that is brought about by conditions that affect the quarternary structure of the oligomer.

In some embodiments, said cross-linking agent as used in a further step of arresting the oligomerization in a specific oligomeric state in a method of the invention is an aldehyde. Non-limiting examples of aldehydes that can function as a cross-linking agent are glutaraldehyde, glyoxal, formaldehyde (including formalin and paraformaldehyde), and combinations thereof.

In preferred embodiments, said cross-linking agent used in a further step of arresting the oligomerization in a method of the invention is glutaraldehyde.

In some embodiments, the cross-linking agent is added in an amount of 0.002-20% v/v with respect to the total volume wherein crosslinking agents, preferably 0.02-2% v/v, more preferably 0.1-0.4% v/v, even more preferably around 0.2% v/v.

In some embodiments, a further step of arresting the oligomerization in a specific oligomeric state in a method of the invention involves incubation. In preferred embodiments, an incubation time of at least 10 seconds is selected; preferably, the incubation time is at least 1 minute; more preferably, the incubation time is at least 10 minutes; even more preferably, the incubation time is at least 20 minutes; most preferably, the incubation time is around 30 minutes. In some embodiments, incubation as part of a further step of arresting the oligomerization in a specific oligomeric state in a method of the invention takes place at temperatures between and including 0-100°C, preferably between and including 10-50°C, more preferably between and including 20-45°C, even more preferably around 37°C.

In some embodiments, the cross-linking agent is added as a solution, wherein the solution further comprises at least one of the compounds selected from the group comprising water, methanol, ethanol, acetone, acetonitrile, and other cross-linking agents.

In some embodiments, the protein oligomer in a stabilized quaternary conformation and specific oligomeric state as resulting from a further step of arresting the oligomerization of said protein oligomer by addition of a cross-linking agent is separated or purified from the crosslinking agent after cross-linking has taken place. Preferably, said separation or purification is performed using buffer-exchange. Exemplary conditions for purifying microtubules is a buffer-exchange into MRB80 buffer by two rounds of centrifugation in an amicon 100 KDa cut-off filter.

A method for preparing a conformation-specific antibody against an oligomeric protein in a specific oligomeric state in accordance with the present invention comprises a further step of using the protein oligomer in said stabilized quaternary conformation and specific oligomeric state of step (b) as a conformation -specific antigen of the protein oligomer to immunize a non-human mammal, optionally in combination with an adjuvant, to thereby obtain antibodies against said conformation-specific antigen. Such methods are well known to one of skill in the art.

In some embodiments, a further step of using the protein oligomer in said stabilized quaternary conformation and specific oligomeric state of step (b) as a conformation-specific antigen of the protein oligomer to immunize a non-human mammal further comprises the steps of: i) injecting a non-human mammal with the conformation-specific antigen obtained in step b); ii) selecting a B cell clone from the serum of said non-human mammal that produces an antibody against said conformation-specific antigen; iii) optionally converting said B cell clone into a hybridoma; and iv) having said B cell clone or said hybridoma produce the conformation-specific antibody. Preferably, said steps are carried out in the order they are presented in.

In some embodiments, step ii) further comprises the step of selecting a B cell clone that produces an antibody with a binding affinity indicated by a KD value of less than IO ⁶.

A method for preparing a conformation-specific antibody against an oligomeric protein in a specific oligomeric state in accordance with the present invention comprises a further step of selecting an antibody obtained in step (c) that binds to said conformation-specific antigen but not to the protein subunits, to thereby obtain a conformation-specific antibody against said oligomeric protein in a specific oligomeric state.

In some embodiments, a further step of selecting an antibody obtained in step (c) that binds to said conformation-specific antigen but not to the protein subunits comprises selecting a conformation-specific antibody that binds to an epitope of the protein oligomer with a binding affinity indicated by a KD value of less than IO ⁶ M, and does not bind to the protein subunits or binds to said subunits with a binding affinity indicated by a KD value of less than IO^-6 M. Preferably, a conformation-specific antibody is selected that binds to an epitope of the protein oligomer with a binding affinity indicated by a KD value of less than IO ⁷ M, more preferably by a KD value of less than IO ⁸ M, even more preferably by a KD value of less than IO ⁹ M, still more preferably by a KD value of less than 10 ¹⁰ M.

The invention provides an antibody obtained by a method of the invention. The invention provides a pharmaceutical composition comprising an antibody obtained by a method of the invention and at least one pharmaceutically acceptable adjuvant. In some embodiments, the adjuvant is selected from the group comprising incomplete Freund’s adjuvant, aluminum hydroxide, modified muramyldipeptide, dimethyl dioctadecyl ammonium bromide, MF59, SAF, aluminum salts, calcium salts and liposomes.

The invention provides a diagnostic composition comprising an antibody obtained by a method of the invention, preferably for use in the detection of a microtubule in cancer cells.

The invention also provides a use of an antibody obtained by a method of the invention, for measuring taxane loading of microtubules in a tissue sample or body fluid sample of a subject, preferably a subject suffering from cancer and treated with said taxane. In some embodiments, said taxane is selected from the group comprising paclitaxel, paclitaxel derivatives, docetaxel, cabazitaxel, tesetaxel, paclitaxel poliglumex, nab- paclitaxel and larotaxel.

Further embodiments will become apparent from the below Examples.

EXAMPLES

Example 1: Generation of fixed GmpCpp polymerized microtubules

An amount of 1 mg of Cycled Tubulin™ (bovine, PurSolutions LLC, Nashville, TN; Cat# 032005) was polymerized at a final concentration of 20 pM of tubulin, together with 0.7 mM GmpCpp (Jena Biosciences), in MRB80 buffer (80 mM PIPES, pH 6.8; ImM EGTA; 4 mM MgCL) for 40 minutes at 37°C. The solution was then centrifuged in a Beckman Airfuge for 10 minutes at 90,000 rpm. The resultant pellet of polymerized microtubules was re-suspended in 200 pl of MRB80 buffer and then Glutaraldehyde (EMS) was added to a final concentration of 0.2% w/v to the solution for 30 minutes at 37°C for fixation. The solution was then buffer-exchanged into pure MRB80 buffer by two rounds of centrifugation in an Amicon 100 KDa cut-off filter, into a final volume of 300-400 pl. The solution was snap frozen in liquid nitrogen and used as MT seeds. The seeds were optionally biotinylated for use in ELISA experiments.

Example 2, ELISA-based screening for anti-MT antibodies.

A dedicated new screening method was developed for detection of newly produced anti-MT antibodies. This screening method was subsequently used to screen serum of immunized mice for putative conformation-specific anti-MT antibodies (see Example 3), and supernatants of hybridomas prepared from B cells of selected antibody -pro during mice (see Example 4).

The screening method used in vitro GmpCpp-generated biotinylated MT seeds immobilized on Streptavidin-coated plastic 96-well plates. These plates were screened with conventional ELISA using an HRP based colorimetric readout. Briefly, the procedure was as follows. Nunc- Immuno Maxisorp 96-well ELISA plates (Nunc, #7350083) were coated with 5 pg/ml Streptavidin (Invitrogen, #A2666) in PBS and incubated overnight at 4°C. The following day GmpCpp -tubulin seeds freshly prepared as described by Leslie & Galjart (Methods Cell Biol. 2013;115:109-24) were first diluted 100 times in MRB80 buffer (80 mM PIPES, pH 6.8; ImM EGTA; 4 mM MgCL) warmed to 37°C, an amount of 50 pL of seeds was added to each well of the plates, and the plate was incubated at 37°C for 15-20 minutes. The solution was then removed by aspiration, and 100 pL of 4% w/v PFA (in PBS, pH 7.2) was added to each well for 10 minutes at 37°C. The PFA was removed and plates were washed twice in PBS (100 pL per well), then blocked with 2% w/v BSA (Sigma)+ 2% w/v milk in PBS containing 0.1% v/v Tween-20 (PBST) for 2 hours at 37°C. An amount of 50 jiL of undiluted hybridoma supernatant was then added to the wells, and the plate was incubated 30 minutes at 37°C. As a positive control, mouse anti-beta-tubulin (Sigma, #T8328) was added at a dilution of 1:500. After primary incubation, plates were washed 3 times with 100 pL of PBST per well each time. Secondary antibody (Dako polyclonal goat anti-mouse secondary; HRP- conjugated, # P0447), was added at a dilution of 1:2500 (50 jiL per well), and the plate was incubated for 30 minutes at 37°C. Plates were then washed 3 times with 100 pL of PBST per well each time. 50 pL of BM Blue POD substrate (Roche, #11484281001) was added per well and incubated at 37°C for 10 minutes. The reaction was then quenched with 10 pL IM H2SO4 per well, taking care to avoid any bubbles, and absorption was measured immediately on the Biotek 800 TS microplate reader at 450 nm. Wells without seeds, and wells lacking any primary antibody, were used as negative controls.

A control experiment was performed using Mouse monoclonal beta tubulin antibody (Sigma, T8328, 1:500) as the primary anti-MT antibody and Rabbit Polyclonal anti -Mouse IgG-HRP (Dako, 1:5000) as the secondary antibody for detection of binding. Results of a representative control experiment are provided in Figure 1. In this Figure, the X-axis shows raw absorbance at 450 nm averaged from triplicate wells. The experiment showed that the ELISA assay is useful for screening for antibodies that specifically detect MTs.

Example 3. Immunization of mice, screening serum from immunized mice for putative conformation-specific anti-MT antibodies, and production of hybridomas

Antibodies against specific tubulin conformations were produced by an external provider (Harbour Biomed). For this, five mice were immunized with GmpCPP-stabilized (G-TUB) microtubules (MTs, see Example 1) as antigens. Following each immunization, an amount of 1 jiL of serum of each immunized mouse was screened for the presence of anti-MT antibodies using the method of Example 2, described above. All mice were then subjected to subsequent rounds of immunization, and screening for anti-MT antibodies. Following five rounds of immunizations and screening serially diluted antisera, two G-TUB mice were selected as the most promising candidates for follow-up. These two mice (#4 and #5) showed a clear improvement in the immune response upon increasing immunizations, notably a strong improvement in signal between the 5th and 6th round of immunization (see Figure 2).

In total, 7 rounds of immunizations were performed on the two selected mice, and hybridomas of B-cell clones of these mice were prepared using conventional techniques for monoclonal antibody production. A total of 2,600 B-cell hybridomas from the two mice were screened using the ELISA protocol of Example 2. In total, sixteen positive hybridoma supernatants were identified. These supernatants of these hybridoma were subsequently used for experimentation.

Example 4, IF -based characterization of antibodies

Immuno(fluorescent) staining experiments with these hybridoma supernatants were subsequently performed on HELA cells to investigate the performance of the antibody produced by the various clones. For this, PFA- fixed HELA cells were incubated with the hybridoma supernatants, and MT binding by the monoclonal antibodies was detected by using a secondary antibody that was fluorescently labeled.

It was found that at least three hybridoma clones produced monoclonal anti-MT antibodies that showed clear immunostaining of MTs or specific MT subsets in HELA cells. An example of this staining is shown in Figure 3, wherein three hybridoma supernatants from G-TUB mice (clone 27G7, top left and top right; clone 50C12, bottom left; clone 15B8, bottom right) are shown. Monoclonal antibody 27G7 was shown to be one of the most promising candidate antibodies for detection of specific MT conformations, and experiments were continued with this monoclonal antibody in order to demonstrate that this antibody in fact does recognize a specific conformation present in MTs.

Example 5. Conformation-specific staining by antibody 27G7

Antibody 27G7 was used for immunostaining of MTs in HeLa cells. For this, HeLa cells were fixed in PFA as described above and incubated with either antibody 27G7 (undiluted or 1:10 diluted) or with a regular anti- beta-tubulin antibody (Sigma #T8328; beta-tub) as control. Following fluorescent staining by secondary anti-mouse antibodies (green fluorescence) and DAPI counterstaining, CLSM images were captured as displayed in Figure 4A. The green fluorescence in Figure 4A represents the pattern obtained with both antibodies. Cells were also stained with DAPI to mark nuclei (blue fluorescence).

It is clear from Figure 4A that antibody 27G7 binds to a subset of MTs, as compared to beta-tub. Most notably, 27G7 fails to stain the centrosomal MTs, the MTs that originate near the nucleus (the very dense MT network in the beta-tub image in panel Al, marked by asterisks).

Further, it is noted that the IF staining with 27G7 has less background compared to beta-tub, indicating that 27G7 recognizes MTs and not soluble tubulin, as opposed to beta-tub.

Still further, both antibodies stain MTs in mitotic cells and midzone MTs at the end of mitosis (arrows in panels Al and A2).

It was also noted that different dilutions of 27G7 give rise to slightly different MT patterns. Without wishing to be bound by theory, the inventors consider that, while the epitope-recognizing domains in a dimeric antibody are positioned approximately 10 nm apart, which is close to the longitudinal spacing of two tubulins in a protofilament in a MT, epitopes recognized by 27G7 at the end of MTs are spaced correctly for the antibody to recognize two epitopes with one dimer, resulting in the creation of a high affinity binding site that is recognized at 1:10 dilution. By contrast, within the MT lattice the epitope is not ideally spaced and hence the 27G7 antibody recognizes only one epitope per antibody dimer. This is a lower affinity binding site, that comes up at lower dilutions (undiluted).

Further experimental results of differential fixation methods are displayed in Figure 4B. Figure 4B shows fluorescence CLSM images of HeLa cells either fixed with PFA or with methanol (MetOH) at -20°C, and incubated either with 27G7 (panels Bl, B2, B4) or with regular anti-tubulin antibodies (panel B3; beta-tub). 27G7 was used undiluted. The green fluorescence represents the pattern obtained with 27G7 antibodies, the red fluorescence in panel B3 represents the pattern obtained with beta-tub antibody. In panels Bl and B2, cells were also stained with DAPI to mark nuclei (blue fluorescence).

It can be seen that 27G7 stains only a subset of MTs, as compared to beta-tub. Most notably, the MT pattern stained by 27G7 differs depending on the fixation used (comparing MT patterns in panels Bl and B2). Further it was noted that the MetOH fixation provides for improved staining with regular anti-tubulin antibodies because of a lower background signal whereas the MT signal is maintained (comparing panel B3 with panel Al of Figure 4A). This can be explained by the fact that MetOH fixation allows soluble tubulin to “leak out of cells”, hence providing for a lower background with beta-tub staining. In this double staining experiment (panels B3 and B4) 27G7 clearly fails to stain the centrosome and centrosomal MTs (the very dense MT network in the beta-tub image in panel B3, the position of the centrosome is marked by the asterisk).

Example 6. Use of conformation-specific antibody 27G7 for monitoring MT oligomerization-modulating events In order to show the unique utility of antibodies capable of recognizing specific conformations in an oligomeric protein, the present inventors produced a monoclonal antibody that recognizes structures in the MT conformation that are the result of polymerization of the oligomer in the presence of a oligomerization-modulating compound such as GmpCPP. This antibody, and the production of its antigen is described in Example 1-5. In this Example, it is shown that such an antibody is capable of recognizing structures in the MT conformation that are the result of an altered polymerization of the oligomer due to the occurrence of oligomerizationmodulating events.

In this Example, the inventors have produced embryonic stem (ES) cells comprising a knockout mutation in the Clasp 2 gene. For this, standard methods well known to one in the art of genetic modification were employed. CLASP2 plays in important role in MT formation. CLASP2 is a MT-growth promoting protein that acts at the ends of growing MTs. CLASP2 has also been shown to be involved in repair of the MT lattice.

Wild type (WT) and Clasp2 knockout (KO) embryonic stem (ES) cell colonies were grown in standard media, fixed with PF A, incubated with 27G7 antibodies and the 27G7 binding pattern was detected by using secondary fluorescent anti-mouse antibodies.

The results of these colony stainings is displayed in Figure 5. Figure 5 A shows two colonies (one on the left, and one on the right), each with about 6 ES cells wherein the secondary fluorescent antibodies are visible as “inverted” staining, i.e. black against a white background.

It is apparent from these images that the staining of MTs by 27G7 is increased in Clasp2 KO compared to WT. The quantification of this phenomenon is shown in Figure 5B wherein the secondary fluorescent staining (as fluorescence intensity, FI) was measurements in several ES cell colonies (measurements based on 3 imaging experiments). Midzone MTs, observed at the end of mitosis (cytokinesis) are extremely apparent (arrows). The data indicate that 27G7 specifically stains “unrepaired” MTs, these are increased in the Clasp 2 KO ES cells, due to absence of CLASP2. Based on these outcomes it is concluded that 27G7 recognizes a specific conformation in MTs that is the result from an oligomerization-modulating event such as a mutation in a gene involved in MT formation and maintenance.

Claims

Claims

1. A method for preparing a conformation-specific antibody against a microtubule in a specific oligomeric state, the method comprising the steps of: a) providing tubulin protein subunits of a microtubule under an in vitro condition and effecting oligomerization of said tubulin protein subunits into a microtubule in the presence of an oligomerization-modulating compound that alters the oligomerization rate of microtubule and/or its oligomeric state relative to said in vitro condition wherein said oligomerizationmodulating compound is absent, wherein said ohgomerization-modulating compound is selected from a protein interacting with microtubule or tubulin, a nucleoside phosphate or an hydrolysis-resistant analogue thereof, or a (de)stabilizing small molecule; b) arresting the oligomerization of said microtubule in a specific oligomeric state by addition of a cross-linking agent to thereby provide said microtubule in a stabilized quaternary conformation and/or specific oligomeric state; c) using the microtubule in said stabilized quaternary conformation and/or specific oligomeric state of step (b) as a conformation-specific antigen of said microtubule to immunize a non-human mammal, optionally in combination with an adjuvant, to thereby obtain antibodies against said microtubule conformation-specific antigen; d) selecting an antibody obtained in step (c) that binds to said microtubule conformation-specific antigen but not to said tubulin protein subunits, to thereby obtain a conformation-specific antibody against said microtubule in a specific oligomeric state.

2. The method according to claim 1, wherein said ohgomerization- modulating compound is selected from the group comprising GTP, dGTP, GDP, dGDP, ATP, dATP, ADP, dADP, CTP, dCTP, CDP, dCDP, TTP, dTTP, TDP, dTDP, UTP, dUTP, m5UTP, UDP, dUTP, m5UDP, GMPCPP, GTPyS, GTPaS, GpCpp, GppCp, GppNHp, paclitaxel, paclitaxel derivatives, docetaxel, cabazitaxel, tesetaxel, paclitaxel poliglumex, nab-paclitaxel, larotaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, ixabepilone, laulimalide, peloruside A, discodermolide, taccalonolide AF, taccalonolide AJ, taccalonolide A, taccalonolide E, taccalonolide Al-epoxide, zampanolide, sagopilone, dictyostatin, cyclostreptin, eleutherobin, sarcodictyin A, sarcodictyin B, patupilone, rhazinilam, XMAP215, CLASP- 1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP 170, doublecourtin, and other proteins that stabilize microtubules, or a protein comprising one or more mutations with respect to stabilizing proteins XMAP215, CLASP- 1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP 170 or doublecourtin, wherein said protein affects the oligomerization state in a similar manner as said stabilizing proteins.

3. The method according to claim 1 or claim 2, wherein said oligomerization-modulating compound is GMPCPP.

4. A method for preparing a conformation-specific antibody against an oligomeric protein in a specific oligomeric state, the method comprising the steps of: a) providing protein subunits of an oligomeric protein under an in vitro condition and effecting oligomerization of said protein subunits into a protein oligomer in the presence of an oligomerization-modulating compound that alters the oligomerization rate of said protein oligomer and/or its oligomeric state relative to said in vitro condition wherein said oligomerization-modulating compound is absent, wherein said oligomerization-modulating compound is selected from a protein interacting with said oligomeric protein or its protein subunits, a nucleoside phosphate or an hydrolysis-resistant analogue thereof, or a (de)stabilizing small molecule; b) arresting the oligomerization of said protein oligomer in a specific oligomeric state by addition of a cross-linking agent to thereby provide said protein oligomer in a stabilized quaternary conformation and/or specific oligomeric state; c) using the protein oligomer in said stabilized quaternary conformation and specific oligomeric state of step (b) as a conformation-specific antigen of said protein oligomer to immunize a non-human mammal, optionally in combination with an adjuvant, to thereby obtain antibodies against said conformation-specific antigen; d) selecting an antibody obtained in step (c) that binds to said conformationspecific antigen but not to said protein subunits, to thereby obtain a conformation-specific antibody against said oligomeric protein in a specific oligomeric state.

5. The method according to any one of the preceding claims, wherein step c) comprises the following steps: i) injecting a non-human mammal with the conformation-specific antigen obtained in step b); ii) selecting a B cell clone from the serum of said non-human mammal that produces an antibody against said conformation-specific antigen; iii) optionally converting said B cell clone into a hybridoma; and iv) having said B cell clone or said hybridoma produce the conformationspecific antibody.

6. The method according to claim 5, wherein step ii) further comprises the step of selecting a B cell clone that produces an antibody with a binding affinity indicated by a KD value of less than IO ⁶ M.

7. The method according to any one of the preceding claims, wherein said conformation-specific antibody binds to an epitope of the protein oligomer with a binding affinity indicated by a KD value of less than IO ⁶ M, and does not bind to the protein subunits or binds to said subunits with a binding affinity indicated by a KD value of more than IO ⁶ M.

8. The method according to any one of the preceding claims, wherein said oligomeric protein is a constituent of the cytoskeleton.

9. The method according to any one of the preceding claims, wherein said cross-linking agent is an aldehyde.

10. The method according to any one of the preceding claims, wherein said cross-linking agent is glutaraldehyde.

11. The method according to any one of the preceding claims, wherein at least one protein subunit of the oligomeric protein comprises at least one amino acid sequence mutation relative to the native protein subunit that affects the quaternary conformation and/or specific oligomeric state of the oligomer.

12. An antibody obtained by the method of any one of claims 1-11.

13. A pharmaceutical composition comprising the antibody according to claim 12 and at least one pharmaceutically acceptable adjuvant.

14. A diagnostic composition comprising the antibody according to claim 12, preferably for use in the detection of a microtubule in cancer cells.

15. Use of a method according to any one of claims 1-11 or an antibody according to claim 12, for measuring taxane loading of microtubules in a tissue sample or body fluid sample of a subject, preferably a subject suffering from cancer and treated with said taxane.

16 A method of detecting or monitoring an oligomerizationmodulating event of a protein oligomer, preferably in vitro in a cell, the method comprising staining the protein oligomer by an antibody obtainable by the method of any one of claims 1-11, and comparing the staining result with a control staining not subjected to or affected by the oligomerizationmodulating event, preferably wherein the ohgomerization-modulating event is a mutation in a gene, or regulatory element thereof, affecting the expression or expression product, more preferably wherein said gene is the gene for proteins that stabilize microtubules, most preferably XMAP215, CLASP-1, CLASP-2, MAP2, tau, MAP4, STOP, CLIP170 or doublecourtin.