WO2011083141A2 - Method for generation of immunoglobulin sequences by using lipoprotein particles - Google Patents

Method for generation of immunoglobulin sequences by using lipoprotein particles Download PDF

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Publication number
WO2011083141A2
WO2011083141A2 PCT/EP2011/050157 EP2011050157W WO2011083141A2 WO 2011083141 A2 WO2011083141 A2 WO 2011083141A2 EP 2011050157 W EP2011050157 W EP 2011050157W WO 2011083141 A2 WO2011083141 A2 WO 2011083141A2
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sequences
cell
amino acid
immunoglobulin
cxcr4
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PCT/EP2011/050157
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French (fr)
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WO2011083141A3 (en
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Maria Gonzalez Pajuelo
Guy Hermans
Peter Vanlandschoot
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Ablynx Nv
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a method for generating immunoglobulin sequences against cell-associated antigens, more particularly, antigens that are membrane-anchored.
  • the invention also provides immunoglobulin sequences obtainable by the method of the invention.
  • the present invention relates to the generation of immunoglobulin sequences by using lipoprotein particles.
  • the present invention relates to generation of immunoglobulin sequences in camelids, preferably directed against cell- associated antigens, in particular antigens with multiple transmembrane spanning domains, including GPCRs and ion channels, by using lipoprotein particles.
  • the invention relates to novel immunoglobulin sequences, constructs and uses thereof.
  • Enveloped viruses have been shown to expand their tropism by acquiring envelope proteins of other viruses during co-infection of a cell by different enveloped viruses. This process is called phenotypic mixing or pseudotyping. Pseudotyped viruses can be generated experimentally by expressing a different viral envelope protein in a (retro) viral infected cell. In addition to other viral membrane proteins, viruses can also incorporate host cell membrane proteins, including viral receptor proteins.
  • MHC class I MHC class II, ICAM-1, I.CAM-2, ICAM-3, CR3, CR4, CD4, CD44, CD46, CD55, CD59, CD73 and CD71 have been found in HIV-L Viruses that incorporate host membrane proteins of interest can be generated by over expressing this protein of interest in a (retro) viral infected cell.
  • retroviruses have been used widely to generate and produce pseudotyped viruses, because of the capacity of retroviral GAG core proteins to assemble into core particles and to bud from the cell, incorporating host membrane proteins during this budding process.
  • retroviral GAG core proteins only expression of retroviral GAG core proteins is sufficient to induce formation of pseudotyped viruses.
  • Pseudotyped viruses or particles are produced by inducing cells to express high levels of the protein of interest and a retroviral GAG core protein. A number of factors influence the uptake efficiency of host proteins like surface density of the membrane protein, membrane location or structural configuration. Optimization or control of particle production is therefore possible for example by using different promoters to express GAG core proteins and/or the protein of interest, by harvesting at the peak of protein expression.
  • Lipoprotein particles also called pseudotyped viruses
  • Lipoprotein particles have been recognized as very valuable tools to study or interfere with viral replication or to use them for gene therapy source of homogenous membrane proteins. Lipoprotein particles are also useful to study the structure and function of the membrane protein that is incoiporated. This especially true for complex membrane proteins like G coupled protein receptors (GPCRs), transporters and ion channels. Indeed, incorporation of such complex membrane proteins has been demonstrated for example for CCR5, CXCR4 and MCAT- 1. Recently, GPCR lipoprotein particles have been shown to be applicable in biosensor assays and a fluorescence polarization molecular binding assay. Such types of assays have been very difficult to achieve with, cell membrane fractions.
  • Immunoglobulin sequences such as antibodies and antigen binding fragments derived therefrom are widely used to specifically target their respective antigens in research and therapeutic applications.
  • the generation of antibodies involves the immunization of experimental animals, fusion of antibody producing cells to create hybridomas and screening for the desired specificities.
  • antibodies can be generated by screening of nai ' ve or synthetic libraries e.g. by phage display.
  • An important class of potential therapeutic targets are cell associated antigens, including transmembrane antigens, in particular transmembrane antigens with multiple membrane spanning domains.
  • Cell -associated, and especially membrane bound antigens are difficult to obtain in their natural conformation because they are embedded within, or anchored in the cell membrane.
  • immunoglobulin sequences against epitopes present in the natural conformation i.e. conformational epitopes, which are present in vivo, it is however essential to immunize with the target antigen in the correct conformation.
  • conformational epitopes are of paramount importance for creating pharmaceutically active immunoglobulin sequences.
  • an immunoglobulin sequence specifically interacting with the natural ligand binding epitope of a GPCR will likely affect its activity, and thus provide a pharmacological effect.
  • Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.
  • such an approach will not provide antibodies to conformation dependent epitopes, as such epitopes cannot be reproduced by short synthetic peptides.
  • the present invention relates to the following.
  • a method for the generation of immunoglobulin sequences that can bind to and/or have affinity for a cell-associated antigen comprising the steps of:
  • said antigen in its natural conformation selected from cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring enriched antigen, liposomes, lipoprotein particles or virus particles expressing the cell associated antigen; and c) screening a set, collection or library of immunoglobulin sequences derived from said non- human animal for amino acid sequences that can bind to and/or have affinity for said cell- associated antigen and wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles.
  • said lipoprotein particles are derived from the same cells as used for the cell based immunization or boost, e.g.
  • human cell line "HEK293” or derivatives thereof is produced using retrovirus structures (e.g. proteins from the mouse retrovirus "murine leukemia vims (MLV)” and enables structurally intact cellular proteins to be purified away from said cell.
  • retrovirus structures e.g. proteins from the mouse retrovirus "murine leukemia vims (MLV)” and enables structurally intact cellular proteins to be purified away from said cell.
  • said cell-associated antigen is selected from transmembrane antigens, transmembrane antigens with multiple spanning domains, such as GPCRs or ion channels.
  • said non-human animal can be selected from vertebrate, shark, mammal, lizard, camelid, llama, preferably camelids and llama.
  • the immunoglobulin sequences are light chain variable domain sequences (e.g. a V L -sequence), or heavy chain variable domain sequences (e.g. a
  • the immunoglobulin sequences can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody.
  • the immunoglobulin sequences can be domain antibodies, or amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, or amino acid sequences that are suitable for use as single domain antibodies, "dAbs", or amino acid sequences that are suitable for use as dAbs, or Nanobodies®, including but not limited to V HH sequences, and preferably are Nanobodies®.
  • vaccination can be performed by a needle-free jet injection, by a ballistic method, by needle-mediated injections such as Tattoo, by topical application of the DNA onto the skin in patches or by any of these administration methods followed by in vivo electroporation, and furthermore includes vaccination performed by intradermal, intramuscular or subcutaneous administration of DNA.
  • the set, collection or library of immunoglobulin sequences can be obtained from the blood of said non-human mammal.
  • said cell-associated antigen can be expressed on any cell background which allows expression of the native conformation of the antigen.
  • Examples of such cell backgrounds that are used in immunization, boost and/or lipoprotein particle generation are Cho, Cos7, Hek293, or cells of camelid origin.
  • said cell-associated antigen is a membrane-spanning antigen, including but not limited to an antigen selected from CXC chemokine receptors such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and/or CXCR7, in particular CXCR4 and its human variant.
  • the set, collection or library of immunoglobulin sequences can be expressed on a set, collection or sample of cells or viruses ⁇ e.g. such as bacteriophages) and said set, collection or sample of cells or viruses is screened for cells or viruses that express an amino acid sequence that can bind to and/or have affinity for the lipoprotein particle (e.g. lipoprotein particle of same cell background as cell based immunization and/or whole cell boost) comprising said cell-associated antigen.
  • a lipoprotein particle e.g. lipoprotein particle of same cell background as cell based immunization and/or whole cell boost
  • the set, collection or library of immunoglobulin sequences can be encoded by a set, collection or library of nucleic acid sequences and said set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode an immunoglobulin sequence that can bind to and/or have affinity for the lipoprotein particle (e.g. lipoprotein particle of same cell background as cell based immunization and/or whole cell boost) comprising said cell-associated antigen.
  • the lipoprotein particle e.g. lipoprotein particle of same cell background as cell based immunization and/or whole cell boost
  • the immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen can be purified and/or isolated.
  • the invention also relates also to immunoglobulin obtainable by a method as described herein, and compositions comprising the said immunoglobulin sequences.
  • Figure 1 shows that no specific binding of antibodies from llamas immunized with CXCR4 cells to CXCR4 ⁇ particles could be observed.
  • FIG. 2 shows that CXCR4+ lipoprotein particles are recognized by purified CXCR4-specific Nanobodies® 238D2 and 238D4.
  • Figure 3 sho s the detection of CXCR4+ lipoprotein particles by periplasmic extracts of CXCR4-specific Nanobodies® 238D2 and 238D4.
  • Figure 4 shows that CXCR4+ lipoprotein particles are recognized by phages that display 238D2 and 238D4.
  • Figures 5a-e show that selections with library 218 on CXCR4+ particles result in isolation of large numbers of Nanobodies ⁇ binding to CXCR4+ particles.
  • Figure 6 shows that selections on CXCR4- particles do not yield Nanobodies® binding to CXCR4+ and CXCR4- particles.
  • Figures 7a-e show that selections with library 217 on CXCR4+ particles result in isolation of large numbers of Nanobodies® binding to CXCR4+ particles.
  • Figures 8a-c show that Nanobodies® selected on CXCR4+ particles specifically bind CXCR4-HEK293T cells.
  • FIG. 9 Number of peripheral white blood cells (WBC) (A) and mobilized stem cells (B) at several timepoints after the administration of Mozobil® or the 238D2-20GS-238D4. An increased number is observed after compound administration with a peak at 3-6 hours post- administration for 238D2-20GS-238D4 and Mozobil®.
  • WBC peripheral white blood cells
  • B mobilized stem cells
  • immunoglobulin sequence whether used herein to refer to a heavy chain antibody or to a conventional 4 ⁇ chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or V ⁇ VL domains, respectively).
  • antigen-binding molecules or antigen-binding protein are used interchangeably with immunoglobulin sequence, and include Nanobodies®.
  • the immunoglobulin sequences are light chain variable domain sequences (e.g. a VL-sequence), or heavy chain variable domain sequences (e.g. a V[[-sequence); more specifically, the immunoglobulin sequences can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody.
  • the immunoglobulin sequences can be domain antibodies, or amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, or amino acid sequences that are suitable for use as single domain antibodies, "dAbs", or amino acid sequences that are suitable for use as dAbs, or Nanobodies®, including but not limited to V H H sequences, and preferably are Nanobodies®.
  • the immunoglobulin sequences provided by the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers).
  • the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e.
  • Such a protein or polypeptide may also be in essentially isolated form (as defined herein).
  • the invention includes immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences.
  • the invention also includes fully human, humanized or chimeric immunoglobulin sequences.
  • the invention comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized Dab as described by Ward et al (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996)).
  • the invention comprises fused immunoglobulin sequences, e.g.
  • a multivalent and/ or muitispecific construct for multivalent and muitispecific polypeptides containing one or more V H H domains and their preparation, reference is also made to Conrath et al, J. Biol. Chem., Vol 276, 10. 7346-7350, 2001, as well as to for example WO 96/34103 and WO 99/23221), and immunoglobulin sequences comprising tags or other functional moieties, e.g. toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present invention.
  • the amino acid sequence and structure of an immunoglobulin sequence, m particular a Nanobody® can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as “Framework region 1" or “FR1”; as “Framework region 2" or “FR2”; as “Framework region 3" or “FR3”; and as “Framework region 4" or “FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's”, which are referred to in the art as “Complementarity Determining Region l”or “CDR1 "; as “Complementarity Determining Region 2' ' or “CDR2”; and as “Complementarity Determining Region 3" or “CDR3", respectively.
  • CDR's complementary determining regions
  • the total number of amino acid residues in a Nanobody® can be in the region of 1 10- 120, is preferably 1 12-115, and is most preferably 1 13. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a Nanobody® are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.
  • immunoglobulin sequences refers to both the nucleic acid sequences coding for an immunoglobulin molecule, and the immunoglobulin polypeptide per se. Any more limiting meaning will be apparent from the particular context.
  • sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.
  • nucleic acid molecule of the invention may either relate to the nucleic acid for genetic vaccination, or the nucleic acid encoding the immunoglobulin sequences of the invention, or both, as will be apparent from the specific context. Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general, background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Dreg Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst.
  • the invention relates to immunoglobulin sequences that can bind to and/or have affinity for an antigen as defined herein.
  • binding to and/or having affinity for a certain antigen has the usual meaning in the art as understood e.g. in the context of antibodies and their respective antigens.
  • the term "binds to and/or having affinity for” means that the immunoglobulin sequence specifically interacts with an antigen, and is used interchangeably with immunoglobulin sequences "against" the said antigen.
  • the term "specificity” refers to the number of different types of antigens or antigenic determinants to which a particular immunoglobulin sequence, antigen-binding molecule or antigen-binding protein (such as a Nanobody® or a polypeptide of the invention) can bind.
  • the specificity of an antigen-binding protein can be determined based on affinity and/or avidity.
  • the affinity represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD) is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen -binding protein: the lesser the value of the KD, the stronger the bindi g strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (K A ). which is 1/K D ).
  • affinity can be determined in a manner known per se, depending on the specific antigen of interest, Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a Nanobody® or polypeptide of the invention.) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule.
  • immunoglobulin sequences of the present invention will bind to their antigen with a dissociation constant (3 ⁇ 4) of 10 "5 to 10 "12 moles/liter or less, and preferably IO "7 to 10 "12 moles/liter or less and more preferably 10 ⁇ 8 to 1CT 12 moles/liter (i.e. with an association constant ( A ) of 10 5 to i0 12 liter/ moles or more, and preferably 10 7 to 10 12 liter/moles or more and more preferably 10 8 to iO 12 liter/moles),
  • Any K D value greater than 10 "4 mol/liter (or any K A value lower than 10 4 M "1 ) liters/mol is generally considered to indicate non-specific binding.
  • a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
  • Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme
  • EIA immunoassays
  • sandwich competition assays and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • the dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more then 10 "4 moles/liter or 10 "3 moles/liter (e.g., of 10 "2 moles/liter).
  • the affinity denotes the strength or stability of a molecular interaction.
  • the affinity is commonly given as by the K D , or dissociation constant, which has units of mol/liter (or M).
  • the affinity can also be expressed as an association constant, K A , which equals 1/KD and has units of (mol/liter) "] (or M " ').
  • K D dissociation constant
  • K A association constant
  • K D for biological interactions, such as the binding of the immunoglobulin sequences of the invention to the cell associated antigen as defined herein, which are considered meaningful (e.g. specific) are typically in the range of 10 "10 M (0.1 nM.) to 10 "5 M ( 10000 nM), The stronger an interaction is, the lower is its KD.
  • the off-rate koff has units s "! (where s is the SI unit notation of second).
  • the on-rate k on has units M " Y ⁇
  • the on-rate may vary between 10 2 ' V 1 to about 10 7 M " V ⁇ approaching the diffusion-limited association rate constant for bi molecular interactions.
  • the off-rate is related to the half-life of a given molecular interaction by the relation .
  • the affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et ah, Intern. Immunology, 13, 1551 -1559, 2001) where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k on , k off ineasurements and hence D (or KA) values.
  • SPR surface plasmon resonance
  • the measured D may correspond to the apparent KD if the measuring proces somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent D may be measured if one molecule contains more than one recognition sites for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules.
  • the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule.
  • a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated ELISA detection).
  • the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B.
  • an IC5 0 value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved.
  • Ko ref the D of the reference molecule, is known, as well as the total concentration c ref of the reference molecule, the apparent K D for the interaction A-B can be obtained from following formula: K D
  • “conformational epitope” denotes an epitope that comprises amino acids which are not within a single consecutive stretch of the primary sequence of the antigen.
  • amino acids which may be spaced apart in the primary sequence are brought into proximity to each other and thereby participate in the formation of an epitope.
  • an antigen comprises three amino acid loops, residues on each one of these loops may participate in the formation of a single epitope.
  • an epitope may be formed by amino acids on different domains or subunits. Complete or partial denaturing of the protein by appropriate conditions, i.e.
  • the present invention is directed to immunoglobulin sequences against conformational epitopes.
  • the invention concerns immunoglobulin sequences against conformational epitopes on cell-associated antigens as defined herein, which may preferably be camelid immunoglobulin sequences, including Nanobodies®.
  • cell-associated antigen means antigens that are firmly anchored in or located within the membranes of a cell (including membranes of subcellular compartments and organelles), and includes antigens that have a single or multiple transmembrane regions.
  • the term refers to antigens exhibiting membrane-dependent conformational epitopes.
  • the term refers to antigens having conformational epitopes as defined herein.
  • the term encompasses transmembrane antigens, transmembrane antigens with multiple membrane spanning domains such as GPCRs or ion channels.
  • the invention in particular relates to ceil associated antigens wherein the conformation dependent epitope is dependent on the correct anchoring and/or location in the membrane.
  • the invention provides immunoglobulin sequences against such conformation dependent epitopes.
  • the invention relates to antigens that are integral membrane proteins having one, or more preferably multiple membrane spanning domains. These antigens will reside in and operate within a cell's plasma membrane, and/or the membranes of subcellular compartments and organelles. Many transmembrane proteins, such as transmembrane receptors comprise two or more subunits or domains, which functionally interact with one another.
  • Integral membrane proteins comprise three distinct parts or domains, i.e. an extracellular (or extracompartmental) domain, a transmembrane domain and an intracellular (or
  • a protein having multiple transmembrane domains will typically also have multiple extra- and intra cellular/compartmental domains.
  • a seven transmembrane receptor will comprise seven transmembrane domains.
  • cell, associated antigen as understood herein is intended to exclude antigens that are only loosely associated, i.e. that are not firmly anchored or located within a membrane.
  • An antigen is firmly anchored if it comprises at least one domain or part that extends into the membrane.
  • the invention excludes antigens that have a membrane insertion via a lipid tail, but no transmembrane domain.
  • the conformation of the hydrophilic portion or domain of the protein will not depend on the membrane environment. It will, for example, be possible to express a recombinant protein lacking the lipid tail, which is in the proper conformation, i.e. expresses the conformational epitopes also present if the antigen is associated with the membrane via the lipid tail.
  • any other proteins which are only loosely associated are excluded from the i vention in a particular embodiment.
  • "Loosely associated" in this connection means proteins which exhibit their natural conformation even in the absence of membrane, i.e. their natural conformation is not dependent on the anchoring or embedding within a membrane.
  • Typical examples of cell associated antigens according to the invention comprise seven membrane domain receptors, including G-protein coupled receptors, such as CXC chemokine receptors, Adrenergic receptor, Olfactory receptors, Receptor tyrosine kinases, such as
  • Epidermal growth factor receptor Epidermal growth factor receptor, Insulin Receptor, Fibroblast growth factor receptors, High affinity neurotrophin receptors, and Eph Receptors, fntegrms, Low Affinity Nerve Growth Factor Receptor, NMDA receptor, Several Immune receptors including Toil-like receptor, T cell receptor and CD28.
  • the term "cell-associated antigen” is intended to include, and also refer to, any part, fragment, subunit, or domain of said cell associated antigen. Any subsection of the cell associated antigen falls within the scope of the present invention, provided it represents a conformational epitope of interest. If for example the epitope of interest is located in a binding site of a receptor, or the pore of an ion channel, any fragment(s) of the cell associated antigen capable of forming said epitope are included in the invention. Preferably, those parts, domains, fragments or subunits will be those parts of the cell associated antigen which are responsible for the membrane-dependent conformation. If for example a protein comprises several transmembrane domains, linked by extended intracellular loops, it is envisaged that such loops are in pari or fully omitted, without influencing the extracellular conformational epitopes.
  • the present invention relates to immunoglobulin sequences directed to cell associated antigens in their natural conformation.
  • natural conformation means that the protein exhibits its secondary and/or tertiary structure, in particular its membrane dependent secondary and/or tertiary structure.
  • the natural conformation describes the protein in a non-denatured form, and describes a conformation wherein the conformational epitopes, in particular the membrane dependent conformational epitopes, are present.
  • the protein will have the conformation that is present when the protein is integrated into or firmly attached to a membrane.
  • Antigens can be obtained in their natural conformation when present in cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring antigen, liposomes, or virus particles expressing the cell associated antigen.
  • antigen may be enriched by suitable means.
  • Said cell-associated antigen can be expressed on any suitable cell allowing expression of the antigen in its native or natural conformation, encompassing, but not limited to Cho, Cos7, Hek293,or cells of camelid origin.
  • the cell associated antigen of the present invention is preferably a druggable membrane protein, in particular a druggable membrane protein having multiple membrane spanning domains.
  • the target is a GPCR or an ion channel.
  • ion channels that represent cell associated antigens according to the present invention are provided in the following. Also listed are therapeutic effects (with activating (agonistic) or inhibiting/blocking (antagonistic, inverse agonistic) properties) of immunoglobulin sequences specifically recognizing such ion channels.
  • Two-P potassium channels such as K 2P 1.1 , K 2P 2.1 , K 2P 3.1 , K 2P 3.1, K 2P 4.1, K 2P 5.1, K 2P 6.1 , K 2P 7.1 , K 2P 9.1, K 2P 10.3 , K 3P I2.
  • I, 2 pl3.1, K 2P 15.1 , K 2P 16.1 , K 2P 17.1 and K 2P 18.1 which can all be screened using electrophysiological assays such as FLIPR or patch-clamp.
  • CatSper channels see Clapham and Garbers, Pharmacological Reviews, 57, 4, 451 (2005)), such as CatSper- 1 and CatSper-2 (both involved in fertility and sperm motility), CatSper-3 and CatSper-4, which can all be screened using
  • electrophysiological assays such as FLIPR, patch-clamp or calcium imaging
  • TPC1 and TPC2 Two-pore channels (see Clapham and Garbers, Pharmacological Reviews, 57, 4, 451 (2005)), such as TPC1 and TPC2.
  • CNGB3 which can be screened using techniques such as patch-clamp and calcium imaging
  • Hyperpolarization-activated cyclic nucleotide-gated channels such as HCN1, HCN2, HCN3, HCN4 (all regarded as promising pharmacological targets for development of drags for cardiac arrhythmias and ischemic heart disease), which can be screened using techniques such as voltage-clamp.
  • Inwardly rectifying potassium channels see ubo et al., Pharmacological Reviews, 57, 4, 509 (2005)), such as K ir l . l , ir 21.
  • Openers of Kc a 2.2 have been proposed for cerebellar ataxia;
  • Kc a 2.2 modulators of which may be useful in the treatment of various diseases such as myotonic muscular dystrophy, gastrointestinal dysmotility, memory disorders, epilepsy, narcolepsy and alcohol intoxication.
  • Openers of Kc a 2.2 have been proposed for cerebellar ataxia;
  • KQ,2.2 - modulators of which may be useful in the treatment of various diseases such as myotonic muscular dystrophy, gastrointestinal dysmotility, memory disorders, epilepsy, narcolepsy, hypertension and urinary incontinence;
  • Kc a 4.1 , ⁇ 3 ⁇ 4 3 ⁇ 4 4.2 and Kc a 5.1 all of which can be screened using
  • Potassium channels see Shieh et al., Pharmacological Reviews, 57, 4, 557 (2005) and Guiman et al., Pharmacological Reviews, 57, 4, 473 (2005) ), including:
  • voltage-gated calcium channels such as Kvl . l, Kvl .2, Kvl.3, vl.4, Kvl .5, Kvl.6 and Kv.17;
  • beta-subunits of v channels such as vBeta- I , KvBeta-2 and KvBeta-3;
  • Stew-like channels such as v.3.1, v3.2. Kv3.3 and Kv3.4;
  • SteWike channels such as Kv4.1, Kv4.2, Kv4.3, v5.1, Kv6.1, Kv6.2, Kv8.1 , Kv9.1 , Kv9.2, v9.3, KH1 and KH2;
  • Ether-a-go-go-c neh such as EAG, HERG, BEC1 and BEC2;
  • MinK-type channels such as MinK, MiRPl and MiRP2;
  • KvLQT -type channels such as vLQTI, KvLQT2, vLQT3, KvLQT4, KvLQTS
  • Inwardly rectifying potassium channels such as those mentioned above; Sulfonylurea receptors such as the sulfonylurea receptors 1 and 2; Large conductance calcium-activated channels such as Slo and the Beta- subunits of BKcai
  • Small conductance calcium-activated channels such as SKI, SK2 and SK3;
  • Two-pore potassium channels such as TWIK1 , TREK, TASK, TASK2, TWIK2, TOSS, TRAAK and CTBAK1 ;
  • Potassium channels are implicated in a wide variety of diseases and disorders such as cardiac diseases (such as an-hythmia), neuronal diseases, neuromuscular disorders, hearing and vestibular diseases, renal diseases, Alzheimer's disease, and metabolic diseases; and are targets for active compounds in these diseases.
  • cardiac diseases such as an-hythmia
  • neuronal diseases neuromuscular disorders, hearing and vestibular diseases, renal diseases, Alzheimer's disease, and metabolic diseases
  • Parkinson's disease and metabolic diseases
  • Tables 3 and 4 of the Shieh review also mention a number of known openers and blockers, respectively, of various potassium channels and the disease indications for which they have been used/proposed.
  • Ca 2 1- modulators of which have been proposed as analgesics for mflammatory pain
  • Ca v 2.2 - - modulators of which have been proposed as analgesics for pain such as inflammatoiy pain, postsurgical pain, thermal hyperalgesia, chronic pain and mechanical allodynia
  • pain such as inflammatoiy pain, postsurgical pain, thermal hyperalgesia, chronic pain and mechanical allodynia
  • Ca v 3.3 which has been proposed as a target for the treatment of thalamic oscillations; and Ca v l. l, Ca v 1.4, Ca v 2.3, Ca v 3.1,; all of which can be screened using techniques such as patch-clamp, voltage-clamp and calcium imaging.
  • TRP Transient receptor potential
  • TRPC channels such as TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and
  • TRPV channels such as TRPV1, TRPV2, TRPV3, TRPV4, TRPV5 and TRPV6; TRPM channels such as TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6,
  • TRPP channels such as ⁇ 1, , PKD2L1 and PKD2L2, which are involved in
  • TRPML channels such as mucolipin 1, mucolipin 2 and mucolipin 3 ;
  • Na v l.l , Na v 1.2 and Na v l .3 - which are a target for drugs for the prevention and treatment of epilepsy and seizures;
  • Na v 1.4 - which is a target for local anaesthetics for the treatment of
  • Na v l .6 - which is a target for antiepileptic and analgesic drugs
  • GPCRs that represent cell associated antigens according to the present invention are provided in the following. Also listed are some exemplary therapeutic effects (with activating (agonistic) or inhibiting/blocking (antagonistic, inverse agonistic) properties) of immunoglobulin sequences of the present invention that are directed against these GPCRs.
  • GPCRs are involved in a wide area variety of physiological processes. Some examples of their physiological roles include:
  • CC chemokine receptor and/or CXC chemokine receptors bind ligands that mediate intercellular communication between cells of the immune system
  • receptors such as histamine receptors bind inflammatory mediators and engage target cell types in the inflammatory response
  • said cell- associated antigen is a membrane- spanning antigen, including but not limited to an antigen selected from CXCR7, CXCR4 and P2X7.
  • an antigen selected from CXCR7, CXCR4 and P2X7 selected from CXCR7, CXCR4 and P2X7.
  • natural conformation there may be different specific three dimensional conformations that are encompassed by the term "natural conformation". If, for example, a protein has two or more different conformations whilst being in a membrane environment, all these conformations will be considered “natural conformations”. This is exemplified by receptors changing their conformation by activation, e.g. the different activation states of rhodopsin induced by light, or ion channels showing a "closed” or “open” conformation.
  • the invention encompasses immunoglobulin sequences to any one of these different natural conformations, i.e. to the different kinds of conformational epitopes that may be present.
  • nucleic acid of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA.
  • the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).
  • the nucleic acid of the invention is in essentially isolated from, as defined herein.
  • the nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.
  • nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the cell associated antigen or immunoglobulin sequences of the invention, and/or can be isolated from a suitable natural source.
  • nucleotide sequences encoding naturally occurring V HH domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog.
  • nucleic acid of the invention also several nucleotide sequences, such as at. least one nucleotide sequence encoding a Nanobody® and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.
  • nucleic acids of the invention may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or li gated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more
  • mismatched primers using for example a sequence of a naturally occurring GPCR as a template.
  • the nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art.
  • Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein.
  • suitable regulatory elements such as a suitable promoter(s), enhancer(s), terminator(s), etc.
  • Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention”.
  • the genetic constructs of the invention may be DNA or RNA, and are preferably double- stranded DNA.
  • the genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism, or in a form suitable for genetic immunization.
  • the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon.
  • the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).
  • a genetic construct of the invention comprises a) at least one nucleic acid of the invention; operably connected to
  • one or more regulatory elements such as a promoter and optionally a suitable
  • regulatory element in which the terms "regulatory element”, “promoter”, “terminator” and “operably connected” have their usual meaning in the art (as further described herein); and in which said "further elements” present in the genetic constructs may for example be 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration.
  • suitable elements for such genetic constructs will be clear to the skilled person, and may for instance depend upon the type of construct used, the intended host cell, host organism or animal to be immunized; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g.
  • regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments may be used in an essentially analogous manner.
  • said at least one nucleic acid of the invention and said regulatory elements, and optionally said one or more further elements are selected from the genetic constructs of the invention.
  • a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription, and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of said promoter).
  • a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription, and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of said promoter).
  • the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism.
  • a promoter, enhancer or terminator should be '"operable" in the intended host cell or host organism, by which is meant that ⁇ for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked (as defined herein).
  • promoters for some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention - such as terminators, transcriptional and/or translational enhancers and/or integration factors - reference is made to the general handbooks such as Sanibrook et al. and Ausubel et al. mentioned above, as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO
  • the genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.
  • the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se.
  • suitable expression vectors are those used in the Examples below, as well as those mentioned herein.
  • the nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the Nanobody® or polypeptide of the invention, or for genetic vaccination.
  • Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
  • the immunoglobulin sequences, Nanobody ⁇ or polypeptide of the invention is glycosylated. According to another non-limiting embodiment of the invention, the immunoglobulin sequences, Nanobody ⁇ or polypeptide of the invention is non-glycosylated.
  • genetic vaccination mcludes any known methods or means to transfer a nucleic acid sequence, e.g. a DNA sequence, into a target animal that is suitable for inducing an immune response to a protein encoded by said nucleic acid sequence.
  • genetic vaccination can be performed by a needle-free jet injection, by a ballistic method, by needle-mediated injections such as tattoo, by topical application of the DNA onto the skin in patches or by any of these administration methods followed by in vivo electroporation, and furthennore includes vaccination performed by intradermal, intramuscular or subcutaneous administration of DNA.
  • cell based immunization includes any known methods or means to immunize animals with cells, e.g. cells expressing the antigen of interest.
  • cell based immunizations can be performed by transiently expressing the antigen or target of interest on the cells used to immunize or by stably transfected cells expressing the antigen or target of interest.
  • lipoprotein particles are membrane-enveloped virus- like particles (VLPs) containing high concentrations of structurally-intact membrane proteins on their surface (see J Biomol Screen 13(5), 2008, p 424 to 429; WO2005/042695).
  • non-human animal includes, but is not limited to vertebrate, shark, mammal, lizard, camelid, llama, preferably camelids and most preferably llama or alpaca.
  • CXCR4 includes, but is not limited to mouse, and/or human CXCR4 and most preferred human CXCR4, i.e. GenBank entry: AF005058.1 (SEQ ID NO: 1 :
  • cross-block In the context of the present invention, the terms “cross-block”, “cross-blocked” and “cross- blocking” are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a polypeptide of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target.
  • the extend to which an amino acid sequence or other binding agents of the invention is able to interfere with the binding of another to target, and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays.
  • One particularly suitable quantitative assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology.
  • Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequence or another binding agents in terms of their binding to the target.
  • the following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the invention. It will be appreciated that the assay can be used with any of the amino acid sequence or other binding agents described herein.
  • the Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations.
  • the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target.
  • test amino acid sequences (termed A* and B*) to be assessed for their ability to cross- block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture.
  • concentrations on a binding site basis the molecular- weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence.
  • the concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip.
  • the amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis).
  • Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix.
  • the test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target.
  • a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such thai during the assay and in the presence of a second amino acid sequence or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1 % (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1 % (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination.
  • the Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CMS Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version (R & D Systems, Minneapolis, MN, USA; 2005 cat# 1406-ST-025).
  • an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence.
  • the cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface.
  • C-terminal His-tagged target could alternatively be used.
  • various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin).
  • the general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules.
  • the plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target.
  • the amount of bound target is then measured using a reagent that is appropriate to detect the target.
  • An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence.
  • the first amino acid sequence e.g.
  • an Ab-X is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added.
  • An excess amount of the second amino acid sequence, i.e. Ab-Y is then added to the ELISA plate such that the moles of Ab-Y antigen binding sites per well are at least 10 fold higher than the moles of Ab-X antigen binding si tes that were used, per well, during the coating of the ELISA plate, antigen is then added such that the moles of antigen added per well are at least 25-fold lower than the moles of Ab-X antigen binding sites that were used for coating each well.
  • the ELISA plate is washed and a reagent for detecting the target is added to measure the amount of target specifically bound by the coated anti-antigen amino acid sequence (in this case Ab-X).
  • the background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence (in this case Ab-Y), antigen buffer only (i.e. no target) and target detection reagents.
  • the positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence buffer only (i.e. no second solution phase amino acid sequence), target and target detection reagents.
  • the ELISA assay may be mn in such a manner so as to have the positive control signal be at least
  • the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution.
  • Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal (i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti- target amino acid sequence (i.e. the positive control wells).
  • the present invention relates to a method for the generation of immunoglobulin sequences that can bind to and/or have affinity for a cell-associated antigen, as defined herein.
  • the method comprises, but is not limited, to the following steps:
  • the method of the present invention includes screening a set, collection or library of immunoglobulin sequences derived from said non-human animal for amino acid sequences that can bind to and/or have affinity for said cell-associated antigen and wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles as defined herein, in one particular embodiment, the immunization, boosting and/or screening is done in the same cell background.
  • One particular advantage of the present invention resides in the fact that it provides a robust method for generating immunoglobulin sequences thai produces superior results, i.e. obtain more variants of functional binders, to complex antigens such as GPCRs and ion channels. In particular, there is no requirement when working in the same background for immunization and screening to counter select with lipoparticles without the antigen of interest.
  • the method also results in high frequency isolation of immunoglobulm sequences directed to complex antigens.
  • the present invention is advantageous as compared to prior art methods that lack such robust and high performance applicability.
  • the present invention provides an improved method for generating
  • immunoglobulin sequences against cell -associated antigens which, according to one specific embodiment, is without the need for counter selection with lipoparticles without the antigen of interest, by inducing an immune response via cell based immunization or DNA vaccination and subsequent screening with lipoprotein particles for immunoglobulin sequences that can bind the cell-associated antigen.
  • immunoglobulin sequences e.g. Nanobodies®
  • Nanobodies® immunoglobulin sequences
  • the affinity of the identified binders was high and for some of the newly found immunoglobulin sequences even higher than 2 binders found with the standard approach (see Examples).
  • the present invention provides a method for the generation of immunoglobulin sequences, including Nanobodies®, against a cell-associated antigen comprising the steps of:
  • genetic vaccination or cell based immunization suitable for inducing an immune response in the animal is performed. More specifically, the
  • immunization must be suitable to induce an immune response as reflected in the generation of immunoglobulin sequences in the animal.
  • the detection of an antibody response in the serum of the animal is also referred to as "serum conversion".
  • the skilled person can monitor the immunization success by determining the antibody response by routine means. Thus, the skilled person can readily determine the adequate dosage and frequency that is required for inducing an appropriate antibody response.
  • the immunization will induce an adequate antibody litre.
  • the antibody titre will correspond to the number of specific antibody producing cells, which will allow the generation of immunoglobulin sequences by isolation and/or screening.
  • Serum titres can be determined by conventional methods, including e.g.
  • the antigen is enriched in any of the cell based immunization preparations, in order to strengthen the immune response.
  • recombinant expression in cells using highly efficient promoters can be used to increase the quantity of antigen per cell.
  • the cells expressing the antigen of interest can be camelid derived cells, preferably immortalized camelid derived cells. The cells will be genetically modified to express the said antigen.
  • the invention also encompasses the use of an adjuvant commonly used in order to enhance an immune response in the context of vaccination.
  • the protein preparation may also be in a physical form that enhances the immune response, such as e.g. a gel or emulsion.
  • an adjuvant include Stimune or Specol (CEDI Diagnostics, Lelystad, The Netherlands), Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, TiterMax (Gold),
  • MPL monophosphoryl lipid A
  • Alum Alum
  • QuilA CpG DNA
  • a further aspect of importance for the present invention is the breadth of the antibody repertoire obtained by the methods of the invention.
  • the antibody response is directed to both linear and conformational epitopes, and importantly is directed to membrane dependent conformational epitopes.
  • the present invention relates to a method suitable for obtaining an antibody response of an adequate litre and breadth in the non-human animals.
  • immunoglobulin sequences can in one embodiment of the invention be directly isolated from said animal, i.e. without protein boost, by methods as described herein. Detection of antibody responses can be done by routine means, such as ELISA, RIA, FACS, or any other method for detecting antibodies. Protein boost
  • the method also includes boosting the animal with a suitable source of protein, in particular it is envisaged to boost the animal with compositions that comprise the cell associated antigen as defined herein, in particular a transmembrane antigen, in its natural conformation.
  • compositions may comprise cells expressing the antigen, or fragments or derivatives of the cell, such as membrane fractions, isolated organelles, or other suitable preparations.
  • viruses, liposomes, micelles, lipoprotein particles or other systems that are suitable for containing the cell associated antigen in its natural conformation.
  • the antigen can be expressed on a homologous cell.
  • the antigen for immunization of a camelid, can be expressed on a carnelid cell.
  • the camelid immune system will be tolerant to the camelid cell, i.e. it will not mount an immune response to most of the antigens comprised in this cell.
  • a heterologous antigen including but not limited to cell associated antigens as defined herein, is artificially introduced into said cell, the immune system of the animal will mount an immune response specifically directed to said antigen.
  • This has the advantage that the immune response will be mainly directed to the antigen of interest, i.e. it will be characterized by an enhanced specificity towards this antigen.
  • camel derived cells can be used for immunization of llama, and vice versa, in view of their close relationship.
  • Any suitable cell mat is homologous to the animal to be immunized can be used.
  • camelid cells can be used for immunization of camelids, e.g. llama cells for immunization of llama.
  • Suitable cells will comprise, but are not limited to, cells that are spontaneously immortal, e.g. cancer cells or undifferentiated cells, such as embryo-derived cells.
  • Suitable cells also encompass cells immortalized artificially by known means. Cells can advantageously be treated prior to administration to the animals, such that then- proliferation in vivo is reduced or eliminated.
  • Suitable treatments comprise, but are not limited to chemical and physical treatments.
  • One specific example of a suitable physical treatment is irradiation with X rays such that the cells can no longer proliferate.
  • the protein is enriched in any of the above prepai-ations, in order to strengthen the immune response.
  • recombinant expression in cells using highly efficient promoters can be used to increase the quantity of antigen per cell.
  • the cells expressing the antigen of interest can be camelid derived cells, preferably immortalized camelid derived cells, e.g. HEK293, HEK293T, HE 293H.
  • the cells will be genetically modified to express the said antigen.
  • the invention also encompasses the use of an adjuvant commonly used in order to enhance an immune response in the context of vaccination.
  • the protein preparation may also be in a physical, form that enhances the immune response, such as e.g. a gel or emulsion.
  • an adjuvant include Stimune or Specol (CEDI Diagnostics, Lelystad, The Netherlands), Freund's Complete Adjuvant, Freund's incomplete Adjuvant, TiterMax (Gold), monophosphoryl lipid A (MPL), Alum, QuilA, CpG DNA.
  • the present invention comprises a single or multiple boosts with the said source of protein in its natural conformation (optionally using an adjuvant).
  • the protein boosts will be performed at suitable intervals, which can be determined by routine means, e.g. by monitoring the immunoglobulin response in the animals.
  • the boost can be performed by different routes of administration, including, but not limited to, intradermal, subcutaneous, or intramuscular administration. Screening/isolating immunoglobulin sequences
  • Isolation includes a) the separation of sequences from the animal, e.g. by sampling suitable tissues, and b) the singling out of specific sequences e.g. by screening, i.e. the isolation of "hits" of specific binders.
  • VHH libraries obtained form the animals immunized in accordance with the present invention, may be used, such as VHH libraries obtained from V H H libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.
  • the invention includes the isolation of material from the animal which comprises immunoglobulin sequences, such as, but not limited to, antibody producing cells.
  • material from the animal which comprises immunoglobulin sequences, such as, but not limited to, antibody producing cells.
  • PBMCs peripheral blood monocytes
  • Other material includes peripheral blood lymphocytes (PBLs), peripheral lymph nodes, in paiticular lymph nodes draining the site of immunization, the spleen, bone marrow, or other immunologically relevant materials.
  • B-cell containing blood samples can be collected, and peripheral, blood lymphocytes (PBLs) can be purified by standard methods.
  • PBLs peripheral, blood lymphocytes
  • a density gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) can be employed according to the manuf cturer' s instructions.
  • any of the above described material including e.g. PBLs isolated from the animal will comprise a multitude of immunoglobulin sequences, i.e. a set, collection or library of immunoglobulin sequences.
  • immunoglobulin sequences i.e. expressed on PBMCs
  • the desired immunoglobulin specificities can be directly isolated, e.g. by immunopanning of the cells.
  • immunoglobulin sequences can be isolated, transferred, and expressed on a set, collection or sample of cells or viruses.
  • the genetic material can be isolated and processed further by suitable means to isolate such sequences that code for the immunoglobulin sequences of the desired specificity.
  • suitable means e.g. the nucleic acid sequences encoding the said multiplicity of immunoglobulin sequences can be extracted from the material by suitable means, and transferred into a recipient cell or virus for expression.
  • suitable techniques for extraction of immunoglobulin sequences and manipulating these sequences for expression e.g. in an expression library in cells or viruses.
  • Some non-limiting examples comprise the generation of an expression library in e.g. E. coli or bacteriophages.
  • total RNA can be extracted from the said material.
  • the total RNA can be converted into cDNA by known means.
  • immunoglobulin sequences such as e.g. the Nanobody® repertoire, can be amplified by routine means, including e.g. PCR, or nested PCR methods as known in the art (see patent references above).
  • Nucleic acid molecules comprising immunoglobulin sequences can be digested by use of suitable restriction enzymes, optionally followed by purification e.g. by gel electrophoresis.
  • the digested sequences can be ligated into corresponding restriction sites in a suitable genetic construct, such as a vector or plasmid.
  • suitable vectors include phage display vectors, e.g. pAX50.
  • pAX50 contains the LacZ promoter, a coliphage pill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site (harboring the Sfil and BstELl restriction sites) and a chimeric leader sequence consisting of gene3 and Erwinia caroiovora pelB motifs.
  • This display vector allows the production of phage particles, expressing the individual Nanobodies® as a fusion protein with the genelll product.
  • the ligated nucleic acid molecule can be used to obtain a library, e.g. by transformation of a suitable host organism, like E. coli.
  • suitable techniques of transformation e.g. chemical methods, electroporation, and others.
  • a library of a suitable size e.g. 1E7 to 1E8, can be obtained.
  • a set, collection or library of cells or viruses is screened for lipoprotein particles that express immunoglobulin sequences that can bind to and/or have affinity for said cell-associated antigen, more specifically, a nucleic acid sequence that encodes the immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen can be purified and/or isolated from the cell or virus, followed by expression of said amino acid sequence.
  • the present invention also encompasses suitable screening step(s), to select and isolate the immunoglobulin sequences directed to the antigen of interest (or nucleic acid sequences encoding the same) from a multitude of sequences present in the non-human animal.
  • suitable screening step(s) to select and isolate the immunoglobulin sequences directed to the antigen of interest (or nucleic acid sequences encoding the same) from a multitude of sequences present in the non-human animal.
  • suitable techniques including phage display, immunopanning, etc.
  • the invention aiso relates to combinations of known metliods. Suitable combinations will be apparent to the skilled person.
  • the library of phages expressing immunoglobulin sequences can be selected by a single round, or multiple rounds of panning on a suitable source of lipoprotein particles comprising cell-associated antigen, including, but not limited to lipoprotein particles comprising highly enriched antigen.
  • the antigen of interest within the lipoprotein particles are typically enriched 10 to 100 fold, preferably 50 to 100 fold, compared with cells or membrane preps (measuring specific membrane protein per total protein concentration).
  • the concentration of the antigen of interest compared to the total protein concentration within the lipoprotein particles is approximately 1% or less, more preferably 5% or less when measured by sypro staining (see e.g. Sypro protein detection staining kit).
  • the output can be recloned as a pool into a suitable expression vector for further selection and/or processing.
  • the immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen can be purified and/or isolated.
  • further characterization of the immunoglobulin sequences e.g. binding affinity, or avidity measurements, can be advantageously be performed by the use of lipoprotein particles in immunoglobulin phage ELISA, and immunoglobulin periplasmic ELISA.
  • the skilled person can use standard techniques for such characterizations.
  • the skilled person can use standard techniques for production of immunoglobulins.
  • the said immunoglobulin sequence can be produced, e.g.
  • the cell or virus can be used directly, or the nucleic acid encoding the immunoglobulin sequence can be transferred into a suitable expression system, including a suitable host cell.
  • Host cells include mammalian systems, such as CHO cells, eukaryotic systems such as insect cells or fungi, including e.g. Pichia pastoris, and prokaryotic systems such as E. coli.
  • suitable expression vectors and tools for use in expressing immunoglobulin sequences in these host systems are known in these host systems.
  • the immunoglobulin sequences, Nanobodies ⁇ and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from, the description herein. The skilled person will understand which of the specific examples are suitable for the generation and/or screening of sets, collections or libraries of immunoglobulin sequences, or for the production of immunoglobulin sequences after selection of antigen specific sequences.
  • the polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments).
  • one particularly useful method for preparing a polypeptide of the invention generally comprises the steps of: the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said Nanobody® or polypeptide of the invention (also referred to herein as a "nucleic acid of the invention", this term is also used for the genetic constructs for vaccination, as will be apparent from the specific context), optionally followed by: isolating and/or purifying the Nanobody® or polypeptide of the invention thus obtained.
  • the produced immunoglobulins can be purified by standard techniques, including precipitation, affinity chromatography, size exclusion chromatography, ion exchange chromatography, HPLC, filtration, and other known purification methods.
  • immunoglobulin sequences can be further characterized by known methods, e.g. to determine their epitope specificity, binding kinetics, etc.
  • the invention also relates to immunoglobulin sequences, i.e. the polypeptide molecules, obtainable by a method as described herein, and compositions comprising the said immunoglobulin sequences.
  • Such compositions comprise compositions for research purposes as well as pharmaceutical compositions for use in therapy.
  • the skilled person is familiar with standard techniques and formulations for therapeutic application of immunoglobulin sequences.
  • the method of the present invention encompasses the purification of specific immunoglobulin sequences and their formulation as a pharmaceutical composition.
  • the present invention provides immunoglobulin sequences in essentially isolated form, e.g. in a form that is at least 90% pure, at least 95% pure, at least 98%, at least 99%, or at least 99.99% pure.
  • purity means that no sequences of other immunoglobulins are present in the preparation.
  • purity means that no contaminants from the producing organism are present in the composition.
  • the present invention also encompasses immunoglobulin sequences that are derivatives of the immunoglobulin sequences obtainable by the methods disclosed herein.
  • the invention encompasses humanized immunoglobulin sequences obtainable by methods known in the art.
  • the invention encompasses camelized immunoglobulin sequences, also obtainable by methods known in the art.
  • the invention also encompasses known structural variants of immunoglobulin sequences.
  • Immunoglobulin sequences obtainable by the methods
  • conformational epitopes and in particular membrane-dependent conformational epitopes are of particular interest as targets for immunoglobulin sequences.
  • the pore of an ion channel represents a target of primary therapeutic importance.
  • the present invention provides for the generation of immunoglobulin sequences to such kind of conformational epitope.
  • the invention relates also to immunoglobulin sequences that were obtained by using the method described herein.
  • the invention relates to a Nanobody® (as defined herein) against CXCR4, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:
  • CDR1 is chosen from the group consisting of:
  • amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1.2 to 21 ; c) amino acid sequences that have 3, 2, or i amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 12 to 21 ;
  • CDR2 is chosen from the group consisting of:
  • amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 32 to 41 ;
  • amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 32 to 41;
  • CDR3 is chosen from the group consisting of:
  • amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 52 to 71 ;
  • amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 52 to 71 ;
  • the invention relates to a Nanobody® (as defined herein) against human CXCR4, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRI to CDR3 respectively), in which:
  • CDRl is chosen from the group consisting of:
  • amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 12 to 21 ;
  • amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 12 to 21 ;
  • CDR2 is chosen from the group consisting of:
  • amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 32 to 41; f) amino acici sequences that have 3, 2, or 3. amino acid difference with at least one of the amino acid sequences of SEQ ID NO' s: 32 to 41;
  • CDR3 is chosen from the group consisting of:
  • amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 52 to 71 ;
  • amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO' s: 52 to 71 ;
  • Nanobody ⁇ of the invention contains one or more CDRl sequences according to b) and/or c):
  • any amino acid substitution in such a CDR according to b) and/or c) is preferably, and compared to the corresponding CDR according to a), a conservative amino acid substitution (as defined herein);
  • the CDR according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to a);
  • the CDR according to b) and/or c) may be a CDR that is derived from a CDR according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.
  • Nanobody ⁇ of the invention contains one or more CDR2 sequences according to e) and/or f):
  • any amino acid substitution in such a CDR according to e) and/or f) is preferably, and compared to the corresponding CDR according to d), a conservative amino acid substitution (as defined herein);
  • the CDR according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to d);
  • the CDR according to e) and/or f) may be a CDR that is derived from a CDR according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.
  • Nanobody® of the invention contains one or more CDR3 sequences according to h) and/or i):
  • any amino acid substitution in such a CDR according to h) and/or i) is preferably, and compared to the corresponding CDR according to g), a conservative amino acid substitution (as defined herein);
  • the CDR according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to g);
  • the CDR according to h) and/or i) may be a CDR that is derived from a CDR according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.
  • Nanobody® of the invention that comprises one or more CD l sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e), f), h) or i), respectively.
  • Nanobodies® of the invention comprising one or more of the CDR's explicitly listed above are particularly preferred; Nanobodies® comprising two or more of the CDR' s explicitly listed above are more particularly preferred; and Nanobodies® comprising three of the CDR' s explicitly listed above are most particularly preferred.
  • CDR sequences are particularly preferred, but non-limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table A-l below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies® of the invention.
  • Table A-l lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies® of the invention.
  • a combination of CDRl, CDR2 and CDR3 sequences that occur in the same clone i.e. CDRl , CDR2 and CDR3 sequences that are mentioned on the same line in Table A-1
  • will usually be preferred although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table A- 1).
  • CDR sequences and framework sequences that occur in the same clone i.e. CDR sequences and framework sequences that are mentioned on the same line in Table A-1
  • CDR sequences and framework sequences that are mentioned on the same line in Table A-1 will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table A- 1, as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein).
  • each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR's; in which:
  • any amino acid substitution in such a CDR is preferably, and compared to the
  • any such CDR sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR sequence mentioned in Table A-1 ;
  • any such CDR sequence is a CDR that is derived by means of a technique for affinity maturation known per se, and in particular starting from the corresponding CDR sequence mentioned in Table A- 1.
  • At least one of the CDR1 , CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A- 1 ; or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% "sequence identity" (as defined herein) with at least one of the CD 1 , CDR2 and CDR3 sequences, respectively, listed in Table A ⁇ l ; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with at least one of the CDR1 , CDR2 and CDR3 sequences, respectively, listed in Table A- 1.
  • a CDR1 sequence is chosen from suitable CDR1 sequences (i.e. as defined herein)
  • a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein)
  • a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively.
  • the CDR sequences are preferably chosen such that the Nanobodies® of the invention bind to GPCRs with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a K A - value (actual or apparent), a k on -rate and/or a k 0 irrate, or alternatively as an IC5 0 value, as further described herein) that is as defined herein.
  • At least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table
  • A-l or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-l; and/or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table A-l .
  • At least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-l or from the group consisting of CDR1 , CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-l ; and/or from the group consisting of the CDR I, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" with at least one of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-1.
  • At least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1, respectively; and at least one of the CD l and CDR2 sequences present is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table A-1 or from the group of CDRl and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at
  • all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group of CDRl, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-1 ; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
  • CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-1.
  • at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at ieast one of the corresponding CDR sequences, respectively, listed in Table A-1 ; and/or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table A-L
  • the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table A- l .
  • At least one and preferably both of the CDR l and CDR2 sequences present are suitably chosen from the groups of CDRl and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDRl and CDR2 sequences, respectively, listed in Table A- l ; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CD l and CDR2 sequences, respectively, listed in Table A- L
  • the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-l .
  • the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A- l ; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table A-l ,
  • the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table A- l, and either the CDRl sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table A-l ,
  • the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A- l ; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table A-l .
  • a CDR in a Nanobody® of the invention is a CDR sequence mentioned in Table A-1 or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table A-1, that at least one and preferably both of the other CDR's are suitably chosen from the CDR sequences that belong to the same combination in Table A-1 (i.e.
  • a Nanobody ⁇ of the invention can for example comprise a CDRl sequence that has more than 80 % sequence identity with one of the CD l sequences mentioned in Table A-1, a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination), and a CDR3 sequence.
  • Nanobodies® of the invention may for example comprise: (1 ) a
  • Nanobodies® of the invention may for example comprise:
  • a CDRI sequence that has more than 80 % sequence identity with one of the CDRI sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table A- 1. that belongs to the same combination; and a CDR3 sequence that has more than 80 % sequence identity with the CDR3 sequence mentioned in Table A- 1 that belongs to the same combination; (2) a CDRI sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed in Table A-1 (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).
  • Nanobodies® of the invention may for example comprise: (1 ) a CDRI sequence that has more than 80 % sequence identity with one of the CDRI sequences mentioned in Table A- 1; the CDR2 sequence listed in Table A-1 that belongs to the same combination; and a CDR3 sequence mentioned in Table A-] that belongs to a different combination; or (2) a CDRI sequence mentioned in Table A- 1 ; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than. 80% sequence identity with the CDR3 sequence listed in Table A- 1 that belongs to the same or a different combination.
  • Nanobodies® of the invention may for example comprise a CDRI sequence mentioned in Table A-1, a CDR2 sequence that has more than 80 % sequence identity with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and the CDR3 sequence mentioned in Table A-1 that belongs to the same combination.
  • the CDRI, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDRI , CDR2 and CDR3 sequences, respectively, listed in Table A-1.
  • CDRI has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and/or
  • CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues; and/or
  • CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues.
  • the invention relates to a Nanobody ⁇ in which the CDR sequences (as defined herein) have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 81, more preferably SEQ ID NO: 72 to 77, 79, 80, even more preferably SEQ ID NO: 74, 76, 77, 79, 80, listed in Table A-2,
  • the invention relates to a Nanobody® with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 81, more preferably SEQ ID NO: 72 to 77, 79, 80, listed in Table A-2.
  • the invention relates to a Nanobody ⁇ with the amino acid sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 81 , more preferably SEQ ID NO: 72 to 77, 79, 80, listed in Table A-2.
  • Method for the generation of immunoglobulin sequences that can bind to and/or have affinity for a cell-associated antigen comprising the steps of:
  • cell-associated antigen is selected from transmembrane antigens, including transmembrane antigens with multiple spanning domains, including but not limited to GPCRs or ion channels.
  • non-human animal is selected from vertebrates such as sharks, lizards, and mammals, more specifically camelids such as llama and alpaca.
  • immunoglobulin sequences are light chain variable domain sequences, or heavy chain variable domain sequences.
  • immunoglobulin sequences are heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain, variable domain sequences that are derived from a heavy chain antibody.
  • immunoglobulin sequences are domain antibodies, or amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, or amino acid sequences that are suitable for use as single domain antibodies, "dAbs", or amino acid sequences that are suitable for use as dAbs, or Nanobodies®, including but not limited to V H H sequences or amino acid sequences that are suitable for use as Nanobodies ⁇ .
  • the said cell-associated antigen is expressed in high concentration on lipoprotein particles, e.g. in a concentration of 0.1 ug/ml or higher, more preferably in a concentration of 0.2 ug/ml or higher, even more preferably in a concentration of 1 ug/ml or higher.
  • said cell-associated antigen is expressed on any cell or lipoprotein particle which allows expressing of the target in its native conformation such as but not limiting to a cell selected from Cho, Cos7, Hek293 t or camelid derived cells such as Llama derived or Alpaca derived cell and/or a lipoprotein selected from virus-like particles.
  • said cell-associated antigen is a membrane-spanning antigen such as e.g. a GPCR and/or ion channel.
  • antigen is selected from CXCR7, CXCR4 and P2X7.
  • nucleic acid sequence that encodes the amino acid sequence that can bind to and/or has affinity for said cell-associated antigen is purified and/or isolated from the cell or virus, followed by expression of said amino acid sequence.
  • any of aspects 1 to 1.6 wherein the set, collection or libraiy of immunoglobulin sequences is encoded by a set, collection or library of nucleic acid sequences and said set. Collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode an immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen.
  • Immunoglobulin obtainable by a method of any one of aspects 3 to 19 such as e.g. a Nanobody® with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 82, more preferably SEQ ID NO: 74, 76, 77, 79, 80, 82.
  • Composition comprising the immunoglobulin sequence according to aspect 20.
  • Method for stem-cell mobilization comprising administering, to a subject in need thereof, a pharmaceutically active amount of i) at least one immunoglobulin directed against CXCR4, e.g. human CXCR4, ii) compound or construct comprising an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) bispecific or multispecific construct comprising at ieast an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) bispecific or multispecific construct comprising at ieast an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) compound or construct comprising an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) bispecific or multispecific construct comprising at ieast an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) compound or construct comprising an immunoglobulin directed against CXCR4, e.g.
  • immunoglobulin directed against CXCR4, e.g. human CXCR4, or iv) composition comprising said immunoglobulin, compound or construct, bispecific or multispecific construct.
  • composition comprising said immunoglobulin, compound or construct, bispecific or multispecific construct in the manufacture of a medicament for prevention and/or treatment of stem-cell mobilization.
  • immunoglobulin directed against CXCR4, e.g. human CXCR4, or iv) composition comprising said immunoglobulin, compound or construct, bispecific or multispecific construct for use in the prevention and/or treatment of stem-cell mobilization.
  • Method for stem-cell mobilization comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one immunoglobulin according to aspect 20, compound or construct comprising an immunoglobulin according to aspect 20, bispecific or multispecific construct comprising at least an immunoglobulin according to aspect 20, or composition according to aspect 21.
  • An immunoglobulin according to aspect 20 compound or construct comprising an immunoglobulm according to aspect 20, bispecific or multispecific construct comprising at least an immunoglobulin according to aspect 20, or composition according to aspect 21 for use in the prevention and/or treatment of stem-cell mobilization.
  • Example 1 CXCR4-specific immune responses are not detectable in serum of llama immunized with HEK293-CXCR4 cells using CXCR4 lipoprotein particles.
  • Llama 217 and 218 were immunized with HEK293T cells transiently expressing
  • Example 2 CXCR4+ lipoprotein particles are recognized by purified CXCR4-specific Nanobodies® 238D2 and 238D4
  • Nanobodies® 238D2 or 238D4 were tested for binding to CXCR4+ and CXCR4- (null) lipoprotein particles. Wells were coated with 10, 1 and 0.1 U of the particles. After blocking with 4% Marvel in PBS, 100, 10 and 1 nM of Nanobodies® 238D2, 238D4 and of an irrelevant control Nanobody® (g l202E6) were added to the wells. Other positive and negative controls included the use of 10 nM 12G5 (a CXCR4- specific mouse monoclonal, antibody) and 10 nM OKT3 (a mouse monoclonal antibody that recognizes CD3), respectively.
  • 10 nM 12G5 a CXCR4- specific mouse monoclonal, antibody
  • 10 nM OKT3 a mouse monoclonal antibody that recognizes CD3
  • Bound Nanobodies® were detected by adding 1 ug/ml mouse anti-myc (Roche Cat 11667149001). All mouse antibodies were detected by rabbit anti-mouse-HRP, followed by TMB substrate. Binding of Nanobodies® 238D2 and 238D4 was detected clearly to 10 U of CXCR4+ particles but not to 10 U of CXCR4- particles ( Figure 2).
  • Figure 2 shows twelve sets of bar graphs, with each set of bar graphs depicting seven data points: Null Lip (10U, 1U, 0.1U) and CXCR4 Lip (10U, 1.U, 0..1.U) and NC, from left to right. The y-axis indicates the OD at 450 nm.
  • Figure 3 shows seven sets of bar graphs, with each set of bar graphs depicting five data points: Lipo Null (10U, 1 U) and Lipo CXCR4 (10U, 1U) and NC, from left to right. The y-axis indicates the OD at 450 nm.
  • Example 4 CXCR4+ lipoprotein particles are recognized by phages that display 238D2 and 238D4.
  • Phages displaying 238D2 and 238D4 were produced and purified. Wells were coated with 2 U of the CXCR4+parti.cles. After blocking with 4% Marvel in PBS, phage diluted 100-fold in 2% Marvel/PBS were added to the wells. Phage displaying 212C 12 (an unrelated Nanobody®) or no phage addition were used as control. Phages binding to CXCR4+p articles were detected by an anti-M13-HRP antibody. A clear signal was observed with 238D2 and 238D4 phages while no interaction was observed for the irrelevant Nanobody® used as negative control ( Figure 4). The y-axis indicates the OD at 450 nm
  • Example 5 Selections with library 218 on CXCR4+ particles results in isolation of large numbers of Nanobodies® binding to CXCR4+ particles.
  • Nanobodies® 238D2, 238D4 and 238C4 were completely identical to the amino acid of 238D2 and the amino acid sequences of clones 281B5, 281H10, 28 I E11 , 281F4 were completely identical to the amino acid sequence of 238D4 ( Figure 5d).
  • the amino acid sequences of 281E10 and 281B6 were identical to the amino acid sequence of 238C4.
  • the amino acid sequence of the 24 other Nanobodies® showed very high similarity to the amino acid of 238D4 ( Figure 5d).
  • a total of five 238D4 subfamilies could be defined.
  • Five more families were defined (Families 281A4, 281 C4, 281F6, 281F1 1 and 281F12) which consist of Nanobodies® that display very low CXCR4+ specific binding. These Nanobodies® were produced, purified and tested for binding to CXCR4+ and CXCR4- particles. A clear dose-dependent interaction was observed only with Nanobody® 281F12. No binding to either particle was observed for the other purified Nanobodies® ( Figure 5e).
  • Figure 5e shows sixteen sets of bar graphs, with each set of bar graphs depicting five data points: 200 nM, 100 nM, 10 nM, 1 nM and 0, from left to right
  • the y-axis indicates the OD at 450 nm.
  • Periplasmic extracts were prepared, diluted 10-fold and added to wells coated with 2 U of CXCR4+ and CXCR4- particles. As positive controls periplasmic extracts of 238D2, 238D4 and 281A12 were used. Periplasmic extract of 2E6 was used as a negative control. Bound Nanobodies® were detected with mouse anti-myc followed by rabbit anti-mouse-HRP and TMB.
  • Example 7 Selections with library 217 on CXCR4+ particles results in isolation of large numbers of Nanobodies® binding to CXCR4+ particles.
  • Nanobody® sequences of 34 clones were obtained (data not all shown, for some see Table A-2). Thirty Nanobodies ⁇ had the same amino acid sequence and a family 283B6 was defined. One more Nanobody® 283E2 also belonged to this family as only one amino acid difference was observed. Nanobody® 283F1 which also showed specific binding to CXCR4+ particles had a different amino acid sequence; family 283F1 was defined.
  • Nanobodies® 283C2 and 283C5 which did not bind particles belonged clearly to other families, although their CDR2 sequences showed very high similarity to the CDR2 sequences of families 283E6 and 283FL
  • phages displaying these Nanobodies® were produced and purified. Binding of these phages to CXCR4+ particles was competed with purified 238D2 and 238D4. Phages displaying 238D2 and 238D4 were used as controls. The assay was performed in duplicate, allowing detection of either phage (anti-M 13-HRP) or Nanobody® (anti-myc, anti mouse -HRP). As depicted in figure 7d and 7e, binding of phage to CXCR4+ particles was inhibited completely upon co-incubation with purified 238D4.
  • Figure 7d shows seven sets of bar graphs, with each set of bar graphs depicting three data points: 283D2 Nb, 283D4 Nb, and No Nb, from left to right.
  • the y ⁇ axis indicates the OD at 450 nm.
  • Figure 7e shows two sets of bar graphs, with each set of bar graphs depicting seven data points: 283d2 phage, 283 D4 phage, 283 B6 phage, 283 E2 phage.
  • 283 Fl phage, 212-C12 phage and no phage from left to right.
  • the y-axis indicates the OD at 450 nm.
  • Nanobodies ⁇ 283B6 and 283E2 indeed bind to CXCR4 on the particles. Moreover the data indicates that 283B6 and 283E2 bind to a similar or overlapping region also recognized by 238D2 and 238D4.
  • Nanobodies® were identified which were previously not isolated using membrane extracts.
  • Example 8 Binding of Nanobodies® selected on CXC 4+ particles to HEK293T- CXCR4 expressing cells
  • Nanobodies® selected on CXCR4+ particles recognize cell- surface-native form of the receptor.
  • a Flow cytometry experiment was performed where the Nanobodies® were tested for specific binding to HEK293T-CXCR4 expressing cells.
  • FACSArray FACS system Only live, intact cells were included in the binding analysis by pre ⁇ gating on intact cells using a first forward/side scatter dot plot defined gate and then subgated further on a TOPR03 fluorescence negative population within the first.
  • the PE fluorescence intensity of these pre-gated cells was expressed as the median channel number of the PE detector histogram, as an indicator of Nanobody® binding intensity.
  • Negative controls included stainings with irrelevant specificity Nanobody ⁇ cione 212C12 followed by anti myc tag and goat anti mouse, or no Nanobody ⁇ followed by anti ⁇ myc and goat anti mouse, or mouse anti human CXCR7 monoclonal staining followed by goat anti mouse secondary ( Figure 8a).
  • vss Four cynomolgus monkeys were administered with AMD3100 or 238D2-20GS-238D4 in a staggered administration scheme.
  • AMD3100 Malignant® or Plerixafor, Genzyme
  • 238D2-20GS-238D4 was administered by a 30 minute infusion in several doses (0.1 , 1, 10, 25 mg/kg) in D-PBS (Gibco).
  • the relative number of mobilized stem cells in the peripheral blood was determined by a dual-platform flow cytometric analysis of CD34+ cells, according to the ISHAGE guidelines (Sutherland DR et al.: The IS H AGE guidelines for CD34+ cell determination by flow cytometry. J. Hematother 5:213-226 (1996); Gratama JW et al: Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. Cytometry 34: 128- 142 (1998); Barnett D et al: Guideline for the flow cytometric enumeration of CD34+ haematopoietic stem cells. Clin. Lab. Haem.21 :301-308 (1999).

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Abstract

The present invention relates to a method for generating immunoglobulin sequences against cell-associated antigens, more particularly, antigens that are membrane-anchored. The invention also provides immunoglobulin sequences obtainable by the method of the invention. Specifically, the present invenlion relates to the generation of immunoglobulin sequences by using lipoprotein particles. More specifically, the present invention relates to generation of immunoglobulin sequences in camelids, preferably directed against cell-associated antigens, in particular antigens with multiple transmembrane spanning domains, including GPCRs and ion channels, by using lipoprotein particles. Furthermore, the invention relates to novel immunoglobulin sequences, constructs and uses thereof.

Description

Method for generation of immunoglobulin sequences by using lipoprotein particles
Field of the invention
The present invention relates to a method for generating immunoglobulin sequences against cell-associated antigens, more particularly, antigens that are membrane-anchored. The invention also provides immunoglobulin sequences obtainable by the method of the invention. Specifically, the present invention relates to the generation of immunoglobulin sequences by using lipoprotein particles. More specifically, the present invention relates to generation of immunoglobulin sequences in camelids, preferably directed against cell- associated antigens, in particular antigens with multiple transmembrane spanning domains, including GPCRs and ion channels, by using lipoprotein particles. Furthermore, the invention relates to novel immunoglobulin sequences, constructs and uses thereof.
Technological Background
Enveloped viruses have been shown to expand their tropism by acquiring envelope proteins of other viruses during co-infection of a cell by different enveloped viruses. This process is called phenotypic mixing or pseudotyping. Pseudotyped viruses can be generated experimentally by expressing a different viral envelope protein in a (retro) viral infected cell. In addition to other viral membrane proteins, viruses can also incorporate host cell membrane proteins, including viral receptor proteins. For example MHC class I, MHC class II, ICAM-1, I.CAM-2, ICAM-3, CR3, CR4, CD4, CD44, CD46, CD55, CD59, CD73 and CD71 have been found in HIV-L Viruses that incorporate host membrane proteins of interest can be generated by over expressing this protein of interest in a (retro) viral infected cell.
Especially retroviruses have been used widely to generate and produce pseudotyped viruses, because of the capacity of retroviral GAG core proteins to assemble into core particles and to bud from the cell, incorporating host membrane proteins during this budding process. In fact, only expression of retroviral GAG core proteins is sufficient to induce formation of pseudotyped viruses. Pseudotyped viruses or particles are produced by inducing cells to express high levels of the protein of interest and a retroviral GAG core protein. A number of factors influence the uptake efficiency of host proteins like surface density of the membrane protein, membrane location or structural configuration. Optimization or control of particle production is therefore possible for example by using different promoters to express GAG core proteins and/or the protein of interest, by harvesting at the peak of protein expression. In addition some cellular membrane proteins are excluded from incorporation, due to exclusion from lipid rafts, cholesterol-rich domains where GAG core particles bud form the cell. As a result the membrane lipid and protein content of pseudotyped particles is different from the lipid and protein content of the plasma membrane.
Lipoprotein particles (also called pseudotyped viruses) have been recognized as very valuable tools to study or interfere with viral replication or to use them for gene therapy source of homogenous membrane proteins. Lipoprotein particles are also useful to study the structure and function of the membrane protein that is incoiporated. This especially true for complex membrane proteins like G coupled protein receptors (GPCRs), transporters and ion channels. Indeed, incorporation of such complex membrane proteins has been demonstrated for example for CCR5, CXCR4 and MCAT- 1. Recently, GPCR lipoprotein particles have been shown to be applicable in biosensor assays and a fluorescence polarization molecular binding assay. Such types of assays have been very difficult to achieve with, cell membrane fractions.
Immunoglobulin sequences, such as antibodies and antigen binding fragments derived therefrom are widely used to specifically target their respective antigens in research and therapeutic applications. Typically, the generation of antibodies involves the immunization of experimental animals, fusion of antibody producing cells to create hybridomas and screening for the desired specificities. Alternatively, antibodies can be generated by screening of nai've or synthetic libraries e.g. by phage display.
Summary of the invention
The generation of immunoglobulin sequences, such as Nanobodies®, has been described extensively in various publications, among which WO 94/04678, Hamers-Casterman et al. 1 93 and Muyldermans et al. 2001 can be exemplified. In these methods, camelids are immunized with the target antigen in order to induce an immune response against said target antigen. The repertoire of Nanobodies® obtained from said immunization is further screened for Nanobodies® that bind the target antigen. In these instances, the generation of antibodies requires purified antigen for immunization and/or screening. Antigens can be purified from natural sources, or in the course of recombinant production.
An important class of potential therapeutic targets are cell associated antigens, including transmembrane antigens, in particular transmembrane antigens with multiple membrane spanning domains. Cell -associated, and especially membrane bound antigens, however, are difficult to obtain in their natural conformation because they are embedded within, or anchored in the cell membrane. In order to obtain immunoglobulin sequences against epitopes present in the natural conformation, i.e. conformational epitopes, which are present in vivo, it is however essential to immunize with the target antigen in the correct conformation. Such conformational epitopes are of paramount importance for creating pharmaceutically active immunoglobulin sequences. For example, an immunoglobulin sequence specifically interacting with the natural ligand binding epitope of a GPCR will likely affect its activity, and thus provide a pharmacological effect.
Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens. However, such an approach will not provide antibodies to conformation dependent epitopes, as such epitopes cannot be reproduced by short synthetic peptides.
Therefore, for these cell-associated antigens, immunization with whole cells carrying the antigen and subsequent screening of the repertoire of Nanobodies® induced in this way for Nanobodies® that bind the cell-associated antigen is an option (as was done e.g. in WO
05/044858; WO 07/042289; US 61/004,332). However, such cells express a multitude of antigens, resulting in an antibody response that is largely directed to antigens of no interest. Hence, the antibody response obtainable by this approach is characterized by a low specificity, and in particular by a very low frequency of the antibodies of interest amongst all antibodies generated. Hence this approach is not sufficient for the efficient generation of antibodies to the target antigen of interest. Hence, the art provides no satisfactory method to generate specific antibody responses and subsequent screening of suitable breadth against conformational epitopes, in particular of membrane associated antigens. It is the objective of the present invention to overcome these shortcomings of the art. In particular it is an objective of the present invention to provide a method for creating immunoglobulin sequences against complex antigens, like cell associated antigens that exhibit conformational epitopes.
The above mentioned problems are overcome by the present invention. It has been found that i) cell based immunization can result in an antibody response of good specificity and acceptable breadth against conformational epitopes, i.e. against cell associated antigens in their natural conformation; and that ii) screening by using lipoprotein particles (e.g. in same cell background as the immunization) comprising the target of interest in high concentration can result in an efficient generation of antibodies to the target antigen of interest.
The present invention relates to the following.
A method for the generation of immunoglobulin sequences that can bind to and/or have affinity for a cell-associated antigen comprising the steps of:
a) cell based immunization or genetic vaccination of a non-human animal with a nucleic acid encoding said cell-associated antigen or a domain or specific part of said cell associated antigen; and
b) boosting the animal with said antigen in its natural conformation selected from cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring enriched antigen, liposomes, lipoprotein particles or virus particles expressing the cell associated antigen; and c) screening a set, collection or library of immunoglobulin sequences derived from said non- human animal for amino acid sequences that can bind to and/or have affinity for said cell- associated antigen and wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles. In a particular embodiment of the invention, said lipoprotein particles are derived from the same cells as used for the cell based immunization or boost, e.g. human cell line "HEK293" or derivatives thereof, and is produced using retrovirus structures (e.g. proteins from the mouse retrovirus "murine leukemia vims (MLV)" and enables structurally intact cellular proteins to be purified away from said cell.
In a further particular embodiment of the invention, said cell-associated antigen is selected from transmembrane antigens, transmembrane antigens with multiple spanning domains, such as GPCRs or ion channels.
According to the invention said non-human animal can be selected from vertebrate, shark, mammal, lizard, camelid, llama, preferably camelids and llama.
In one embodiment of the invention, the immunoglobulin sequences are light chain variable domain sequences (e.g. a VL-sequence), or heavy chain variable domain sequences (e.g. a
Vn-sequence); more specifically, the immunoglobulin sequences can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. According to the invention, the immunoglobulin sequences can be domain antibodies, or amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, or amino acid sequences that are suitable for use as single domain antibodies, "dAbs", or amino acid sequences that are suitable for use as dAbs, or Nanobodies®, including but not limited to VHH sequences, and preferably are Nanobodies®.
According to the invention, vaccination can be performed by a needle-free jet injection, by a ballistic method, by needle-mediated injections such as Tattoo, by topical application of the DNA onto the skin in patches or by any of these administration methods followed by in vivo electroporation, and furthermore includes vaccination performed by intradermal, intramuscular or subcutaneous administration of DNA. The set, collection or library of immunoglobulin sequences can be obtained from the blood of said non-human mammal.
In the present invention, said cell-associated antigen can be expressed on any cell background which allows expression of the native conformation of the antigen. Examples of such cell backgrounds that are used in immunization, boost and/or lipoprotein particle generation are Cho, Cos7, Hek293, or cells of camelid origin. Preferably, said cell-associated antigen is a membrane-spanning antigen, including but not limited to an antigen selected from CXC chemokine receptors such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and/or CXCR7, in particular CXCR4 and its human variant.
The set, collection or library of immunoglobulin sequences can be expressed on a set, collection or sample of cells or viruses {e.g. such as bacteriophages) and said set, collection or sample of cells or viruses is screened for cells or viruses that express an amino acid sequence that can bind to and/or have affinity for the lipoprotein particle (e.g. lipoprotein particle of same cell background as cell based immunization and/or whole cell boost) comprising said cell-associated antigen.
According to the invention, the set, collection or library of immunoglobulin sequences can be encoded by a set, collection or library of nucleic acid sequences and said set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode an immunoglobulin sequence that can bind to and/or have affinity for the lipoprotein particle (e.g. lipoprotein particle of same cell background as cell based immunization and/or whole cell boost) comprising said cell-associated antigen.
According to the invention, the immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen can be purified and/or isolated.
The invention also relates also to immunoglobulin obtainable by a method as described herein, and compositions comprising the said immunoglobulin sequences. Brief description of the figures
Figure 1: shows that no specific binding of antibodies from llamas immunized with CXCR4 cells to CXCR4÷ particles could be observed.
Figure 2: shows that CXCR4+ lipoprotein particles are recognized by purified CXCR4- specific Nanobodies® 238D2 and 238D4.
Figure 3: sho s the detection of CXCR4+ lipoprotein particles by periplasmic extracts of CXCR4-specific Nanobodies® 238D2 and 238D4.
Figure 4: shows that CXCR4+ lipoprotein particles are recognized by phages that display 238D2 and 238D4.
Figures 5a-e: show that selections with library 218 on CXCR4+ particles result in isolation of large numbers of Nanobodies© binding to CXCR4+ particles.
Figure 6: shows that selections on CXCR4- particles do not yield Nanobodies® binding to CXCR4+ and CXCR4- particles.
Figures 7a-e: show that selections with library 217 on CXCR4+ particles result in isolation of large numbers of Nanobodies® binding to CXCR4+ particles.
Figures 8a-c: show that Nanobodies® selected on CXCR4+ particles specifically bind CXCR4-HEK293T cells.
Figure 9: Number of peripheral white blood cells (WBC) (A) and mobilized stem cells (B) at several timepoints after the administration of Mozobil® or the 238D2-20GS-238D4. An increased number is observed after compound administration with a peak at 3-6 hours post- administration for 238D2-20GS-238D4 and Mozobil®.
Detailed description of the invention
The present invention encompasses, but is not limited to, the subject matter of the appended claims. A) Definitions
Unless indicated or defined otherwise, ail terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" ( 2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, "Genes II", John Wiley & Sons, New York, N.Y., ( 1985); Old et al, "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd edition, University of California Press, Berkeley, CA (1981 ); Roitt et al„ "Immunology" (6th. Ed.),
Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt' s Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing/Churchill Livingstone, New York (2005), as well as to the general background art cited herein;
Unless indicated otherwise, the term "immunoglobulin sequence" - whether used herein to refer to a heavy chain antibody or to a conventional 4~chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or V^ VL domains, respectively). The terms antigen-binding molecules or antigen-binding protein are used interchangeably with immunoglobulin sequence, and include Nanobodies®.
In one embodiment of the invention, the immunoglobulin sequences are light chain variable domain sequences (e.g. a VL-sequence), or heavy chain variable domain sequences (e.g. a V[[-sequence); more specifically, the immunoglobulin sequences can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. According to the invention, the immunoglobulin sequences can be domain antibodies, or amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, or amino acid sequences that are suitable for use as single domain antibodies, "dAbs", or amino acid sequences that are suitable for use as dAbs, or Nanobodies®, including but not limited to VHH sequences, and preferably are Nanobodies®.
The immunoglobulin sequences provided by the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than cell, associated antigens), so as to provide a monovalent, multivalent or muitispecific polypeptide of the invention, respectively, all as described herein. Such a protein or polypeptide may also be in essentially isolated form (as defined herein).
The invention includes immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The invention also includes fully human, humanized or chimeric immunoglobulin sequences. For example, the invention comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized Dab as described by Ward et al (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996)). Moreover, the invention comprises fused immunoglobulin sequences, e.g. forming a multivalent and/ or muitispecific construct (for multivalent and muitispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al, J. Biol. Chem., Vol 276, 10. 7346-7350, 2001, as well as to for example WO 96/34103 and WO 99/23221), and immunoglobulin sequences comprising tags or other functional moieties, e.g. toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present invention. The amino acid sequence and structure of an immunoglobulin sequence, m particular a Nanobody® can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region l"or "CDR1 "; as "Complementarity Determining Region 2'' or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively.
The total number of amino acid residues in a Nanobody® can be in the region of 1 10- 120, is preferably 1 12-115, and is most preferably 1 13. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a Nanobody® are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.
As used herein, the term "immunoglobulin sequences" refers to both the nucleic acid sequences coding for an immunoglobulin molecule, and the immunoglobulin polypeptide per se. Any more limiting meaning will be apparent from the particular context.
All these molecules are also referred to as "polypeptide of the invention", which is synonymous with "immunoglobulin sequences" of the invention. In addition, the term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation. In the following, reference to a "nucleic acid molecule" of the invention may either relate to the nucleic acid for genetic vaccination, or the nucleic acid encoding the immunoglobulin sequences of the invention, or both, as will be apparent from the specific context. Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general, background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Dreg Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods, 2001 , 248(1-2), 31-45 ; Schmitz et al, Placenta, 2000, 21 Suppl. A, S 106- 12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.
The invention relates to immunoglobulin sequences that can bind to and/or have affinity for an antigen as defined herein. In the context of the present invention, "binding to and/or having affinity for" a certain antigen has the usual meaning in the art as understood e.g. in the context of antibodies and their respective antigens. in particular embodiments of the invention, the term "binds to and/or having affinity for" means that the immunoglobulin sequence specifically interacts with an antigen, and is used interchangeably with immunoglobulin sequences "against" the said antigen. The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular immunoglobulin sequence, antigen-binding molecule or antigen-binding protein (such as a Nanobody® or a polypeptide of the invention) can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen -binding protein: the lesser the value of the KD, the stronger the bindi g strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA). which is 1/KD). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest, Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a Nanobody® or polypeptide of the invention.) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule. Typically, immunoglobulin sequences of the present invention (such as the amino acid sequences, Nanobodies® and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (¾) of 10"5 to 10"12 moles/liter or less, and preferably IO"7 to 10"12 moles/liter or less and more preferably 10~8 to 1CT12 moles/liter (i.e. with an association constant ( A) of 105 to i012 liter/ moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to iO12 liter/moles),
and/or
bind to cell associated antigens as defined herein with a kon-rate of between 102 M'V1 to about 1.07 M"V!, preferably between 103 M"V! and 107 IvI'V1, more preferably between 104 M"V and IO7 M"V!, such as between 105 M"V! and 107 IVrV1;
and/or
bind to cell associated antigens as defined herein with a k0fr rate between Is4 (ti/2=0.69 s) and 10"6 s"1 (providing a near irreversible complex with a t of multiple days), preferably between 10"2 s"1 and 10~6 s"' , more preferably between IO"3 s"1 and 10"6 s"1, such as between 10" 4 s"1 and l0"V.
Any KD value greater than 10"4 mol/liter (or any KA value lower than 104 M"1) liters/mol is generally considered to indicate non-specific binding.
Preferably, a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme
immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more then 10"4 moles/liter or 10"3 moles/liter (e.g., of 10"2 moles/liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant (KA), by means of the relationship [¾ =
The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the KD, or dissociation constant, which has units of mol/liter (or M). The affinity can also be expressed as an association constant, KA, which equals 1/KD and has units of (mol/liter)"] (or M" '). In the present specification, the stability of the interaction between two molecules (such as an amino acid sequence, immunoglobulin sequence,
Nanobody® or polypeptide of the invention and its intended target) will mainly be expressed in terms of the KD value of their interaction; it being clear to the skilled person that in view of the relation KA =1/KD, specifying the strength of molecular interaction by its KD value can also be used to calculate the corresponding KA value. The ¾- value characterizes the strength of a molecular" interaction also in a thermodynamic sense as it is related to the free energy (DG) of binding by the well known relation DG=RT.ln(Ko) (equivalently DG=
~RT.ln(KA)), where R equals the gas constant, T equals the absolute temperature and In denotes the natural logarithm. The KD for biological interactions, such as the binding of the immunoglobulin sequences of the invention to the cell associated antigen as defined herein, which are considered meaningful (e.g. specific) are typically in the range of 10"10M (0.1 nM.) to 10"5M ( 10000 nM), The stronger an interaction is, the lower is its KD.
The KD can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as koff, to the rate of its association, denoted koa (so that KD =k0ff/kon and KA = koi off)- The off-rate koff has units s"! (where s is the SI unit notation of second). The on-rate kon has units M"Y\
As regards immunoglobulin sequences of the invention, the on-rate may vary between 102 ' V1 to about 107 M"V\ approaching the diffusion-limited association rate constant for bi molecular interactions. The off-rate is related to the half-life of a given molecular interaction by the relation
Figure imgf000015_0001
. The off-rate of immunoglobulin sequences of the invention may vary between 10"6 s"1 (near irreversible complex with a t of multiple days) to 1 s~* (t1/2=0.69 s).
The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et ah, Intern. Immunology, 13, 1551 -1559, 2001) where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding kon, koffineasurements and hence D (or KA) values. This can for example be performed using the well-known Biacore instruments.
It will also be clear to the skilled person that the measured D may correspond to the apparent KD if the measuring proces somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent D may be measured if one molecule contains more than one recognition sites for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules.
Another approach that may be used to assess affinity is the 2-step ELiSA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J. Immunol. Methods, 77, 305- 19, 1985). This method establishes a solution phase binding equilibrium measurement and avoids possible artefacts relating to adsorption of one of the molecules on a support such as plastic.
However, the accurate measurement of D may be quite labour-intensive and as
consequence, often apparent D values are determined to assess the binding strength of two molecules. It should be noted that as long as all measurements are made in a consistent way (e.g. keeping the assay conditions unchanged) apparent KD measurements can be used as an approximation of the true D and hence in the present document D and apparent KD should be treated with equal importance or relevance.
Finally, it should be noted that in many situations the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule. For example, to assess the binding strength between molecules A and B, one may e.g. use a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B. As a result an IC50 value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved. Provided Ko ref, the D of the reference molecule, is known, as well as the total concentration cref of the reference molecule, the apparent KD for the interaction A-B can be obtained from following formula: KD
=IC5o (l+cref Ko ref). Note that if cref « D K ¾ ~ IC50. Provided the measurement of the IC50 is performed in a consistent way (e.g. keeping cref fixed) for the binders that are compared, the strength or stability of a molecular interaction can be assessed by the IC50 and this measurement is judged as equivalent to KD or to apparent KD throughout this text.
In the context of the present invention, "conformation dependent epitope", or
"conformational epitope" denotes an epitope that comprises amino acids which are not within a single consecutive stretch of the primary sequence of the antigen. In other words, due to the secondary and/or tertiary structure of a protein target, amino acids which may be spaced apart in the primary sequence are brought into proximity to each other and thereby participate in the formation of an epitope. If for example an antigen comprises three amino acid loops, residues on each one of these loops may participate in the formation of a single epitope. The same applies to antigens comprising more than one domain or subunit. In this case, an epitope may be formed by amino acids on different domains or subunits. Complete or partial denaturing of the protein by appropriate conditions, i.e. the partial or full destruction of secondary and/or tertiary structures, will also partly or fully destroy conformational epitopes. The skilled person will understand that the precise conditions under which a conformational epitope is destroyed by denaturing a protein will depend on the nature of the protein and the specific circumstances.
In a preferred embodiment, the present invention is directed to immunoglobulin sequences against conformational epitopes. In particular, the invention concerns immunoglobulin sequences against conformational epitopes on cell-associated antigens as defined herein, which may preferably be camelid immunoglobulin sequences, including Nanobodies®.
In the context of the present invention, "cell-associated antigen" means antigens that are firmly anchored in or located within the membranes of a cell (including membranes of subcellular compartments and organelles), and includes antigens that have a single or multiple transmembrane regions. In other words, the term refers to antigens exhibiting membrane-dependent conformational epitopes. In particular, the term refers to antigens having conformational epitopes as defined herein. The term encompasses transmembrane antigens, transmembrane antigens with multiple membrane spanning domains such as GPCRs or ion channels. Amongst all these antigens the skilled person knows a range of druggable target antigens, which represent a preferred cell associated antigen of the present invention. The invention in particular relates to ceil associated antigens wherein the conformation dependent epitope is dependent on the correct anchoring and/or location in the membrane. Thus, the invention provides immunoglobulin sequences against such conformation dependent epitopes.
In a preferred embodiment the invention relates to antigens that are integral membrane proteins having one, or more preferably multiple membrane spanning domains. These antigens will reside in and operate within a cell's plasma membrane, and/or the membranes of subcellular compartments and organelles. Many transmembrane proteins, such as transmembrane receptors comprise two or more subunits or domains, which functionally interact with one another.
Integral membrane proteins comprise three distinct parts or domains, i.e. an extracellular (or extracompartmental) domain, a transmembrane domain and an intracellular (or
intracompartmental) domain. A protein having multiple transmembrane domains will typically also have multiple extra- and intra cellular/compartmental domains. For example, a seven transmembrane receptor will comprise seven transmembrane domains.
Thus, the term cell, associated antigen as understood herein is intended to exclude antigens that are only loosely associated, i.e. that are not firmly anchored or located within a membrane. An antigen is firmly anchored if it comprises at least one domain or part that extends into the membrane.
In one embodiment, the invention excludes antigens that have a membrane insertion via a lipid tail, but no transmembrane domain. In this instance, the conformation of the hydrophilic portion or domain of the protein will not depend on the membrane environment. It will, for example, be possible to express a recombinant protein lacking the lipid tail, which is in the proper conformation, i.e. expresses the conformational epitopes also present if the antigen is associated with the membrane via the lipid tail. Similarly, any other proteins which are only loosely associated are excluded from the i vention in a particular embodiment. "Loosely associated" in this connection means proteins which exhibit their natural conformation even in the absence of membrane, i.e. their natural conformation is not dependent on the anchoring or embedding within a membrane.
Typical examples of cell associated antigens according to the invention comprise seven membrane domain receptors, including G-protein coupled receptors, such as CXC chemokine receptors, Adrenergic receptor, Olfactory receptors, Receptor tyrosine kinases, such as
Epidermal growth factor receptor, Insulin Receptor, Fibroblast growth factor receptors, High affinity neurotrophin receptors, and Eph Receptors, fntegrms, Low Affinity Nerve Growth Factor Receptor, NMDA receptor, Several Immune receptors including Toil-like receptor, T cell receptor and CD28.
As used herein, the term "cell-associated antigen" is intended to include, and also refer to, any part, fragment, subunit, or domain of said cell associated antigen. Any subsection of the cell associated antigen falls within the scope of the present invention, provided it represents a conformational epitope of interest. If for example the epitope of interest is located in a binding site of a receptor, or the pore of an ion channel, any fragment(s) of the cell associated antigen capable of forming said epitope are included in the invention. Preferably, those parts, domains, fragments or subunits will be those parts of the cell associated antigen which are responsible for the membrane-dependent conformation. If for example a protein comprises several transmembrane domains, linked by extended intracellular loops, it is envisaged that such loops are in pari or fully omitted, without influencing the extracellular conformational epitopes.
In particular, the present invention relates to immunoglobulin sequences directed to cell associated antigens in their natural conformation. In the context of the present invention, "natural conformation" means that the protein exhibits its secondary and/or tertiary structure, in particular its membrane dependent secondary and/or tertiary structure. In other words, the natural conformation describes the protein in a non-denatured form, and describes a conformation wherein the conformational epitopes, in particular the membrane dependent conformational epitopes, are present. Specifically, the protein will have the conformation that is present when the protein is integrated into or firmly attached to a membrane. Antigens can be obtained in their natural conformation when present in cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring antigen, liposomes, or virus particles expressing the cell associated antigen. In any of these embodiments, antigen may be enriched by suitable means. Said cell-associated antigen can be expressed on any suitable cell allowing expression of the antigen in its native or natural conformation, encompassing, but not limited to Cho, Cos7, Hek293,or cells of camelid origin. The cell associated antigen of the present invention is preferably a druggable membrane protein, in particular a druggable membrane protein having multiple membrane spanning domains. In one embodiment of the invention, the target is a GPCR or an ion channel. Specific, non limiting examples of ion channels that represent cell associated antigens according to the present invention are provided in the following. Also listed are therapeutic effects (with activating (agonistic) or inhibiting/blocking (antagonistic, inverse agonistic) properties) of immunoglobulin sequences specifically recognizing such ion channels.
1. Two-P potassium channels (see Goldstein et al., Pharmacological Reviews, 57, 4, 527 (2005)), such as K2P1.1 , K2P2.1 , K2P3.1 , K2P3.1, K2P4.1, K2P5.1, K2P6.1 , K2P7.1 , K2P9.1, K2P10.3 , K3PI2. I, 2pl3.1, K2P15.1 , K2P16.1 , K2P17.1 and K2P18.1 , which can all be screened using electrophysiological assays such as FLIPR or patch-clamp.
2. CatSper channels (see Clapham and Garbers, Pharmacological Reviews, 57, 4, 451 (2005)), such as CatSper- 1 and CatSper-2 (both involved in fertility and sperm motility), CatSper-3 and CatSper-4, which can all be screened using
electrophysiological assays such as FLIPR, patch-clamp or calcium imaging
techniques.
3. Two-pore channels (see Clapham and Garbers, Pharmacological Reviews, 57, 4, 451 (2005)), such as TPC1 and TPC2.
4. Cyclic nucleotide-gated channels (see Hofman et al., Pharmacological Reviews, 57,
4, 455 (2005), such as CNGA-1, CNGA-2, CNGA-3, CNGA-4A, CNGB 1 and
CNGB3, which can be screened using techniques such as patch-clamp and calcium imaging
5. Hyperpolarization-activated cyclic nucleotide-gated channels (see Hofman et al., Pharmacological Reviews, 57, 4. 455 (2005)), such as HCN1, HCN2, HCN3, HCN4 (all regarded as promising pharmacological targets for development of drags for cardiac arrhythmias and ischemic heart disease), which can be screened using techniques such as voltage-clamp. 6. Inwardly rectifying potassium channels (see ubo et al., Pharmacological Reviews, 57, 4, 509 (2005)), such as Kirl . l , ir21. Kir2.2, ir2.3, Kjr2.4, Kir3.1 , Kir3.2, Kir3.3, ii-3.4, Kif3.4, Kir4.2, Kjr5.1, Kjr6.1 (a target for antihypertensive agents and coronary vasodilators), jr6.2 (the target for pentholamme; its subunit SUR1 is a target for the treatment of diabetes and PHHI) and Kir7.1 (which is a possible site for side-effects of calcium channel blockers), which can be screened using techniques such as voltage- clamp.
7. Calcium-activated potassium channels (see Wei et al.. Pharmacological Reviews, 57, 4, 463 (2005)), such as
Kca l .l - openers of which may be useful in the treatment of stroke, epilepsy, bladder over-reactivity, asthma, hypertension, gastric hypermotility and psychoses;
I¾a .1 - modulators of which may be useful in the treatment of various diseases such as myotonic muscular dystrophy, gastrointestinal dysmotility, memory disorders, epilepsy, narcolepsy and alcohol intoxication. Openers of Kca2.2 have been proposed for cerebellar ataxia;
ca2.2 - modulators of which may be useful in the treatment of various diseases such as myotonic muscular dystrophy, gastrointestinal dysmotility, memory disorders, epilepsy, narcolepsy and alcohol intoxication. Openers of Kca2.2 have been proposed for cerebellar ataxia;
KQ,2.2 - modulators of which may be useful in the treatment of various diseases such as myotonic muscular dystrophy, gastrointestinal dysmotility, memory disorders, epilepsy, narcolepsy, hypertension and urinary incontinence;
Kca3.1 - blockers of which may be useful in the treatment of sickle cell anemia, diarrhea, as immunosuppressants, EAE, the prevention of restenosis and angiogenesis, the treatment of brain injuries and the reduction of brain oedema. Openers if Kca3.1 have been proposed for the treatment of cystic fibrosis and COPD;
as well as Kca4.1 , Ι¾¾4.2 and Kca5.1 ; all of which can be screened using
electrophysiological techniques or techniques such as patch-clamp or voltage-clamp. 8. Potassium channels (see Shieh et al., Pharmacological Reviews, 57, 4, 557 (2005) and Guiman et al., Pharmacological Reviews, 57, 4, 473 (2005) ), including:
voltage-gated calcium channels such as Kvl . l, Kvl .2, Kvl.3, vl.4, Kvl .5, Kvl.6 and Kv.17;
voltage- and cGMP-gated calcium channels such as Kvl.l 0;
beta-subunits of v channels such as vBeta- I , KvBeta-2 and KvBeta-3;
Stei?- like channels such as v2.1 and Kv2.2;
Stew-like channels such as v.3.1, v3.2. Kv3.3 and Kv3.4;
SteWike channels such as Kv4.1, Kv4.2, Kv4.3, v5.1, Kv6.1, Kv6.2, Kv8.1 , Kv9.1 , Kv9.2, v9.3, KH1 and KH2;
Ether-a-go-go-c neh such as EAG, HERG, BEC1 and BEC2;
MinK-type channels such as MinK, MiRPl and MiRP2;
KvLQT -type channels such as vLQTI, KvLQT2, vLQT3, KvLQT4, KvLQTS
Inwardly rectifying potassium channels such as those mentioned above; Sulfonylurea receptors such as the sulfonylurea receptors 1 and 2; Large conductance calcium-activated channels such as Slo and the Beta- subunits of BKcai
Small conductance calcium-activated channels such as SKI, SK2 and SK3;
Intermediate conductance calcium-activated channels such as IKC l ;
Two-pore potassium channels such as TWIK1 , TREK, TASK, TASK2, TWIK2, TOSS, TRAAK and CTBAK1 ;
all of which can be screened using electrophysiological techniques or techniques such as patch-clamp or voltage-clamp. Potassium channels are implicated in a wide variety of diseases and disorders such as cardiac diseases (such as an-hythmia), neuronal diseases, neuromuscular disorders, hearing and vestibular diseases, renal diseases, Alzheimer's disease, and metabolic diseases; and are targets for active compounds in these diseases. Reference is again made to the reviews by Shieh et al. and by Gutman et al. (and the further prior ait cited therein) as well as to the further references cited in the present specification. Tables 3 and 4 of the Shieh review also mention a number of known openers and blockers, respectively, of various potassium channels and the disease indications for which they have been used/proposed.
9. Voltage-gated calcium channels (see Catterall et al., Pharmacological Reviews, 57, 4, 411 (2005)), such as:
Cav1.2 - modulators of which are useful as Ca2÷ antagonists;
Cavl .3 - modulators of which have been proposed for modulating the heart rate, as antidepressants and as drugs for hearing disorders;
Ca 2, 1- modulators of which have been proposed as analgesics for mflammatory pain; Cav2.2 - - modulators of which have been proposed as analgesics for pain such as inflammatoiy pain, postsurgical pain, thermal hyperalgesia, chronic pain and mechanical allodynia;
Cav3.2- which has been proposed as a target for epilepsy, hypertension and angina pectoris;
Cav3.3 - which has been proposed as a target for the treatment of thalamic oscillations; and Cavl. l, Cav1.4, Cav2.3, Cav3.1,; all of which can be screened using techniques such as patch-clamp, voltage-clamp and calcium imaging.
10. Transient receptor potential (TRP) channels (see Clapham et al., Pharmacological Reviews, 57, 4, 427 (2005)) such as:
TRPC channels such as TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and
TRPC7;
TRPV channels such as TRPV1, TRPV2, TRPV3, TRPV4, TRPV5 and TRPV6; TRPM channels such as TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6,
TRPM7 and TRPM 8;
TRPA1 ;
TRPP channels such as ΡΚΌ1, , PKD2L1 and PKD2L2, which are involved in
polycystic kidney disease;
TRPML channels such as mucolipin 1, mucolipin 2 and mucolipin 3 ;
which can be screened using techniques such as patch-clamp and calcium imaging.
11. Voltage-gated sodium channels (see Catterall et al, Pharmacological Reviews, 57, 4, 397 (2005)), such as:
Navl.l , Nav1.2 and Navl .3 - which are a target for drugs for the prevention and treatment of epilepsy and seizures; Nav1.4 - which is a target for local anaesthetics for the treatment of
myotonia;
Nav 1.5 - which is a target for anti rrhythmic drags;
Navl .6 - which is a target for antiepileptic and analgesic drugs;
Navl .7, Navl .8 and Navl .9 - which are potential targets for local
anaesthetics;
all of which can be screened using voltage clamp or techniques involving voltage- sensitive dyes.
Specific, non limiting examples of GPCRs that represent cell associated antigens according to the present invention are provided in the following. Also listed are some exemplary therapeutic effects (with activating (agonistic) or inhibiting/blocking (antagonistic, inverse agonistic) properties) of immunoglobulin sequences of the present invention that are directed against these GPCRs.
GPCRs are involved in a wide area variety of physiological processes. Some examples of their physiological roles include:
1. Behavioral and mood regulation: receptors in the mammalian brain bind several
different neurotransmitters, including serotonin, dopamine, GABA, and glutamate . Regulation of immune system activity and inflammation:chemokine receptors
including CC chemokine receptor and/or CXC chemokine receptors bind ligands that mediate intercellular communication between cells of the immune system;
receptors such as histamine receptors bind inflammatory mediators and engage target cell types in the inflammatory response
3. Autonomic nervous system transmission: both the sympathetic and parasympathetic nervous systems are regulated by GPCR pathways, responsible for control of many automatic functions of the body such as blood pressure, heart rate, and digestive processes
. The visual sense: the opsins use a photoisomerization reaction to translate
electromagnetic radiation into cellular signals
5. The sense of smell: receptors of the olfactory epithelium bind odorants (olfactory
receptors) and pheromones (vomeronasal receptors) Preferably, said cell- associated antigen is a membrane- spanning antigen, including but not limited to an antigen selected from CXCR7, CXCR4 and P2X7. The skilled person will appreciate that there may be different specific three dimensional conformations that are encompassed by the term "natural conformation". If, for example, a protein has two or more different conformations whilst being in a membrane environment, all these conformations will be considered "natural conformations". This is exemplified by receptors changing their conformation by activation, e.g. the different activation states of rhodopsin induced by light, or ion channels showing a "closed" or "open" conformation. The invention encompasses immunoglobulin sequences to any one of these different natural conformations, i.e. to the different kinds of conformational epitopes that may be present.
A "nucleic acid" of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism). According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein.
The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.
The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the cell associated antigen or immunoglobulin sequences of the invention, and/or can be isolated from a suitable natural source. To provide analogs, nucleotide sequences encoding naturally occurring VHH domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at. least one nucleotide sequence encoding a Nanobody® and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner. Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or li gated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more
"mismatched" primers, using for example a sequence of a naturally occurring GPCR as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et ai. and Ausubel et al.. mentioned above, as well as the Examples below.
The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention".
The genetic constructs of the invention may be DNA or RNA, and are preferably double- stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism, or in a form suitable for genetic immunization. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. in particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).
In a preferred but non-limiting embodiment, a genetic construct of the invention comprises a) at least one nucleic acid of the invention; operably connected to
b) one or more regulatory elements, such as a promoter and optionally a suitable
terminator;
and optionally also
c) one or more further elements of genetic constructs known per se;
in which the terms "regulatory element", "promoter", "terminator" and "operably connected" have their usual meaning in the art (as further described herein); and in which said "further elements" present in the genetic constructs may for example be 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to the skilled person, and may for instance depend upon the type of construct used, the intended host cell, host organism or animal to be immunized; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g. via constitutive, transient or inducible expression); and/or the transformation/vaccination technique to be used. For example, regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in an essentially analogous manner.
Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the invention and said regulatory elements, and optionally said one or more further elements, are
"operably linked" to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered "operably linked" to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription, and/or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of said promoter). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.
Preferably, the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism.
For instance, a promoter, enhancer or terminator should be '"operable" in the intended host cell or host organism, by which is meant that {for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked (as defined herein).
For some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention - such as terminators, transcriptional and/or translational enhancers and/or integration factors - reference is made to the general handbooks such as Sanibrook et al. and Ausubel et al. mentioned above, as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO
97/42320, WO 98/06737, WO 98/21355, US-A~6,207,41G, US-A- 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Reference is also made to the general background art cited above and the further references cited herein.
The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.
Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the Examples below, as well as those mentioned herein. The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the Nanobody® or polypeptide of the invention, or for genetic vaccination. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
According to one non-limiting embodiment of the invention, the immunoglobulin sequences, Nanobody© or polypeptide of the invention is glycosylated. According to another non- limiting embodiment of the invention, the immunoglobulin sequences, Nanobody© or polypeptide of the invention is non-glycosylated.
In the context of the present invention, "genetic vaccination" mcludes any known methods or means to transfer a nucleic acid sequence, e.g. a DNA sequence, into a target animal that is suitable for inducing an immune response to a protein encoded by said nucleic acid sequence.
The skilled person knows standard ways of genetic vaccination. According to the invention, genetic vaccination can be performed by a needle-free jet injection, by a ballistic method, by needle-mediated injections such as tattoo, by topical application of the DNA onto the skin in patches or by any of these administration methods followed by in vivo electroporation, and furthennore includes vaccination performed by intradermal, intramuscular or subcutaneous administration of DNA.
In the context of the present invention, "cell based immunization" includes any known methods or means to immunize animals with cells, e.g. cells expressing the antigen of interest. The skilled person knows standard ways of cell based immunizations. According to the invention, cell based immunizations can be performed by transiently expressing the antigen or target of interest on the cells used to immunize or by stably transfected cells expressing the antigen or target of interest. In the context of the present invention, "lipoprotein particles" are membrane-enveloped virus- like particles (VLPs) containing high concentrations of structurally-intact membrane proteins on their surface (see J Biomol Screen 13(5), 2008, p 424 to 429; WO2005/042695). In the context of the present invention, "non-human animal" includes, but is not limited to vertebrate, shark, mammal, lizard, camelid, llama, preferably camelids and most preferably llama or alpaca.
In the context of the present invention, "CXCR4" includes, but is not limited to mouse, and/or human CXCR4 and most preferred human CXCR4, i.e. GenBank entry: AF005058.1 (SEQ ID NO: 1 :
MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFN IFLPTIYSIIFLTGIVGNGLVI LVMGYQ KLRSMTD YRLHLSVADLLFVITLPFWAVDAVANWYFGNFLC AVHVI YTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIF AN V SE ADDRYICDRF YPNDL WV VVFQFQHIM V GLILPGI V ILS C YCIIISKLSHS KGHQ RKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVH WISITEALAFFH CCLNPILYAFLGA FKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS)
In the context of the present invention, the terms "cross-block", "cross-blocked" and "cross- blocking" are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a polypeptide of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target. The extend to which an amino acid sequence or other binding agents of the invention is able to interfere with the binding of another to target, and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequence or another binding agents in terms of their binding to the target. The following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the invention. It will be appreciated that the assay can be used with any of the amino acid sequence or other binding agents described herein. The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations. Thus in one cross-blocking assay, the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target. Typically 200- 800 resonance units of the target would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used). Two test amino acid sequences (termed A* and B*) to be assessed for their ability to cross- block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture. When calculating the concentrations on a binding site basis the molecular- weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence. The concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip. The amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix. The test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target.
Typically this is done by treating the chip with 30 mM HC1 for 60 seconds. The solution of A* alone is then passed over the target-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound target. The solution of B* alone is then passed over the target-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each amino acid sequence when passed over the target surface alone. If the actual recorded binding of the mixture is less than this theoretical maximum then the two amino acid sequences are cross-blocking each other. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such thai during the assay and in the presence of a second amino acid sequence or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1 % (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1 % (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination. The Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CMS Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version (R & D Systems, Minneapolis, MN, USA; 2005 cat# 1406-ST-025). In this particular format, an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface. In addition to the example given using N-terminal His- tagged antigen, C-terminal His-tagged target could alternatively be used. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin).
The following generally describes an ELISA assay for determining whether an amino acid sequence or other binding agent directed against a target cross-blocks or is capable of cross- blocking as defined herein. It will be appreciated that the assay can be used with any of the amino acid sequences (or other binding agents such as polypeptides of the invention) described herein. The general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules. The plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target. The amount of bound target is then measured using a reagent that is appropriate to detect the target. An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence. In the instance where the first amino acid sequence, e.g. an Ab-X, is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of the second amino acid sequence, i.e. Ab-Y, is then added to the ELISA plate such that the moles of Ab-Y antigen binding sites per well are at least 10 fold higher than the moles of Ab-X antigen binding si tes that were used, per well, during the coating of the ELISA plate, antigen is then added such that the moles of antigen added per well are at least 25-fold lower than the moles of Ab-X antigen binding sites that were used for coating each well. Following a suitable incubation period the ELISA plate is washed and a reagent for detecting the target is added to measure the amount of target specifically bound by the coated anti-antigen amino acid sequence (in this case Ab-X). The background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence (in this case Ab-Y), antigen buffer only (i.e. no target) and target detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence buffer only (i.e. no second solution phase amino acid sequence), target and target detection reagents. The ELISA assay may be mn in such a manner so as to have the positive control signal be at least
6 times the background signal. To avoid any artefacts (e.g. significantly different affinities between Ab-X and Ab-Y for target) resulting from the choice of which amino acid sequence to use as the coating amino acid sequence and which to use as the second (competitor) amino acid sequence, the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution. Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal (i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti- target amino acid sequence (i.e. the positive control wells).
B) The methods of the present invention
The present invention relates to a method for the generation of immunoglobulin sequences that can bind to and/or have affinity for a cell-associated antigen, as defined herein. The method comprises, but is not limited, to the following steps:
a) immunization of a non-human animal;
a. by cell based immunization wherein said cells expressing the cell-associated antigen or a domain or specific part of said cell associated antigen; or b. genetic vaccination with a nucleic acid encoding said cell-associated antigen or a domain or specific part of said cell associated antigen; and
b) boosting the animal with said antigen in its natural conformation selected from cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring enriched antigen, liposomes, lipoprotein particles or virus particles expressing the cell associated antigen; and c) screening a set, collection or library of immunoglobulin sequences derived from said non- human animal for amino acid sequences that can bind to and/or have affinity for said cell- associated antigen and wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles.
Thus, in general terms the method of the present invention includes screening a set, collection or library of immunoglobulin sequences derived from said non-human animal for amino acid sequences that can bind to and/or have affinity for said cell-associated antigen and wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles as defined herein, in one particular embodiment, the immunization, boosting and/or screening is done in the same cell background.
One particular advantage of the present invention resides in the fact that it provides a robust method for generating immunoglobulin sequences thai produces superior results, i.e. obtain more variants of functional binders, to complex antigens such as GPCRs and ion channels. In particular, there is no requirement when working in the same background for immunization and screening to counter select with lipoparticles without the antigen of interest.
Advantageously, the method also results in high frequency isolation of immunoglobulm sequences directed to complex antigens.
Hence, the present invention is advantageous as compared to prior art methods that lack such robust and high performance applicability. In particular there is no teaching in the art for such a robust and well performing method for the generation of immunoglobulin sequences against complex targets in animals such as camelids, in particular llama.
Specifically, the present invention provides an improved method for generating
immunoglobulin sequences against cell -associated antigens, which, according to one specific embodiment, is without the need for counter selection with lipoparticles without the antigen of interest, by inducing an immune response via cell based immunization or DNA vaccination and subsequent screening with lipoprotein particles for immunoglobulin sequences that can bind the cell-associated antigen.
It has also been surprisingly found, that when using the antigen enriched lipoprotein particles for panning, immunoglobulin sequences (e.g. Nanobodies®) were identified which were previously isolated by another method, i.e. a method using antigen enriched cell based immunization and panning by using antigen enriched membrane extracts from different cell background, but more importantly completely new specific immunoglobulin sequences (Nanobodies®) were isolated (see Examples). Moreover, the affinity of the identified binders was high and for some of the newly found immunoglobulin sequences even higher than 2 binders found with the standard approach (see Examples). This underlines the particular advantage of the present invention of resulting in obtaining more variety and more specific high affinity immunoglobulin sequences, and allowing for more efficient screening and isolation of specific immunoglobulin sequences. It was unforeseeable from the prior art that such advantages can be obtained by using the methods of the invention, in particular when the lipoprotein particles were generated in the same background as the cell based and/or cell based (or membrane based) boost.
In an alternative embodiment, the present invention provides a method for the generation of immunoglobulin sequences, including Nanobodies®, against a cell-associated antigen comprising the steps of:
a) immunization of a non-human animal;
a. by cell based immunization wherein said cells expressing the cell-associated
antigen or a domain or specific part of said cell associated antigen; or b. genetic vaccination with a nucleic acid encoding said cell-associated antigen or a domain or specific part of said cell associated antigen; and
b) boosting the animal with said antigen in its natural conformation selected from cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring enriched antigen, liposomes, lipoprotein particles or vims particles expressing the cell associated antigen; and c) screening a set, collection or library of immunoglobulin sequences derived from said non- human animal for amino acid sequences that can bind to and/or have affinity for said cell- associated antigen and wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles; and/or wherein the lipoprotein particles are generated from the same cells as used in a) and/or b). Immunization
In the method of the invention, genetic vaccination or cell based immunization suitable for inducing an immune response in the animal is performed. More specifically, the
immunization must be suitable to induce an immune response as reflected in the generation of immunoglobulin sequences in the animal. The detection of an antibody response in the serum of the animal is also referred to as "serum conversion". The skilled person can monitor the immunization success by determining the antibody response by routine means. Thus, the skilled person can readily determine the adequate dosage and frequency that is required for inducing an appropriate antibody response.
Preferably, the immunization will induce an adequate antibody litre. The antibody titre will correspond to the number of specific antibody producing cells, which will allow the generation of immunoglobulin sequences by isolation and/or screening. However, it is considered that the method of the present invention allows for the successful isolation of high affinity immunoglobulin sequences even when there is only a low serum or undetectable antibody titre. Serum titres can be determined by conventional methods, including e.g.
ELISA or FACS.
Preferably the antigen is enriched in any of the cell based immunization preparations, in order to strengthen the immune response. For example, recombinant expression in cells using highly efficient promoters can be used to increase the quantity of antigen per cell. In one embodiment, when using camelids as the non-human animal, the cells expressing the antigen of interest can be camelid derived cells, preferably immortalized camelid derived cells. The cells will be genetically modified to express the said antigen.
Moreover, the skilled person will understand that the invention also encompasses the use of an adjuvant commonly used in order to enhance an immune response in the context of vaccination. The protein preparation may also be in a physical form that enhances the immune response, such as e.g. a gel or emulsion. Specific, non-limiting examples of an adjuvant include Stimune or Specol (CEDI Diagnostics, Lelystad, The Netherlands), Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, TiterMax (Gold),
monophosphoryl lipid A (MPL), Alum, QuilA, CpG DNA.
A further aspect of importance for the present invention is the breadth of the antibody repertoire obtained by the methods of the invention. In particular, it is one aspect of the present invention that the antibody response is directed to both linear and conformational epitopes, and importantly is directed to membrane dependent conformational epitopes. Thus, the present invention relates to a method suitable for obtaining an antibody response of an adequate litre and breadth in the non-human animals.
When a suitable antibody response has been confirmed in the animal, immunoglobulin sequences can in one embodiment of the invention be directly isolated from said animal, i.e. without protein boost, by methods as described herein. Detection of antibody responses can be done by routine means, such as ELISA, RIA, FACS, or any other method for detecting antibodies. Protein boost
The method also includes boosting the animal with a suitable source of protein, in particular it is envisaged to boost the animal with compositions that comprise the cell associated antigen as defined herein, in particular a transmembrane antigen, in its natural conformation. Such compositions may comprise cells expressing the antigen, or fragments or derivatives of the cell, such as membrane fractions, isolated organelles, or other suitable preparations. Also envisaged are viruses, liposomes, micelles, lipoprotein particles or other systems that are suitable for containing the cell associated antigen in its natural conformation.
In one aspect of the invention the antigen can be expressed on a homologous cell. For example, for immunization of a camelid, the antigen can be expressed on a carnelid cell. The camelid immune system will be tolerant to the camelid cell, i.e. it will not mount an immune response to most of the antigens comprised in this cell. However, if a heterologous antigen, including but not limited to cell associated antigens as defined herein, is artificially introduced into said cell, the immune system of the animal will mount an immune response specifically directed to said antigen. This has the advantage that the immune response will be mainly directed to the antigen of interest, i.e. it will be characterized by an enhanced specificity towards this antigen. The skilled person will appreciate that this approach can be used for related species. For example, camel derived cells can be used for immunization of llama, and vice versa, in view of their close relationship.
Any suitable cell mat is homologous to the animal to be immunized can be used. For example, camelid cells can be used for immunization of camelids, e.g. llama cells for immunization of llama. Suitable cells will comprise, but are not limited to, cells that are spontaneously immortal, e.g. cancer cells or undifferentiated cells, such as embryo-derived cells. Suitable cells also encompass cells immortalized artificially by known means. Cells can advantageously be treated prior to administration to the animals, such that then- proliferation in vivo is reduced or eliminated. Suitable treatments comprise, but are not limited to chemical and physical treatments. One specific example of a suitable physical treatment is irradiation with X rays such that the cells can no longer proliferate. Preferably the protein is enriched in any of the above prepai-ations, in order to strengthen the immune response. For example, recombinant expression in cells using highly efficient promoters can be used to increase the quantity of antigen per cell. In one embodiment, when using camelids as the non-human animal, the cells expressing the antigen of interest can be camelid derived cells, preferably immortalized camelid derived cells, e.g. HEK293, HEK293T, HE 293H. The cells will be genetically modified to express the said antigen.
Moreover, the skilled person will understand that the invention also encompasses the use of an adjuvant commonly used in order to enhance an immune response in the context of vaccination. The protein preparation may also be in a physical, form that enhances the immune response, such as e.g. a gel or emulsion. Specific, non-limiting examples of an adjuvant include Stimune or Specol (CEDI Diagnostics, Lelystad, The Netherlands), Freund's Complete Adjuvant, Freund's incomplete Adjuvant, TiterMax (Gold), monophosphoryl lipid A (MPL), Alum, QuilA, CpG DNA. The present invention comprises a single or multiple boosts with the said source of protein in its natural conformation (optionally using an adjuvant). The protein boosts will be performed at suitable intervals, which can be determined by routine means, e.g. by monitoring the immunoglobulin response in the animals. The boost can be performed by different routes of administration, including, but not limited to, intradermal, subcutaneous, or intramuscular administration. Screening/isolating immunoglobulin sequences
The immunization and boost as described herein will induce an immune response in the animal. Then, a set, collection or library of immunoglobulin sequences is isolated from the animals. "Isolation" includes a) the separation of sequences from the animal, e.g. by sampling suitable tissues, and b) the singling out of specific sequences e.g. by screening, i.e. the isolation of "hits" of specific binders.
The skilled person is well acquainted with techniques for establishing sui table sets, collection or libraries of immunoglobulin sequences, and screening thereof for the sequences of interest. The skilled person can make general reference to the techniques described, in for example WO 02/085945 and in WO 04/049794. Reference can also be made to techniques and methods described in WO 99/37681 , WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi -synthetic libraries derived from e.g. VHH libraries, obtained form the animals immunized in accordance with the present invention, may be used, such as VHH libraries obtained from VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.
The invention includes the isolation of material from the animal which comprises immunoglobulin sequences, such as, but not limited to, antibody producing cells. For example, peripheral blood monocytes (PBMCs) can be isolated by conventional means. Other material includes peripheral blood lymphocytes (PBLs), peripheral lymph nodes, in paiticular lymph nodes draining the site of immunization, the spleen, bone marrow, or other immunologically relevant materials.
In one specific, non-limiting example, B-cell containing blood samples can be collected, and peripheral, blood lymphocytes (PBLs) can be purified by standard methods. For example, a density gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) can be employed according to the manuf cturer' s instructions.
Any of the above described material, including e.g. PBLs isolated from the animal will comprise a multitude of immunoglobulin sequences, i.e. a set, collection or library of immunoglobulin sequences. Amongst this multitude of immunoglobulin sequences, e.g. expressed on PBMCs, the desired immunoglobulin specificities can be directly isolated, e.g. by immunopanning of the cells. Alternati vely, nucleic acid sequences coding for the set, collection or library of
immunoglobulin sequences can be isolated, transferred, and expressed on a set, collection or sample of cells or viruses.
The genetic material can be isolated and processed further by suitable means to isolate such sequences that code for the immunoglobulin sequences of the desired specificity. To this end, e.g. the nucleic acid sequences encoding the said multiplicity of immunoglobulin sequences can be extracted from the material by suitable means, and transferred into a recipient cell or virus for expression. The skilled person is familiar with, suitable techniques for extraction of immunoglobulin sequences and manipulating these sequences for expression, e.g. in an expression library in cells or viruses. Some non-limiting examples comprise the generation of an expression library in e.g. E. coli or bacteriophages.
In one specific, non-limiting example, total RNA can be extracted from the said material. The total RNA can be converted into cDNA by known means. Using this cDNA, immunoglobulin sequences, such as e.g. the Nanobody® repertoire, can be amplified by routine means, including e.g. PCR, or nested PCR methods as known in the art (see patent references above).
Nucleic acid molecules comprising immunoglobulin sequences can be digested by use of suitable restriction enzymes, optionally followed by purification e.g. by gel electrophoresis. The digested sequences can be ligated into corresponding restriction sites in a suitable genetic construct, such as a vector or plasmid. Non-limiting examples of suitable vectors include phage display vectors, e.g. pAX50. pAX50 contains the LacZ promoter, a coliphage pill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site (harboring the Sfil and BstELl restriction sites) and a chimeric leader sequence consisting of gene3 and Erwinia caroiovora pelB motifs. This display vector allows the production of phage particles, expressing the individual Nanobodies® as a fusion protein with the genelll product. The ligated nucleic acid molecule can be used to obtain a library, e.g. by transformation of a suitable host organism, like E. coli. The skilled person knows suitable techniques of transformation, e.g. chemical methods, electroporation, and others. Thus, a library of a suitable size, e.g. 1E7 to 1E8, can be obtained.
In one embodiment, libraries can be rescued by growing the bacteria to logarithmic phase (e.g. OD600= 0.5), followed by infection with helper phage to obtain recombinant phage expressing the repertoire of cloned immunoglobulin sequences on tip of the phage as a pill fusion protein, the obtained phage can be stored, e.g. after filter sterilization, for further use, e.g. at 4°C.
A set, collection or library of cells or viruses is screened for lipoprotein particles that express immunoglobulin sequences that can bind to and/or have affinity for said cell-associated antigen, more specifically, a nucleic acid sequence that encodes the immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen can be purified and/or isolated from the cell or virus, followed by expression of said amino acid sequence.
Thus, the present invention also encompasses suitable screening step(s), to select and isolate the immunoglobulin sequences directed to the antigen of interest (or nucleic acid sequences encoding the same) from a multitude of sequences present in the non-human animal. The skilled person is well aware of a multitude of suitable techniques, including phage display, immunopanning, etc. Of course the invention aiso relates to combinations of known metliods. Suitable combinations will be apparent to the skilled person.
In one specific embodiment, the library of phages expressing immunoglobulin sequences can be selected by a single round, or multiple rounds of panning on a suitable source of lipoprotein particles comprising cell-associated antigen, including, but not limited to lipoprotein particles comprising highly enriched antigen. In a specific embodiment the antigen of interest within the lipoprotein particles are typically enriched 10 to 100 fold, preferably 50 to 100 fold, compared with cells or membrane preps (measuring specific membrane protein per total protein concentration). In a further specific embodiment the concentration of the antigen of interest compared to the total protein concentration within the lipoprotein particles is approximately 1% or less, more preferably 5% or less when measured by sypro staining (see e.g. Sypro protein detection staining kit). After a round of selection, e.g. by immunopannmg, the output can be recloned as a pool into a suitable expression vector for further selection and/or processing.
According to the invention, the immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen can be purified and/or isolated. According to the invention, further characterization of the immunoglobulin sequences, e.g. binding affinity, or avidity measurements, can be advantageously be performed by the use of lipoprotein particles in immunoglobulin phage ELISA, and immunoglobulin periplasmic ELISA. The skilled person can use standard techniques for such characterizations. The skilled person can use standard techniques for production of immunoglobulins. Thus, after a cell, virus or nucleic acid sequence encoding the immunoglobulin sequence of interest has been identified by a screening method, the said immunoglobulin sequence can be produced, e.g. by means of recombinant expression. For this purpose, the cell or virus can be used directly, or the nucleic acid encoding the immunoglobulin sequence can be transferred into a suitable expression system, including a suitable host cell. Host cells include mammalian systems, such as CHO cells, eukaryotic systems such as insect cells or fungi, including e.g. Pichia pastoris, and prokaryotic systems such as E. coli. The skilled person knows suitable expression vectors and tools for use in expressing immunoglobulin sequences in these host systems.
The immunoglobulin sequences, Nanobodies© and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from, the description herein. The skilled person will understand which of the specific examples are suitable for the generation and/or screening of sets, collections or libraries of immunoglobulin sequences, or for the production of immunoglobulin sequences after selection of antigen specific sequences. For example, the polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). As will be clear to the skilled person, one particularly useful method for preparing a polypeptide of the invention generally comprises the steps of: the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said Nanobody® or polypeptide of the invention (also referred to herein as a "nucleic acid of the invention", this term is also used for the genetic constructs for vaccination, as will be apparent from the specific context), optionally followed by: isolating and/or purifying the Nanobody® or polypeptide of the invention thus obtained.
Moreover, the produced immunoglobulins can be purified by standard techniques, including precipitation, affinity chromatography, size exclusion chromatography, ion exchange chromatography, HPLC, filtration, and other known purification methods.
Furthermore, the immunoglobulin sequences can be further characterized by known methods, e.g. to determine their epitope specificity, binding kinetics, etc.
Thus, the invention also relates to immunoglobulin sequences, i.e. the polypeptide molecules, obtainable by a method as described herein, and compositions comprising the said immunoglobulin sequences. Such compositions comprise compositions for research purposes as well as pharmaceutical compositions for use in therapy. The skilled person is familiar with standard techniques and formulations for therapeutic application of immunoglobulin sequences. Thus, in one aspect the method of the present invention encompasses the purification of specific immunoglobulin sequences and their formulation as a pharmaceutical composition. The present invention provides immunoglobulin sequences in essentially isolated form, e.g. in a form that is at least 90% pure, at least 95% pure, at least 98%, at least 99%, or at least 99.99% pure. In one non-limiting embodiment, purity means that no sequences of other immunoglobulins are present in the preparation. In a further non-limiting embodiment purity means that no contaminants from the producing organism are present in the composition.
The present invention also encompasses immunoglobulin sequences that are derivatives of the immunoglobulin sequences obtainable by the methods disclosed herein. For example, the invention encompasses humanized immunoglobulin sequences obtainable by methods known in the art. Moreover, the invention encompasses camelized immunoglobulin sequences, also obtainable by methods known in the art. The invention also encompasses known structural variants of immunoglobulin sequences.
Immunoglobulin sequences obtainable by the methods
In the context of transmembrane proteins, and in particular proteins with multiple transmembrane domains, conformational epitopes, and in particular membrane-dependent conformational epitopes are of particular interest as targets for immunoglobulin sequences. For example, the pore of an ion channel represents a target of primary therapeutic importance. However, by use of conventional approaches, it is nearly impossible to generate immunoglobulin sequences that recognize such a target. The present invention provides for the generation of immunoglobulin sequences to such kind of conformational epitope.
In a preferred but non-limiting aspect, the invention relates also to immunoglobulin sequences that were obtained by using the method described herein. In particular, the invention relates to a Nanobody® (as defined herein) against CXCR4, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:
CDR1 is chosen from the group consisting of:
a) the amino acid sequences of SEQ ID NO ' s : 12 to 21 ;
b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1.2 to 21 ; c) amino acid sequences that have 3, 2, or i amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 12 to 21 ;
and/or
CDR2 is chosen from the group consisting of:
d) the amino acid sequences of SEQ ID NO's: 32 to 41 ;
e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 32 to 41 ;
0 amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 32 to 41;
and/or
CDR3 is chosen from the group consisting of:
g) the amino acid sequences of SEQ ID NO' s: 52 to 71 ;
h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 52 to 71 ;
i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 52 to 71 ;
or any suitable fragment of such an amino acid sequence.
In particular, according to this preferred but non-limiting aspect, the invention relates to a Nanobody® (as defined herein) against human CXCR4, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRI to CDR3 respectively), in which:
CDRl is chosen from the group consisting of:
a) the amino acid sequences of SEQ ID NO's: 12 to 21 ;
b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 12 to 21 ;
c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 12 to 21 ;
and
CDR2 is chosen from the group consisting of:
d) the amino acid sequences of SEQ ID NO's: 32 to 41 ;
e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 32 to 41; f) amino acici sequences that have 3, 2, or 3. amino acid difference with at least one of the amino acid sequences of SEQ ID NO' s: 32 to 41;
and
CDR3 is chosen from the group consisting of:
g) the amino acid sequences of SEQ ID NO' s: 52 to 71 ;
h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 52 to 71 ;
i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO' s: 52 to 71 ;
or any suitable fragment of such an amino acid sequences.
As generally mentioned herein for the amino acid sequences of the invention, when a Nanobody© of the invention contains one or more CDRl sequences according to b) and/or c):
i.) any amino acid substitution in such a CDR according to b) and/or c) is preferably, and compared to the corresponding CDR according to a), a conservative amino acid substitution (as defined herein);
and/or
ii) the CDR according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to a);
and/or
iii) the CDR according to b) and/or c) may be a CDR that is derived from a CDR according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.
Similarly, when a Nanobody© of the invention contains one or more CDR2 sequences according to e) and/or f):
i) any amino acid substitution in such a CDR according to e) and/or f) is preferably, and compared to the corresponding CDR according to d), a conservative amino acid substitution (as defined herein);
and/or ii) the CDR according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to d);
and/or
iii) the CDR according to e) and/or f) may be a CDR that is derived from a CDR according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.
Also, similarly, when a Nanobody® of the invention contains one or more CDR3 sequences according to h) and/or i):
i) any amino acid substitution in such a CDR according to h) and/or i) is preferably, and compared to the corresponding CDR according to g), a conservative amino acid substitution (as defined herein);
and/or
ii) the CDR according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to g);
and/or
iii) the CDR according to h) and/or i) may be a CDR that is derived from a CDR according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.
It should be understood that the last three paragraphs generally apply to any
Nanobody® of the invention that comprises one or more CD l sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e), f), h) or i), respectively.
Of the Nanobodies® of the invention, Nanobodies® comprising one or more of the CDR's explicitly listed above are particularly preferred; Nanobodies® comprising two or more of the CDR' s explicitly listed above are more particularly preferred; and Nanobodies® comprising three of the CDR' s explicitly listed above are most particularly preferred.
Some particularly preferred, but non-limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table A-l below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies® of the invention. As will be clear to the skilled person, a combination of CDRl, CDR2 and CDR3 sequences that occur in the same clone (i.e. CDRl , CDR2 and CDR3 sequences that are mentioned on the same line in Table A-1 ) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table A- 1). Also, a combination of CDR sequences and framework sequences that occur in the same clone (i.e. CDR sequences and framework sequences that are mentioned on the same line in Table A-1 ) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table A- 1, as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein).
Also, in the Nanobodies® of the invention that comprise the combinations of CDR' s mentioned in Table A- 1 , each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR's; in which:
i) any amino acid substitution in such a CDR is preferably, and compared to the
corresponding CDR sequence mentioned in Table A-1 , a conservative amino acid substitution (as defined herein);
and/or
ii) any such CDR sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR sequence mentioned in Table A-1 ;
and/or
iii) any such CDR sequence is a CDR that is derived by means of a technique for affinity maturation known per se, and in particular starting from the corresponding CDR sequence mentioned in Table A- 1.
However, as will be clear to the skilled person, the (combinations of) CDR sequences, as well as (the combinations of) CDR sequences and framework sequences mentioned in Table A- 1 will generally be preferred.
Table A-ϊ CDR's and framework sequences of Nanobodies® against human CXCR4
Figure imgf000050_0001
LVQTGGSLRL 7 7 KEREFVA 7 ALYGDSVKD 7 YLQMN MKPEDT 7 LRRFEY 7 TVSS
SCAASGGTFN AVYTCAA DS
EVQLVESGGG RFTISRDNAKNTV KFVNTD
LVQAGDSL L 1 2 WFRQAPG 3 AIGWG PSKTN YA YLQMNTLKPEDTA STWSRS 6 WGQGTQ
SCAASGRAFS 8 RYA G 8 KEREFVA 8 DSVKG 8 VYSCAA S EMYTY 8 VTVSS
EVQLVASGGG RFTSSRDNAKNLA
LVQAGGSLRL 1 2 WYRQAPG 3 D!SSGGSTNYAD YLQ NSLKPEDTA 5 RTSGWR 6 WGQGTQ
SCAVSGTTFS 9 VATLG QQ ALVA 9 SVRG 9 VYYCNA 9 TRSNY 9 TVSS
EVQLVESGGG RFT1SRDNAKNLA
LVGAGGSiRL 2 3 WYRQAPG 4 DISSGGSTNYAD 5 YLQMNSLKP EDTA 6 RTSGWR 7 WGQGTQ
SCAVSGTTFS 0 VATLG 0 QQRALVA 0 SVRG 0 VYYCNA 0 TRSNY 0 VTVSS
EVQLVESGGG RFTISRDNT NTV GR1GQR
LVQAGGSLRL 2 3 WYRQAPG 4 SISSGGRINYAD 5 HLQWINSLEPEDTA 6 TLTFTPD 7 WGQGTQ
SCVASVN!FG 1 SiAMA 1 KQRNLVA 1 SRKG i VYYCAA 1 Y 1 TVSS
1
Thus, in the Nanobodies® of the invention, at least one of the CDR1 , CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A- 1 ; or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% "sequence identity" (as defined herein) with at least one of the CD 1 , CDR2 and CDR3 sequences, respectively, listed in Table A~l ; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with at least one of the CDR1 , CDR2 and CDR3 sequences, respectively, listed in Table A- 1.
In this context, by "suitably chosen" is meant that, as applicable, a CDR1 sequence is chosen from suitable CDR1 sequences (i.e. as defined herein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively. More in particular, the CDR sequences are preferably chosen such that the Nanobodies® of the invention bind to GPCRs with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a KA- value (actual or apparent), a kon-rate and/or a k0irrate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
In particular, in the Nanobodies® of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table
A-l or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-l; and/or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table A-l .
Preferably, in the Nanobodies® of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-l or from the group consisting of CDR1 , CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-l ; and/or from the group consisting of the CDR I, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" with at least one of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-1.
In particular, in the Nanobodies® of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1, respectively; and at least one of the CD l and CDR2 sequences present is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table A-1 or from the group of CDRl and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table A-1.
Most preferably, in the Nanobodies® of the invention, all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group of CDRl, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-1 ; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
Even more preferably, in the Nanobodies® of the invention, at least one of the CDRl ,
CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-1. Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at ieast one of the corresponding CDR sequences, respectively, listed in Table A-1 ; and/or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table A-L
In particular, in the Nanobodies® of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table A- l .
Preferably, in this aspect, at least one and preferably both of the CDR l and CDR2 sequences present are suitably chosen from the groups of CDRl and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDRl and CDR2 sequences, respectively, listed in Table A- l ; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CD l and CDR2 sequences, respectively, listed in Table A- L
Even more preferably, in the Nanobodies© of the invention, at least two of the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table A-l . Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A- l ; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table A-l ,
In particular, in the Nanobodies® of the invention, at least the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table A- l, and either the CDRl sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table A-l , Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A- l ; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table A-l . Even more preferably, in the Nanobodies® of the invention, all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
Also, generally, the combinations of CDR's listed in Table A-1 (i.e. those mentioned on the same line in Table A-1) are preferred. Thus, it is generally preferred that, when a CDR in a Nanobody® of the invention is a CDR sequence mentioned in Table A-1 or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table A-1, that at least one and preferably both of the other CDR's are suitably chosen from the CDR sequences that belong to the same combination in Table A-1 (i.e. mentioned on the same line in Table A-1) or are suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR sequence(s) belonging to the same combination and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the CDR sequence(s) belonging to the same combination. The other preferences indicated in the above paragraphs also apply to the combinations of CDR's mentioned in Table A-1.
Thus, by means of non-limiting examples, a Nanobody© of the invention can for example comprise a CDRl sequence that has more than 80 % sequence identity with one of the CD l sequences mentioned in Table A-1, a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination), and a CDR3 sequence.
Some preferred Nanobodies® of the invention may for example comprise: (1 ) a
CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table A-1 ; a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination); and a CDR3 sequence that has more than 80 % sequence identity with one of the CDR3 sequences mentioned in Table A-1 (but belonging to a different combination); or (2) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table A-1 ; a CDR2 sequence, and one of the CDR3 sequences listed in Table A-1 ; or (3) a CDR I sequence; a CDR2 sequence that has more than 80% sequence identity with one of the CDR2 sequence listed in Table A- 1; and a CDR3 sequence that has 3, 2 or 1 amino acid differences with the CDR3 sequence mentioned in Table A- 1 that belongs to the same combination as the CDR2 sequence.
Some particularly preferred Nanobodies® of the invention may for example comprise:
(1 ) a CDRI sequence that has more than 80 % sequence identity with one of the CDRI sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table A- 1. that belongs to the same combination; and a CDR3 sequence that has more than 80 % sequence identity with the CDR3 sequence mentioned in Table A- 1 that belongs to the same combination; (2) a CDRI sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed in Table A-1 (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).
Some even more preferred Nanobodies® of the invention may for example comprise: (1 ) a CDRI sequence that has more than 80 % sequence identity with one of the CDRI sequences mentioned in Table A- 1; the CDR2 sequence listed in Table A-1 that belongs to the same combination; and a CDR3 sequence mentioned in Table A-] that belongs to a different combination; or (2) a CDRI sequence mentioned in Table A- 1 ; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than. 80% sequence identity with the CDR3 sequence listed in Table A- 1 that belongs to the same or a different combination.
Particularly preferred Nanobodies® of the invention may for example comprise a CDRI sequence mentioned in Table A-1, a CDR2 sequence that has more than 80 % sequence identity with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and the CDR3 sequence mentioned in Table A-1 that belongs to the same combination.
In the most preferred Nanobodies® of the invention, the CDRI, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDRI , CDR2 and CDR3 sequences, respectively, listed in Table A-1.
According to another preferred, but non-limiting aspect of the invention (a) CDRI has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues; and/or (c) CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues.
In another preferred, but non-limiting aspect, the invention relates to a Nanobody© in which the CDR sequences (as defined herein) have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 81, more preferably SEQ ID NO: 72 to 77, 79, 80, even more preferably SEQ ID NO: 74, 76, 77, 79, 80, listed in Table A-2,
In another preferred, but non-limiting aspect, the invention relates to a Nanobody® with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 81, more preferably SEQ ID NO: 72 to 77, 79, 80, listed in Table A-2.
In another preferred, but non-limiting aspect, the invention relates to a Nanobody© with the amino acid sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 81 , more preferably SEQ ID NO: 72 to 77, 79, 80, listed in Table A-2.
Table A-2:
SEQ Alternative Amino Acid Sequences
ID names
NO:
EVQLVES GGGLVQTGGSLRLSC A AS GFTFS S Y AM S W VRQ AP
281D1 1, GKGLEWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNMLYLQ
72 238D2 MYSLKPEDTAVYYCA SRVSRTGLYTYDNRGQGTQVTVSS
EVQLMESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRR APGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKN
281B5, TLYLQMNSL PEDTAVYTCAASAIGSGALRRFEYDYSGQGT
73 238D4 QVTVSS
EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRA PGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNT LYLQMNSL PEDTAVYTCAASAIGSGALRRFEYDYSGQGTQ
74 281A5 VTVSS
75 281 E10, EVQLVESGGGLVQAGGSLRLSC ASGGTFNNYAMGWFRRA 238C4 PGKEREFVAAITRSGVRSGVSAIYGDSV DRFTISRDNV T LYLQMNTLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQ VTVSS
EVQLVESGGGLVQAGGSLRLSCAASGGTFNNYAMGWFRRA PG EREFV AAISRSG VRTGV S AL YG DS VKDRFTIS RDNA KNT L YLQMNKMKPEDT A V YTC A AS A IGSGALRRFE YDS SGQGT
76 281D4 QVTVSS
EVQLVESGGGLVQTGGSLRLSCAASGGTFNNYAMGWFRRA PGKEREFV AAISRSGVRTGVSALYGDSVKDRFTISRDNAKNT LYLQMNKMKPEDTAVYTCAASAIGSGALRRFEYDSSGQGT
77 281A6 QVTVSS
EVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQA PGKEREFV AA1GWGPS TNYADSVKGRFTISRDNAKNTVYL QMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGT
78 281F12 QVTVSS
EVQLVASGGGLVQAGGSLRLSCAVSGTTFSVATLGWYRQA PGQQRALVADISSGGSTNYADSVRGRFTISRDNAKNLAYLQ
79 283B6 MNSLKPEDTAVYYCNARTSGWRTRSNYWGQGTQVTVSS
EVQLVESGGGLVQAGGSLRLSCAVSGTTFSVATLGWYRQA PGQQRALVADISSGGSTNYADSVRGRFflSRDNAK LAYLQ
80 283E2 MNSLKPEDT A V Y Y CN ARTSGWRTRSN YWGQGTQVT V S S
EVQLVESGGGL V Q AGGS LRLSC VA S VNiFGS IAM AWYRQAP GKQRNLV AS IS S GGRINY ADSRKGRFTISRDNTKJ TVHLQM
81 283F1 NSLEPEDTAVYYCAAGRIGQRTLTFTPDYWGQGTQ VTVSS
The general principles of the present invention as set forth above will now be exemplified by reference to specific experiments and examples. However, the invention is not to be understood as being limited thereto.
The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. Preferred aspects:
Method for the generation of immunoglobulin sequences that can bind to and/or have affinity for a cell-associated antigen comprising the steps of:
a. immunization of a non-human animal;
i. by cell based immunization wherein said cells expressing the cell-associated antigen or a domain or specific part of said cell associated antigen; or ii. genetic vaccination with a nucleic acid encoding said cell-associated antigen or a domain or specific part of said cell associated antigen; and b. boosting the animal with said antigen in its natural conformation selected, from cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring enriched antigen, liposomes, lipoprotein particles or virus particles expressing the cell associated antigen; and
c. screening a set, collection or library of immunoglobulin sequences derived from said non-human animal for amino acid sequences that can bind to and/or have affinity for said cell-associated antigen and wherein the said cell-associated antigen is expressed in high concentration on. lipoprotein particles.
The method according to aspect 1, wherein said cell-associated antigen is selected from transmembrane antigens, including transmembrane antigens with multiple spanning domains, including but not limited to GPCRs or ion channels.
The method according to aspect 1 or 2, wherein said non-human animal is selected from vertebrates such as sharks, lizards, and mammals, more specifically camelids such as llama and alpaca.
The method according to any one of aspects 1 to 3, wherein the non-human animal is a camelid or llama.
The method according to any one of aspects 1 to 4, wherein said immunoglobulin sequences are light chain variable domain sequences, or heavy chain variable domain sequences.
The method according to aspect 5, wherein the immunoglobulin sequences are heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain, variable domain sequences that are derived from a heavy chain antibody.
The method according to any one of aspects 1 to 6, wherein the immunoglobulin sequences are domain antibodies, or amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, or amino acid sequences that are suitable for use as single domain antibodies, "dAbs", or amino acid sequences that are suitable for use as dAbs, or Nanobodies®, including but not limited to VHH sequences or amino acid sequences that are suitable for use as Nanobodies©.
The method according to aspect 7, wherein the immunoglobulin sequences are Nanobodies©.
The method according to any one of aspects 1 to 8, wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles, e.g. in a concentration of 0.1 ug/ml or higher, more preferably in a concentration of 0.2 ug/ml or higher, even more preferably in a concentration of 1 ug/ml or higher.
The method according to any one of aspects 1 to 9, wherein the lipoprotein particles are generated from the same cells as used in a) and/or b).
The method according to any one of aspects 1 to 10, wherein the set, collection or library of immunoglobulin sequences is obtained from the blood, lymph node, spleen, bone marrow or any tissue harbouring cells encoding these immunoglobulin sequences of said non-human mammal. The method according to any one of aspects 1 to 11, wherein said cell-associated antigen is expressed on any cell or lipoprotein particle which allows expressing of the target in its native conformation such as but not limiting to a cell selected from Cho, Cos7, Hek293t or camelid derived cells such as Llama derived or Alpaca derived cell and/or a lipoprotein selected from virus-like particles.
The method according to any one of aspects 1 to 12, wherein said cell-associated antigen is a membrane-spanning antigen such as e.g. a GPCR and/or ion channel.
The method according to any one of aspects 1 to 13, wherein said antigen is selected from CXCR7, CXCR4 and P2X7.
The method according to any of aspects 1 to 14, wherein the set. collection or library of immunoglobulin sequences is expressed on a set, collection or sample of cells or viruses or lipoprotein particles and said set, collection or sample of cells or viruses or lipoprotein particles is screened for cells that express an amino acid sequence that can bind to and/or have affinity for said cell-associated antigen.
The method according to aspect 15, wherein a nucleic acid sequence that encodes the amino acid sequence that can bind to and/or has affinity for said cell-associated antigen is purified and/or isolated from the cell or virus, followed by expression of said amino acid sequence.
The method according to any of aspects 1 to 1.6, wherein the set, collection or libraiy of immunoglobulin sequences is encoded by a set, collection or library of nucleic acid sequences and said set. collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode an immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen. The method according to aspect 17, wherein the nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for said cell-associated antigen are purified and/or isolated, foilowed by expressing said amino acid sequence.
The method according to any one of aspects 1 to 18, wherein the immunoglobulin sequence that can bind to and/or has affinity for said cell -associated antigen is purified and/or is isolated.
Immunoglobulin obtainable by a method of any one of aspects 3 to 19 such as e.g. a Nanobody® with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 82, more preferably SEQ ID NO: 74, 76, 77, 79, 80, 82.
Composition comprising the immunoglobulin sequence according to aspect 20.
Method for stem-cell mobilization, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of i) at least one immunoglobulin directed against CXCR4, e.g. human CXCR4, ii) compound or construct comprising an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) bispecific or multispecific construct comprising at ieast an
immunoglobulin directed against CXCR4, e.g. human CXCR4, or iv) composition comprising said immunoglobulin, compound or construct, bispecific or multispecific construct.
Use of i) at least one immunoglobulin directed against CXCR4, e.g. human CXCR4, ii) compound or construct comprising an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) bispecific or multispecific construct comprising at least an immunoglobulin directed against CXCR4, e.g. human CXCR4, or iv) composition comprising said immunoglobulin, compound or construct, bispecific or multispecific construct in the manufacture of a medicament for prevention and/or treatment of stem-cell mobilization. i) An immunoglobulin directed against CXCR4, e.g. human CXCR4, ii) compound or construct comprising an immunoglobulin directed against CXCR4, e.g. human CXCR4, iii) bispecific or multispecific construct comprising at least an
immunoglobulin directed against CXCR4, e.g. human CXCR4, or iv) composition comprising said immunoglobulin, compound or construct, bispecific or multispecific construct for use in the prevention and/or treatment of stem-cell mobilization.
Method for stem-cell mobilization, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one immunoglobulin according to aspect 20, compound or construct comprising an immunoglobulin according to aspect 20, bispecific or multispecific construct comprising at least an immunoglobulin according to aspect 20, or composition according to aspect 21.
Use of at least one immunoglobuli according to aspect 20, compound or construct comprising an immunoglobulin according to aspect 20, bispecific or multispecific construct comprising at least an immunoglobulin according to aspect 20, or composition according to aspect 21 in the manufacture of a medicament for prevention and/or treatment of stem-cell mobilization.
An immunoglobulin according to aspect 20, compound or construct comprising an immunoglobulm according to aspect 20, bispecific or multispecific construct comprising at least an immunoglobulin according to aspect 20, or composition according to aspect 21 for use in the prevention and/or treatment of stem-cell mobilization.
Examples
Example 1 : CXCR4-specific immune responses are not detectable in serum of llama immunized with HEK293-CXCR4 cells using CXCR4 lipoprotein particles.
Llama 217 and 218 were immunized with HEK293T cells transiently expressing
CXCR4. Pre-immune sera and sera taken 4 days after the last immunization was serially diluted and added to CXCR4+ and CXCR4- lipoprotein particles coated plates (2'U/well). Bound llama antibodies were detected with goat an ti- llama (IgG) conjugated with HRP followed by TMB substrate. No specific binding of antibodies from llamas immunized with CXCR4 cells to CXCR4+ particles could be observed (Figure 1). The y-axis indicates the OD at 450 nm. indeed, similar OD values of serum antibodies binding to CXCR4+ and CXCR4- particles were obtained.
Example 2: CXCR4+ lipoprotein particles are recognized by purified CXCR4-specific Nanobodies® 238D2 and 238D4
Purified Nanobodies® 238D2 or 238D4 were tested for binding to CXCR4+ and CXCR4- (null) lipoprotein particles. Wells were coated with 10, 1 and 0.1 U of the particles. After blocking with 4% Marvel in PBS, 100, 10 and 1 nM of Nanobodies® 238D2, 238D4 and of an irrelevant control Nanobody® (g l202E6) were added to the wells. Other positive and negative controls included the use of 10 nM 12G5 (a CXCR4- specific mouse monoclonal, antibody) and 10 nM OKT3 (a mouse monoclonal antibody that recognizes CD3), respectively. Bound Nanobodies® were detected by adding 1 ug/ml mouse anti-myc (Roche Cat 11667149001). All mouse antibodies were detected by rabbit anti-mouse-HRP, followed by TMB substrate. Binding of Nanobodies® 238D2 and 238D4 was detected clearly to 10 U of CXCR4+ particles but not to 10 U of CXCR4- particles (Figure 2). Figure 2 shows twelve sets of bar graphs, with each set of bar graphs depicting seven data points: Null Lip (10U, 1U, 0.1U) and CXCR4 Lip (10U, 1.U, 0..1.U) and NC, from left to right. The y-axis indicates the OD at 450 nm. Specific binding to wells coated with 1U of CXCR4 particles was only obtained with 238D4. An interaction with 10 U CXCR4+ particles was observed with concentrations as low as 10 nM for 238D2 and 1 nM for 238D4. No interaction of control Nanobody® 2E6 with CXCR4+ and CXCR4- particles was observed. Finally, 12G5 interacted with CXCR4+ particles only. O T3 did not bind to either particle.
Figure 3: Detection of CXCR4+ lipoprotein particles by periplasmic extracts of CXCR4-specific Nanobodies® 238D2 and 238D4
Wells were coated with 10, 1 and 0.1 U of the CXCR4+ and CXCR4-particles (null). After blocking with 4% Marvel in PBS, periplasmic extracts diluted 10-fold in 2%
Marvel/PBS were added to the wells. Periplasmic extracts were prepared for CXCR4 Nanobodies® 238D2, 238D4, 238C5 and the irrelevant control Nanobody® 2E6. Other positive and negative controls included the use of 10 nM 12G5 (a CXCR4-specific mouse monoclonal antibody) and 10 nM O T3 (a mouse monoclonal antibody that recognizes CD3), respectively. Bound Nanobodies® were detected by adding 1 ug/ml mouse anti-myc (Roche Cat 1 1667149001). All mouse antibodies were detected by rabbit anti-mouse-HRP, followed by TMB substrate, 12G5 interacted with CXCR4+ particles only. OKT3 did not bind to either particle. A clear signal was observed with 238D2 and 238D4 periplasmic extracts added to wells coated with I 0U of CXCR4+ particles. No binding of Nanobodies® to particles was observed when 238D5 periplasmic extracts were used. No interaction with periplasmic extracts of control Nanobody® 2E6 with CXCR4+ and CXCR4- particles was detected (Figure 3). Figure 3 shows seven sets of bar graphs, with each set of bar graphs depicting five data points: Lipo Null (10U, 1 U) and Lipo CXCR4 (10U, 1U) and NC, from left to right. The y-axis indicates the OD at 450 nm.
Example 4: CXCR4+ lipoprotein particles are recognized by phages that display 238D2 and 238D4.
Phages displaying 238D2 and 238D4 were produced and purified. Wells were coated with 2 U of the CXCR4+parti.cles. After blocking with 4% Marvel in PBS, phage diluted 100-fold in 2% Marvel/PBS were added to the wells. Phage displaying 212C 12 (an unrelated Nanobody®) or no phage addition were used as control. Phages binding to CXCR4+p articles were detected by an anti-M13-HRP antibody. A clear signal was observed with 238D2 and 238D4 phages while no interaction was observed for the irrelevant Nanobody® used as negative control (Figure 4). The y-axis indicates the OD at 450 nm Example 5: Selections with library 218 on CXCR4+ particles results in isolation of large numbers of Nanobodies® binding to CXCR4+ particles.
Because functional CXCR4 Nanobodies® (238D2 and 238D4) were isolated previously only from library 218, this library was used first for panning on lipoprotein particles. For the first round of selections, wells were not coated (NC) or coated with 10 U of CXCR4+ or 10 U of CXCR4- particles (null). After blocking with 4% Marvel/PBS, phages from library 218 were diluted 30-fold and added to the wells. After 2.5 h at room
temperature, phages were removed and wells were washed 20 times with PBS. Bound phages were eluted with trypsin and phage titers were determined (Figure 5a). Compared to the panning on NC wells, a clear 10-fold enrichment was observed when panning was performed on CXCR4+ wells. However, a very similar enrichment was observed after panning on CXCR4- wells, indicating only very limited CXCR4-specific enrichment (Figure 5a).
Outputs from the panning on CXCR4+ particles (21 SRI) were used for a second round of selection. Wells were not coated (NC) or coated with 10 and 1 1) of CXCR4+ or 10 U CXCR4- particles (null). After blocking with 4% Marvel/PBS, 21 SRI phages were diluted 10-fold and added to the wells. After 2.5 h at room temperature, phages were removed and wells were washed 20 times with PBS. Bound phages were eluted with trypsin and phage titers were determined (Figure 5b). A clear 100,000-fold enrichment was observed when panning was performed on 10 U CXCR4+ particles while 1 , 000-fold enrichment was observed after panning on 1 U of CXCR4+ particles. Only 1 ,000 to 10,000-fold enrichment was obtained on 10 U CXCR4- particles, which indicates a clear specific enrichment on 10 U CXCR4+ particles (Figure 5b).
From the 218RII a total of 44 monoclonal clones were selected; 22 were derived from the selections on 10 U CXCR4+ particles and 22 from the selections on 1 U CXCR4+ particles. Periplasmic extracts were prepared, diluted 10-fold and added to wells coated with 2 U of CXCR4+ and CXCR4- particles. As positive controls periplasmic extracts of 238D2 and 238D4 were used. Periplasmic extract of 2E6 was used as a negative control. Bound Nanobodies® were detected with mouse anti-myc followed by rabbit anti-mouse-HRP and TMB. Of the 22 clones derived from the 218RI1 selection on 10 U CXCR4+, 17 showed binding to CXCR4+ particles only, with OD values higher than the one obtained with 238D2. Three clones displayed specific binding to CXCR4+ particles as well, but OD values were lower than the one obtained for 238D2. Two clones did not bind to CXCR4+ and CXCR4- particles (Figure 5c). Of the 22 clones derived from the R21 811 selection on 1 U CXCR4+, 17 showed binding to CXCR4+ particles only, with OD values higher than the one obtained with 238D2. Three clones displayed specific binding to CXCR4+ particles as well, but OD values were lower than the one obtained for 238D2. Two clones did not bind to CXCR4+ and CXCR4- particles (Figure 5c).
The coding sequences of all 44 clones were PCR amplified and PCR products were sequenced. Forty Nanobody® sequences were obtained (data not shown for all, see Table A- 2 for selected Nanobodies®). AH 31 Nanobodies® that showed a high specific binding to CXCR4+ particles, showed high sequence similarity to one the previously isolated
Nanobodies® 238D2, 238D4 and 238C4. In fact, the amino acid sequence of clone 281D1 1 was completely identical to the amino acid of 238D2 and the amino acid sequences of clones 281B5, 281H10, 28 I E11 , 281F4 were completely identical to the amino acid sequence of 238D4 (Figure 5d). The amino acid sequences of 281E10 and 281B6 were identical to the amino acid sequence of 238C4. The amino acid sequence of the 24 other Nanobodies® showed very high similarity to the amino acid of 238D4 (Figure 5d). A total of five 238D4 subfamilies (subfamilies 238D4, 281A5, 238C4, 281D4 and 281 A6, Figure 5d) could be defined. Five more families were defined (Families 281A4, 281 C4, 281F6, 281F1 1 and 281F12) which consist of Nanobodies® that display very low CXCR4+ specific binding. These Nanobodies® were produced, purified and tested for binding to CXCR4+ and CXCR4- particles. A clear dose-dependent interaction was observed only with Nanobody® 281F12. No binding to either particle was observed for the other purified Nanobodies® (Figure 5e). Figure 5e shows sixteen sets of bar graphs, with each set of bar graphs depicting five data points: 200 nM, 100 nM, 10 nM, 1 nM and 0, from left to right The y-axis indicates the OD at 450 nm.
Overall these data demonstrate that highly CXCR4-specific enrichment occurred when using the CXCR4+ particles for panning. Nanobodies® were identified which were previously isolated using membrane extracts, but more importantly completely new CXCR4 specific Nanobodies® were isolated when using the CXCR4+ particles. Example 6: Selections on CXCR4- particles does not yield Nanobodies® binding to CXCR4+ and CXCR4- particles
From the 218R1I on 10U CXCR4- particles, 1 1 clones were selected. Periplasmic extracts were prepared, diluted 10-fold and added to wells coated with 2 U of CXCR4+ and CXCR4- particles. As positive controls periplasmic extracts of 238D2, 238D4 and 281A12 were used. Periplasmic extract of 2E6 was used as a negative control. Bound Nanobodies® were detected with mouse anti-myc followed by rabbit anti-mouse-HRP and TMB. While a specific binding of Nanobodies® 238D2, 238D4 and 283 A12 to CXCR4-S- particles was again obtained, no binding to CXCR4+ and CXCR4- particles was observed for the 1 1 clones selected on the CXCR4- particles (Figure 6). The y-axis indicates the OD at 450 nm. This data further indicates that strong CXCR4-specific enrichments occurred when using the CXCR4+ particles for panning.
Example 7: Selections with library 217 on CXCR4+ particles results in isolation of large numbers of Nanobodies® binding to CXCR4+ particles.
From the selections with library 237 on membrane extracts, no CXCR4-specific Nanobodies® could be isolated. Following the successful panning on the CXCR4+ particles with library 218, library 217 was now tested. For the first round of selections, wells were not coated (NC) or coated with 10 U of CXCR4+ or 10 U of CXCR4- particles (null). After blocking with 4% Marvel/PBS phages from library 218 were diluted 10-fold and added to the wells. After 2.5 h at room temperature phages were removed and wells were washed 20 times with PBS. Bound phages were eluted with trypsin and phage titers were determined (Figure 7a). Compared to the panning on NC wells, a clear 10-fold enrichment was observed when panning was performed on CXCR4+ wells. However, a very similar enrichment was observed after panning on CXCR4- wells, indicating only very limited CXCR4-specific enrichment (Figure 7a).
Outputs from the panning on CXCR4+ particles (217RT) were used for a second round of selection. Wells were not coated (NC) or coated with 10 and 1. U of CXCR4+ or 10 U CXCR4- particles (null). After blocking with 4% Marvel/PBS, 218RI phages were diluted 30-fold and added to the wells. After 2.5 h at room temperature phages were removed and wells were washed 20 times with PBS. Bound phages were eluted with trypsin and phage titers were determined. With R2171, a clear 100,000-fold enrichment was observed when panning was performed on 10 U CXCR4+ particles while a 10, 000-fold enrichment was observed after panning on 1 U of CXCR4 - particles. Although enrichment was also obtained on 10 U CXCR4- particles this was only 1000-fold, which indicates a clear specific enrichment on 10 and 1 U CXCR4+ particles (Figure 7b).
From the R2 I 7RII selection a total of 42 monoclonal clones were selected; 21 derived from the selections on 1 U CXCR4+ particles and 21 from the selections on 1 U CXCR4+ particles. Periplasmic extracts were prepared, diluted 10-fold and added to wells coated with 2 U of CXCR4+ and CXCR4- particles. As positive controls periplasmic extracts of 238D2, 238D4 and 281A12 were used. Periplasmic extract of 281A1 and E6 were used as negative controls. Bound Nanobodies© were detected with mouse anti-myc followed by rabbit anti- mouse-FTRP and TMB. Of the 21 clones derived from the R217II selection on 10 U
CXCR4+, 20 showed binding to CXCR4+ particles only, with OD values higher than the one obtained with 238D2. No binding to either particle was observed for the one remaining clone. Of the 21 clones derived from the R217II selection on 1 U CXCR4+, 20 showed binding to CXCR4+ particles only, with OD values higher than the one obtained with 238D2. No binding to either particle was observed for the one remaining clone (Figure 7c).
The coding sequences of all 42 clones were PCR amplified and PCR products were sequenced. Nanobody® sequences of 34 clones were obtained (data not all shown, for some see Table A-2). Thirty Nanobodies© had the same amino acid sequence and a family 283B6 was defined. One more Nanobody® 283E2 also belonged to this family as only one amino acid difference was observed. Nanobody® 283F1 which also showed specific binding to CXCR4+ particles had a different amino acid sequence; family 283F1 was defined.
Nanobodies® 283C2 and 283C5 which did not bind particles belonged clearly to other families, although their CDR2 sequences showed very high similarity to the CDR2 sequences of families 283E6 and 283FL
To verify that 283B6, 283E2 and 283F1 bind to CXCR4 on the CXCR4+particies, phages displaying these Nanobodies® were produced and purified. Binding of these phages to CXCR4+ particles was competed with purified 238D2 and 238D4. Phages displaying 238D2 and 238D4 were used as controls. The assay was performed in duplicate, allowing detection of either phage (anti-M 13-HRP) or Nanobody® (anti-myc, anti mouse -HRP). As depicted in figure 7d and 7e, binding of phage to CXCR4+ particles was inhibited completely upon co-incubation with purified 238D4. Binding was also reduced upon co-incubation with purified 238D2. Figure 7d shows seven sets of bar graphs, with each set of bar graphs depicting three data points: 283D2 Nb, 283D4 Nb, and No Nb, from left to right. The y~axis indicates the OD at 450 nm. Figure 7e shows two sets of bar graphs, with each set of bar graphs depicting seven data points: 283d2 phage, 283 D4 phage, 283 B6 phage, 283 E2 phage. 283 Fl phage, 212-C12 phage and no phage, from left to right. The y-axis indicates the OD at 450 nm. This data indicate that Nanobodies© 283B6 and 283E2 indeed bind to CXCR4 on the particles. Moreover the data indicates that 283B6 and 283E2 bind to a similar or overlapping region also recognized by 238D2 and 238D4.
Overall these data demonstrate that from library 217 it was possible to highly CXCR4-specific enrichment occurred when using the CXCR4+ particles for panning.
Nanobodies® were identified which were previously not isolated using membrane extracts.
Example 8: Binding of Nanobodies® selected on CXC 4+ particles to HEK293T- CXCR4 expressing cells
To check whether the Nanobodies® selected on CXCR4+ particles recognize cell- surface-native form of the receptor, a Flow cytometry experiment was performed where the Nanobodies® were tested for specific binding to HEK293T-CXCR4 expressing cells.
C-terminally myc tagged Nanobodies® (1, 10 and 100 nM) were allowed to bind to 10x5 cells of HEK293T-CXCR4 or HEK293T WT cells for 30 minutes at 4°C in 100 ul of FACS buffer (10% FBS (Invitrogen, Cat 10270-306, Lot#41G41170K) in PBS (Invitrogen # 14190)). Bound Nanobodies® were detected with mouse anti-myc tag antibody (Serotec, Cat#MCA2200, Lot #0407) followed by a phycoerythrin (PE) conjugated goat anti-mouse- (Jackson ImmunoResearch Inc. Cat #1 15-1 15-164 Lot #79725). Cells were washed three times using 200 microliter of 4°C FACS buffer in between staining steps. Dead cells were stained selectively by including 1 uM TOPR03 in the FACS buffer used to resuspend the cells following the final centrifugation step (Molecular probes T3605; Lot #413969).
Twenty thousand events were acquired per staining condition using a standard BD
FACSArray FACS system. Only live, intact cells were included in the binding analysis by pre~gating on intact cells using a first forward/side scatter dot plot defined gate and then subgated further on a TOPR03 fluorescence negative population within the first. The PE fluorescence intensity of these pre-gated cells was expressed as the median channel number of the PE detector histogram, as an indicator of Nanobody® binding intensity. Expression of human CXCR4 on HE 293T-pCDNA3.1 hCXCR4 transient transfected cells, as well as lack thereof on WT HEK293T cells, was confirmed in parallel experiments by staining both cell types using the mouse anti human hCXCR4 monoclonal antibody mAbl2G5 (R&D Systems, Cat# MAB 170, Lot #AEI0907031), followed by the same goat secondary antiserum.
Negative controls included stainings with irrelevant specificity Nanobody© cione 212C12 followed by anti myc tag and goat anti mouse, or no Nanobody© followed by anti~myc and goat anti mouse, or mouse anti human CXCR7 monoclonal staining followed by goat anti mouse secondary (Figure 8a).
The results obtained for the different Nanobodies® are shown in Figures 8b and c. All Nanobodies® selected on CXCR4+ particles bound CXCR4~expressing but not WT
HEK293T cells. Based on the results obtained with the different concentration of the Nanobodies®, it seems that clone 238D4, other members of the same family (281A5,
281E10, 281D4 and 281A6) and the Nanobodies® of family 283B6 (283B6 and 283E2) are the highest affinity CXCR4 binding Nanobodies®. Other clones such as 281F12 and 283F1 bound CXCR4-expressing cells with moderate affinity. Clone 238D2 was shown to be the lowest affinity binder.
Table B-l :
Figure imgf000071_0001
+- Low affinity
++- Moderate affinity
+++- High affinity Example 9: Membrane Extract vs. Lipopartides selection summary
Table B-2
Figure imgf000072_0001
These data suggest that the method using CXCR4+particI.es as selection and screening tools can provide more variants of CXCR4 specific Nanobodies® and overall indicate that the methods of the invention are suitable and an improvement over the prior art methods described for immunoglobulin sequences such as Nanobodies©. Example 10: Use of a Nanobody® construct directed against CXCR4 in stem cell mobilization
Table B-3:
Amino acid sequence Name References
EVQLVESGGGLVQTGGSLRLSCAASGFTFSSYAM 238D2-20GS- See also SEQ ID
SWVRQAPGKGLEWVSGIKSSGDSTRYAGSV GR 238D4 NO: 264 in WO
FTISRDNAKN LYLQMYSLKPEDTAVYYCA SR 2009/138519;
VSRTGLYTYDNRGQGTQVTVSSGGGGSGGGGSG SEQ ID NO: 82
GGGSGGGGSEVQLMESGGGLVQAGGSLRLSCAA
SGRTFNNYAMGWFRRAPGKEREFVAAITRSGVR
SGV S AI YGDS V DRFTISRDN AKNTL YLQMNS L
PEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVT
vss Four cynomolgus monkeys were administered with AMD3100 or 238D2-20GS-238D4 in a staggered administration scheme. AMD3100 (Mozobil® or Plerixafor, Genzyme) was given by a subcutaneous bolus injection at 1 mg/kg. 238D2-20GS-238D4 was administered by a 30 minute infusion in several doses (0.1 , 1, 10, 25 mg/kg) in D-PBS (Gibco).
Table B-4:
Figure imgf000073_0001
At several timepoints blood was collected in tubes with 8% EDTA as anti-coagulants. The collection timepoints of the administrations at day 1 and 30 were pre-administration and at 3, 6, 9 and 24 hours post-injection. For the administrations at day 30 and 39 blood was collected pre-administration and at 1.5, 3, 6 and 9 hours post-injection. White blood cells were counted with the ADVIA 120™ Automated Hematology Analyzer (Siemens) and corrected for nucleated red biood cells by a manual blood count. The relative number of mobilized stem cells in the peripheral blood was determined by a dual-platform flow cytometric analysis of CD34+ cells, according to the ISHAGE guidelines (Sutherland DR et al.: The IS H AGE guidelines for CD34+ cell determination by flow cytometry. J. Hematother 5:213-226 (1996); Gratama JW et al: Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. Cytometry 34: 128- 142 (1998); Barnett D et al: Guideline for the flow cytometric enumeration of CD34+ haematopoietic stem cells. Clin. Lab. Haem.21 :301-308 (1999). in summary, 100 μΐ of blood was incubated with 5 μΐ of anti-CD45 FITC (Miltenyi Biotec) and 20 μΐ anti-CD34 PE (BD Pharmingen) or 20 μΐ isotype control mouse IgGl PE (BD Phamiingen) for 15 minutes at room temperature in the dark. Then, red blood cells were lysed by 2 ml ammonium chloride (Stem Cell Technologies) during a 10 minute incubation period at room temperature in the dark. Finally, 1 μg/m3 7-AAD (Sigma Aldrich) was added to stain viable cells and incubated for 10 minutes in the dark. The samples were analyzed by the cytometer within one hour after addition of the lysing reagent. According to the ISHAGE gating strategy, a minimum of 100 CD34+ cells and 75,000 nucleated events were counted. The absolute number of stem cells per μΐ was calculated by the following equation:
CD34+ events - isotype control events x corrected white blood cells x 103/μ1
CD45+ events Stem cell mobilization was observed after 238D2-20GS-238D4 administration induces, even to doses as low as 0.1 mg/kg (Figure 9). The number of mobiiized stem cells returns faster to baseline than with Mozobil©.

Claims

Method for the generation of immunoglobulin sequences that can bind to and/or have affinity for a cell-associated antigen comprising the steps of:
a. immunization of a non -human animal;
iii. by cell based immunization wherein said cells expressing the cell- associated antigen or a domain or specific part of said cell associated antigen; or
iv. genetic vaccination with a nucleic acid encoding said cell-associated antigen or a domain or specific part of said cell associated antigen; and b. boosting the animal with said antigen in its natural conformation selected from cells comprising natural or transfected cells expressing the cell-associated antigen, cell derived membrane extracts, vesicles or any other membrane derivative harbouring enriched antigen, liposomes, lipoprotein particles or virus particles expressing the cell associated antigen; and
c. screening a set, collection or library of immunoglobulin sequences derived from said non-human animal for amino acid sequences that can bind to and/or have affinity for said cell-associated antigen and wherein the said cell- associated antigen is expressed in high concentration on lipoprotein particles.
The method according to claim 1, wherein said cell-associated antigen is selected from transmembrane antigens, including transmembrane antigens with multiple spanning domains, including but not limited to GPCRs or ion channels.
The method according to claim 3 or 2, wherein said non-human animal is selected from vertebrates such as sharks, lizards, and mammals, more specifically camelids such as llama and alpaca.
4. The method according to any one of claims 1 to 3, wherein the non-human animal is a camelid or llama. The method according to any one of claims 1 to 4, wherein said immunoglobulin sequences are light chain variable domain sequences, or heavy chain variable domain sequences.
The method according to claim 5, wherein the immunoglobulin sequences are heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody.
The method according to any one of claims 1 to 6, wherein the immunoglobulin sequences are domain antibodies, or amino acid sequences that are suitable for use as domain antibodies, single domain antibodies, or amino acid sequences that are suitable for use as single domain antibodies, "dAbs", or amino acid sequences that are suitable for use as dAbs, or Nanobodies®, including but not limited to VHH sequences or amino acid sequences that are suitable for use as Nanobodies®.
The method according to claim 7, wherein the immunoglobulin sequences are Nanobodies®.
The method according to any one of claims 1 to 8, wherein the said cell-associated antigen is expressed in high concentration on lipoprotein particles, e.g. in a concentration of 0.1 ug/ml or higher, more preferably in a concentration of 0.2 ug/ml or higher, even more preferably in a concentration of 1 ug/ml or higher.
The method according to any one of claims 1 to 9, wherein the lipoprotein particles are generated from the same cells as used in a) and/or b).
The method according to any one of claims 1 to 10, wherein the set, collection or library of immunoglobulin sequences is obtained from the blood, lymph node, spleen, bone marrow or any tissue harbouring cells encoding these immunoglobulin sequences of said non-human mammal.
12. The method according to any one of claims 1 to 11 , wherein said cell-associated antigen is expressed on any cell or lipoprotein particle which allows expressing of the target in its native conformation such as but not limiting to a cell selected from Cho, Cos7, Hek293, or camelid derived cells such as Llama derived or Alpaca derived cell and/or a lipoprotein selected from virus-like particles.
13. The method according to any one of claims 1 to 12, wherein said cell-associated antigen is a membrane-spanning antigen such as e.g. a GPCR and/or ion channel.
14. The method according to any one of claims 1 to 13, wherein said antigen is selected from CXCR7, CXCR4 and P2X7.
15. The method according to any of claims 1 to 14, wherein the set, collection or library of immunoglobulin sequences is expressed on a set, collection or sample of cells or viruses or lipoprotein particles and said set, collection or sample of cells or viruses or lipoprotein particles is screened for cells that express an amino acid sequence that can bind to and/or have affinity for said cell-associated antigen. 16, The method according to claim 15, wherein a nucleic acid sequence that encodes the amino acid sequence that can bind to and/or has affinity for said cell-associated antigen is purified and/or isolated from the cell or virus, followed by expression of said amino acid sequence. 17. The method according to any of claims 1 to 16, wherein the set, collection or library of immunoglobulin sequences is encoded by a set, collection or library of nucleic acid sequences and said set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode an immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen. The method according to claim 17, wherein the nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for said cell-associated antigen are purified and/or isolated, followed by expressing said amino acid sequence.
The method according to any one of claims 1 to 18, wherein the immunoglobulin sequence that can bind to and/or has affinity for said cell-associated antigen is purified and/or is isolated.
Immunoglobulin obtainable by a method of any one of claims 1 to 19 such as e.g. a Nanobody® with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 72 to 81 , more preferably SEQ ID NO: 74, 76, 77, 79. 80.
Composition comprising the immunoglobulin sequence according to claim 20.
Method for stem-cell mobilization, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one
immunoglobulin according to claim 20, compound or construct comprising an immunoglobulin according to claim 20, bispecific or multispecific construct comprising at least an immunoglobulin according to claim 20, or composition according to claim 21.
Use of at least one immunoglobulin according to aspect 20, compound or construct comprising an immunoglobulin according to claim 20, bispecific or multispecific construct comprising at least an immunoglobulin according to claim 20, or composition according to claim 21 in the manufacture of a medicament for prevention and/or treatment of stem -cell mobilization.
An immunoglobulin according to claim 20, compound or construct comprising an immunoglobulin according to claim 20, bispecific or multispecific construct comprising at least an immunoglobulin according to claim 20, or composition according to claim 21 for use in the prevention and/or treatment of stem-cell mobilization.
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