EP2723772A1 - Gegen ige gerichtete variable immunglobulin-einzeldomänen - Google Patents

Gegen ige gerichtete variable immunglobulin-einzeldomänen

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Publication number
EP2723772A1
EP2723772A1 EP12732607.2A EP12732607A EP2723772A1 EP 2723772 A1 EP2723772 A1 EP 2723772A1 EP 12732607 A EP12732607 A EP 12732607A EP 2723772 A1 EP2723772 A1 EP 2723772A1
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EP
European Patent Office
Prior art keywords
amino acid
seq
ige
polypeptide
sequence
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EP12732607.2A
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English (en)
French (fr)
Inventor
Peter Meerts
Sigrid CORNELIS
Guy Hermans
Jo Vercammen
Frank Kamiel Delphina Verdonck
Stephanie Staelens
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Ablynx NV
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Ablynx NV
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Publication of EP2723772A1 publication Critical patent/EP2723772A1/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to polypeptides and protein constructs that are directed against IgE; to nucleic acids that encode such polypeptides and protein constructs; to
  • compositions and in particular pharmaceutical compositions, that comprise such polypeptides and protein constructs; and to uses of such polypeptides, protein constructs and compositions.
  • the international application WO 04/041865 describes specific protein constructs that are directed against IgE that comprise at least one VHH or humanized VHH against IgE and at least one Nanobody that is directed against a serum protein such as (human) serum albumin, which because of the presence of the serum albumin-binding Nanobody have increased half-life in vivo compared to the corresponding VHH's against IgE alone.
  • a serum protein such as (human) serum albumin
  • VHH's may be humanized and may be suitably linked to one or more VHH's that are directed against a serum protein such as serum albumin, to provide a protein construct that has increased half-life as well as the favorable properties that are associated with VHH's and Nanobodies.
  • Nanobodies against (human) serum albumin include the Nanobody called Alb-1 (SEQ ID NO: 52 in WO 06/122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 06/122787; SEQ ID NO: 174 herein).
  • Alb-1 SEQ ID NO: 52 in WO 06/122787
  • Alb-8 SEQ ID NO: 62 in WO 06/122787; SEQ ID NO: 174 herein.
  • Nanobody® and Nanobodies® are trademarks of Ablynx N. V.J.
  • the use of Nanobodies against (human) serum albumin for extending the half-life of therapeutic moieties such as Nanobodies has been validated by means of clinical trials. For example, the safety, tolerability,
  • Nanobodies against (human) serum albumin is a good and broadly applicable way of extending the half-life of Nanobodies and other therapeutic entities
  • the present inventors have found that when representative Nanobodies according to WO 06/122787 are applied to extending the half-life of Nanobodies that are directed to IgE (in the manner generally described in WO 04/041865 and WO 04/041867), that the constructs thus obtained, even though they are sufficiently biologically active against IgE and have a half-life that is suitable for therapeutic applications, have some properties that would benefit from further improvement.
  • IgE and Alb-8 a representative serum albumin-binding Nanobody according to WO 06/122787, have a limited storage stability that leaves room for further improvement (see for example the results presented in the Experimental Part herein).
  • the invention generally has the objective of providing improved polypeptides and (other) protein constructs that comprise at least one immunoglobulin single variable domain (such as a Nanobody) against IgE and that have an increased half-life in vivo (as further described herein, and compared to the immunoglobulin single variable domain against IgE per se) that are not associated with the same problem(s) as have now been found to occur when anti- IgE constructs of the type generally described in WO 04/041865 and WO 04/041867 are used.
  • immunoglobulin single variable domain such as a Nanobody
  • NExpediteTM technology [NExpediteTM is a trademark of Ablynx N. V.J. WO 08/068280 also generally mentions that the therapeutic moiety may be a Nanobody against IgE as described in WO 04/041867).
  • the inventors have also generated a number of immunoglobulin single variable domains against IgE that have certain advantages compared to the known Nanobodies against IgE (such as those described in WO 04/041865 and WO 04/041867). These improved Nanobodies against IgE can, for example and with advantage, be used as "building blocks" to provide the half-life extended anti-IgE protein constructs described herein.
  • the inventors have also provided a range of different half-life extended anti-IgE proteins constmcts with improved properties compared to the half-life extended anti-IgE constructs described in WO 04/041865, and these improved protein constructs and polypeptides form further aspects of the invention.
  • the invention relates to a polypeptide, protein, construct or compound (also collectively referred to herein as "anti-IgE polypeptides of the invention” or “polypeptides of the invention ”) that comprises or essentially consists of:
  • ISV's immunoglobulin single variable domains
  • Such a polypeptide, protein, construct or compound (and/or the one or more ISV's against IgE present therein) may in particular be such that they are capable of modulating, and in particular inhibiting or blocking (fully or partially) the interaction between (human) IgE and the (human) Fc(epsilon)RI (the high affinity IgE receptor), for example as measured in a suitable assay, such as one of the assays used in the Experimental Part below (for example, the assay used in Example 7).
  • it may be such that it inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 ⁇ 10 M, better than 5.10 "n M, better than 2.10 "n M, or even better than 10 " n M.
  • Such a polypeptide, protein, construct or compound (and/or the one or more ISV's against IgE present therein) may also be such (or in addition be such) that they are capable of modulating, and in particular inhibiting or blocking (fully or partially) that interaction between (human) IgE and the (human) Fc(epsilon)RII (the low affinity IgE receptor), for example as measured in a suitable assay, such as one of the assays used in the Experimental Part below (for example, the assay used in Example 8).
  • a suitable assay such as one of the assays used in the Experimental Part below (for example, the assay used in Example 8).
  • it may be such that it inhibits the
  • HuIgE/HuFc(epsilon)RII interaction for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 ⁇ 8 M, preferably better than 2.10 "8 M, such as better than 10 ⁇ 8 M, better than 5.10 "9 M, better than 2.10 ⁇ 9 M, better than 10 "9 M, better than 5.10 "I0 M, or better than 2.10 "10 M.
  • any such polypeptide, protein, construct or compound (and/or the one or more ISV's against IgE present therein) may be such that it has an IC50 value in the degranulation assay described in Example 11 better than 100 nM, preferably better than 50 nM, more preferably better than 20nM, such as better than 5nM, better than 1 nM, or even better than 0.5 nM.
  • IgE binding ISV may be such that it has an IC50 value in the degranulation assay described in Example 11 better than 100 nM, preferably better than 50 nM, more preferably better than 20nM, such as better than 5nM, better than 1 nM, or even better than 0.5 nM.
  • the one or more immunoglobulin single variable domains or "ISV's" against IgE that are present in the IgE constructs of the invention can be any suitable immunoglobulin single variable domain that is directed against IgE.
  • the ISV against IgE can for example be a domain antibody, single domain antibody, dAbTM, Nanobody, VHH, IgNAR domain or other suitable immunoglobulm single variable domain that is directed against IgE.
  • said one or more ISV's against IgE are Nanobodies (such as VHH's, humanized VHH's or camelized VH's, such as camelized human VH's).
  • the one or more ISV's (and in particular Nanobodies) against IgE present in the anti-IgE polypeptides of the invention may in particular be ISV's or Nanobodies that are cross-reactive between the human IgE Fc sequence of SEQ ID NO: 172 and the and IgE Fc sequence from cynomolgus monkey given in SEQ ID NO: 173.
  • ISV's may have an affinity (as determined by a suitable technique, such as Biacore, for example using the protocol described in Example 12) for cynomolgus IgE that is at least 1%, for example 5%, such as at least 10%, for example at least 25% such as at least 50% or more of the affinity of the ISV for human IgE.
  • the one or more ISV's (and in particular Nanobodies) against IgE present in the anti- IgE polypeptides of the invention may have an affinity for human IgE (as determined using Biacore, for example using the protocol described in Example 12) that is better than 50nM, preferably better than 25nM, (KD1 value - see Example 12) and/or better than 5 nM, preferably better than 2 nM, such as better than 1 nM (KD2 value - see again Example 12); and an affinity for cyno IgE that is better than 50 nM, such as better than 30nM (KDl value) and/or better than 5 nM, preferably better than 2nM, such as better than 1 nM (KD2 value).
  • an affinity for human IgE as determined using Biacore, for example using the protocol described in Example 12
  • an affinity for cyno IgE that is better than 50 nM, such as better than 30nM (KDl value
  • Nanobodies against IgE that can be used in the anti-IgE polypeptides of the invention are the anti-IgE Nanobodies described in WO 04/041865 or WO 04/041867, such as VHH#2C3, VHH#4G12, VHH#2C1 , VHH#2H3, VHH#2D12, VHH#2G4, VHH#4C5, VHH#4A2, VHH#2D4, VHH#2B6 or VHH#2H1 1 (see Table 14 and SEQ ID NO's: 1 to 11 from WO 04/041867), or humanized and/or sequence optimized variants of the same.
  • Nanobodies against IgE that can be used in the anti-IgE polypeptides of the invention are the anti-IgE Nanobodies described herein, such as 39B02 (SEQ ID NO: 114), 39D8 (SEQ ID NO: 1 15), 42D7 (SEQ ID NO: 1 16), 42G5(SEQ ID NO: 117), 36G5 (SEQ ID NO: 118) or humanized and/or sequence-optimized version of the same, and in particular 39D1 1 (SEQ ID NO: 119), a "39D1 1-type sequence” (as defined herein) and in particular a "39D11 -like sequence” (as defined herein), such as a humanized and/or sequence-optimized variant of 39D11, for example of the variants of SEQ ID NO's: 120 to 133, of which IGE026 (SEQ ID NO: 128, also referred to herein as IGE0045 or IgE66G02) is a particularly preferred example.
  • IGE026 is IGE026 with a E to D mutation at position 1.
  • This building block is also referred to herein as IgE66G02(ElD) or IgE66G02 E1D (SEQ ID NO: 129).
  • the amino acid sequence of 39D11 is (with the CDR's underlined and in bold. These CDR's are also given in SEQ ID NO's: 134, 136 and 137, respectively): EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSSTOT GGDITHYADSVKGRFTISRDNANNMLYLQMNSLKPEDTAVYWCATDEDYALG PNEYDYYGQGTQVTVSS [SEQ ID NO: 1 19]
  • IGE026 (also called IgE0045).
  • the amino acid sequence of IGE026 is (with the CDR's underlined and in bold. These CDR's are also given in SEQ ID NO's: 135, 136 and 138, respectively; it being understood that CDR2 is the same for 39D11 and IGE026):
  • a "39D11-type sequence", “39D11-type ISV” or “39D11-type building block” in its broadest sense is an immunoglobulin single variable domain, and in particular a Nanobody, that is such that (i) it competes with 39D11 for binding to (human) IgE (for example, in a suitable binding assay, such as a BIAcore assay, for example the BIAcore assay set up or Alphascreen assay essentially as described in Example 9, but using 39D11 instead of
  • omalizumab binds to the same epitope on (human) IgE as 39D11; and/or (iii) it cross-blocks (as defined in WO 09/068627) the binding of 39D11 to IgE.
  • 39D11-type sequences also preferably are cross reactive between human IgE and cyno IgE, as further described herein.
  • an "39D11-type sequence", “39D11-type ISV” or “39D11-type building block” is preferably further such that it (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 'U M, better than 2.10 "n M, or even better than 10 "n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 'U M, better than 2.10 "n M, or even better than 10 "
  • HuIgE/HuFc(epsilon)RII interaction for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 "S M, preferably better than 2.10 "8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10 ⁇ 9 M, better than 5.10 "10 M, or better than 2.10 "10 M; and/or (iii) has an IC50 value in the degranulation assay described in Example 11 better than 100 nM, preferably better than 50 nM, more preferably better than 20nM, such as better than 5nM, better than 1 nM, or even better than 0.5 nM.
  • a u 39Dll-like sequence ii 39Dll-like sequence
  • 39Dll-like ISV or u 39Dll-like building block is defined as an ISV (as described herein) that comprises:
  • a CDR1 which comprises or essentially consists of either (i) the amino acid sequence
  • SYDMS SEQ ID NO: 134) and/or the amino acid sequence NYDMA (SEQ ID NO: 135) or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYDMS (SEQ ID NO: 134) and/or the amino acid sequence NYDMA (SEQ ID NO: 135); and/or
  • a CDR2 which comprises or essentially consists of either (i) the amino acid sequence
  • SIDTGGDITHYADSVKG (SEQ ID NO: 136) or (ii) an amino acid sequence that has at least
  • DEEYALGPNEFDY (SEQ ID NO: 138) or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and/or the amino acid sequence DEEYALGPNEFDY (SEQ ID NO: 138); or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and/or the amino acid sequence
  • HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "n M, better than 2.10 "n M, or even better than 10 "n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RII interaction (for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 " 8 M, preferably better than 2.10 "8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10 "9 M, better than 5.10 "10 M, or better than 2.10 “10 M; and/or (iii) has an IC50 value in the degranulation assay described in Example 11 better than 100 nM, preferably better
  • CDRl and CDR2 are as defined under a) and b), respectively; or CDRl and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 39D11 -like sequence, CDRl, CDR2 and CDR3 are all as defined under a), b) and c), respectively.
  • CDRl, CDR2 and CDR3 are preferably such that the 39D1 1-like ISV: (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "1 l M, better than 2.10 "1 'M, or even better than 10 "n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "1 l M, better than 2.10 "1 'M, or even better than 10 "n M; and
  • HuIgE/HuFc(epsilon)RII interaction for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 ⁇ 8 M, preferably better than 2.10 ⁇ 8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10 "9 M, better than 5.10 "10 M, or better than 2.10 "10 M; and/or (iii) has an IC50 value in the degranulation assay described in Example 11 better than 100 nM, preferably better than 50 nM, more preferably better than 20nM, such as better than 5nM, better than 1 nM, or even better than 0.5 nM.
  • CDRl may comprise or essentially consist of the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably of the amino acid sequence NYDMA (SEQ ID NO: 135) (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136) (with CDRl and CDR3 being as defined under a) and c), respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and preferably of the amino acid sequence DEEYALGPNEFDY (SEQ ID NO: 138) (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably of the amino acid sequence NYDMA (SEQ ID NO: 135) and CDR2 may comprise or essentially consist of the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136) (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably of the amino acid sequence NYDMA (SEQ ID NO: 135) and CDR3 may comprise or essentially consist of the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and preferably of the amino acid sequence DEEYALGPNEFDY (SEQ ID NO: 138) (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence
  • SIDTGGDITHYADSVKG (SEQ ID NO: 136) and CDR3 may comprise or essentially consist of the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and preferably of the amino acid sequence DEEYALGPNEFDY (SEQ ID NO : 138) (with CDR1 being as defined under a) above).
  • CDR1, CDR2 and CDR3 are preferably such that the 39D1 1 -like ISV (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 ⁇ 10 M, better than 5.10 ⁇ n M, better than 2.10 "n M, or even better than 10 1 M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RII interaction (for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 "8 M, preferably better than 2.10 "8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10 "9 M, better than 5.10 "10 M
  • CDR1 comprises or essentially consists of the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably the amino acid sequence NYDMA (SEQ ID NO: 135)
  • CDR2 comprises or essentially consists of the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136)
  • CDR3 comprises or essentially consists of the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and preferably the amino acid sequence DEEYALGPNEFDY (SEQ ID NO: 138).
  • a "39Dll-like sequence", “39Dll-like ISV” or “39D11- like building blocK” is an ISV that comprises:
  • a CDR1 which is either (i) the amino acid sequence SYDMS (SEQ ID NO: 134) and
  • amino acid sequence NYDMA (SEQ ID NO: 135) or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYDMS (SEQ ID NO: 134) and/or the amino acid sequence NYDMA (SEQ ID NO: 135); and/or
  • a CDR2 which is either (i) the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136) or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136); or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136); and/or
  • a CDR3 which is either (i) the amino acid sequence DEDYALGPNEYDY (SEQ ID NO:
  • amino acid sequence DEEYALGPNEFDY SEQ ID NO: 138
  • amino acid sequence DEDYALGPNEYDY SEQ ID NO: 137
  • amino acid sequence DEEYALGPNEFDY SEQ ID NO: 138
  • amino acid sequence DEDYALGPNEYDY SEQ ID NO: 137
  • amino acid sequence DEEYALGPNEFDY SEQ ID NO: 138
  • amino acid sequence DEDYALGPNEYDY SEQ ID NO: 137
  • amino acid sequence DEEYALGPNEFDY SEQ ID NO: 138
  • amino acid sequence DEEYALGPNEFDY SEQ ID NO: 138
  • the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the 39D11 -like ISV (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "1 *M, better than 2.10 "n M, or even better than 10 "n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RII interaction (for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 " 8 M, preferably better than 2.10 "8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than
  • CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 39D11 -like sequence, CDRl, CDR2 and CDR3 are all as defined under d), e) and f), respectively.
  • CDRl, CDR2 and CDR3 are preferably such that the 39D11-like ISV has (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 " M, preferably better than 6.10 " M, such as better than 10 "10 M, better than 5.10 "U M, better than 2.10 "n M, or even better than 10 " U M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RII interaction (for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 "8 M, preferably better than 2.10 "8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10 "9 M, better than 5.10 "10 M, or better than
  • CDR1 is the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably the amino acid sequence NYDMA (SEQ ID NO: 135) (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136) (with CDR1 and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and preferably the amino acid sequence DEEYALGPNEFDY (SEQ ID NO: 138) (with CDR1 and CDR2 being as defined under d) and e), respectively).
  • CDR1 is the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably the amino acid sequence NYDMA (SEQ ID NO: 135) and CDR2 is the amino acid sequence
  • CDR1 is the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably the amino acid sequence NYDMA (SEQ ID NO: 135) and CDR3 is the amino acid sequence
  • DEEYALGPNEFDY (SEQ ID NO: 138) (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136) and CDR3 is the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and preferably the amino acid sequence DEEYALGPNEFDY (SEQ ID NO: 138) (with CDR1 being as defined under d) above).
  • CDR1, CDR2 and CDR3 are preferably such that the 39D1 1-like ISV (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "n M, better than 2.10 "n M, or even better than 10 "n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RII interaction (for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 "8 M, preferably better than 2.10 “8 M, such as better than 10 "8 M, better than 5.10 ⁇ 9 M, better than 2.10 "9 M, better than 10 "9 M, better than 5.10 "10 M, or
  • CDR1 is the amino acid sequence SYDMS (SEQ ID NO: 134) and preferably the amino acid sequence NYDMA (SEQ ID NO: 135)
  • CDR2 is the amino acid sequence SIDTGGDITHYADSVKG (SEQ ID NO: 136)
  • CDR3 is the amino acid sequence DEDYALGPNEYDY (SEQ ID NO: 137) and preferably the amino acid sequence DEEYALGPNEFDY (SEQ ID NO: 138).
  • the FR1 , FR2, FR3 and FR4 sequences are preferably such that taken together, these framework sequences have at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity with the framework sequences of 39D1 1 (SEQ ID NO's: 139, 141 , 143 and 145, respectively) and/or the framework sequences of IGE026 (SEQ ID NO's: 140, 142, 144 and 146, respectively).
  • each of the FR1 , FR2, FR3 and FR4 sequences in a 39D1 1-like sequence may have between 0 and 7, such as between 0 and 5, such as 1 , 2, 3 or 4 (suitable) amino acid differences with the FR1 , FR2, FR3 and FR4 sequence, respectively, of 39D1 1 and/or with the FR1 , FR2, FR3 and FR4 sequence, respectively, of IGE026.
  • Other suitable framework sequences may essentially be as described on pages 273 to 291 of WO 09/068627 (described in WO 09/068627 for Nanobodies against IL-23). Reference is for example made to Tables A- 10 to A-25 of WO 09/068627, as well as the various humanizing substitutions described in Tables A-6 to A-9 of WO 09/068627.
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 39D1 1 -like ISV (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "IO M, better than 5.10 "n M, better than 2.10 "n M, or even better than 10 " n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RII interaction (for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 " M, preferably better than 2.10 "8 M, such as better than 10 ⁇ 8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10 "9 M, better than 5.10 "10 M, or better than
  • a 39D11 -like sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 39D1 1 (SEQ ID NO: 1 19).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be SYDMS (SEQ ID NO: 134) and preferably NYDMA (SEQ ID NO: 135) (CDR1); SIDTGGDITHYADSVKG (SEQ ID NO: 136) (CDR2); and DEDYALGPNEYDY (SEQ ID NO: 137) and preferably DEEYALGPNEFDY (SEQ ID NO: 138) (CDR3).
  • SYDMS SEQ ID NO: 134
  • NYDMA SEQ ID NO: 135)
  • SIDTGGDITHYADSVKG SEQ ID NO: 136)
  • DEDYALGPNEYDY SEQ ID NO: 137
  • DEEYALGPNEFDY SEQ ID NO: 138
  • the combination of CDR's and frameworks present in such a 39D1 1 -like ISV are preferably such that the resulting 39D1 1 - like ISV (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "1 'M, better than 2.10 "1 l M, or even better than 10 "n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "1 'M, better than 2.10
  • HuIgE/HuFc(epsilon)RII interaction for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 "8 M, preferably better than 2.10 "8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10 "9 M, better than 5.10 "10 M, or better than 2.10 "10 M; and/or (iii) has an IC50 value in the degranulation assay described in Example 1 1 better than 100 nM, preferably better than 50 nM, more preferably better than 20nM, such as better than 5nM, better than 1 nM, or even better than 0.5 nM.
  • any 39D1 1 -like sequences may be a humanized and/or sequence- optimized variant of 39D1 1, as further described herein.
  • These may for example and without limitation contain (any suitable combination of) one or more, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and essentially up to all, of the following mutations compared to the sequence of 39D1 1 : E1D, V5L, E6Q, F29Y, S3 IN, S35A or S36G, G44R, N73K, M77T or M77L, K83R, W91 A, D97E, YlOOeF and/or Q108L (with the numbering according to Kabat, see for example Tables A-5 to A-8 of WO 08/020079; and with each letter denominating an amino acid residue in accordance with the standard one-letter amino acid code, for which reference is made to Table A-2 of WO 08/020079).
  • these may for example and without limitation contain (any suitable combination of) one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and essentially up to all, of the following mutations compared to the sequence of 39D11 : E1D, V5L, E6Q, F29Y, S31N, S35A or S35G, G44R, N75K, M77T or M77L, K83R, W91Y, D97E, YlOOeF and/or Q108L (with the numbering according to Kabat, see for example Tables A- 5 to A-8 of WO 08/020079; and with each letter denominating an amino acid residue in accordance with the standard one-letter amino acid code, for which reference is made to Table A-2 of WO
  • the 39D11-like sequence is selected from any of SEQ ID NO's: 120-133 or an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with any of SEQ ID NO's: 120-133.
  • Preferred 39D11-like sequences are SEQ ID NO's: 128 and 129.
  • 39D11-like sequences also preferably are cross reactive between human IgE and cyno
  • any "39D11-like sequence", “39D1 1 -like sequence”, “39D11-like ISV” or “39D11-like building block” is preferably such that it: (i) inhibits the HuIgE/HuFc(epsilon)RI interaction (for example, in the ELISA assay described in Example 7; in the Alphascreen assay described in Example 6) with an IC50 value better than 8.10 "10 M, preferably better than 6.10 "10 M, such as better than 10 "10 M, better than 5.10 "n M, better than 2.10 “n M, or even better than 10 "n M; and/or (ii) inhibits the HuIgE/HuFc(epsilon)RII interaction (for example, in the ELISA assay described in Example 8) with an IC50 value better than 5.10 " 8 M, preferably better than 2.10 “8 M, such as better than 10 "8 M, better than 5.10 "9 M, better than 2.10 "9 M, better than 10
  • any "39D11-like sequence”, “39D11-like sequence”, “39D11 -like ISV” or “39D11- like building block” is preferably such that it has an IC50 value in the degranulation assay described in Example 11 better than 100 nM, preferably better than 50 nM, more preferably better than 20nM, such as better than 5nM, better than 1 nM, or even better than 0.5 nM.
  • the ISV against IgE may or may not further comprise one or more other groups, residues, moieties or binding units. If present, such further groups, residues, moieties or binding units may or may not provide further functionality to the immunoglobulin single variable domain (and/or to the polypeptide in which it is present) and may or may not modify the properties of the
  • such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the polypeptide is a (fusion) protein or (fusion) polypeptide.
  • additional binding units can be linked to form multispecific polypeptides.
  • the one, two or more immunoglobulin single variable domains and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers.
  • the linkers may also be amino acid sequences, so that the resulting polypeptide is a fusion (protein) or fusion (polypeptide).
  • the one or more further groups, residues, moieties or binding units may be any suitable and/or desired amino acid sequences.
  • the further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the fusion polypeptide, and may or may not add further functionality to the fusion polypeptide.
  • the further amino acid sequence is such that it confers one or more desired properties or functionalities to the fusion polypeptide.
  • Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson (Nature Biotechnology 23: 1126-1136, 2005).
  • such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the (fusion) polypeptide, compared to the ISV per se.
  • Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).
  • a polypeptide is prepared that has an increased half-life, compared to the corresponding ISV against IgE.
  • polypeptides of the invention that comprise such half-life extending moieties for example include, without limitation, polypeptides in which the immunoglobulin single variable domains are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, Domain antibodies, amino acids that are suitable for use as a domain antibody, single domain antibodies, amino acids that are suitable for use as a single domain antibody, "dAb"'s, amino acids that are suitable for use as a dAb, or Nanobodies) that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins (such as IgG), transferrin or one of the other serum proteins listed in WO 04/003019; polypeptides in which the immunoglobulin single variable domain is
  • the polypeptides of the invention may contain, besides the one, two or more immunoglobulin single variable domains against IgE, at least one peptide (as further defined) against human serum albumin.
  • the polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding immunoglobulin single variable domain or polypeptide of the invention per se.
  • the polypeptides of the invention with increased half-life preferably have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the half-life of the corresponding immunoglobulin single variable domain or polypeptide of the invention per se.
  • polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more.
  • polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
  • the further amino acid residues may or may not change, alter or otherwise influence other (biological) properties of the (fusion) polypeptide and may or may not add further functionality to the (fusion) polypeptide.
  • amino acid residues such amino acid residues:
  • a) can comprise an N-terminal Met residue, for example as result of expression in a
  • heterologous host cell or host organism heterologous host cell or host organism.
  • b) may form a signal sequence or leader sequence that directs secretion of the (fusion)
  • polypeptide from a host cell upon synthesis for example to provide a pre-, pro- or prepro- form of the (fusion) polypeptide, depending on the host cell used to express the(fusion) polypeptide).
  • Suitable secretory leader peptides will be clear to the skilled person, and may be as further described herein. Usually, such a leader sequence will be linked to the N- terminus of the (fusion) polypeptide, although the invention in its broadest sense is not limited thereto;
  • c) may form a "tag", for example an amino acid sequence or residue that allows or facilitates the purification of the (fusion) polypeptide, for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g. by chemical or enzymatical cleavage) to provide the polypeptide (for this purpose, the tag may optionally be linked to the amino acid sequence or polypeptide sequence via a cleavable linker sequence or contain a cleavable motif).
  • residues are multiple histidine residues, glutathione residues and a myc-tag such as
  • AAAEQKLISEEDLNGAA SEQ ID NO: 193
  • d) maybe one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups.
  • Suitable amino acid residues and functional groups will be clear to the skilled person and include, but are not limited to, the amino acid residues and functional groups mentioned herein for the derivatives of the polypeptides of the invention.
  • the one or more ISVs may be used as a "binding unit", “binding domain” or “building block” (these terms are used interchangeable) for the preparation of a (fusion) polypeptide, which may optionally contain one or more further binding unit (i.e., against the same or another epitope on IgE and/or against one or more other antigens, proteins or targets than IgE).
  • binding unit i.e., against the same or another epitope on IgE and/or against one or more other antigens, proteins or targets than IgE.
  • the ISVs comprising the CDR sequences as described herein are particularly suited for use as building block or binding unit for the preparation of the (fusion) polypeptides.
  • the present invention also relates to the use of the ISVs as described herein in preparing a multivalent polypeptide.
  • the method for the preparation of a multivalent polypeptide will comprise the linking of an ISV to at least one further binding unit, optionally via one or more linkers.
  • the ISV as defined herein is then used as a binding domain or binding unit in providing and/or preparing the multivalent, polypeptide comprising two (e.g., in a bivalent polypeptide), three (e.g., in a trivalent polypeptide) or more (e.g., in a multivalent polypeptide) binding units.
  • the IVS as described herein may be used as a binding domain or binding unit in providing and/or preparing a multivalent, such as bivalent or trivalent polypeptide comprising two, three or more binding units.
  • the serum albumin binding peptide that may be present in the anti-IgE polypeptides of the invention may generally be as described in WO 08/068280, in WO 09/127691 , and in particular as described in WO 2011/095545. Thus, for a general description of these peptides, reference is made to these applications.
  • these peptides may have a total length of between 5 and 50, preferably between 7 and 40, more preferably between 10 and 35, such as about 15, 20, 25 or 30 amino acid residues and are such that they can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493 (numbering as described in Example 8 of WO 09/127691).
  • V442, S443, T446, L484, L487, H488, K490, T491 and/or V493 numberbering as described in Example 8 of WO 09/127691.
  • WO 08/068280 describes a number of such peptides, including the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO: 19), which is called “17D12" in WO 08/068280 and which is listed in WO 08/068280 as SEQ ID NO: 3.
  • WO 09/127691 describes a number of improved variants of 17D2 which generally have at least 50%, preferably at least 65 %, more preferably at least 70%, even more preferably at least 75%, such as at least 80%, such as at least 90%, but not 100%, sequence identity (as defined herein) with the amino acid sequence of SEQ ID NO: 19 and which can bind better to human serum albumin than the amino acid sequence of SEQ ID NO: 19.
  • some of the preferred peptides according to WO 09/127691 comprise: (i) an Arg (R) residue, in particular an Arg (R) residue that is capable of forming a hydrogen bond with the amino acid residues Asn (N) 133 and Asn (N) 135 of human serum albumin and/or capable of forming electrostatic interactions with the main-chain oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum albumin; and/or
  • Trp (W) residue in particular a Trp (W) residue that is capable of forming electrostatic interactions with the Arg (R) 138 residue of human serum albumin;
  • exemplary peptides described in WO 09/127691 may for example contain one or more of the following features:
  • sequence motif RXWD in which X may be any amino acid sequence but is preferably W, Y, F, S or D; and/or (ii) the sequence motif GGG, preferably the sequence motif FGGG (SEQ ID NO: 24), more preferably the sequence motif DVFGGG (SEQ ID NO: 33), and in particular the sequence motif DVFGGGT (SEQ ID NO: 37);
  • WO 2011/095545 describes some even further improved variants of the peptides described in WO 09/127691, which compared to the peptides described in WO 09/127691 further comprise, upstream (as defined in WO 201 1/095545) of the Arg (R) residue mentioned above (which according to the numbering used in WO 08/068280, WO 09/127691 and WO 2011/095545 is position 3), and in particular upstream of the RXWD motif mentioned above, a stretch of amino acid residues of between 2 and 10 amino acid residues, which comprises at least one hydrophobic and/or aromatic amino acid residue (and for the remainder one or more further suitable amino acid residues, as for example exemplified in WO 2011/095545), in which said at least one hydrophobic amino acid residue may in particular be chosen from L, I, V and/or M and/or in which said at least one aromatic amino acid residue may in particular be chosen from W, Y and/or F.
  • upstream as defined in WO
  • said stretch of amino acids upstream of position 3 is preferably such that at least one of said hydrophobic and/or aromatic amino acid residues can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493.
  • albumin binding peptides described in WO 2011/095545 may contain, upstream of position 3, a stretch of amino acid residues of between 2 and 10 amino acid residues, which comprises at least one W residue and/or at least one Y residue, such that at least one of said W or Y residues can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493.
  • said stretch of amino acid residues may comprise (i) at least two W residues; (ii) at least two Y residues; and/or (iii) at least one W residue and at least one Y residue, such that at least one of said W or Y residues can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493.
  • WO 2011/095545 further describes that it is possible, and may even be advantageous, in the peptides describes therein: (i) to replace an aspartate (D) residue at position 8 by a threonine (T) residue (for example, but without limitation, in amino acid sequences of the invention that do not contain a threonine residue at position 14); and/or (ii) to replace the threonine (T) residue at position 14 with another amino acid residue, such as (for example and without limitation) A, N or D.
  • D aspartate
  • T threonine residue
  • albumin-binding peptides For example, some sequence motifs that may be present in the albumin-binding peptides according to WO 2011/095545 are:
  • RXWDXDVFGGGT (SEQ ID NO: 24 of WO 2011/095545; SEQ ID NO: 94 herein), in which the first (from the N-terminal end) amino acid residue indicated by X is chosen from Y, S or D; and the second amino acid residue indicated by X is chosen from Y or F;
  • RYWDFDVFGGG SEQ ID NO: 31 of WO 201 1/095545; SEQ ID NO: 101 herein
  • RDWDFDVFGGG SEQ ID NO: 28 of WO 201 1/095545; SEQ ID NO: 98 herein
  • RSWDFDVFGGG SEQ ID NO: 29 of WO 2011/095545; SEQ ID NO: 99 herein
  • RYWDFDVFGGG SEQ ID NO: 30 of WO 2011/095545; SEQ ID NO: 100 herein
  • RDWDFDVFGGG SEQ ID NO: 28 of WO 201 1/095545; SEQ ID NO: 98 herein
  • RSWDFDVFGGG SEQ ID NO: 29 of WO 2011/095545; SEQ ID NO: 99 herein
  • RYWDFDVFGGG SEQ ID NO: 30 of WO 2011/095545; SEQ ID NO: 100 herein
  • RYWDYDVFGGGTP SEQ ID NO: 36 of WO 201 1/095545; SEQ ID NO: 106 herein
  • RDWDFDVFGGGTP SEQ ID NO: 37 of WO 2011/095545; SEQ ID NO: 107 herein
  • RSWDFDVFGGGTP SEQ ID NO: 38 of WO 2011/095545; SEQ ID NO: 108 herein
  • RYWDFDVFGGGTP SEQ ID NO: 39 of WO 2011/095545; SEQ ID NO: 109 herein
  • RSWDFDVFGGGTP SEQ ID NO: 38 of WO 2011/095545; SEQ ID NO: 108 herein
  • RYWDFDVFGGGTP SEQ ID NO: 39 of WO 2011/095545; SEQ ID NO: 109 herein
  • RSWDFDVFGGGTP SEQ ID NO: 38 of WO 2011/095545; SEQ ID NO: 108 herein
  • RYWDFDVFGGGTP SEQ ID NO: 39 of WO 2011
  • RYWDYDVFGGGTPV SEQ ID NO: 40 of WO 2011/095545; SEQ ID NO: 110 herein
  • RDWDFDVFGGGTPV SEQ ID NO: 41 of WO 2011/095545; SEQ ID NO: 11 1 herein
  • RSWDFDVFGGGTPV SEQ ID NO: 42 of WO 2011/095545; SEQ ID NO: 112 herein
  • RYWDFDVFGGGTPV SEQ ID NO: 43 of WO 2011/095545; SEQ ID NO: 113 herein
  • RDWDFDVFGGGTPV (SEQ ID NO: 41 of WO 2011/095545; SEQ ID NO: 11 1 herein); RSWDFDVFGGGTPV (SEQ ID NO: 42 of WO 2011/095545; SEQ ID NO: 112 herein) or RYWDFDVFGGGTPV (SEQ ID NO: 43 of WO 2011/095545; SEQ ID NO: 113 herein).
  • WO 201 1/095545 (SEQ ID NO's: 94 to 97 herein) or 32 to 43 of WO 2011/095545 (SEQ ID NO's: 102 to 1 13 herein), in which (i) the threonine (T) residue at position 14 has been replaced by another amino acid residue (preferably but without limitation, A, N or D), and (ii) the aspartate (D) at position 8 has been replaced by a threonine (T).
  • all these sequence motifs may contain one or more other suitable substitutions, such as (for example and without limitation) one or more of the substitutions listed in Table I of WO 2011/095545.
  • WO 2011/095545 and WO 09/127691 also describe that two or more (such as two) of the peptides described therein (which may be the same or different) may be linked to each other (either directly or via a suitable linker, for example a GS-linker such as the 9GS linker of SEQ ID NO: 164 herein) in order to provide a "tandem repeat" of two (or more) such serum albumin- binding peptides.
  • a suitable linker for example a GS-linker such as the 9GS linker of SEQ ID NO: 164 herein
  • these peptides can be linked or fused to a therapeutic moiety, compound, protein or other therapeutic entity in order to increase the half- life thereof.
  • the serum albumin binding peptide or peptides that is/are present in the anti-IgE polypeptides of the invention may generally be as described in WO 08/068280, in WO 09/127691, and in particular as described in WO 2011/095545.
  • the serum albumin-binding peptide(s) that is/are present in the anti- IgE polypeptides of the invention may be a peptide with a total length of between 5 and 50, preferably between 7 and 40, more preferably between 10 and 35, such as about 15, 20, 25 or 30 amino acid residues (as a single repeat), which is such that it can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493 (numbering as described in Example 8 of WO 09/127691 ).
  • the serum albumin-binding peptide(s) that is/are present in the anti-IgE polypeptides of the invention may be such a peptide that has at least 50%, preferably at least 65 %, more preferably at least 70%, even more preferably at least 75%, such as at least 80%, such as at least 90%, but not 100%, sequence identity (as defined herein) with the amino acid sequence of SEQ ID NO: 19 and that can bind better to human serum albumin than the amino acid sequence of SEQ ID NO: 19.
  • serum albumin-binding peptide(s) that is/are present in the anti- IgE polypeptides of the invention may be such a peptide that comprises:
  • an Arg (R) residue in particular an Arg (R) residue that is capable of forming a hydrogen bond with the amino acid residues Asn (N) 133 and Asn (N) 135 of human serum albumin and/or capable of forming electrostatic interactions with the main-chain oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum albumin; and/or
  • Trp (W) residue in particular a Trp (W) residue that is capable of forming electrostatic interactions with the Arg (R) 138 residue of human serum albumin;
  • upstream of said Arg (R) residue a stretch of amino acid residues of between 2 and 10 amino acid residues, which comprises at least one hydrophobic and/or aromatic amino acid residue (and for the remainder one or more further suitable amino acid residues), in which said at least one hydrophobic amino acid residue may in particular be chosen from L, I, V and/or M and/or in which said at least one aromatic amino acid residue may in particular be chosen from W, Y and/or F.
  • said stretch of amino acids upstream of position 3 is preferably such that at least one of said hydrophobic and/or aromatic amino acid residues can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442,
  • the stretch of amino acids upstream of position 3 may be chosen from SEQ ID NO's: 78 to 98 of WO 2011/095545 (SEQ ID NO's: 72 to 92 herein).
  • the serum albumin-binding peptide(s) that is/are present in the anti-IgE polypeptides of the invention may be a peptide: a) with a total length of between 5 and 50, preferably between 7 and 40, more preferably
  • c) which has at least 50%, preferably at least 65 %, more preferably at least 70%, even more preferably at least 75%, such as at least 80%, such as at least 90%, but not 100%, sequence identity (as defined herein) with the amino acid sequence of SEQ ID NO: 19; and d) which is such that can bind better to human serum albumin than the amino acid sequence of SEQ ID O: 19; and
  • e which contains one or more of the following sequence motifs: DYDV (SEQ ID NO: 20), YDVF (SEQ ID NO: 21), DVFG (SEQ ID NO: 22), VFGG (SEQ ID NO: 23), FGGG (SEQ ID NO: 24) and/or GGGT (SEQ ID NO: 25), and in particular one of the sequence motifs
  • DYDVF (SEQ ID NO: 26), YDVFG (SEQ ID NO: 27), DVFGG (SEQ ID NO: 28), VFGGG (SEQ ID NO: 29), FGGGT (SEQ ID NO: 30), DYTVF (SEQ ID NO: 44), YTVFG (SEQ ID NO: 45) or TVFGG (SEQ ID NO: 46); and more in particular one of the sequence motifs D YDVFG (SEQ ID NO: 31), YD VFGG (SEQ ID NO: 32), D VFGGG (SEQ ID NO: 33), VFGGGT (SEQ ID NO: 34), D YTVFG (SEQ ID NO: 47), YTVFGG (SEQ ID NO: 48) or
  • TVFGGG (SEQ ID NO: 49), and even more in particular one of the sequence motifs DYDVFGG (SEQ ID NO: 35), YD VFGGG (SEQ ID NO: 36), D VFGGGT (SEQ ID NO: 37); D YTVFGG (SEQ ID NO: 50), YTVFGGG (SEQ ID NO: 51), TVFGGGT, (SEQ ID NO: 52), DVFGGGA (SEQ ID NO: 53), DVFGGGN (SEQ ID NO: 54), DVFGGGD (SEQ ID NO: 55), TVFGGGA (SEQ ID NO: 56), TVFGGGN (SEQ ID NO: 57) or TVFGGGD
  • SEQ ID NO: 58 such as one of the sequence motifs DYD VFGGG (SEQ ID NO: 38), YD VFGGGT (SEQ ID NO: 39); DYD VFGGGT (SEQ ID NO: 40); D YTVFGGG (SEQ ID NO: 59), YTVFGGGT (SEQ ID NO: 60), YDVFGGGA (SEQ ID NO: 61), YDVFGGGN (SEQ ID NO: 62), YDVFGGGD (SEQ ID NO: 63); YTVFGGGA (SEQ ID NO: 64), YTVFGGGN (SEQ ID NO: 65) or YTVFGGGD (SEQ ID NO: 66);
  • f which contains, upstream of the sequence motif referred to under e), for example at positions 3 to 6, a sequence motif RXWD, in which X may be any amino acid sequence but is preferably W, Y, F, S or D; and/or
  • g) which contains upstream of said sequence motif RXWD a stretch of amino acid residues of between 2 and 10 amino acid residues, which comprises at least one hydrophobic and/or aromatic amino acid residue (and for the remainder one or more further suitable amino acid residues), in which said at least one hydrophobic amino acid residue may in particular be chosen from L, I, V and/or M and/or in which said at least one aromatic amino acid residue may in particular be chosen from W, Y and/or F.
  • said stretch of amino acids upstream of position 3 is preferably such that at least one of said hydrophobic and/or aromatic amino acid residues can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493; and may in particular contain, upstream of position 3, a stretch of amino acid residues of between 2 and 10 amino acid residues, which comprises at least one W residue and/or at least one Y residue, such that at least one of said W or Y residues can bind (in)to a subpocket in (human) serum albumin that comprises (at least) one or more of the following amino acid residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493; and may more in particular comprise (i) at least two W residues of
  • the stretch of amino acids upstream of position 3 may be chosen from SEQ ID NO's: 78 to 98 of WO 2011/095545 (SEQ ID NO's: 72 to 92).
  • the serum albumin-binding peptide(s) that is/are present in the anti-IgE polypeptides of the invention may be a peptide: i) with a total length of between 5 and 50, preferably between 7 and 40, more preferably
  • iii which has at least 50%, preferably at least 65 %, more preferably at least 70%, even more preferably at least 75%, such as at least 80%, such as at least 90%, but not 100%, sequence identity (as defined herein) with the amino acid sequence of SEQ ID NO: 19; and iv) which is such that can bind better to human serum albumin than the amino acid sequence of SEQ ID NO: 19;
  • xi an amino acid sequence chosen from RYWDYDVFGGG (SEQ ID NO: 98);
  • RDWDFDVFGGG (SEQ ID NO: 99); RSWDFDVFGGG (SEQ ID NO: 100) or
  • RYWDFDVFGGG (SEQ ID NO: 101); and in particular chosen from RDWDFDVFGGG (SEQ ID NO: 98); RSWDFDVFGGG (SEQ ID NO: 99) or RYWDFDVFGGG (SEQ ID NO: 100);
  • xii an amino acid sequence chosen from RYWDYDVFGGGT (SEQ ID NO: 102);
  • RDWDFDVFGGGT (SEQ ID NO: 103); RSWDFDVFGGGT (SEQ ID NO: 104) or RYWDFDVFGGGT (SEQ ID NO: 105); and in particular chosen from
  • RDWDFDVFGGGT (SEQ ID NO: 103); RSWDFDVFGGGT (SEQ ID NO: 104) or RYWDFDVFGGGT (SEQ ID NO: 105);
  • xiii) an amino acid sequence chosen from RYWDYDVFGGGTP (SEQ ID NO: 106);
  • RDWDFDVFGGGTP (SEQ ID NO: 107); RSWDFDVFGGGTP (SEQ ID NO: 108) or RYWDFDVFGGGTP (SEQ ID NO: 109); and in particular chosen from
  • RDWDFDVFGGGTP (SEQ ID NO: 107); RSWDFDVFGGGTP (SEQ ID NO: 108) or RYWDFDVFGGGTP (SEQ ID NO: 109);
  • xiv an amino acid sequence chosen from RYWDYDVFGGGTPV (SEQ ID NO: 110);
  • RDWDFDVFGGGTPV SEQ ID NO: 1 11
  • RSWDFDVFGGGTPV SEQ ID NO: 112
  • RYWDFDVFGGGTPV (SEQ ID NO: 113); and in particular chosen from
  • RDWDFDVFGGGTPV (SEQ ID NO: 1 11); RSWDFDVFGGGTPV (SEQ ID NO: 1 12) or RYWDFDVFGGGTPV (SEQ ID NO: 1 13 herein).
  • serum albumin-binding peptide(s) that may be present in the anti-IgE polypeptides of the invention (as a single repeat or tandem repeat) are the sequences of SEQ ID NO's: 2 to 1 15 or 147 to 157 of WO 09/127691.
  • serum albumin-binding peptide(s) that may be present in the anti-IgE polypeptides of the invention (as a single repeat or tandem repeat) are the sequences of SEQ ID NO's: 54 to 74 or 103 to 108 of WO 201 1/095545.
  • serum albumin-binding peptide(s) that may be present in the anti-IgE polypeptides of the invention (as a single repeat or tandem repeat) are the sequences of SEQ ID NO's: 1 to 8.
  • SEQ ID NO's: 9 to 18 are some preferred, but non-limiting examples of tandem repeats of such peptides that can be present in the anti-IgE polypeptides of the invention.
  • the one or more (such as one or two) ISV's against IgE (and in particular Nanobodies against IgE, such as 39D11-type sequences and in particular 39D11 -like sequences) and the one or more (such as one or two) serum albumin binding peptides may be suitably linked to each other, either directly or via one or more suitable spacers or linkers.
  • the linker may be a "GS linker" (i.e. a linker essentially consisting of G and S residues) such as one of the linkers of SEQ ID NO's: 162 to 171 , of which the 9GS, 20GS, 30GS and 35GS linkers may be particularly suited.
  • the use of a 20GS linker may be preferred over a 9GS linker, because in some instances, albumin-binding peptides that are linked via a 20GS linker show improved (i.e., less) susceptibility for
  • anti-IgE polypeptides of the invention may comprise:
  • a single ISV against IgE (and in particular a Nanobody against IgE, such as an 39D11-type sequence and in particular a 39D11 -like sequences) and a single albumin binding peptide as described herein; which may be linked directly to each other or via a suitable linker;
  • a single ISV against IgE and in particular a Nanobody against IgE, such as an 39D11-type sequence and in particular a 39D11 -like sequences
  • a tandem repeat of two single albumin binding peptides as described herein which may the same or different, and which may be linked directly to each other or via a suitable linker, such as the 9GS linker of SEQ
  • ID NO: 164 which may be linked directly to each other or via a suitable linker;
  • a single ISV against IgE and in particular a Nanobody against IgE, such as an 39D11-type sequence and in particular a 39D11 -like sequences
  • two single albumin binding peptides as described herein which may be as described herein, and which may the same or different
  • two ISV's against IgE and in particular a Nanobody against IgE, such as an 39D11-type sequence and in particular a 39D11 -like sequences
  • a single albumin binding peptide as described herein which may be linked directly to each other or via one or more suitable linkers
  • anti-IgE polypeptides of the invention can be schematically represented as follows, in which "[peptide]” represents a serum albumin binding peptide as described herein, “[peptide-peptide]” represents a tandem repeat of two serum albumin binding peptide as described herein (which may be the same or different, and which may be linked directly or via a suitable linker), and "[anti-IgE]” represents a ISV against IgE (and in particular a Nanobody against IgE, such as an 39D11-type sequence and in particular a 39D11-like sequences), and " - " represents a suitable linker (which is optional), with the N- terminus on the left hand side and the C-terminus on the right hand side. It should be noted that when the polypeptides schematically shown below comprise two serum albumin binding peptides or two ISV's against IgE, respectively, that these may be the same or different.
  • the anti-albumin peptide(s) may be at the N-terminus, may be at the C-terminus, or may be part of the linker; or, when more than one albumin-binding peptide is present, any suitable combination thereof.
  • the albumin-binding peptides are at the C-terminus (compare for example the results given in Example 15).
  • the anti-IgE polypeptides of the invention contain one or two, and more preferably only one IS V against IgE, and only one serum albumin binding peptide (or one tandem repeat of two serum albumin binding peptides).
  • the ISV against IgE present in these polypeptides and constructs is preferably a Nanobody against IgE, such as an 39D1 1-type sequence and in particular an 39D11- like sequences, of which IGE026/IGE0045 (SEQ ID NO: 128) is a particularly preferred example.
  • the peptide against serum albumin is preferably chosen from either the "single repeat" peptides of SEQ ID NO's: 1 to 8 or the "tandem repeat" peptides of SEQ ID NO's: 9 to 18.
  • the linker(s) present (if any) are preferably chosen from SEQ ID NO's: 162 to 171, with 9GS, 20GS, 30GS and 35GS being particularly preferred.
  • a polypeptide of the invention is such that, when it is tested in the storage stability assay described in Example 16, that the pre-peak on SE-HPLC for the polypeptide after 1 month storage at 25°C (under the further conditions given in Example 16) is less than 10%, preferably less than 5%; and/or that the pre-peak on SE-HPLC for the polypeptide after 1 month storage at 40°C (under the further conditions given in Example 16) is less than 20%, preferably less than 10%.
  • anti-IgE polypeptides of the invention are given in SEQ ID NO's: 147 to 161 (see also Table A-9).
  • the invention also relates to a polypeptide selected from any of SEQ ID NO's: 147 to 161, or a polypeptide which has at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity with any of SEQ ID NO's: 147 to 161.
  • IGE109 IGE66G02 tlu -20GS-NEXP001-9GS- NEXP002
  • SEQ ID NO: 155 is particularly preferred.
  • one specific aspect of the invention relates to a polypeptide which has at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity with SEQ ID NO: 155.
  • a polypeptide is preferably as further described herein, and preferably according to the preferred aspects mentioned herein.
  • 39D11 and its variants as described herein may also generally have a number of advantages compared to the anti-IgE Nanobodies that have been described in the art, such as those described in WO 04/041867.
  • 39D11 and its variants as described herein may also generally have a number of advantages compared to the anti-IgE Nanobodies that have been described in the art, such as those described in WO 04/041867.
  • 39D1 1, 39D11-type sequences and the 39D11 -like sequences are cross-reactive between human IgE and cynomolgus IgE (as further described herein);
  • 39D1 1, 39D1 1 -type sequences and the 39D1 1 -like sequences may block interaction of Human and cyno IgE both with the high affinity IgE receptor (Fc(epsilon) I) and the low affinity receptor (Fc(epsilon)RII) (see Examples 6, 7 and 8 respectively); and generally have a desirable "balance" between affinity for the human receptor(s) and the cyno receptor(s); and/or
  • 39D1 1, 39D11-type sequences and the 39D11 -like sequences have a desirable "balance" between affinity for the human high affinity IgE receptor and the low affinity IgE receptor (see again the data presented in the Experimental Part);
  • 39D11, the 39D1 1-type sequences and in particular the 39D11 -like sequences described herein may not only be used in the anti-IgE polypeptides of the invention described herein (i.e. in constructs comprising at least one such building block and at least one albumin-binding peptide according to WO 08/068280, WO 09/127691 and WO 2011/095545), but also with advantage either by itself or as a component or building block for other polypeptides, proteins or other compounds or constructs.
  • 39D11, the 39D11-type sequences and in particular the 39D11-like sequences described herein may be used in polypeptides of the type described in WO 04/041867 and/or WO 04/041865 (i.e., instead of one of the anti-lgE Nanobodies described therein).
  • Such polypeptides may also be half-life extended, for example through pegylation or the use of a serum albumin binding Nanobody (including those described in WO 04/041867, WO 04/041865 or WO 06/122787, although it should then be noted that such polypeptides may have a limited stability under storage, as described herein).
  • the invention further relates to:
  • Such a protein or polypeptide may for example also comprise two or three such ISV's against IgE (which may be the same or different), which may be linked to each other either directly or via one or more suitable linkers. It may also be half-life extended (for example through pegylation) or otherwise modified (for example, through some of the modifications generally described in WO 09/068627);
  • a (fusion) protein, polypeptide, construct or compound that comprises or essentially consists of at least one (such as one or two) (i) 39D1 1, a 39D11-type sequence (as defined herein) and in particular of a 39D11-like sequence (as defined herein); and more in particular a protein or polypeptide that comprises or essentially consists of at least one (such as one or two) 39D1 1- like sequences (as defined herein) that is a humanized and/or sequence-optimized variant of 39D1 1 such as one of the variants of SEQ ID NO's: 120 to 133 (of which SEQ ID NO's: 128 and 129 are particularly preferred examples); and (ii) at least one other amino acid sequence, protein, (polypeptide, binding domain, binding unit, group, residue or moiety; suitably linked to each other via one or more suitable linkers or spacers.
  • such as further amino acid sequence may be another ISV (such as another Nanobody) that is directed against IgE (for example, against the same or a different part or epitope on IgE) or against another target (such as for example serum albumin or another serum protein so as to provide for increased half-life or against another therapeutic target so as to provide a bispecific construct).
  • IgE for example, against the same or a different part or epitope on IgE
  • another target such as for example serum albumin or another serum protein so as to provide for increased half-life or against another therapeutic target so as to provide a bispecific construct.
  • target such as for example serum albumin or another serum protein so as to provide for increased half-life or against another therapeutic target so as to provide a bispecific construct.
  • Such polypeptides etc. may for example generally be as described in WO
  • WO 04/041867 and/or WO 04/041865 may for example be bivalent (or multivalent), biparatopic (multiparatopic) and/or bispecific (or multispecific) constructs (as these terms are generally defined in WO 09/068627).
  • the 39D11 -like sequence may generally be as further described herein, and is preferably according to the preferred aspects described herein.
  • the invention relates to a nucleic acid that encodes one of the amino acid sequences or polypeptides against IgE that are described herein.
  • a nucleic acid may for example be in the form of a genetic construct, as for example generally described in WO
  • the invention further relates to methods for preparing the polypeptides, nucleic acids, host cells, and compositions described herein.
  • polypeptides 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 further description herein.
  • 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).
  • Some preferred, but non-limiting methods for preparing the polypeptides and nucleic acids include the methods and techniques described herein.
  • the polypeptides of the invention can generally be prepared by a method which comprises at least the step of suitably linking the immunoglobulin single variable domain against IgE to one or more further binding units, such as the one or more peptides against serum albumin, optionally via the one or more suitable linkers.
  • the immunoglobulin single variable domains and peptides (and linkers) can be coupled by any method known in the art and as further described herein.
  • Preferred techniques include the linking of the nucleic acid sequences that encode the immunoglobulin single variable domains and peptides (and linkers) to prepare a genetic construct that expresses the polypeptide. Techniques for linking amino acids or nucleic acids will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et ah, mentioned above, as well as the Examples below.
  • Polypeptides of the invention can thus be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention.
  • Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.
  • the method for producing a polypeptide of the invention may comprise the following steps:
  • nucleic acid of the invention 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 polypeptide of the invention (also referred to herein as a "nucleic acid of the invention”), optionally followed by:
  • such a method may comprise the steps of:
  • nucleic acid of the invention also relates to a nucleic acid or nucleotide sequence that encodes an ISV or polypeptide as described herein (also referred to as "nucleic acid of the invention").
  • 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.
  • 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.
  • the nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the ISV or polypeptides as described herein, and/or can be isolated from a suitable natural source.
  • nucleic acid of the invention also several nucleotide sequences, such as at least two nucleic acids encoding an immunoglobulin single variable domain or a monovalent polypeptide 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 ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers.
  • 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.
  • 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:
  • one or more regulatory elements such as a promoter and optionally a suitable terminator; and optionally also
  • 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 or host organism; 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 technique to be used.
  • 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 "operably linked" to each other, by which is generally meant that they are in a functional relationship with each other.
  • 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).
  • two nucleotide sequences when 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.
  • 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 ISV or polypeptide as described herein.
  • 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, for example:
  • bacterial strain including but not limited to gram-negative strains such as strains of
  • Escherichia coli of Proteus, for example of Proteus mirabilis; of Pseudomonas, for example of Pseudomonas fluorescens; and gram-positive strains such as strains of Bacillus, for example of Bacillus subtilis or of Bacillus brevis; of Streptomyces, for example of
  • Streptomyces lividans for example of Staphylococcus carnosus
  • Lactococcus for example of Lactococcus lactis
  • a fungal cell including but not limited to cells from species of Trichoderma, for example from Trichoderma reesei; of Neurospora, for example from Neurospora crassa; of Sordaria, for example from Sordaria macrospora; of Aspergillus, for example from Aspergillus niger or from Aspergillus sojae; or from other filamentous fungi;
  • yeast cell including but not limited to cells from species of Saccharomyces, for example of Saccharomyces cerevisiae; of Schizosaccharomyces, for example of Schizosaccharomyces pombe; of Pichia, for example of Pichia pastoris or of Pichia methanolica; of Hansenula, for example of Hansenula polymorpha; of Kluyveromyces , for example of Kluyveromyces lactis; ofArxula, for example of Arxula adeninivorans; of Yarrowia, for example of Yarrowia lipolytica;
  • an amphibian cell or cell line such as Xenopus oocytes
  • an insect-derived cell or cell line such as cells/cell lines derived from lepidoptera, including but not limited to Spodoptera SF9 and Sf21 cells or cells/cell lines derived from Drosophila, such as Schneider and c cells;
  • a mammalian cell or cell line for example a cell or cell line derived from a human, a cell or a cell line from mammals including but not limited to CHO-cells, BHK-cells (for example BHK-21 cells) and human cells or cell lines such as HeLa, COS (for example COS-7) and PER.C6 cells;
  • the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) one of the ISV's, amino acid sequences or polypeptides against IgE that are described herein; and/or that contains a nucleic acid encoding the same.
  • a host or host cell that expresses (or that under suitable circumstances is capable of expressing) one of the ISV's, amino acid sequences or polypeptides against IgE that are described herein; and/or that contains a nucleic acid encoding the same.
  • polypeptides comprising at least one anti-IgE building block as described herein and at least one albumin-binding peptide according to WO 08/068280, WO 09/127691 and WO 2011/095545 may with advantage be expressed, produced or manufactured in a yeast strain, such as a strain of Pichia pastoris.
  • WO 201 1/095545 which also describes the expression/production in Pichia and other hosts/host cells of polypeptides that comprise the albumin binding peptides of WO 2011/095545.
  • ISV's and polypeptides as described herein may also be expressed as so-called “intrabodies”, as for example described in WO 94/02610, WO 95/22618 and US 7,004,940; WO 03/014960; in Cattaneo and Biocca ("Intracellular Antibodies: Development and Applications” Austin and Springer- Verlag, 1997); and in Kontermann (Methods 34: 163-170, 2004).
  • Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.
  • a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the ISV or polypeptide as described herein, e.g., using specific antibodies.
  • the transformed host cell which may be in the form or a stable cell line
  • host organisms which may be in the form of a stable mutant line or strain
  • these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g., under suitable conditions), an ISV or polypeptide as described herein (and in case of a host organism: in at least one cell, part, tissue or organ thereof).
  • the invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention, which may for instance be obtained by cell division or by sexual or asexual reproduction.
  • the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) ISV or polypeptide is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.
  • suitable conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g., when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person.
  • a suitable inducing factor or compound e.g., when the nucleotide sequences of the invention are under the control of an inducible promoter
  • the ISV's or polypeptides may be expressed in a constitutive manner, in a transient manner, or only when suitably induced.
  • ISV or polypeptide may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell/host organism used. Also, the ISV or polypeptide may be glycosylated, again depending on the host cell/host organism used.
  • the ISV or polypeptide as described herein may then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se, such as (preparative)
  • the invention further relates to a product or composition containing or comprising at least one ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein (i.e. directed against IgE/comprising at least one ISV against IgE as described herein), and optionally one or more further components of such compositions known per se, i.e., depending on the intended use of the composition.
  • a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein).
  • Some preferred but non- limiting examples of such products or (pharmaceutical) compositions may generally be as described in WO 04/041867, WO 04/041865, WO 09/068627 or WO 2011/095545.
  • the invention also relates to the use of an ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein, or of a composition comprising the same, in (methods or compositions for) modulating (as generally defined in WO 09/068627) IgE and/or one or more biological actions, mechanisms or responses associated with IgE, either in vitro (e.g., in an in vitro or cellular assay) or in vivo ⁇ e.g., in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a disease or disorder associated with IgE, with increased levels and/or overproduction of IgE or with abnormal sensitivity (such as
  • IgE hypersensitivity
  • the invention also relates to methods for modulating IgE and/or one or more biological actions, mechanisms or responses associated with IgE, either in vitro ⁇ e.g., in an in vitro or cellular assay) or in vivo ⁇ e.g., in a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a disease associated with IgE, with increased levels and/or overproduction of IgE or with abnormal sensitivity (such as hypersensitivity) for IgE), which method comprises at least the step of contacting IgE with an ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein, or with a composition comprising the same, in a manner and in an amount suitable to modulate IgE (or the intended or desired action(s), mechanism(s) or response(s) associated with IgE).
  • the invention also relates to the use of an ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating IgE or one or more biological actions, mechanisms or responses associated with IgE, either in vitro (e.g., in an in vitro or cellular assay) or in vivo (e.g., in a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a disease or disorder associated with IgE, with increased levels and/or overproduction of IgE or with abnormal sensitivity (such as hypersensitivity) for IgE).
  • a composition such as, without limitation, a pharmaceutical composition or preparation as further described herein
  • the invention also relates to an ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein for use in therapy.
  • the invention also relates to the use of an ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein for the treatment of a disease or disorder that can be prevented or treated by administering, to a subject in need thereof, of (a pharmaceutically effective amount of) an ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein (or a suitable composition comprising the same).
  • the invention relates to an ISV, amino acid sequence, polypeptide, protein, construct or other compound as described herein for use in therapy of a disease or disorder associated with IgE, with increased levels and/or overproduction of IgE or with abnormal sensitivity (such as hypersensitivity) for IgE, or more generally any disease or disorder associated with and/or mediated by IgE.
  • IgE mediated diseases and disorders will be clear to the skilled person and may for example include, without limitation: conditions such as asthma, allergic rhinitis, hay fever, conjunctivitis, eczema, utricaria, food allergies and other allergies, including serious and/or life-threatening allergic reactions such as those to insect bites or stings, snake bites etc., as well as to allergic reaction to medication; and more generally any disease or disorder associated with anaphylactic hypersensitivity and/or (atopic) allergy.
  • omalizumab a monoclonal against IgE called omalizumab (Xolair®) is currently marketed by Genentech for IgE mediated disorders. It is envisaged that the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds, as well as compositions comprising the same, can be used in the prevention or treatment of the same diseases and disorders that omalizumab has been approved for prior to the priority date of the present invention and/or will be approved for after the priority date of the present application.
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds, as well as compositions comprising the same may have substantial advantages over omalizumab (see for example the Experimental Part, in which omalizumab (obtamed from a commercial source) and a Fab obtamed through papain digest of commercial omalizumab were used as reference compounds).
  • omalizumab obtamed from a commercial source
  • a Fab obtamed through papain digest of commercial omalizumab were used as reference compounds.
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein may be formulated as a
  • such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
  • parenteral administration such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion
  • topical administration for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
  • suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein.
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079) as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005); or the Handbook of
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv's and diabodies) and other pharmaceutically active proteins.
  • Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra- arterial or intrathecal administration) or for topical (i.e., transdermal or intradermal) administration.
  • Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection.
  • Suitable carriers or diluents for such preparations for example include, without limitation, those mentioned on page 143 of WO 08/020079.
  • aqueous solutions or suspensions will be preferred.
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • the ISV's, amino acid sequences, Nanobodies and polypeptides of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • compositions and preparations should contain at least 0.1 % of the ISV, amino acid sequence, Nanobody or polypeptide as described herein. Their percentage in the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the ISV, amino acid sequence, Nanobody or polypeptide as described herein in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain binders, excipients, disintegrating agents, lubricants and sweetening or flavouring agents, for example those mentioned on pages 143-144 of WO 08/020079.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the ISV's, amino acid sequences, Nanobodies and polypeptides as described herein, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the ISV's, amino acid sequences, polypeptides, proteins, constmcts or other compounds as described may be incorporated into sustained-release preparations and devices.
  • Preparations and formulations for oral administration may also be provided with an enteric coating that will allow the constructs of the invention to resist the gastric environment and pass into the intestines. More generally, preparations and formulations for oral
  • administration may be suitably formulated for delivery into any desired part of the
  • suppositories may be used for delivery into the gastrointestinal tract.
  • ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein may also be administered intravenously or intraperitoneally by infusion or injection, as further described on pages 144 and 145 of WO 08/020079.
  • polypeptides as described herein may be applied in pure form, i.e., when they are liquids.
  • compositions or formulations in combination with a dermatologically acceptable carrier, which may be a solid or a liquid, as further described on page 145 of WO 08/020079.
  • the concentration of the ISV's, amino acid sequences, Nanobodies and polypeptides as described herein in a liquid composition will be from about 0.1- 25 wt-%, preferably from about 0.5-10 wt-%.
  • concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
  • the amount of the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein required for use in treatment will vary not only with the particular ISV, amino acid sequence, polypeptide, protein or construct selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
  • An administration regimen could include long-term, daily treatment.
  • long-term is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication.
  • the invention in another aspect, relates to a method for the prevention and/or treatment of at least one disease or disorder associated with IgE, with increased levels and/or overproduction of IgE or with abnormal sensitivity (such as hypersensitivity) for IgE, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence, polypeptide, protein, construct or compound as described herein, and/or of a pharmaceutical composition comprising the same.
  • prevention and/or treatment not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.
  • the subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being.
  • the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.
  • the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that is associated with IgE, with increased levels and/or overproduction of IgE or with abnormal sensitivity (such as hypersensitivity) for IgE, with its biological or pharmacological activity, and/or with the biological pathways or signalling in which IgE is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein, and/or of a pharmaceutical composition comprising the same.
  • the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be treated by modulating IgE, its biological or pharmacological activity, and/or the biological pathways or signalling in which IgE is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein, and/or of a pharmaceutical composition comprising the same.
  • said pharmaceutically effective amount may be an amount that is sufficient to modulate IgE, its biological or pharmacological activity, and/or the biological pathways or signalling in which IgE is involved; and/or an amount that provides a level of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein that is sufficient to modulate IgE, its biological or
  • the invention furthermore relates to a method for the prevention and/ or treatment of at least one disease or disorder that can be prevented and/or treated by administering an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein, and/or of a pharmaceutical composition comprising the same.
  • the invention relates to a method for the prevention and/or treatment of at least one disease or disorder chosen from the group consisting of the diseases and disorders listed herein, said method comprising administering, to a subject in need thereof, a
  • the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein, and/or of a pharmaceutical composition comprising the same.
  • a pharmaceutically active amount of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein, and/or of a pharmaceutical composition comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used.
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein and/or the compositions comprising the same can for example be administered orally, intraperitoneally (e.g., intravenously, subcutaneously, intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), intranasally, transdermally, topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used.
  • the clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the clinician.
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated.
  • the clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific ISV, amino acid sequence, polypeptide, protein, construct or compound to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.
  • the treatment regimen will comprise the administration of one or more ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses.
  • the specific amount(s) or doses to administer can be determined by the clinician, again based on the factors cited above.
  • the potency of the specific ISV, amino acid sequence, polypeptide, protein, construct or compound to be used, the specific route of administration and the specific pharmaceutical formulation or composition used is particularly important.
  • ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1, 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g., by infusion), as a single daily dose or as multiple divided doses during the day.
  • the clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment.
  • ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e., as a combined treatment regimen, which may or may not lead to a synergistic effect.
  • further pharmaceutically active compounds or principles i.e., as a combined treatment regimen, which may or may not lead to a synergistic effect.
  • the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.
  • the ISV's, amino acid sequences, polypeptides, proteins, constructs or other compounds as described herein may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained.
  • examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.
  • two or more substances or principles When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g., essentially simultaneously, consecutively, or according to an alternating regime).
  • the substances or principles When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.
  • each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect.
  • the effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician.
  • the clinician will also be able, where appropriate and on a case-by- case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.
  • the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.
  • the invention relates to the use of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one disease or disorder that is associated with IgE, with increased levels and/or overproduction of IgE or with abnormal sensitivity (such as hypersensitivity) for IgE; and/or for use in one or more of the methods of treatment mentioned herein.
  • the subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.
  • the invention also relates to the use of an ISV, amino acid sequence, polypeptide, protein, construct or compound as described herein in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an ISV, amino acid sequence, Nanobody or polypeptide of the invention to a patient.
  • the invention relates to the use of an to be used ISV, amino acid sequence, Nanobody or polypeptide as described herein in the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease or disorder that is associated with IgE, with increased levels and/or oveiproduction of IgE or with abnormal sensitivity (such as hypersensitivity) for IgE, and in particular for the prevention and treatment of one or more of such diseases and disorders listed herein.
  • the one or more ISV's, amino acid sequences, Nanobodies or polypeptides as described herein may also be suitably combined with one or more other active principles, such as those mentioned herein.
  • sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “V H H sequence”, “polypeptide sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.
  • a nucleic acid or amino acid is considered to be "(in) (essentially) isolated (form)" - for example, compared to the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component.
  • a nucleic acid or amino acid is considered “(essentially) isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100- fold, and up to 1000-fold or more.
  • a nucleic acid or amino acid that is "in (essentially) isolated form” is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide-gel electrophoresis.
  • nucleotide sequence or amino acid sequence is said to "comprise” another nucleotide sequence or amino acid sequence, respectively, or to “essentially consist of another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein).
  • a polypeptide when said polypeptide is said to comprise an immunoglobulin single variable domain, this may mean that said immunoglobulin single variable domain sequence has been incoiporated into the sequence of the polypeptide, but more usually this generally means that the polypeptide contains within its sequence the sequence of the immunoglobulin single variable domains irrespective of how said polypeptide has been generated or obtained.
  • the first mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g.
  • the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the first mentioned, larger nucleic acid or nucleotide sequence).
  • polypeptide that "essentially consist of an ISV is meant that the immunoglobulin single variable domain either is exactly the same as the polypeptide or corresponds to the polypeptide which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the immunoglobulin single variable domain.
  • amino acid residues such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues
  • the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e., as an "amino acid difference" as defined herein.
  • the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768.
  • the amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.
  • amino acid substitutions which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide.
  • Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB 335768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein.
  • Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Tip.
  • Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into lie or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Tip into Tyr; Tyr into Trp; and/or Phe into Val, into He or into Leu.
  • amino acid difference refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences. More particularly, in the amino acid sequences and/or polypeptides as described herein, the term “amino acid difference” refers to an insertion, deletion or substitution of a single amino acid residue on a position of the specified CDR sequence, compared to the CDR sequence under consideration.
  • amino acid difference can be any one, two, three, four, five, six or maximal seven substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the polypeptide or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the polypeptide as described herein.
  • the resulting polypeptide of the invention should at least bind IgE with the same, about the same, or a higher affinity compared to the polypeptide comprising the one or more CDR sequences without the one, two, three, four, five, six or maximal seven substitutions, deletions or insertions, said affinity as measured by surface plasmon resonance.
  • amino acid sequence under consideration may be an amino acid sequence that is derived from the specified amino acid sequence according by means of affinity maturation using one or more techniques of affinity maturation known per se.
  • deletions and/or substitutions may be designed in such a way that one or more sites for post- translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art.
  • a polypeptide such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, a polypeptide of the invention, or generally an antigen binding molecule or a fragment thereof
  • a polypeptide that can "bind to” or “specifically bind to”, that "has affinity for” and/or that "has specificity for” a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be “against” or “directed against” said epitope, antigen or protem or is a "binding” molecule with respect to such epitope, antigen or protein, or is said to be “anti”- epitope, "anti”-antigen or “anti”-protein (e.g., "anti”-IgE).
  • (cross)-block means the ability of an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent to interfere with the binding of other
  • immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides or binding agents to a given target can be determined using competition binding assays.
  • One particularly suitable quantitative cross-blocking assay uses a BIAcore instrument 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
  • immunoglobulins antibodies, immunoglobulin single variable domains, polypeptides or other binding agents in terms of their binding to the target.
  • the BIAcore instrument (for example the BIAcore 3000) is operated in line with the
  • the target protein e.g. IgE
  • CM5 BIAcore chip using standard amine coupling chemistry to generate a surface that is coated with the target.
  • 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 binding agents (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.
  • the molecular weight of a binding agent is assumed to be the total molecular weight of the binding agent divided by the number of target binding sites on that binding agent.
  • the concentration of each binding agent in the test mix should be high enough to readily saturate the binding sites for that binding agent on the target molecules captured on the BIAcore chip.
  • the binding agents 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 binding agents 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 binding agents 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 binding agent when passed over the target surface alone.
  • a cross-blocking immunoglobulin, antibody immunoglobulin single variable domain, polypeptide or other binding agent according to the invention is one which will bind to the target in the above BIAcore cross-blocking assay such that during the assay and in the presence of a second immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent 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.
  • the BIAcore assay described above is a primary assay used to determine if immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptide or other binding agents cross-block each other according to the invention. On rare occasions particular immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides or other binding agents may not bind to a target coupled via amine chemistry to a CM5 BIAcore chip (this usually occurs when the relevant binding site on the target is masked or destroyed by the coupling to the chip).
  • cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version.
  • an anti-His antibody 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 antibody.
  • 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 antibody 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 following generally describes an ELISA assay for determining whether an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent directed against a target (e.g., IgE) 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 target (e.g., IgE) 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 target (e.g., IgE) 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 target (e.g., IgE) 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 target (e.g., IgE) cross-blocks or is capable of cross-blocking as defined herein. It will be appreciated that the assay can be used with any
  • immunoglobulins antibodies, immunoglobulin single variable domains, polypeptides or other binding agents described herein.
  • the general principal of the assay is to have an
  • immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide 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 immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide is added in solution (i.e. not bound to the ELISA plate).
  • a limited amount of the target is then added to the wells.
  • immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide and the immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide 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 immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide and to also remove the second, solution phase
  • immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide as well as any complexes formed between the second, solution phase immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide and target.
  • the amount of bound target is then measured using a reagent that is appropriate to detect the target.
  • An immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide in solution that is able to cross- block the coated immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide will be able to cause a decrease in the number of target molecules that the coated immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide can bind relative to the number of target molecules that the coated immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide can bind in the absence of the second, solution phase, immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide.
  • the first immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide e.g., an Ab-X
  • the immobilized immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide 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 immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide i.e.
  • Ab-Y is then added to the ELISA plate such that the moles of Ab-Y target binding sites per well are at least 10 fold higher than the moles of Ab-X target binding sites that were used, per well, during the coating of the ELISA plate.
  • Target is then added such that the moles of target added per well are at least 25-fold lower than the moles of Ab-X target 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-target immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide (in this case Ab-X).
  • the background signal for the assay is defined as the signal obtained in wells with the coated immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide (in this case Ab-X), second solution phase immunoglobulin single variable domain or polypeptide (in this case Ab-Y), target buffer only (i.e., without target) and target detection reagents.
  • the positive control signal for the assay is defined as the signal obtained in wells with the coated immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide (in this case Ab-X), second solution phase immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide buffer only (i.e., without second solution phase immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide), target and target detection reagents.
  • the ELISA assay may be run 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 the target) resulting from the choice of which
  • the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide that is coated onto the ELISA plate and Ab-Y is the competitor immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide that is in solution and 2) format 2 is where Ab-Y is the immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide that is coated onto the ELISA plate and Ab-X is the competitor immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide 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 immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide 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 immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide) as compared to the target detection signal obtained in the absence of the solution phase anti-target immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide (i.e., the positive control wells).
  • the target detection signal i.e., the amount of target bound by the coated immunoglobulin, antibody, immunoglobulin single variable domain or polypeptide
  • the "half-life" of a polypeptide of the invention can generally be defined as described in paragraph o) on page 57 of WO 08/020079 and as mentioned therein refers to the time taken for the serum concentration of the polypeptide to be reduced by 50%, in vivo, for example due to degradation of the polypeptide and/or clearance or sequestration of the polypeptide by natural mechanisms.
  • the in vivo half-life of a polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally be as described in paragraph o) on page 57 of WO 08/020079.
  • the half-life can be expressed using parameters such as the tl/2-alpha, tl/2-beta and the area under the curve (AUC).
  • AUC area under the curve
  • immunoglobulin 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 VH/VL domains, respectively).
  • domain (of a polypeptide or protein) as used herein refers to a folded protein structure which has the ability to retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • immunoglobulin domain refers to a globular region of an antibody chain (such as e.g., a chain of a conventional 4-chain antibody or of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are characterized in that they retain the immunoglobulin fold characteristic of antibody molecules, which consists of a two-layer sandwich of about seven antiparallel beta-strands arranged in two beta-sheets, optionally stabilized by a conserved disulphide bond.
  • immunoglobulin variable domain means an immunoglobulin domain essentially consisting of four "framework regions” which are referred to in the art and herein below as “framework region 1" or "FR1”; as “framework region 2" or “FR2”; as
  • immunoglobulin single variable domain defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site.
  • a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • CDRs complementarity determining regions
  • the antigen-binding domain of a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of
  • immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain.
  • the binding site of an immunoglobulin single variable domain is formed by a single VH, VHH or VL domain.
  • the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.
  • the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).
  • a light chain variable domain sequence e.g., a VL-sequence
  • a heavy chain variable domain sequence e.g., a VH-sequence or VHH sequence
  • the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, the immunoglobulin single variable domains 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 single variable domains 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 single variable domain may be a (single) domain antibody (or an amino acid that is suitable for use as a (single) domain antibody), a "dAb” or dAb (or an amino acid that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof.
  • the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof.
  • Nanobody® as defined herein
  • suitable fragment thereof e.g., Nanobody®, Nanobodies® and
  • Nanoclone® are registered trademarks of Ablynx N.V.]
  • VHH domains also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin
  • VHH domain has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “V H domains” or “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "V L domains” or “VL domains”).
  • VHH's and Nanobodies For a further description of VHH's and Nanobodies, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591 , WO 99/37681 , WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1 134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V.
  • Nanobodies in particular VHH sequences and partially humanized Nanobodies
  • Nanobodies including humanization and/or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobodies and their preparations can be found e.g. in WO
  • Nanobodies For a further general description of Nanobodies, reference is made to the prior art cited herein, such as e.g., described in WO 08/020079 (page 16).
  • Domain antibodies also known as “Dab”s, “Domain Antibodies”, and “dAbs” (the terms “Domain Antibodies” and “dAbs” being used as trademarks by the GlaxoSmithKline group of companies) have been described in e.g., EP 0368684, Ward et al. (Nature 341 : 544-546,
  • Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammalians, in particular human 4-chain antibodies.
  • specific selection for such antigen binding properties is required, e.g. by using libraries of human single VH or VL domain sequences.
  • Domain antibodies have, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not require humanization for e.g. therapeutical use in humans.
  • variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629).
  • the term “immunoglobulin single variable domain” or “single variable domain” comprises polypeptides which are derived from a non- human source, preferably a camelid, preferably a camelid heavy chain antibody. They may be humanized, as previously described. Moreover, the term comprises polypeptides derived from non-camelid sources, e.g. mouse or human, which have been “camelized”, as e.g., described in Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13: 475-479, 1995; Prot. Eng. 9: 531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231 : 25-38, 1999).
  • the amino acid residues of a VHH domain are numbered according to the general numbering for V H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids, as shown e.g., in Figure 2 of Riechmann and Muyldermans (J. Immunol. Methods 231 : 25-38, 1999).
  • Alternative methods for numbering the amino acid residues of VH domains which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the present description, claims and figures, the numbering according to Kabat applied to VHH domains as described above will be followed, unless indicated otherwise.
  • the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • the total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • CDR regions may also be done according to different methods.
  • FR1 of a VHH comprises the amino acid residues at positions 1-30
  • CDR1 of a VHH comprises the amino acid residues at positions 31-35
  • FR2 of a VHH comprises the amino acids at positions 36-49
  • CDR2 of a VHH comprises the amino acid residues at positions 50-65
  • FR3 of a VHH comprises the amino acid residues at positions 66-94
  • CDR3 of a VHH comprises the amino acid residues at positions 95-102
  • FR4 of a VHH comprises the amino acid residues at positions 103-113.
  • Immunoglobulin single variable domains such as Domain antibodies and Nanobodies
  • humanized immunoglobulin single variable domains such as Nanobodies (including VHH domains) may be immunoglobulin single variable domains that are as generally defined for in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution.
  • Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V H H sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized V H H sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable
  • an immunoglobulin single variable domain such as a Nanobody (including VHH domains) may be partially humanized or fully humanized.
  • Immunoglobulin single variable domains such as Domain antibodies and Nanobodies (including VHH domains and humanized VHH domains), can also be subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations result in an improved affinity of the resulting immunoglobulin single variable domain for its respective antigen, as compared to the respective parent molecule.
  • Affinity-matured immunoglobulin single variable domain molecules of the invention may be prepared by methods known in the art, for example, as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas et al. (Proc. Nat. Acad. Sci, USA 91 : 3809-3813, 1994), Shier et al. (Gene 169: 147-155, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226: 889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996).
  • one or more immunoglobulin single variable domains may be used as a "binding unit", “binding domain” or “building block” (these terms are used interchangeable) for the preparation of a polypeptide, which may optionally contain one or more immunoglobulin single variable domain or peptide that can serve as a binding unit (i.e., against the same or another epitope on IgE and/or against one or more other antigens, proteins or targets than IgE).
  • Monovalent polypeptides comprise or essentially consist of only one binding unit (such as e.g., immunoglobulin single variable domains).
  • Polypeptides that comprise two or more binding units will also be referred to herein as "multivalent” polypeptides, and the binding units/immunoglobulin single variable domains present in such polypeptides will also be referred to herein as being in a "multivalent format".
  • a "bivalent" polypeptide may comprise two immunoglobulin single variable domains, optionally linked via a linker sequence
  • a "trivalent” polypeptide may comprises three immunoglobulin single variable domains, optionally linked via two linker sequences; etc..
  • the two or more immunoglobulin single variable domains may be the same or different, and may be directed against the same antigen or antigenic determinant (for example against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof.
  • Polypeptides that contain at least two binding units (such as e.g., immunoglobulin single variable domains) in which at least one binding unit is directed against a first antigen (i.e., IgE) and at least binding unit is directed against a second antigen (i.e., different from IgE) will also be referred to as "multispecific" polypeptides, and the binding units (such as e.g., immunoglobulin single variable domains) present in such polypeptides will also be referred to herein as being in a "multispecific format".
  • a "bispecific" polypeptide of the invention is a polypeptide that comprises at least one immunoglobulin single variable domain directed against a first antigen (i.e., IgE) and at least one further immunoglobulin single variable domain directed against a second antigen (i.e., different from IgE), whereas a "trispecific" polypeptide of the invention is a polypeptide that comprises at least one immunoglobulin single variable domain directed against a first antigen (i.e., IgE), at least one further immunoglobulin single variable domain directed against a second antigen (i.e., different from IgE) and at least one further immunoglobulin single variable domain directed against a third antigen (i.e., different from both IgE and the second antigen); etc.
  • Figure 1 Sequence alignment of the various humanized and sequence-optimized variants of 39D11 compared to 39D11.
  • Figure 2 Sequence alignment of the various humanized and sequence-optimized variants of 39D11 compared to the humanized variant IgE009.
  • Figure 3 Study design of the PK/PD study.
  • FIG. 4 Free IgE plasma levels. Levels of free IgE overtime after a single injection of either vehicle, IgE109 or Xolair. Data on the graph represent the median value for each group of animals.
  • CFA Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Sera from blood samples of llamas 002, 004, 193 and 197 were obtained prior to immunization, during the immunization protocol and after completion of the immunizations.
  • Human IgE was coated onto Nunc Maxisorb plates at 2 microgram/ml, blocked with 1% casein in PBS and incubated with serial dilutions of pre- and post-immune llama sera. Plate- immobilized llama IgG was detected using HRP conjugated goat-anti-llama IgG (Bethyl Labs, Montgomery, TX) and TMB chromogen according to standard methods. Comparison of optical density values clearly indicated immunization induced a humoral immune response against IgE in all four animals.
  • Peripheral blood mononuclear cells were prepared from the blood samples using Ficoll- Hypaque according to the manufacturer's instructions. Total RNA extracted from these cells and from lymph nodes was used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector derived of pUCl 19, containing the LacZ promoter, a coliphage pill protein coding sequence, a resistance gene for ampicillin or carbenicillin, and a multicloning site harbours several restriction sites. In frame with the Nanobody® coding sequence, the vector codes for a C-tenninal c-myc tag and a (His)6 tag.
  • the signal peptide is the gen3 leader sequence which translocates the expressed Nanobody® to the periplasm.
  • Phage was prepared according to standard protocols (Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1st edition (October 28, 1996)) and stored after filter sterilization at 4°C until further use. In total, 4 phage libraries were constructed (002, 004, 193 and 197), with library sizes between 3.5xl0 6 and 28xl0 6 , and a percentage of insert ranging from 96 to 100%.
  • the cyno IgE sequence (SEQ ID NO: 173) was amplified by RT-PCR using different primers (SEQ ID NO's: 189 to 192). The obtained fragments were cloned in a cloning vector and sequenced.
  • the cyno IgE c(epsilon)2-c(epsilon)3-c(epsilon)4 fragment was cloned in an expression vector derived from pCIneo which contained the human cytomegalovirus (CMV) immediate-early
  • the vector coded for a C-temiinal (His) 10 tag.
  • the cis-acting viral DNA element, oriP locus allowed for episomal replication in HEK- EBNA cells, expressing the Epstein-Barr virus nuclear antigen- 1.
  • Phage display was used to enrich IgE-specific Nanobodies. Phages were prepared according to standard methods from libraries obtained from llamas No. 002, 004, 193 and 197. Phage libraries were used for two rounds (R1/R2) of selection on plate-immobilized cyno IgE Fc. Cyno IgE c(epsilon)2-c(epsilon)3-c(epsilon)4-Fc was immobilized at concentrations varying from 0.2 to 2 microgram/ml on Nunc Maxisorp ELISA plates. Plate-immobilized phages were retrieved using trypsin elution or Fc(epsilon)RIcc elution.
  • periplasmic extracts were analyzed for their ability to block the interaction of human IgE (Diatec, Oslo,
  • Cyno cross-reactive inhibiting Nanobodies were selected and sequenced. Sequence analysis revealed 1 Family (named family 12) which was subdivided into two subfamilies 12.1 (comprising clones 39D1 1, 39D8, 42D7 and 42G5; SEQ ID NO's: 119, 1 15, 116 and 117, respectively) and 12.2 (clone 36G5; SEQ ID NO: 1 18) and a unique clone (39B02; SEQ ID NO: 114). The corresponding DNA sequences are given in SEQ ID NO's: 183 to 188. This low diversity indicates that it is very difficult to generate and identify cyno-cross reactive Nanobodies which block the interaction of IgE with Fc(epsilon)RI(alpha).
  • Off-rate constants (koff) of individual inhibitory Nanobody clone periplasmic extracts were determined by surface plasmon resonance on a Biacore T100 instrument. Human IgE (Diatec, Oslo, Norway) was amine-coupled to a CM5 sensor chip at a density of 3100 RU.
  • Nanobody binding and off-rate was assessed at a single dilution of periplasmic extract.
  • the omalizumab Fab fragment was tested at a single concentration of lOOnM.
  • Each sample was injected 2 minutes at a flow rate of 45 ⁇ /min to allow binding to chip-bound antigen.
  • binding buffer without periplasmic extracts was sent over the chip at the same flow rate to allow spontaneous dissociation of bound Nanobody.
  • Analyte remaining bound after the monitored dissociation phase was removed by injecting regeneration solution (4.5M MgCl 2 ). Data of the best representatives per subfamily and the unique clone 39B2 (all selected for purification and more detailed analysis) are shown in Table 1.
  • Table 1 Off rate values of Nanobodies (periplasmic extracts).
  • Nanobodies were expressed in the periplasmic space of E.coli as c-myc, His6- tagged proteins in a culture volume of 250 mL. Expression was induced in high density cultures by addition of 1 mM IPTG and allowed to continue for 4h at 37°C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets and resuspension in dPBS. These extracts were used as starting material for affinity chromatography using HisTrap crude columns (GE Healthcare). Nanobodies were eluted from the column with 250 mM imidazole and subsequently desalted towards dPBS.
  • Example 6 Inhibition of HulgE or cvno IgE Fc (ce2-ce3-ce4) /HuFc(epsilon)RIfalpha)Fc interaction in Alphascreen
  • Table 2 IC50 values for competition between the human IgE receptor Fc(epsilon)RI (alpha) and anti-IgE Nanobodies to bind human IgE, their 95% confidence intervals (CI95) and percentage inhibition at 250 nM Nanobody as determined by Alphascreen. NA: not applicable.
  • Table 3 IC50 values for competition between the human IgE receptor Fc(epsilon)RI (alpha) and anti-IgE Nanobodies to bind cyno IgE Fc (Ce2-ce3-ce4), their 95% confidence intervals (CI95) and percentage inhibition at 250 nM Nanobody as determined by Alphascreen. NA: not applicable.
  • Nanobodies were analyzed for their ability to block the interaction of HuIgE (Diatec, Oslo, Norway) or cyno IgE (plasma) with Fc(epsilon)RI. To this end, ELISA plates were coated with Fc(epsilon)RI (0.1 microgram/ml), then blocked. Serial Nanobody dilutions were pre- incubated with 50 pM HuIgE or 1/50 cyno plasma dilution, followed by addition of the pre- incubated mixture to the Fc(epsilon)RI coated ELISA plates. Binding of HuIgE or cyno IgE to the immobilized Fc(epsilon)RI was detected by using goat anti-human IgE-HRP (KPL,
  • Nanobodies to bind human IgE Fc (Ce2-ce3-ce4), their 95% confidence intervals and percentage inhibition at 500 nM Nanobody as determined by ELISA.
  • Table 5 IC50 values for competition between the human IgE receptor Fc(epsilon)RI(alpha) and anti-IgE Nanobodies to bind cyno IgE Fc (Ce2-ce3-ce4), their 95% confidence intervals and percentage inhibition at 500 nM Nanobody as determined by ELISA. NA: not applicable.
  • Example 8 Inhibition of HuIgE/HuFc(epsilon)RII interaction in ELISA
  • Nanobodies were analyzed for their ability to block the interaction of HulgE (Diatec, Oslo, Norway) with recombinant Fc(epsilon)RII (R&D Systems, Minneapolis, MN). To this end, ELISA plates were coated with Fc(epsilon)RII, then blocked. Serial Nanobody dilutions were pre-incubated with 20 nM HulgE, followed by addition of the pre-incubated mixture to the
  • Nanobodies 39D11 and 36G5 had a similar potency and efficacy (100% block) as compared to Reference Fab, whereas Nanobody 39B2 was 10-fold less potent and only reached around 80% block at 500 nM Nanobody concentration.
  • Table 6 IC50 values for competition between the human IgE receptor Fc(epsilon)RII and anti-IgE Nanobodies to bind human IgE, their 95% confidence intervals as determined by
  • Nanobodies were analyzed for their ability to block the interaction of HulgE (Diatec, Oslo, Norway) with Omalizumab. To this end, an alphascreen assay (Perkin Elmer, Waltham, MA) was set up. In brief, serial dilution series of individual Nanobodies were incubated with O.lnM biotinylated hulgE, O.lnM Omalizumab, streptavidin coated donor beads and anti-human Fc Nanobody coupled acceptor beads. The Reference Fab fragment, known to inhibit the
  • HuIgE/Omalizumab interaction was used as a positive control. Assays were read in an Envision alphascreen option fitted multimode reader (Perldn Elmer, Waltham, MA). Individual clones were scored as putative HuIgE/Omalizumab interaction inhibitors if their presence decreased the fluorescent signal of the acceptor beads. The results are shown in Table 7 and indicate that all tested Nanobodies except 39B2 compete with Omalizumab for IgE binding. The bottom plateau levels of the Nanobody inhibition curves are significantly higher (% inhibition maximum 79.9%) in comparison to that of Reference Fab or IgE (% inhibition maximum 96.9 and 99.6, respectively), suggesting that the Omalizumab en Nanobody epitopes are only partially overlapping.
  • Table 7 IC50 values for competition between omalizumab and anti-IgE Nanobodies to bind human IgE, their 95% confidence intervals and percentage inhibition at 250 nM Nanobody as determined by Alphascreen. NA: not applicable.
  • Example 10 Inhibition of HuIgE/HuFc(epsilon)RI and HuFc(epsilon)RII interaction in FACS Nanobodies 39D11 and 36G5 were tested in FACS competition assay for their ability to inhibit IgE-binding to Fc(epsilon)RI on RBL Fc(epsilon)RI( alpha) stable transfectants and Fc(epsilon)RII on Raji cells. To this end serial dilutions of Nanobody were added to the cells followed by addition of human IgE (7.5 nM for RBL Fc(epsilon)RI assay; 10 nM for Raji cell assay). Presence of putative interaction inhibitors would result in decreased MCF signals.
  • Table 8 indicate that both Nanobodies block IgE binding to Fc(epsilon)RI and
  • Fc(epsilon)RII comparable to the positive control Reference Fab. All three tested molecules reached more than 90% inhibition at 1 ⁇ concentrations.
  • Table 8 IC50 values for competition between anti-IgE Nanobodies and Fc(epsilon)RI or Fc(epsilon)RII (expressed on transfected RBL and endogenously expressed on Raji cells, respectively) to bind human IgE and their 95% confidence intervals as determined by FACS.
  • Assay setup was based on a protocol supplied with xCELLigence RTCA instrument (Roche).
  • the System measures electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture E-Plates.
  • the real time impedance measurement provides quantitative information about the morphological status of the cells, including degranulation, without incorporation of labels.
  • RBL cells transfected with Fc(epsilon)RIalpha were overnight incubated in E-plates and sensitized with anti-DNP chimeric IgE (50ng/ml; Serotec) for 1 hr. Then, degranulation was triggered by adding NIP-BSA (50 ng ml; Biosearch Technologies).
  • the activation/degranulation of the basophils can be detected in real time on the xCELLigence System measuring impedance (measure every minute during 8 hrs). The maximum cell index signal per well was used to calculate EC50 and IC50 values. A correlation was shown with histamine release in supernatant (LDN histamine Research ELISA).
  • Nanobodies 39D08, 39D1 1 , 36G5 and 39B2 were tested in degranulation assay for their ability to inhibit IgE-mediated degranulation. To this end serial dilutions of Nanobody or control antibody were pre-incubated with human IgE and then added to the cells. Results are shown in Table 9. Nanobody 39B2 only reached 25% block of degranulation at a Nanobody concentration of 500 nM, whereas the other 3 Nanobodies and Reference Fab blocked more than 95% at this concentration. The potency of the Nanobodies was 3- to 40-fold weaker in comparison with the Reference Fab control.
  • Table 9 IC50 values for blocking IgE-mediated degranulation of RBL- Fc(epsilon)RI(alpha) transfected cells by anti-IgE Nanobodies or control Fab, and their 95% confidence intervals as determined by impedance measurement.
  • Nanobodies were determined by surface plasmon resonance (SPR) on a Biacore 3000 instrument.
  • SPR surface plasmon resonance
  • Human IgE Diatec, Oslo, Norway
  • cyno IgE Fc Ce2-ce3-ce4
  • ethanolamine a reactive organic compound
  • Nanobodies were injected at different concentrations. Each sample was injected at a flow rate of 45 ⁇ /min to allow for binding to chip-bound antigen, followed by binding buffer without Nanobody at the same flow rate to allow for spontaneous dissociation. Analyte remaining bound after the monitored dissociation phase was removed by injecting regeneration solution (4.5M MgCk).
  • Binding curves at different concentrations were used to calculate the kinetic parameters kon-values (k a ), k 0f rvalues (kd) and K D .
  • Kinetic parameters were determined using heterogeneous ligand fit since a two phase interaction was observed. The obtained results are presented in Table 10.
  • Table 10 Kinetic parameters for binding of anti-IgE Nanobodies to human IgE and eyno IgE Fc.
  • the IgE-specific Nanobody 39D1 1 was the most potent cyno cross-reactive Nanobody identified during screening. It was selected and further pursued for sequence optimization and affinity maturation.
  • variants Based on alignment of 39D11 sequence with VH3-23/JH5 human germline, it was decided to generate 4 variants: IGE009, IGEOl 0, IGEOl 1 and IGE012 (SEQ ID NO's: 120 to 123, respectively). All variants include the three mutations V5L, K83R and Q108L. In variants IGEOl 1 and IGE012, a unique mutation W91Y is included. In all four variants, the methionine at position 77 is substituted by threonine (IGE009 and IGEOl 1) or leucine (IGEOl 0 and IGEOl 2). Sequence alignment of sequence optimized variants with 39D11 is shown in Figure 1.
  • An error-prone library was generated by amplifying IGE009 under error-prone conditions and cloning the fragments into a phage display vector.
  • the obtained library was subjected to multiple rounds of in solution panning using gradually decreasing concentrations (10-0.001 nM) of biotinylated human IgE.
  • Outputs from the phage selections were analyzed for off rate on ProteOn. Up to 6-fold improved off-rates relative to 39D1 1 were measured for the best clones.
  • Sequence analysis identified mutations (both in the frameworks and the CDR's) with positive effect on off-rate. These were: E6Q; F29Y; G30D; S31N or S31P; S35G or S35A; G44R; N75K; D97E and Y100eF.
  • Nb-9GS-ALB8 formats were made containing the parental IgE-specific Nanobody 39D1 1 or its humanized variant IGE009 (constructs IGE020 and IGE019 respectively, SEQ ID NO's: 175 and 176). Alignment of the affinity matured variants with 39D1 1 en IGE009 is shown in Figures 1 and 2 respectively.
  • Off rates were analysed on BIAcore. Binding profiles and off rates are shown in Table 12. Off rate analysis on BIAcore shows decreased off rates for affinity matured variants compared to IGE009 (up to 25-fold on human IgE and up to 15-fold on cyno IgE-Fc). The binding patterns are still biphasic but the percentage of the fast off rate is significantly reduced after affinity maturation (certainly on human IgE).
  • Nanobodies to human IgE and cyno IgE-Fc are described in detail below.
  • Affinity matured variants were tested in degranulation assay in the presence or absence of purified human serum albumin (Sigma, A8763). Calculated IC50s and CI95 are shown in Table 13. The obtained results clearly indicate that a number of variants outperform Omalizumab in degranulation assay. Binding of HSA does not influence the potency of the Nanobody constructs.
  • Table 13 IC50 values for blocking IgE-mediated degranulation of RBL-Fc(epsilon)RI(aIpha) transfected cells by anti-IgE Nanobodies or control Mab/Fab, and their 95% confidence intervals as determined by impedance measurement.
  • Nanobodies IGE026, IGE027 and 39D11 were determined on human IgE in solution using KinExA technology.
  • the obtained affinity of the Nanobodies IGE026 and IGE027 was 30 to 50-fold improved compared to that of 39D11 (Table 14).
  • Table 14 KD values for IgE binding.
  • Nanobodies (2.5 to 200 nM) was injected at a flow rate 45 ⁇ /min. Affinity constants were determined and are listed in Table 15. The HSA-specific Nanobody ALB 8 was included for comparison. In general, a lower affinity was observed. Table 15: KD values of formatted Nanobodies to serum albumin
  • Examples 1 to 13 above illustrate the favourable properties of the anti-IgE Nanobodies (including 39D11, the 39D11-type sequences and the 39D11-like sequences) per se or when used as building blocks in order to provide multivalent, multispecific and/or biparatopic constructs.
  • the anti-IgE Nanobodies of the invention are combined with the half-life extending peptides that are preferred according to the invention.
  • the IgE binding Nanobody building block of IGE026/ALB8 i.e. IgE66G02 (SEQ ID NO's: 128 or 129) was genetically fused at its N- or C-terminus to one or more HSA binding NExpedite peptides via a short or long GlySer- linker, resulting in 15 variants (IGE100-105, IGE107-1 13, EXP476, EXP483, SEQ ID NO's: 147 to 161).
  • the constructs were cloned in an in-house Pichia pastoris expression vector derived from pPICZa (Invitrogen) which contains the AOX1 promotor for tightly regulated, methanol induced expression of Nanobodies® in Pichia pastoris, a resistance gene for ZeocinTM, a multicloning site and the alpha- factor secretion signal. Expression and purification of the constructs was performed essentially as described in Example 20 of US 201 1/0243954.
  • Table 17 IC50 values for blocking IgE-mediated degranulation of RBL-Fe(epsilon)RI (alpha) transfected cells by IGE-NExpcdite Nanobodies or control Mab and their 95% confidence intervals as determined by impedance measurement.
  • IGE-NExpedite Nanobodies were incubated with 3.4pM bio-HSA, in presence or absence of hlgE, prior to the addition of EXP413 conjugated acceptor beads and streptavidin donor beads (Perkin Elmer). Fluorescence was measured by reading plates on the EnVision Multilabel Plate Reader (Perlcin Elmer) using an excitation wavelength of 680 nm and an emission wavelength of 520 nm. Decrease in fluorescence signal indicates that the binding of bio-HSA to Nb2112-9GS-EXP89D03 is blocked by IGE-NExpedite Nanobody.
  • EXP413, EXP486 (Nb2112-9GS-EXP89D3-9GS-EXP89D3) and EXP553 were included in the assays.
  • the data are shown in Table 18. If a single NExpedite peptide is at the N-terminus of the IGE Nanobody (IGE102, IGE103), the IC50 is markedly increased, suggesting the Nanobody hampers binding of the NExpedite peptide to HSA.
  • the IC50's are comparable to the IC50's of their respective Nb21 12- NExpedite controls: compare EXP476 and IGE100 with EXP413, compare EXP483, IGE101 and IGE 107 with EXP486, compare IGEl 09 with EXP553. Furthermore, binding of hlgE to the Nanobody does not influence the HSA binding of the NExpedite peptide(s). Table 18: IC50 values for competition between Nb2112-9GS-EXP89D3 and IGE-NExpedite
  • Example 16 Storage stability: comparison of IgE-NExpedite constructs and construct comprising an albumin-binding Nanobody
  • the storage stability of the representative IgE-NExpedite constructs was compared with corresponding constructs in which the albumin-binding NExpedite peptide was replaced with an albumin-binding Nanobody (Alb-8).
  • the constructs used were IGE026/ALB-8 (SEQ ID NO: 178) and IGE109 (SEQ ID NO: 155).
  • constructs were formulated at 50mg/ml in D-PBS buffer and stored for 1 month at the indicated temperature in plastic PCR tubes in the dark.
  • the percentage of pre-peak (corresponding to the amount of dimer formed) was determined using SE-HPLC.
  • SE-HPLC was performed using aBioSep SEC- 2000 column (Phenomenex) and D-PBS as running buffer at a flow rate of 0.2 ml/min. 10 g material was injected and data was analysed using Chromeleon software.
  • SE-HPLC was performed using a SEC-5 column (Agilent) and 1 OmM Na-Phosphate (pH 7.5), 125mM Arginine.HCl, 10% Ethanol as running buffer at a flow rate of 0.5 ml/min. 10 ⁇ g material was injected and data was analysed using Chromeleon software. The results are shown in Table 19.
  • Table 19 Percentage of pre-peak on SE-HPLC after 1 month storage at indicated temperatures.
  • EXP483 (SEQ ID NO: 161), IGEIOO (SEQ ID NO: 147) and IGEl Ol (SEQ ID NO: 148).
  • the same SE-HPLC protocol was used as for IGE109 above. The results are shown in Table 20.
  • Table 20 Percentage of pre-peak on SE-HPLC after storage at indicated temperatures.
  • Example 17 In vivo PK/PD analysis of IgE 109 in cynomolgus monkey
  • PK, PD and immunogenicity markers were measured at different time points after a single administration of IgE or Xolair in cynomolgus monlceys, screened for moderate to high IgE plasma levels.
  • Modelling analysis resulting in the determination of the in vivo K D is performed using a published model for Xolair (Hayashi et al, British Journal of Clinical Pharmacology, 2006).
  • Table 21 Animal treatment groups for the PK/PD study. NA: not applicable.
  • S.c subcutaneous injection, i.v.: intravenous injection.
  • the dose volume for each animal is based on the most recent body weight measurement.
  • Table A-2 Amino acid sequences of IgE binding Nanobodies.
  • IGE009 120 EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAP
  • IGE010 121 EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAP
  • IGE01 1 122 EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAP
  • IGE026 128 EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYDMAWVRQAP
  • IGE027 130 EVQLLQSGGGLVQPGGSLRLSCAASGFTFGNYDMAWVRQAP
  • IGE028 131 EVQLLQSGGGLVQPGGSLRLSCAASGFTFGNYDMGWVRQAP
  • IGE029 132 EVQLLQSGGGLVQPGGSLRLSCAASGFTFGSYDMAWVRQAP
  • Table A-6 Serum albumin binding peptides for use in the polypeptides of the invention.
  • Table A- 7 Sequence motifs present in the serum albumin binding peptides.
  • sequence motif 59 DYTVFGGG sequence motif 60 YTVFGGGT sequence motif 61 YDVFGGGA sequence motif 62 YDVFGGGN sequence motif 63 YDVFGGGD sequence motif 64 YTVFGGGA sequence motif 65 YTVFGGGN sequence motif 66 YTVFGGGD sequence motif 67 TAFGGG
  • Table A-9 Sequences of some anti-IgE polypeptides of the invention.
  • IGE100 147 DVQLLESGGGLVQPGGSLRLSCAASGFTFGNYDMA
  • IGE103 150 WEQDRDWDFDVFGGGTPGGGGSGGGSWWEQDR
  • IGE104 151 WaveQDRDWDFDVFGGGTPGGGGSGGGSWWEQDR (EXP89D03- DWDFDVFGGGTPGGGGSGGGGSGGGGSGGGGSEVQ 9GS-EXP89D03- LLESGGGLVQPGGSLRLSCAASGFTFGNYDMAWVRQ 9GS-IgE66G02) APGKRPEWVSSIDTGGDITHYADSVKGRFTISRDNAK
  • IGE108 154 DVQLLESGGGLVQPGGSLRLSCAASGFTFGNYDMA IGE66G02 E1D - WVRQAPGKRPEWVSSIDTGGDITHYADSVKGRFTISR 20GS-NEXP001 - DNAKNTLYLQMNSLRPEDTAVYWCATDEEYALGPN 9GS- NEXP001) EFDYYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGS Name SEQ ID NO: SEQUENCE
  • IGE109 155 DVQLLESGGGLVQPGGSLRLSCAASGFTFGNYDMA IGE66G02 E1D - WVRQAPGKRPEWVSSIDTGGDITHYADSVKGRFTISR 20GS-NEXP001 - DNAKNTLYLQMNSLRPEDTAVYWCATDEEYALGPN 9GS- NEXP002) EFDY YGQGTL VTVS SGGGG SGGGG SGGGG SGGGGS
  • EXP476 160 EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYDMAW
  • EXP483 161 EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYDMAW
  • VFGGGTPGGGGSGGGSWWEQDRDWDFD VFGGGTP Table A-10: Sequence of Alb-8 Nanobody and fusion proteins with Alb-8 Nanobody.
  • Alb-8 (Alb- 1 1) 174 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV QAPGKGLE
  • IGE019 175 EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGP
  • IGE/ALB-8-020 AVYWCATDEDYALGPNEYDYYGQGTQVTVSSGGGGSGGGSEVQL
  • IGE025/ALB-8 177 EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYDMAWVRQAPGKGP IGE/ALB-8-025 EWVSSIDTGGDITHYADSVKGRFTISRDNAKNTLYLQMNSLRPEDT
  • IGE028/ALB-8 180 EVQLLQSGGGLVQPGGSLRLSCAASGFTFGNYDMGWVRQAPGKRP IGE/ALB-8-028 EWVSSIDTGGDITHYADSVKGRFTISRDNAKNTLYLQMNSLRPEDT
  • IGE029/ALB-8 181 EVQLLQSGGGLVQPGGSLRLSCAASGFTFGSYDMAWVRQAPGKRP IGE/ALB-8-029 EWVSSIDTGGDITHYADSVKGRFTISRDNAK TLYLQMNSLRPEDT
  • IGE030/ALB-8 182 EVQLLQSGGGLVQPGGSLRLSCAASGFTYGNYDMAWVRQAPGKR IGE/ALB-8-030 PEWVSSIDTGGDITHYADSVKGRFTISRDNAKNTLYLQMNSLRPED
  • TIGGSLSRSSQGTLVTVSS Table A-11 Primers used for the amplification of the cyno IgE sequence.
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WO2012175740A1 (en) 2012-12-27

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