WO2011083140A1 - Domaines variables simples d'immunoglobuline dirigés contre le cxcr4 doués d'une meilleure activité thérapeutique et produits de recombinaison les comprenant - Google Patents

Domaines variables simples d'immunoglobuline dirigés contre le cxcr4 doués d'une meilleure activité thérapeutique et produits de recombinaison les comprenant Download PDF

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WO2011083140A1
WO2011083140A1 PCT/EP2011/050156 EP2011050156W WO2011083140A1 WO 2011083140 A1 WO2011083140 A1 WO 2011083140A1 EP 2011050156 W EP2011050156 W EP 2011050156W WO 2011083140 A1 WO2011083140 A1 WO 2011083140A1
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sequence
amino acid
isv
construct
polypeptide
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PCT/EP2011/050156
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Maarten Dewilde
Maria Gonzalez Pajuelo
Peter Vanlandschoot
Karen Cromie
Els Pattyn
Benedikte Serruys
Catelijne Stortelers
Beatrijs Strubbe
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Ablynx Nv
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to amino acid sequences that are immunoglobulin single variable domains (as defined herein) directed against CXCR4.
  • the invention further relates to compounds, constructs, proteins or polypeptides that comprise or essentially consist of one or more such amino acid sequences.
  • WO 09/138519 describes a number of amino acid sequences and in particular VHHs and constructs thereof that are directed against human CXCR4 (see for example the amino acid sequences mentioned such as SEQ ID NO: 238 and SEQ ID NO: 239 in Table B-1.1 of WO 09/138519).
  • WO 09/138519 also describes multivalent, multispecific and/or biparatopic constructs (as defined in WO 09/138519) that are directed against human CXCR4. Reference is for example made to the constructs referred to in Example 4 of WO 09/138519 such as SEQ ID NO: 264 in Table B-5 of WO 09/138519).
  • amino acid sequence against human CXCR4 from WO 09/138519 is the sequence called 238D2 (see SEQ ID NO: 238 in WO 09/138519 and SEQ ID NO: 445 herein)
  • CXCR4 from WO 09/138519 is the sequence called 238D4 (see SEQ ID NO: 239 in WO 09/138519 and SEQ ID NO: 447 herein)
  • WO 09/138519 further gives some non-limiting examples of multivalent, multispecific and/or biparatopic constructs that comprise 238D2 and/or 238D4 (see for example SEQ ID NO's: 261 to 266 in WO 09/138519 and in particular 238D2-20GS-238D4).
  • Nanobody® is as defined in WO 09/138519, and thus in a specific aspect generally denotes a VHH, a humanized VHH or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as e.g., optimized for chemical stability and/or solubility, maximum overlap with known human framework regions and maximum expression).
  • a sequence optimized VHH such as e.g., optimized for chemical stability and/or solubility, maximum overlap with known human framework regions and maximum expression.
  • V H H'S and Nanobodies® For a further description of V H H'S and Nanobodies®, reference is made to the review article by Muyldermans in Reviews in Molecular Biotechnology (74(2001), 277-302); 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 1134231 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.
  • Nanobodi.es® in particular VHH sequences and partially humanized. Nanobodies®
  • amino acid residues of a Nanobody® are numbered according to the general numbering for VH 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 in the article of Riechmann and
  • FR1 of a Nanobody® comprises the amino acid residues at positions 1-30
  • CDRl of a Nanobody® comprises the amino acid residues at positions 31-35
  • FR2 of a Nanobody® comprises the amino acids at positions 36-49
  • CDR2 of a Nanobody® comprises the amino acid residues at positions 50-65
  • FR3 of a Nanobody® comprises the amino acid residues at positions 66-94
  • CDR3 of a Nanobody® comprises the amino acid residues at positions 95-102
  • FR4 of a Nanobody® comprises the amino acid residues at positions 103-113.
  • the total number of amino acid residues in each of the CDR's 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.
  • position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa
  • position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa
  • position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa
  • position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.
  • the anti-human CXCR4 amino acid sequences and constructs from WO 09/138519, from US 61/358,495, US 61/358,495 and from PCT/EP2010/064766 show excellent biological activity and other desired properties.
  • this does not mean that either alternative anti-human CXCR4 amino acid sequences and constructs and/or an anti- human CXCR4 amino acid sequences and constructs that would have (even further) improved properties would not be a valuable addition to the art.
  • the invention provides such alternative and/or improved anti human CXCR4 amino acid sequences (also referred to herein as “amino acid sequence (s) of the invention” or “ Immunoglobulin Single Variable Domains (ISVs) of the invention ' ”) and constructs comprising the same (also referred to herein as “constructs of the invention” or “polypeptides of the invention”).
  • the invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as “nucleic acids of the invention” or “nucleotide sequences of the invention”): to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.
  • the invention provides: - amino acid sequences binding CXCR-4, which are an alternative to the sequences from WO 09/138519 and US 61/358,495 in that, for example and without limitation, they can bind to a different epitope, domain or (extracellular) loop of CXCR-4 than the sequences 238D2 or 238D4 and their improved variants; and/or which are an alternative to the sequences from WO 09/138519 and US 61/358,495 in that, for example and without limitation, they belong to a different family (as described herein) than the sequences 238D2 or 238D4 and their improved variants;
  • sequences 238D2 and/or 238D4 and their improved variants in that, for example and without limitation, they show greater potency in a relevant assay (for example but without limitation, in the Jurkat assay described in Example 1, C-4; and/or the cAMP inhibition assay described in Example 1, C-3);
  • constructs, compounds and polypeptides comprising one or more of such amino acid sequences, as further described herein.
  • the invention provides the following amino acid sequences: 4CXCR010E09 (SEQ ID NO: 189, also referred to herein as “10E9”), 4CXCR281E10 (SEQ ID NO: 190, also referred to herein as "281E1.0”),
  • 4CXCR010E12 (SEQ ID NO: 191, also referred to herein as “10E12"), 4CXCR010A10 (SEQ ID NO: 192, also referred to herein as “10A10"), 4CXCR010G10 (SEQ ID NO: 193, also referred to herein as “10G10"), 4CXCR014A02 (SEQ ID NO: 194, also referred to herein as “14A2”), 4CXCR015A01 (SEQ ID NO: 195, also referred to herein as "15 ⁇ ), 4CXCR015H03 (SEQ ID NO: 196, also referred to herein as "15H3”) and 4CXCR283B06 (SEQ ID NO: 197, also referred to herein as "283B6”), as well as humanized and/or otherwise sequence-optimized variants thereof (as further described herein), of which: - the amino acid sequences 4CXCR010E09 (SEQ ID NO
  • amino acid sequences 4CXCR010E12 (SEQ ID NO: 191), 4CXCR010A10 (SEQ ID NO: 192), 4CXCR010G10 (SEQ ID NO: 193), 4CXCR014A02 (SEQ ID NO: 194), 4CXCR015A01 (SEQ ID NO: 195), 4CXCR015H03 (SEQ ID NO: 196) and 4CXCR283B06 (SEQ ID NO: 197), belong to different families and/or bind to different epitopes, domains or loops on CXCR-4 and/or have different "footprints" (see Example 1, D-4 and Example 1.9 of PCT/EP2010/064766,) than 238D2 and 238D4 (see Table C-2), and which each also belong to different families and/or bind to different epitopes from each other (except for 4CXCR283B06 and 4CXCR014A02, which belong to the same family).
  • amino acid sequences provided by the invention are immunoglobulin single variable domains (or ISV's" for short) or amino acid sequences that, under suitable conditions (such as physiological conditions), are capable of forming an immunoglobulin single variable domain, or are suitable antigen- binding fragments of the same (as further described herein).
  • amino acid sequences of the invention comprise or essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which the framework sequences and CDR's are as defined herein.
  • Each amino acid sequence/ISV of the invention is such that it comprises an immunoglobulin fold or may be an amino acid sequence that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold ⁇ i.e., by folding), and in particular such that it forms (or under such suitable conditions is capable of fonning), an immunoglobulin single variable domain (i.e., an immunoglobulin variable domain that comprises a functional antigen binding site and does not require an interaction with another immunoglobulin variable domain (such as a VH-VL interaction) to form a functional antigen binding site).
  • suitable conditions such as physiological conditions
  • an immunoglobulin single variable domain i.e., an immunoglobulin variable domain that comprises a functional antigen binding site and does not require an interaction with another immunoglobulin variable domain (such as a VH-VL interaction) to form a functional antigen binding site.
  • any ISV as described herein 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 a suitable fragment thereof.
  • the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a VH sequence that is derived from a human antibody) or it may be a so- called VHH-sequence (as defined herein) that is derived from a so-called “heavy chain antibody” (as defined herein).
  • any ISV described herein may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb” (or an amino acid sequence that is suitable for use as a d Ab) or a Nanobody® (as defined herein, and including but not limited to a VFIH sequence); other single variable domains, or any suitable fragment of any one thereof.
  • dAb single domain antibody
  • dAb's reference is for example made to Ward et al.
  • the ISV's of the invention can either be used per se -i.e., in the form of a (monovalent) protein or polypeptide that comprises or essentially consists of such a preferred immunoglobulin single variable domains)- and/or that can be used as building blocks for making compounds or constructs that comprise one or more of ISV's of the invention and one or more further binding domains, binding units and/or other functional groups or functionalities.
  • the ISV's of the invention may be used as building blocks for providing a range of different multivalent (such as bi- or trivalent), multispecific (such as such as bi- or trispecific) or multiparatopic (such as biparatopic) constructs as further described herein, which may also have a tailored or increased half-life or other desirable properties and/or functionalities.
  • multivalent such as bi- or trivalent
  • multispecific such as such as bi- or trispecific
  • multiparatopic such as biparatopic constructs as further described herein, which may also have a tailored or increased half-life or other desirable properties and/or functionalities.
  • the amino acid sequences/ISV's of the invention are also generically referred to herein as "building blocks” and specifically referred to herein as "building blocks of the invention”.
  • 10E9-tvpe sequences a "10E9-type sequence", "10E9-type ISV or "10E9- type building block” is defined as an ISV (as described herein) that has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0 10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM), and that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR010E09 (SEQ ID NO: 189); and/or (ii) competes with 4CXCR010E09 (SEQ ID NO: 189) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010E09
  • a 10E9-type sequence belongs to the same family as 4CXCR010E09 (SEQ ID NO: 189).
  • a 10E9-type sequence also has an IC 5 o in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than lOOOnM, such as better than 750nM, such as better than 500nM.
  • 10E9-type sequences are 4CXCR010E09 (SEQ ID NO: 189) and the "10E9-like sequences", u I0E9-like ISV or ii 10E9-like building blocks ' " described herein.
  • 281E10-type sequences a "281EW-type sequence''.
  • "28IE10-type ISV or "281E10-type building block” is defined as an ISV (as described herein) that has an IC 5 0 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM), and that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR281E10 (SEQ ID NO: 190); and/or (ii) competes with 4CXCR281E10 (SEQ ID NO: 190) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in
  • Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR281E10 (SEQ ID NO: 190) to human CXCR-4.
  • a 281E10-type sequence belongs to the same family as 4CXC 281E10 (SEQ ID NO: 190).
  • a 281E10-type sequence also has an IC 5 o in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than lOOOnM, such as better than 750nM, such as better than 500nM.
  • 281E10-type sequences are 4CXCR281E10 (SEQ ID NO: 190) and the "281E10-iike sequences", "281E10-Iike ISV" of u 281E10-Hke building blocks described herein.
  • 10E12-tvne sequences a i ⁇ 10E12-type sequence
  • 10E12-type IS I or "10E12-type building block” is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as
  • 4CXCR010E12 (SEQ ID NO: 191); and/or (ii) competes with 4CXCR010E12 (SEQ ID NO : 191) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010E12 (SEQ ID NO: 191) to human CXCR-4.
  • such a 10E12-type sequence belongs to the same family as
  • 10E12-type sequence has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a 10E12-type sequence further has an IC 50 in cAMP assay of Example 1 , C-3; of that is either (i) better than the IC 5 0 value of 238D2 in the same assay and/or (ii) better than lOOOnM, such as better than 750nM, such as better than 500nM.
  • 10E12- type sequences are 4CXCR010E12 (SEQ ID NO: 191) and the ii 10E12-like sequences", "I0E12-like ISV” or ⁇ WEH-like building blocks" described herein.
  • lOAlO-type sequences a "JOAIO-npe sequence", "1 OA 10 -type ISV or “lOAlO-type building block” is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as
  • 4CXCR010A10 (SEQ ID NO: 192); and/or (ii) competes with 4CXCR010A10 (SEQ ID NO: 192) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010A10 (SEQ ID NO: 192) to human CXCR-4.
  • such a 10A10-type sequence belongs to the same family as
  • 10A10-type sequence has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl.0 "9 M (7.0E-9M or 7.0 nM). in particular better than 5.0xlO "9 M (5.0E-9M or 5.0 nM).
  • a 10A10-type sequence further has an IC50 in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than !OOOnM, such as better than 750nM, such as better than 500nM.
  • 10A10-type sequences are 4CXCR010A10 (SEQ ID NO: 192) and the "WAlO-like sequences", “lOAJO-like ISV” or “JOAW-like building blocks” described herein.
  • lOGlO-type sequences a "lOGlO-rype sequence", ''lOGlO-type ISV or "WGJO-type building block” is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as
  • 4CXCR010G10 (SEQ ID NO: 193); and/or (ii) competes with 4CXCR010G10 (SEQ ID NO: 193) for binding to huma CXCR-4 (in particular-, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010G10 (SEQ ID NO: 193) to human CXCR-4.
  • a lOGlO-type sequence belongs to the same family as
  • lOGlO-type sequence has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 ' M (5.0E-9M or 5.0 nM).
  • a lOGlO-type sequence further has an IC50 in cAMP assay of Example 1, C-3; of that is either (i) better
  • 14A2-type sequences a "l4A2-t)pe sequence", J4A2-type ISV" or "14A2-type building block' ' ' is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR014A02 (SEQ ID NO: 194); and/or (ii) competes with 4CXCR014A02 (SEQ ID NO: 194) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR014A02 (SEQ ID NO: 194) to human CXCR-4.
  • such a 14A2-type sequence belongs to the same family as 4CXCR014A02 (SEQ ID NO: 194).
  • 14A2-type sequence has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xlG "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a 14A2-type sequence further has an IC 50 in cAMP assay of Example 1, C-3; of that is either (i) better than the IC 50 value of 238D2 in the same assay and/or (ii) better than ⁇ , such as better than 750nM, such as better than 500nM.
  • Some preferred, but non-limiting examples of 14A2-type sequences are 4CXCR014A02 (SEQ ID NO: 194) and the "14A2-like sequences", u I4A2-like ISV or "14A2-like building blocks" described herein. [Note: because 14A2 and 283B6 belong to the same family and have an essentially similar footprint, it is expected that a substantial part of the 283B6-type sequences will also be 14A2-type sequences and visa-versa].
  • ISA l-type sequence a "ISA l-type sequence", 'J5A J-type ISV or ISAl-iype building block” is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR015A01 (SEQ ID NO: 195); and/or (ii) competes with 4CXCR015A01 (SEQ ID NO: 195) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3). and/or cross-blocks (as defined herein) the binding of 4CXCR015A01 (SEQ ID NO: 195) to human CXCR-4.
  • such a 15Al-type sequence belongs to the same family as 4CXCR015A01 (SEQ ID NO: 195). Most preferably, 15Al-type sequence has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • _a 15Al-type sequence further has an IC 50 in cAMP assay of.Example 1, C-3; of that is either (i) better than the IC 5 o value of 238D2 in the same assay and/or (ii) better than 1 OOOnM, such as better than 750nM, such as better than 500nM.
  • Some preferred, but non-limiting examples of 15Al-type sequences are 4CXCR015A01 (SEQ ID NO: 195) and the "15A1-Uke sequences", "lSAl-lihe ISV or "15AI ⁇ like building blocks" described herein.
  • 15H3-type sequences a "15H3-type sequence", "15H3-type ISV or "15H3-lype building block” is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR015H03 (SEQ ID NO: 196); and/or (ii) competes with 4CXCR015H03 (SEQ ID NO: 196) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross -blocks (as defined herein) the binding of 4CXCR015H03 (SEQ ID NO: 196) to human. CXCR-4.
  • such a 15H3-type sequence belongs to the same family as 4CXCR0151103 (SEQ ID NO: 196).
  • 15H3-type sequence has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO "9 M (5.0E-9M or 5.0 nM).
  • a 15H3-type sequence further has an IC 50 in .
  • cAMP assay of Example 1, C-3 of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than !OOOnM, such as better than 750nM, such as better than 500nM.
  • 15H3-type sequences are 4CXCR015H03 (SEQ ID NO: 196) and the "15H3-Iite sequences", "ISHJ-like ISV or i l 15H3-like building blocks" described herein.
  • 283B6-type sequences a "283B6-iype sequence", Li 283B6 ⁇ type ISV or "283B6-type building block "1 is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as
  • 4CXCR283B06 (SEQ ID NO: 197); and/or (ii) competes with 4CXCR283B06 (SEQ ID NO: 197) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR283B06 (SEQ ID NO: 197) to human CXCR-4.
  • such a 283B6-type sequence belongs to the same family as
  • 283B6-type sequence has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a 283B6-type sequence further has an IC50 in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than ⁇ , such as better than 750nM, such as better than 500nM.
  • 283B6-type sequences are 4CXCR283B06 (SEQ ID NO: 197) and the "283B6-like sequences ' ', li 283B6-like ISV or ii 283B6-like building blocks" described herein. [Note: because 14A2 and 283B6 belong to the same family and have an essentially similar footprint, it is expected that a substantial part of the 283B6-type sequences will also be 14A2-type sequences and visa- ersa].
  • 10E9-like sequences a "10E9-like sequence", "J 0E9-like LSI” or J 0E9- 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 SYAMG 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 SYAMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence TISWRGDRKYYSESVKD 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 TISWRGDRKYYSESVKD; 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 TISWRGDRKYYSESVKD; and/or
  • a CDR3 which comprises or essentially consists of either (i) the amino acid sequence DRAPYGSGSPDVKQYEH 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 DRAPYGSGSPDVKQYEH; 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 DRAPYGSGSPDVKQYEH; in which the framework sequences present in such an ISV are as further described herein, and in which.
  • CDRI, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0 10 ⁇ 9 M (5.0E-9M or 5.0 nM).
  • CDRi and CDR2 are as defined under a) and b), respectively; or CDRI 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 10E9-like sequence, CDRI, CDR2 and CDR3 are all as defined under a), b) and c), respectively.
  • CDRI, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC 5 o in the Jurkat assay of Example I, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO "9 M (5.0E- 9M or 5.0 nM).
  • CDR1 may comprise or essentially consist of the amino acid sequence SYAMG (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 TISWRGDRKYYSESVKD (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 DRAPYGSGSPDVKQYEH (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence SYAMG
  • CDR2 may comprise or essentially consist of the amino acid sequence
  • CDRl may comprise or essentially consist of the amino acid sequence SYAMG and CDR3 may comprise or essentially consist of the amino acid sequence DRAPYGSGSPDVKQYEH (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence TISWRGDRKYYSESVKD and CDR3 may comprise or essentially consist of the amino acid sequence DRAPYGSGSPDVKQYEH (with CDRl being as defined under a) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO "9 M (5.0E-9M or 5.0 nM).
  • a ii J0E9-Iike sequence iL 10E9-like ISV" or "10E9-like building block"' is an ISV that comprises:
  • a CDRl which is either (i) the amino acid sequence SYAMG 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 SYAMG; and/or
  • TISWRGDRKYYSESVKD 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 TISWRGDRKYYSESVKD; 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 TISWRGDRKYYSESVKD; and/or
  • DRAPYGSGSPDVKQYEH 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 DRAPYGSGSPDVKQYEH; 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 DRAPYGSGSPDVKQYEH; in which 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 10E9-Hke ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • 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 10E9-like sequence, CDRl, CDR2 and CDR3 are all as defined under d). e) and f), respectively. Again, in such an 10E9-like sequence.
  • CDRl, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence SYAMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • CDRl is the amino acid sequence SYAMG and CDR2 is the amino acid sequence TISWRGDRKYYSESVKD (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence SYAMG and
  • CDR3 is the amino acid sequence DRAPYGSGSPDVKQYEH (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence
  • TISWRGDRKYYSESVKD and CDR3 is DRAPYGSGSPDVKQYEH (with CDRl being as defined under d) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC 50 in the Jurkat assay of Example 1,
  • C-4 of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • CDR1 is the amino acid sequence SYAMG
  • CDR2 is the amino acid sequence TI S WRGDRK Y Y S ES V KD
  • CDR3 is the amino acid sequence DRAPYGSGSPDV QYEH.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%, such, as at least 95%o sequence identity with the framework sequences of 10E9 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 10E9 while disregarding the CDR ' s in the calculation).
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 10E9-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a 10E9-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 4CXCR010E09 (SEQ ID NO: 189).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be SYAMG (CDR1); TISWRGDRKYYSESVKD (CDR2); and
  • DRAPYGSGSPDVKQYEH CDR3
  • the combination of CDR's and frameworks present in such a 10E9-like ISV are preferably such that the resulting 10E9- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xI0 "9 M (5.0E-9M or 5.0 nM).
  • any 10E9 ⁇ like sequence may be a humanized sequence, as further described herein.
  • 281 El Mike sequences: a "281 El 0-like sequence", "281 El 0-like ISV or
  • "281 El 0-like building block” is defined as an ISV (as described herein) that comprises: a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence NYAMG 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 NYAMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AITRSGVRSGVSAIYGDSVKD 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 AITRS G VRS G VS AI YGDS V D ; 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 AITRSGVRSGVSAIYGDSVKD; and/or
  • a CDR3 which comprises or essentially consists of either (i) the amino acid sequence SAIGSGALRRFEYDY 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 SAIGSGALRRFEYDY; 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 SAIGSGALRRFEYDY; in which the framework sequences present in such an IS V are as further described herein, and in which CDRl , CDR2 and CDR3 are preferably such that the 281E10-like ISV has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 " M (5.0E-9M or 5.0 nM).
  • 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 28 IE 3.0-like sequence, CDRl, CDR2 and CDR3 are all as defined under a), b) and c), respectively.
  • an 281 El 0-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 281 El 0-like ISV has an IC50 in the Jurkat assay of Example 1, C- 4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 n ).
  • CDRl may comprise or essentially consist of the amino acid sequence NYAMG (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 AITRSGVRSGVSAIYGDSVKD (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 SAIGSGALRRFEYDY (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence NYAMG and CDR2 may comprise or essentially consist of the amino acid sequence AITRSG VRS G VS AI YGD S VKD (with CDR3 being as defined under c) above); and/or CDR1 may comprise or essentially consist of the amino acid sequence NYAMG and CDR3 may comprise or essentially consist of the amino acid sequence SAIGSGALRRFEYDY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence
  • AITRSGVRSGVSAIYGDSVKD and CDR3 may comprise or essentially consist of the amino acid sequence SAIGSGALRRFEYDY (with CDR1 being as defined under a) above).
  • CDRL CDR2 and CDR3 are preferably such that the 281E10-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • a "281E10-like sequence", "I&IEIQ-Vike ISV or u 281E10-like building block” is an ISV that comprises:
  • a CDR1 which is either (i) the amino acid sequence NYAMG 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 NYAMG; and/or
  • AITRSGVRSGVSAIYGDSVKD or (ii) an amino acid sequence that has at least 80%, suc as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AITRSGVRSGVSAIYGDSVKD; or (iii) ell! 3. ⁇ 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 AITRSGVRSGVSAIYGDSVKD; and/or
  • SAIGSGALRRFEYDY 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 SAIGSGALRRFEYDY; 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 SAIGSGALRRFEYDY; in which the framework sequences present in such an ISV are as further described herein, and in which CDR1, CDR2 and CDR3 are preferably such that the 281E10-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • 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 281 El 0-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 281E10-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO "9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence NYAMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • CDR3 is the amino acid sequence SA1GSGALRRFEYDY (with CDRl and CDR2 being as defined under d) and e), respectively).
  • CDRl is the amino acid sequence
  • NYAMG and CDR2 is the amino acid sequence AITRSGVRSGVSAIYGDSVKD (with CDR3 being as defined under f) above): and/or CDRl is the amino acid sequence NYAMG and CDR3 is the amino acid sequence SAIGSGALRRFEYDY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence
  • AITRSGVRSGVSAIYGDSVKD and CDR3 is SAIGSGALRRFEYDY (with CDRl being as defined under d) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 281 El 0-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence NYAMG
  • CDR2 is the amino acid sequence AITRSGVRSGVSAIYGDSVKD
  • CDR3 is the amino acid sequence SAIGSGALRRFEYDY.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%o, such as at least 95% sequence identity with the framework sequences of 281 El 0 (which, for example, can be determined by detennining the overall degree of sequence identity of a given sequence with the sequence of 28 IE 10 while disregarding the CDR's in the calculation).
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 281E10-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 '9 M (5.0E-9M or 5.0 nM).
  • a 281E10-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 4CXCR281 E10 (SEQ ID NO: 190).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be NYAMG (CDR1); AITRS GVRSGVS AI YGD S VKD (CDR2); and
  • any 281 El 0-like sequence may be a humanized sequence, as further described herein.
  • 10E12-Iike sequences; a ⁇ JOE J 2 -like sequence", 10E12-like ISV" or “lOEll-Uke building block” is defined as an ISV (as described herein) that comprises: a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence SYDMA 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 SYDMA; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AIRWTGSSTYYADSVKG 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 AIRWTGSSTYYADSVKG; 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 AIRW
  • a CDR3 which comprises or essentially consists of either (i) the amino acid sequence RDTGRYRSRTYDY 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 RDTGRYRSRTYDY; 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 RDTGRYRSRTYDY; in which 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 10E12-like ISV has an IC 50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 (5.0E-9M or 5.0 nM).
  • 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 10E12-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 10E12-like ISV has an IC 5 o in the Jurkat assay of Example 1, C- 4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or S.O nM).
  • CDRl may comprise or essentially consist of the amino acid sequence S YDMA (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 AIRWTGSSTYYADSVKG (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 RDTGRYRSRTYDY (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence SYDMA and CDR2 may comprise or essentially consist of the amino acid sequence
  • CDRl may comprise or essentially consist of the amino acid sequence SYDMA and CDR3 may comprise or essentially consist of the amino acid sequence RDTGRYRSRTYDY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence AIRWTGSSTYYADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence RDTGRYRSRTYDY (with CDRl being as defined under a) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an IC 5 o in the Jurkat assay of Example 1 , C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO '9 M (5.0E-9M or 5.0 nM).
  • a "WEH-Iike sequence", i 10El2-like ISV or "10E12- like building block” IS Xl ISV that comprises:
  • a CDRl which is either (i) the amino acid sequence SYDMA 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 SYDMA; and/or
  • AIRWTGS STY Y AD S V G 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 AIRWTGSSTYYADSVKG; 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 AIRWTGSSTYYADSVKG; and/or
  • a CDR3 which is either (i) the amino acid sequence RDTGRYRSRTYDY 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 RDTGRYRSRTYDY; 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
  • RDTGRYRSRTYDY in which 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 10E12-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • 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 10E12-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 10E12-like ISV has an iC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • CDR1 is the amino acid sequence SYDMA (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • CDR3 is the amino acid sequence RDTGRYRSRTYDY (with
  • CDR1 and CDR2 being as defined under d) and e), respectively).
  • CDR1 is the amino acid sequence SYDMA and CDR2 is the amino acid sequence AIRWTGSSTYYADSVKG (with CDR3 being as defined under f) above); and/or CDR1 is the amino acid sequence SYDMA and CDR3 is the amino acid sequence RDTGRYRSRTYDY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence AIRWTGSSTYYADSVKG and CDR3 is RDTGRYRSRTYDY (with CDR1 being as defined under d) above).
  • CDR1, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 " M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 ⁇ 9 M (5.0E-9M or 5.0 nM).
  • CDR1 is the amino acid sequence SYDMA
  • CDR2 is the amino acid sequence AIRWTGSSTYYADSVKG
  • CDR3 is the amino acid sequence
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the 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 10E12 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 10E12 while disregarding the CDR's in the calculation).
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 10E12-like ISV has an IC 5 0 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO "9 M (7.0E-9M or 7.0 nM). in particular better than 5.0xl0 '9 M (5.0E-9M or 5.0 nM).
  • a 10E12-Iike 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 4CXCR010E12 (SEQ ID NO: 191).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be SYDMA (CDRl); AIRWTGSSTYYADSV G (CDR2); and
  • the combination of CDR's and frameworks present in such a 10E12-like ISV are preferably such that the resulting 10E12-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0 10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • any I0E12-like sequence may be a humanized sequence, as further described herein.
  • lOAI O-Hke sequences a '"lOAlO-like sequence", ' ⁇ ⁇ -lik ISV" or "WAlO- ike building block” is defined as an ISV (as described herein) that comprises: a) a CDRl which comprises or essentially consists of either (i) the amino acid sequence DTGTMA 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 DTGTMA; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AINSGTTNYADSVKG 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 AINSGTTNYADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5,
  • a CDR3 which comprises or essentially consists of either (i) the amino acid sequence RVSGWRTRYDY 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 RVSGWRTRYDY; 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 RVSGWRTRYDY; in which 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 lOAlO-like ISV has an IC50 in the Jurkat assay of Example I , C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO "9 M (5.0E-9M or 5.0 nM).
  • 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 10A10-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 l OAlO-like ISV has an IC50 in the Jurkat assay of Example 1 , C- 4; of better than 7.0x10 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or S.O nM).
  • CDRl may comprise or essentially consist of the amino acid sequence DTGTMA (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 AINSGTTN Y ADS V G (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 RVSGWRTRYDY (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence DTGTMA and CDR2 may comprise or essentially consist of the amino acid sequence
  • CDRl may comprise or essentially consist of the amino acid sequence DTGTMA and CDR3 may comprise or essentially consist of the amino acid sequence RVSGWRTRYDY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence AINSGTTNYADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence RVSGWRTRYDY (with CDRl being as defined under a) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 10A10-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • a "lOAlO-like sequence", “lOAlO-like ISV " or "10A10- like building block? ' ' is an ISV that comprises:
  • a CDRl which is either (i) the amino acid sequence DTGTMA 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 DTGTMA; and/or
  • AINSGTTNYADSVKG 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 AINSGTTNYADSVKG; 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 AIN S GTTN Y AD S VKG; and/or
  • a CDR3 which is either (i) the amino acid sequence RVSGWRTRYDY or
  • 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
  • RVSGWRTRYDY 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
  • RVSGWRTRYDY in which 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 1 OA 10-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0 ⁇ 10 "9 ⁇ (7.0E-9M or 7.0 nM), in particular better than 5.0 10 "9 M (5.0E-9M or 5.0 nM).
  • 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 1 OA 10-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 1 OA 1.0-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO " M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • CD l is the amino acid sequence DTGTMA (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • CDR3 is the amino acid sequence RVSGWRTRYDY (with CDRl and CDR2 being as defined under d) and e), respectively).
  • CDRl is the amino acid sequence DTGTMA and CDR2 is the amino acid sequence AINSGTTNYADSVKG (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence DTGTMA and CDR3 is the amino acid sequence RVSGWRTRYDY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence AINSGTTNYADSVKG and CDR3 is RVSGWRTRYDY (with CDRl being as defined under d) above).
  • CDR1 , CDR2 and CDR3 are preferably such that the 10A10 ike ISV has an IC 50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0 "9 M (5.0E-9M or 5.0 nM).
  • CDR1 is the amino acid sequence DTGTMA
  • CDR2 is the amino acid sequence AIN SGTTNY ADS V G
  • CDR3 is the amino acid sequence RVSGWRTRYDY.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the 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 10A10 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 1 OA 10 while disregarding the CDR's in the calculation).
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 10A10-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • a 10A10-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 4CXCR010A10 (SEQ ID NO: 192).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be DTGTMA (CDR1 ); AINSGTTN YAD S VKG (CDR2); and
  • RVSGWRTRYDY (CDR3).
  • CDR3 CDR3
  • the combination of CDR's and frameworks present in such a l OAl O-like ISV are preferably such that the resulting lOAlO-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xl0 "9
  • any 10A10-like sequence may be a humanized sequence, as further described herein.
  • lOGl O-like sequences a "JOGIO-Iike sequence", "lOGlO-lih ISV or l OGlO-like building block” is defined as an ISV (as described herein) that comprises: a) a CDRl which comprises or essentially consists of either (i) the amino acid sequence SYAMA 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 SYAMA; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AIRWSGGRATKYADSVRG 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 AIRWSGGRATKYADSVRG; or (iii) an amino acid sequence that has only 7, 6, 5,
  • 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 lOGlO-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 lOGlO-like ISV has an IC 50 in the Jurkat assay of Example 1, C- 4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • CDRl may comprise or essentially consist of the amino acid sequence SYAMA (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 AIRWSGGRATKYADSVRG (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 QTYYRSGLASTRDFDS (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence SYAMA and CDR2 may comprise or essentially consist of the amino acid sequence
  • AIRWS GGRATKY AD S VRG (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence SYAMA and CDR3 may comprise or essentially consist of the amino acid sequence
  • QTYYRSGLASTRDFDS (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence
  • AIRWSGGRATKYADSVRG and CDR3 may comprise or essentially consist of the amino acid, sequence QTYYRSGLASTRDFDS (with CDRl being as defined under a) above).
  • CDRl , CDR2 and CDR3 are preferably such that the lOGlO-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • a "lOGlO-like sequence", “lOGlO-like ISV or “lOGJO- like building block” is an ISV that comprises:
  • a CDRl which is either (i) the amino acid sequence SYAMA 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 SYAMA; and/or
  • AIRWSGGRATKYADSVRG 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 AIRWSGGRATKYADSVRG; 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 AIRWSGGRATKYADSVRG; and/or
  • QTYYRSGLASTRDFDS 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 QTYYRSGLASTRDFDS; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3.
  • CDRl and CDR2 are as defined under d) and e), respectively; or CD l 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 lOGlO-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 lOGlO-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO ⁇ 9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence SYAMA (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • AIRWSGGRATKYAD S VRG (with CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence QTYYRSGLASTRDFDS (with CDRl and CDR2 being as defined under d) and e), respectively).
  • CDRl is the amino acid sequence SYAMA
  • CDR2 is the amino acid sequence AIRWSGGRATKYADSVRG (with
  • CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence
  • SYAMA and CDR3 is the amino acid sequence QTYYRSGLASTRDFDS (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence
  • AIRWSGGRATKYADSVRG and CDR3 is QTYYRSGLASTRDFDS (with CDRl being as defined under d) above). Again, in such lOGlO-like sequences, CDRl, CDR2 and
  • CDR3 are preferably such that the lOGlO-like ISV has an IC 5 o in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO ⁇ 9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence SYAMA
  • CDR2 is the amino acid sequence AIRWSGGRATKYADSVRG
  • CDR3 is the amino acid sequence QTYYRSGLASTRDFDS.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the 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 10G10 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 10G10 while disregarding the CDR' s in the calculation). Again, the combination of CDR's and.
  • frameworks present in a given sequence are preferably such that the resulting 10G10-like ISV has an IC50 in the Jurkat assay of Example I , C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0 "9 M (5.0E-9M or 5.0 nM).
  • a 10G10-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 4CXCR010G10 (SEQ ID NO: 193).
  • the CDR' s may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be SYAMA (CDR1); AIRWSGGRATKYADSVRG (CDR2); and
  • CDR3 QTYYRSGLASTRDFDS
  • the combination of CDR's and frameworks present in such a 10G10-like ISV are preferably such that the resulting l OGJ O-like ISV has an IC 5 0 in the Jurkat assay of Example 1 , C ⁇ 4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO "9 M (5.0E-9M or 5.0 nM).
  • any lOGlO-Iike sequence may be a humanized sequence, as further described herein.
  • 14A2-Iike sequences a ii 14A2-like sequence
  • HA 2-1 ike ISV or "14A2- 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 IRNMG 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 IRNMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence TISSGGNKDYTDAVKD 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 TISSGGNKDYTDAVKD; 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 TISSGGNKDYTDAVKD; and/or c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence EAGTGWATRRGYTY or (ii) an amino acid sequence that has at least 80%, such as at least 80%
  • CDR2 and CDR3 are preferably such that the 14A2 ⁇ like ISV has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 ⁇ 9 M (5.0E-9M or 5.0 nM).
  • CDR1 and CDR2 are as defined under a) and b), respectively; or CDR1 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 14A2-like sequence, CDR1, CDR2 and CDR3 are all as defined under a), b) and c), respectively.
  • CDR1, CDR2 and CDR3 are preferably such that the 14A2-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0 l0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E- 9M or 5.0 nM).
  • CDR1 may comprise or essentially consist of the amino acid sequence IRNMG (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 T1S S GGN D YTD A VKD (with CDR1 and CDR3 being as defined under a) and c), respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence EAGTGWATRRGYTY (with CDR1 and CDR2 being as defined under a) and b), respectively).
  • CDR1 may comprise or essentially consist of the amino acid sequence IRNMG and CDR2 may comprise or essentially consist of the amino acid sequence
  • CDR1 may comprise or essentially consist of the amino acid sequence IRNMG and CDR3 may comprise or essentially consist of the amino acid sequence EAGTGWATRRGYTY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence TISSGGNKDYTDAVKD and CDR3 may comprise or essentially consist of the amino acid sequence EAGTGWATRRGYTY (with CDRl being as defined under a) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 14A2-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • a "14A2-Hke sequence", "14A2-like ISV or ii 14A2-like building block” is an ISV that comprises:
  • a CDRl which is either (i) the amino acid sequence IRNMG 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 IRNMG; and/or
  • TISSGGNKDYTDAV D 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 TISSGGNKDYTDAVKD; 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 TISSGGNKDYTDAVKD; and/or
  • EAGTGWATRRGYTY 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 EAGTGWATRRGYTY; 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 EAGTGWATRRGYTY; in which 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 14A2-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x1.0 '9 M (5.0E-9M or 5.0 nM).
  • 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 14A2-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 14A2-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 '9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence IRNMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • CDR3 is the amino acid sequence EAGTGWATRRGYTY (with CDRl and CDR2 being as defined under d) and e), respectively).
  • CDRl is the amino acid sequence IRNMG and CDR2 is the amino acid sequence TISSGGNKDYTDAVKD (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence IRNMG and CDR3 is the amino acid sequence EAGTGWATRRGYTY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence TISSGGNKDYTDAVKD and CDR3 is EAGTGWATRRGYTY (with CDRl being as defined under d) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 14A2- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence IRNMG
  • CDR2 is the amino acid sequence TISSGGNKDYTDAVKD
  • CDR3 is the amino acid sequence EAGTGWATRRGYTY.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the 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 14A2 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 14A2 while disregarding the CDR's in the calculation).
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 14A2-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xlO ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a 14A2-Iike 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 4CXCR014A02 (SEQ ID NO: 194).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be IR MG (CDRl); TISSGGNKDYTDAVKD (CDR2); and
  • EAGTGWATRRGYTY CDR3
  • the combination of CDR's and frameworks present in such a 14A2-Iike ISV are preferably such that the resulting 14A2- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 '9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • any 14A2-like sequence may be a humanized sequence, as further described herein.
  • 15A1-Iike sequences a "ISAl-lih sequence", “15Al-like ISV” or “15A1- like building block” is defined as an ISV (as described herein) that comprises:
  • a CDRl which comprises or essentially consists of either (i) the amino acid sequence RAAMG 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 RAAMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence CALSSAGSALTADSVKG 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 CALSSAGSALTADSVKG; 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 CALSSAGSALTADSVKG; and/or
  • a CDR3 which comprises or essentially consists of either (i) the amino acid sequence GGYCTRAGVYPY 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 GGYCTRAGVYPY; 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 GGYCTRAGVYPY; in which 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 15Al-like ISV has an IC 5 0 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0 lO "9 M (5.0E-9M or 5.0 nM).
  • 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 15Al-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 15Al-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-
  • CDRl may comprise or essentially consist of the amino acid sequence RAAMG (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 CALSSAGSALTADSVKG (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 GGYCTRAGVYPY (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence RAAMG and CDR2 may comprise or essentially consist of the amino acid sequence CALSSAGSALTADSVKG
  • CDRl may comprise or essentially consist of the amino acid sequence RAAMG and CDR3 may comprise or essentially consist of the amino acid sequence GGYCTRAGVYPY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence CALSSAGSALTADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence GGYCTRAGVYPY (with CDRl being as defined under a) above).
  • CDRl, CDR2 and CDR3 are preferably such that the 15Al-like ISV has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a ⁇ JSAl-like sequence is an ISV that comprises:
  • a CDRl which is either (i) the amino acid sequence RAAMG 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 RAAMG; and/or
  • CALSSAGSALTADSVKG 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 CALSSAGSALTADSV G; 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 CALSSAGSALTADSVKG; and/or
  • a CDR3 which is either (i) the amino acid sequence GGYCTRAGVYPY or
  • 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
  • GGYCTRAGVYPY 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
  • GGYCTRAGVYPY in which 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 15A1 -like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • 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 15Al-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 15A1-Iike ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 '9 M (7.0E-9M or 7.0 nM), in particular better than 5-OxlO "9 M (5.0E-9M or 5.0 nM).
  • CDRl is the amino acid sequence RAAMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • CDR3 is the amino acid sequence GGYCTRAGVYPY (with CDRl and CDR2 being as defined under d) and e), respectively).
  • CDRl is the amino acid sequence RAAMG and CDR2 is the amino acid sequence CALSSAGSALTADSVKG (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence RAAMG and CDR3 is the amino acid sequence GGYCTRAGVYPY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence CALSSAGSALTADSVKG and CDR3 is GGYCTRAGVYPY (with CDR1 being as defined under d) above).
  • CDR1, CDR2 and CDR3 are preferably such that the 15Al-like ISV has an IC 50 in the Jurkat assay of Example 1 , C-4; of better than 7.0 10 "9 M (7.0E- 9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • CDR1 is the amino acid sequence RAAMG
  • CDR2 is the amino acid sequence CALSSAGSALTADSVKG
  • CDR3 is the amino acid sequence GGYCTRAGVYPY.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the 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 15A1 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 15A1 while disregarding the CDR's in the calculation).
  • the combination of CDR ' s and frameworks present in a given sequence are preferably such that the resulting 1 Al-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xlO "9 M (7.0E-9M. or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • a 15A1-Iike 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 4CXCR015A01 (SEQ ID NO: 195).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be RAAMG (CDR1); CALSSAGSALTADSVKG (CDR2); and
  • the combination of CDR' s and frameworks present in such a 15Al-like ISV are preferably such that the resulting 15A.1- Hke ISV has an IC 5 0 in the Jurkat assay of Example L C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0 '9 M (5.0E-9M or 5.0 nM).
  • any 15A.l-like sequence may be a humanized sequence, as further described herein.
  • 15H3-iike sequences a "15H3-like sequence", “J5H3-like ISV 7 or "15 IB- like building block ' " is defined as an ISV (as described herein) that comprises: a) a CDRl which comprises or essentially consists of either (i) the amino acid sequence TYTMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid.
  • a CDR2 which comprises or essentially consists of either (i) the amino acid sequence VINWNGDRTNYADSVKG 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 VINWNGDRTNYADSVKG; 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 VINWNGDRTNYADSVKG; and/or c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence RPRFTAEPMTSRRYQY or (ii) an amino acid sequence that has at least 80%.
  • 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 15H3-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 15H3-Iike ISV has an IC 5 o in the Jurkat assay of Example 1 , C-4; of better than 7.0xl O ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl O ⁇ 9 M (5.0E- 9M or S.O nM).
  • CDRl may comprise or essentially consist of the amino acid sequence TYTMG (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 VINWNGDRTNYADSVKG (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 RPRFTAEPMTSRRYQY (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence TYTMG and CDR2 may comprise or essentially consist of the amino acid sequence
  • CDRl may comprise or essentially consist of the amino acid sequence TYTMG and CDR3 may comprise or essentially consist of the amino acid sequence
  • RPRFTAEPMTSRRYQY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence
  • VINWNGDRTNYADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence RPRFTAEPMTSRRYQY (with CDRl being as defined under a) above).
  • CDRL CDR2 and CDR3 are preferably such that the 15H3-like ISV has an IC 0 in the Jurkat assay of Example 1, C-4: of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a "ISFB-like sequence", "15H3-like ISV or ⁇ ISHS- ke building block ' is an ISV that comprises:
  • a CDRl which is either (i) the amino acid sequence TYTMG 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 TYTMG; and/or
  • VINWNGDRTNYADSVKG 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 VINWNGDRTNYADSVKG; 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 VINWNGDRTNYADSVKG; and/or
  • RPRFTAEPMTSRRYQY 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%o sequence identity with the amino acid sequence RPRFTAEPMTSRRYQY; 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 RPRFTAEPMTSRRYQY; in which 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 15H3-like ISV has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • 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 15H3-like sequence, CDRl . CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 15H3-like sequence, CDRl , CDR2 and CDR3 are preferably such that the 15H3-like ISV has an IC 50 in the
  • CDRl is the amino acid sequence TYTMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • CDRl is the amino acid sequence TYTMG
  • CDR2 is the amino acid sequence VINWNGDRTNYADSVKG (with
  • CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence TYTMG and CDR3 is the amino acid sequence RPRFTAEPMTSRRYQY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence
  • VINWNGDRTNYADSVKG and CDR3 is RPRFTAEPMTSRRYQY (with CDRl being as defined under d) above). Again, in such 15H3-Iike sequences, CDRl, CDR2 and
  • CDR3 are preferably such that the 15H3-like ISV has an.
  • CDRl is the amino acid sequence TYTMG
  • CDR2 is the amino acid sequence VINW GDRTNYADSVKG
  • CDR3 is the amino acid sequence RPRFTAEPMTSRRYQY.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the 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 15H3 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 15H3 while disregarding the CDR's in the calculation).
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 15H3-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10 '9 M (7.0E-9M or 7.0 nM). in particular better than 5.0xl0 ⁇ 9 M (5.0E-9M or 5.0 nM).
  • a 15H3-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 4CXCR015H03 (SEQ ID NO: 196).
  • the CDR's may be according to the specifically preferred aspect described above, and. may in particularly (but without limitation) be TYTMG (CDR1); VINWNGDRTNYADSVKG (CDR2); and
  • RPRFTAEPMTSRRYQY CDR3
  • the combination of CDR's and frameworks present in such a 15H3-like ISV are preferably such that the resulting 15H3- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • any 15H3-like sequence may be a humanized sequence, as further described herein.
  • 283B6-like sequences a ⁇ 283B6-like sequence'', "283B6-like ISV' or Li 283B6-like building block" is defined as an ISV (as described herein) that comprises: a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence VATLG 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 VATLG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence DISSGGSTNYADSVRG or (ii) an amino acid sequence that has at least 80%, such as at least 85%o, for example at least 90% or more than 95%o sequence identity with the amino acid sequence DISSGGSTNYADSVRG; 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 acid sequence DISSGGSTNYADSVRG
  • 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 283B6-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 283B6-like ISV has an IC50 in the Jurkat assay of Example 1 , C- 4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0 "9 M (5.0E-9M or 5.0 nM).
  • CDRl may comprise or essentially consist of the amino acid sequence VATLG (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 DISSGGSTNYADSVRG (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 RTSGWRTRSNY (with CDRl and CDR2 being as defined under a) and b), respectively).
  • VATLG amino acid sequence
  • DISSGGSTNYADSVRG with CDRl and CDR3 being as defined under a) and c)
  • CDR3 may comprise or essentially consist of the amino acid, sequence RTSGWRTRSNY (with CDRl and CDR2 being as defined under a) and b), respectively).
  • CDRl may comprise or essentially consist of the amino acid sequence VATLG and CDR2 may comprise or essentially consist of the amino acid sequence
  • DISSGGSTNYADSVRG (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence VATLG and CDR3 may comprise or essentially consist of the amino acid sequence RTSGWRTRSNY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence DISSGGSTNYADSVRG and CDR3 may comprise or essentially consist of the amino acid, sequence RTSGWRTRSNY (with CDR.1 being as defined under a) above).
  • CDR1, CDR2 and CDR3 are preferably such that the 283B6-like ISV has an IC 5 o in the Jurkat assay of Example 1, C-4; of better than 7.0xlO "9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0 "9 M (5.0E-9M or 5.0 nM).
  • a "283B6-!ike sequence", "283B6-like ISV ' or " 5556- like building block' ' ' is an ISV that comprises:
  • a CDR1 which is either (i) the amino acid sequence VATLG 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 VATLG; and/or
  • DISSGGSTNYADSVRG 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 DISSGGSTNYADSVRG; 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 DISSGGSTNYADSVRG; and/or
  • a CDR3 which is either (i) the amino acid sequence RTSGWRTRSNY 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
  • RTSGWRTRSNY 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
  • RTSGWRTRSNY in which the framework sequences present in such an ISV are as further described herein, and in which CDR1, CDR2 and CDR3 are preferably such that the 283B6-like ISV has an.
  • CDR1 and CDR2 are as defined under d) and e), respectively; or CDR1 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 283B6-like sequence, CDR1, CDR2 and CDR3 are all as defined under d), e) and f), respectively.
  • CDR1, CDR2 and CDR3 are preferably such that the 283B6-Iike ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xl0 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • CDR1 is the amino acid sequence VATLG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence
  • DISSGGSTNYADSVRG (with CDR1 and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence RTSGWRTRSNY (with CDR1 and CDR2 being as defined under d) and e), respectively).
  • CDR1 is the amino acid sequence VATLG and CDR2 is the amino acid sequence DISSGGSTNYADSVRG (with CDR3 being as defined under f) above); and/or CDR1 is the amino acid sequence VATLG and CDR3 is the amino acid sequence RTSGWRTRSNY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence DISSGGSTNYADSVRG and CDR3 is
  • CDR1 , CDR2 and CDR3 are preferably such that the 283B6-like ISV has an IC5o in the Jurkat assay of Example 1, C-4; of better than 7.0xl0 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO "9 M (5.0E-9M or 5.0 nM).
  • CDR1 is the amino acid sequence VATLG
  • CDR2 is the amino acid sequence DISSGGSTNYADSVRG
  • CDR3 is the amino acid sequence RTSGWRTRSNY.
  • the framework sequences may be as further described herein.
  • the framework sequences are such that the 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 283B6 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 283B6 while disregarding the CDR's in the calculation).
  • the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 283B6-like ISV has an IC 50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10 ⁇ 9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10 "9 M (5.0E-9M or 5.0 nM).
  • a 283B6-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 4CXCR283B06 (SEQ ID NO: 197).
  • the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be VATLG (CDR1); DISSGGSTNYADSVRG (CDR2); and
  • CDR3 RTSGWRTRSNY
  • frameworks present in such a 283B6-Iike ISV are preferably such that the resulting
  • 283B6-like ISV has an IC 50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xl0 "9 M (7.0E-9M or 7.0 nM), in particular better than S.OxlO "9 M (5.0E-9M or 5.0 nM).
  • any 283B6-like sequence may be a humanized sequence, as further described herein.
  • Constructs, proteins or polypeptides containing one or more building blocks according to the invention may be as further described herein, and form further aspects of the invention. Some specific but-non-limiting examples of constructs, proteins or polypeptides of the invention are given below.
  • the building blocks may be linked to each other either directly or via one or more suitable linkers and the building blocks may be in any suitable order in respect of each other (i.e., towards the N-terminus or towards the C- terminus).
  • each. 10E9-type sequence. 281E10-type sequence, 10E12-type sequence, !OAlO-type sequence, lOGlO-type sequence, 14 A2 -type sequence, 15Al -type sequence, 15H3-type sequence, 283B6-type sequence, 10E9-like sequence, 281E10-like sequence, 10E12-like sequence, lOAlO-like sequence, lOGlO-like sequence, 14 A2 -like- sequence, 15Al.-like sequence, 15H3 -like sequence and 283B6-like sequence (when present in such a construct, protein or and polypeptide) is as defined herein, and the preferences described herein for each such sequence/ISV also apply when such a sequence/ISV is present in a construct, protein or polypeptide described herein).
  • sequence/ISV when such a sequence/ISV is present in such a construct, protein or polypeptide, it is preferably such that (i.e., has a combination of CDR's such that and/or has a combination of CDR's and. framework sequences such that) the sequence/ISV, when measured, in the corresponding monovalent format, has an IC 50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM).
  • a construct, compound, protein or polypeptide of the invention contains at least one ISV of the invention.
  • a construct, compound, protein or polypeptide comprises two or more ISV's that are directed against CXCR-4, preferably all these ISV's are ISV's of the invention.
  • a construct, compound, protein or polypeptide of the invention comprises, besides the at least one ISV of the invention, other ISV's that are directed against CXCR-4.
  • ISV's are the Nanobodies® against CXCR-4 that are described in WO 09/138519 and US application 61/358,495, such as 238D2, 238D4 and sequence-optimized and/or humanized variants thereof; and/or the Nanobodies® against CXCR-4 of SEQ ID NO's:371 to 458, or sequence-optimized and/or humanized variants thereof.
  • the compounds, construct, protein or polypeptide of the invention are preferably such that they have IC50 in the Jurkat assay of Example 1, C-4; of better than 20.0 nM, preferably better than ⁇ , more preferably better than 7.0 nM, even more preferably better than 5.0 nM, such as better than 3.0 nM.
  • the ISV's, compounds, construct, protein or polypeptide of the invention are such that they are inverse agonists of CXCR-4, in particular in the assay described in Example 1 , D-2 using the mutant CXCR-4 Nl 19S and/or in the assay described in Example 1 , D-2 using the truncated version of the mutant CXCR-4 Nl 19S described by Zhang et al (see Example 1 , D-2).
  • the ISV's, compounds, construct, protein or polypeptide of the invention are such that they are not inverse agonists of CXCR-4.
  • polypeptides of the invention are the sequences of SEQ ID NO: 198-370, or the corresponding polypeptides in which one or both of the ISV's present therein have been suitably sequence-optimized and/or humanized.
  • ISV's of the invention are preferred: a 10E9-type sequence, 10E12-type sequence. l OG! O-type sequence, 15H3-type sequence, 283B6-type sequence, with the following ISV's of the invention being more preferred: a 10E9-like sequence, 10E12-like sequence, lOGlO-like sequence, 15H3-like sequence or 283B6-like sequence; and the following ISV's of the invention being particularly preferred: 10E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of 10G10; 15H3 or (preferably) a humanized (and/or sequence optimized) variant of 15H3; and/or 283B6 or (preferably) a humanized (and/or sequence optimized
  • 15Al-type sequence, 15Al-like sequence, 10A10-type sequence, lOAlO-like sequence, 14A2-type sequence, 14A2-like sequence, 281E10-type sequence, or 281E10-like sequence will usually be less preferred.
  • constructs and polypeptides comprising one or more ISV's of the invention in which each ISV of the invention is a 10E9-type sequence, a 10E12-type sequence, a lOGlO-type sequence, a 15H3-type sequence and/or a 283B6-type sequence; and in particular a 10E9-like sequence, a 10E12-like sequence, a lOGlO-like sequence, a 15H3- like sequence and/or a 283B6-like sequence are particularly preferred.
  • all ISV's of the invention that are present in a construct or polypeptide of the invention are a 10E9-type sequence, a 10E12-type sequence, a lOGlO-type sequence, a 15H3-type sequence and/or a 283B6-type sequence; and in particular a 10E9-like sequence, a 10E12-like sequence, a lOGlO-like sequence, a 15H3-like sequence and/or a 283B6-like sequence.
  • each of the constructs below may optionally also contain one or more further binding domains, binding units, amino acid sequences or other
  • the constructs, proteins or polypeptides may have been provided with an increased half-life, for example by functionalisation and/or by including in the construct a moiety or binding unit that increases the half- life of the construct.
  • functionalisation, moieties or binding units will be clear to the skilled person and may for example be as described herein, and for example may include pegylation, fusion to serum albumin, or fusion to a peptide or binding unit that can bind, to a serum protein such as serum albumin.
  • the "serum-albumin binding peptide or binding domain” may be any suitable serum-albumin binding peptide or binding domain capable of increasing the half-life of the construct (compared to the same construct without the serum-albumin binding peptide or binding domain), and may in particular be serum albumin binding peptides as described in WO 2008/068280 by Ablynx N.V. (and in particular WO 2009/127691 and the non- prepublished US application 61/301 ,819, both by Ablynx N.V.), or a serum-albumin binding ISV (such as a serum-albumin binding Nanobody; for example Alb-1 or a humanized version of Alb-1 such as Alb-8, for which reference is for example made to WO 06/122787).
  • serum-albumin binding peptide or binding domain may be any suitable serum-albumin binding peptide or binding domain capable of increasing the half-life of the construct (compared to the same construct without the serum-albumin binding peptide or binding domain), and may in particular be serum albumin binding
  • the half-life of a construct or polypeptide of the invention can (and preferably is) suitably "tailored" for the intended (therapeutic and/or diagnostic) application and/or to obtain the best balance between the desired therapeutic and/or pharmacological effect and possible undesired side-effects.
  • a relatively short half-life in man e.g.
  • the half-life is between 5 to 70 hours, between 10 to 80 hours, between 10 to 70 hours, between 10 to 60 hours, between 20 to 70 hours, between 20 to 60 hours, between 30 to 60 hours, between 20 to 50 hours, or between 30 to 50 hours, all inclusive of the end values of the range of hours; or approximately 5, 6, 7, 8.
  • construct or polypeptide of the invention when a construct or polypeptide of the invention is to be used for the prevention and/or treatment of cancer (as described herein), it will generally be preferred to have a longer half-life (e.g., 7-23 days), but it should be noted that when a construct or polypeptide of the invention is to be used for the prevention and/or treatment of cancer, the construct or polypeptide of the invention may also mobilize stem cells and leukocytes.
  • the half-life for a construct or polypeptide of the invention to be used for the prevention and/or treatment of cancer is between 7 to 20 days, between 10 to 23 days, between 10 to 20 days, between 10 to 18 days, between 12 to 20 days, between 12 to 18 days, between 12 to 16 days, between 14 to 18 days, or between 14 to 16 days, all inclusive of the end values of the range of days; or approximately 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 or 23 days.
  • the use of a construct or polypeptide of the invention with a somewhat shorter half-life may allow the clinician to exert more control over the treatment and to set a treatment and/or dosage regimen which provides an optimal balance between treating the cancer to be treated and avoiding undesired or excessive stem cell and leukocyte mobilization.
  • the half-life for a construct or polypeptide of the invention requiring balancing between treatment of cancer and avoiding undesired or excessive stem cell and leukocyte mobilization is between 3 to 8 days, between 4 to 9 days, between 4 to 8 days, between 4 to 7 days, between 5 to 8 days, between 5 to 7 days, all inclusive of the end values of the range of days; or approximately 3, 4. 5, 6, 7, 8 or 9 days.
  • - a construct, protein or polypeptide essent tally consisting of a 283B6-like sequence; Monovalent constructs with a serum albumin binding moiety for extended half-life a construct, protein or polypeptide essentially consisting of a 10E9-type sequence and a serum -albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 281E10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 10E12-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 10A10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); - a construct, protein or polypeptide essentially consisting of a 10G10-type sequence and a serum
  • Biparatopic constructs a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence and at least one more (such as one or two more) binding domain, or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • constructs, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 1 OA 10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • constructs protein or polypeptide comprising or essentially consisting of a 10G10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed, against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 283B6- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • constructs, protein or polypeptide comprising or essentially consisting of a 281E10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1 - like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;
  • construct protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4:
  • a construct, protein or polypeptide comprising or essentially consisting of two 10E9- type sequences (which may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of two 281E10-type sequences (which may be the same or different);
  • constructs protein or polypeptide comprising or essentially consisting of two 10E12-type sequences (which may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of two l OA!O-type sequences (which may be the same or different);
  • constructs protein or polypeptide comprising or essentially consisting of two 15Al-type sequences (which may be the same or different);
  • constructs protein or polypeptide comprising or essentially consisting of two 15H3-type sequences (which may be the same or different);
  • construct protein or polypeptide comprising or essentially consisting of two 283B6-type sequences (which may be the same or different);
  • protei or polypeptide comprising or essentially consisting of two 10E9- like sequences (which may be the same or different);
  • constructs protein or polypeptide comprising or essentially consisting of two 281E10-like sequences (which may be the same or different);
  • constructs protein or polypeptide comprising or essentially consisting of two 10A10-like sequences (which may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of two 15H3-like sequences (which may be the same or different);
  • a -construct, protein or polypeptide comprising or essentially consisting of two 283B6-Iike sequences (which may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of a I0E9- like sequence and a (different) 10E9-type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10- Hke sequence and a (different) 281E10-type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E 12- like sequence and a (different) 10E12-type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- like sequence and a (different) 10A10-type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 1GG10- like sequence and a (different) lOGlO-type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and a (different) 14A2-type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- like sequence and a (different) 15Al -type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and a (different) 15H3-type sequence;
  • construct, protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and a (different) 283B6-type sequence;
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 281E10-type sequence (and preferably a 281E10-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 10E12-type sequence (and preferably a 10E12-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 10A10-type sequence (and preferably a 10A10-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a lOGlO-type sequence (and preferably a lOG!O-like sequence); a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 15Al-type sequence (and preferably a 15A1 -like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and. preferably a 10E9-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281 ⁇ 10 ⁇ type sequence (and preferably a 281E10-like sequence) and a 10E12-type sequence (and preferably a 10E12-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281E10-iike sequence) and a !OAlO-type sequence (and preferably a 10A10-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281E10-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-iike sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10 type sequence (and preferably a 281E10-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281E10-like sequence) and a 15Al-type sequence (and preferably a 15 A 1 -like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E30 ⁇ type sequence (and preferably a 281E10-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281 ⁇ 10 ⁇ type sequence (and preferably a 281E10-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence); a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a ! OAlO-type sequence (and preferably a l OAl O-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E 12-Iike sequence) and a 15Al-type sequence (and preferably a 5Al-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-Iike sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a lOAlO-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10 type sequence (and preferably a lOAlO-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence):
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10 type sequence (and preferably a lOAlO-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10 type sequence (and preferably a 10A10-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10 ⁇ 10 ⁇ type sequence (and preferably a 10A10-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence); .
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);
  • a construct, protein, or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 15Al-type sequence (and preferably a 15A1-Iike sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2 ⁇ Hke sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence (and preferably a 15H3-like sequence) and a 283B6 ⁇ type sequence (and preferably a 283B6-like sequence);
  • Preferred biparatopie constructs a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 281E10 or a humanized variant of 281E10;
  • construct protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10EI2 or a humanized variant of 10E12;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10A10 or a humanized variant of 10A10;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10G10 or a humanized variant of 10G10;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15A1 or a humanized variant of 15 Al ;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15H3 or a humanized variant of 15H3;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 283B6 or a humanized variant of 283B6;
  • a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281E10 and 10E12 or a humanized variant of 10E12;
  • a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281E10 and 1 OA 10 or a humanized variant of 10A10;
  • a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281E10 and 10G10 or a humanized variant of 10G10;
  • a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281 E 10 and 14 A2 or a humanized variant of 14 A2 ;
  • a construct, protein or polypeptide comprising or essentially consisting of 281 El 0 or a humanized variant of 281 El 0 and 15A1 or a humanized variant of 15A1 ;
  • a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 El 0 and 15H3 or a humanized variant of 15H3 ;
  • - a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281E10 and 283B6 or a humanized variant of 283B6; a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 1 OA 10 or a humanized valiant of 10A10;
  • a construct, protein or polypeptide comprising or essentially consisting of 1.0E12 or a humanized variant of 10E12 and 10G10 or a humanized variant of 10G10;
  • a construct, protein or polypeptide comprising or essentially consisting of 30E12 or a humanized variant of 10E12 and 14A2 or a humanized, variant of 14A2;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 15A1 or a humanized variant of 15A1 ;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant, of 10E 12 and 15H3 or a humanized variant of 15H3 ;
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 283B6 or a humanized variant of 283B6;
  • a construct, protein or polypeptide comprising or essentially consisting of ⁇ 0A10 or a humanized variant of 10A10 and 10G 10 or a humanized variant of 10G10;
  • a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 10A10 and 14A2 or a humanized variant of 14A2;
  • a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 10A10 and 15A1 or a humanized variant of 15A1 ;
  • a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 0A10 and 15H3 or a humanized variant of 15H3 ;
  • a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 1.0A10 and 283B6 or a humanized variant of 283B6;
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 14A2 or a humanized variant of 14A2;
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 15A1 or a humanized variant of 15A1;
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 1 OG10 and 15H3 or a humanized variant of 15H3;
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 283B6 or a humanized variant of 283B6;
  • a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15A1 or a humanized variant of 15A1; a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15H3 or a humanized variant of 15H3;
  • a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 283B6 or a humanized variant of 283B6;
  • a construct, protein or polypeptide comprising or essentially consisting of 15A1 or a humanized variant of 15A1 and 283B6 or a humanized variant of 283B6;
  • a construct, protein or polypeptide comprising or essentially consisting of 15H3 or a humanized variant of 15H3 and 283B6 or a humanized variant of 283B6;
  • Bivalent constructs a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10E9 or (preferably) a humanized variant of 10E9 and a second ISV which is either 10E9 or (preferably) a humanized variant of 10E9 (in which the first and second ISV may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 281 E10 or (preferably) a humanized, variant of 281E10 and a second ISV which is either 281 El 0 or (preferably) a humanized variant of 281 El 0 (in which the first and second ISV may be the same or different);
  • first ISV which is either 10E12 or (preferably) a humanized variant of 10E12
  • second ISV which is either I0E12 or (preferably) a humanized variant of 10E12 (in which the first and second ISV may be the same or different)
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10A10 or (preferably) a humanized variant of 1 OA 10 and a second ISV which is either 10A10 or (preferably) a humanized variant of 1 OA 10 (in which the first and second ISV may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10G10 or (preferably) a humanized variant of 10G10 and a second ISV which is either 10G10 or (preferably) a humanized variant of 10G10 (in which the first and second ISV may be the same or different); a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 14A2 or (preferably) a humanized variant of 14A2 and a second ISV which is either 14A2 or (preferably) a humanized variant of 1.4A2 (in which the first and second ISV may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 15A1 or (preferably) a humanized variant of 15A1 and a second ISV which is either 15A1 or (preferably) a humanized variant of 15A1 (in which the first and second ISV may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 15H3 or (preferably) a humanized variant of 15H3 and a second ISV which is either 15H3 or (preferably) a humanized variant of 15H3 (in which the first and second ISV may be the same or different);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 283B6 or (preferably) a humanized variant of 283B6 and a second ISV which is either 283B6 or (preferably) a humanized variant of 283B6 (in which the first and second ISV may be the same or different);
  • Biparatopic constructs with a serum albumin binding moiety for extended half-life a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or pol y peptide comprising or essentially consisting of a 10E12- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serara-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum- albumin binding peptide or binding domain (as further described herein):
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence and at least one more (such as one or two more) bmding domain or binding unit (for example, one or more other ISV's) directed, against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 283B6- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • construct protein or polypeptide comprising or essentially consisting of a 10G10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum -albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);
  • constructs protein or polypeptide comprising or essentially consisting of two 10E9- type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two lOAlO-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two lOG!O-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two 14A2-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • constructs protein or polypeptide comprising or essentially consisting of two 15Ai-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • constructs protein or polypeptide comprising or essentially consi sting of two 15H3-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • constructs, protein or polypeptide comprising or essentially consisting of two 283B6-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two 10E9- like sequences (which may be the same or different) and a serum -albumin binding peptide or binding domain (as further described herein);
  • constructs protein or polypeptide comprising or essentially consisting of two 281E10-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two iOE12-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two 10A10-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of two lOGlO-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two 14A2-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of two 15Al-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • constructs protein or polypeptide comprising or essentially consisting of two 15H3-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • constructs protein or polypeptide comprising or essentially consisting of two 283B6-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- like sequence and a (different) 10E9-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10- like sequence and a (different) 281 El 0-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- like sequence and a (different) 10E12-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a lOAiO- like sequence and a (different) 10A10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10- like sequence and a (different) 10G10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and a (different) 14A2-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 15A1- like sequence and a (different) 15Al-type sequence and a serum -albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and a (different) 15H3-type sequence and a serum-albumin binding peptide ' or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and a (different) 283B6-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 281E10-type sequence (and preferably a 281E10-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 10E12-type sequence (and preferably a 10E12-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and. preferably a 10E9-like sequence) and a lOAlO-type sequence (and preferably a 10A10-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a l OGlO-type sequence (and preferably a !OGlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9 ike sequence) and a 283B6-type sequence (and preferably a 283B6-Iike sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0- type sequence (and preferably a 281E10-like sequence) and a 10E12-type sequence (and preferably a 10E12-Iike sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10 type sequence (and preferably a 281E10-like sequence) and a lOAlO-type sequence (and preferably a lOAlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0 type sequence (and preferably a 281E10-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0 type sequence (and preferably a 281E10-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum -albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0 type sequence (and preferably a 281E10-like sequence) and a 15Al-type sequence (and preferably a 15A1-Iike sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 281E10 type sequence (and preferably a 281E10-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281 E10-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a lOAlO-type sequence (and preferably a !OAlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E 12- type sequence (and preferably a 10E12-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a consu-uct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum- albumin binding peptide or binding domain (as further described herein);
  • construct protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein):
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 1 OA 10- type sequence (and preferably a lOAlO-like sequence) and a lOGlO-type sequence (and preferably a lOGl O-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a lOAlO-iike sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a 10A10-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a lOAlO-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a 10A10-like sequence) and. a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a 1 OGlO-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a !OGlO-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 10G1O type sequence (and preferably a lOGlG-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a semm-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a lOGlO type sequence (and preferably a lOG!O-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and. a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-iike sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence (and preferably a 15H3-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);
  • Preferred biparatopic constructs with a serum albumin binding moiety for extended half-life a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 281 El 0 or a humanized variant of 281 El 0 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10E12 or a humanized variant of 10E12 and a serum- albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10A10 or a humanized variant of 10A10 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10G10 or a humanized variant of 10G10 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 E 10 and 1 OE 12 or a humanized variant of 1 OE 12 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281E10 and 10A10 or a humanized variant of 10A10 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 El 0 and 10G10 or a humanized variant of 10G10 and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 El 0 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281E10 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain, (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 281 El 0 or a humanized variant of 281E10 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 281 El 0 or a humanized variant of 281E10 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein):
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 10A10 or a humanized variant of 10A10 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E 12 or a humanized variant of 10E12 and 10G10 or a humanized variant of 10G10 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 1.0E12 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 10A10 and 10G10 or a humanized variant of 10G10 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 10A10 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 1 OA 10 and 15 A 1 or a humanized variant of 15 A 1 and a serum- albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 10A10 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 10A10 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 30G10 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and. 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 15A1 or a humanized variant of 1.5A1 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of 15A1 or a humanized variant of 15A1 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 15H3 or a humanized variant of 15H3 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);
  • Bivalent constructs with a serum albumin binding moiety for extended half-life a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10E9 or (preferably) a humanized variant of 10E9 and a second ISV which is either 10E9 or (preferably) a humanized, variant of 10E9 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 281E10 or (preferably) a humanized variant of 281E10 and a second ISV which is either 281E10 or (preferably) a humanized variant of 281E10 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10E12 or (preferably) a humanized variant of 10E12 and a second ISV which is either 10E12 or (preferably) a humanized variant of 10E12 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10A10 or (preferably) a humanized variant of 10A10 and a second ISV which is either 10A10 or (preferably) a humanized variant of 10A10 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10G10 or (preferably) a humanized variant of 10G10 and a second ISV which is either 10G10 or (preferably) a humanized variant of lOGlO (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 14A2 or (preferably) a humanized variant of 14A2 and a second ISV which is either 14A2 or (preferably) a humanized variant of 14A2 (in which the first and second 1SV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first IS V which is either 15A1 or (preferably) a humanized variant of 15A1 and a second ISV which is either 15A1 or (preferably) a humanized variant of 15A1 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 15H3 or (preferably) a humanized variant of 15H3 and a second ISV which is either 1 H3 or (preferably) a humanized variant of 15H3 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);
  • a construct, protein or polypeptide comprising or essentially consisting of a first ISV which, is either 283B6 or (preferably) a humanized variant of 283B6 and a second ISV which is either 283B6 or (preferably) a humanized variant of 283B6 (in which the first and second ISV may be the same or different) and a seram-albumin binding peptide or binding domain (as further described herein).
  • the invention generally relates to: a construct, protein or polypeptide comprising a 10E9-type sequence; and preferably a 10E9-like sequence; and in particular 10E9 or (preferably) a humanized variant of 10E9;
  • a construct, protein or polypeptide comprising a 281E10-type sequence; and preferably a 281E10-like sequence; and in particular 281E10 or (preferably) a humanized variant of 281 El 0;
  • a construct, protein or polypeptide comprising a 10E12-type sequence; and preferably a 10E12-Iike sequence; and in particular 10E12 or (preferably) a humanized variant of 10E12;
  • a construct, protein or polypeptide comprising a 14A2-type sequence; and preferably a 14A2-like sequence; and in particular 14A2 or (preferably) a humanized variant of 14A2;
  • a construct, protein or polypeptide comprising a 35Al-type sequence; and preferably a 15 A 1 -like sequence; and in particular 15A1 or (preferably) a humanized variant of 15A1;
  • 15H3-like sequence preferably a 15H3-like sequence: and in particular 15H3 or (preferably) a humanized variant of 15H3;
  • constructs, protein or polypeptide comprising a 283B6-type sequence; and preferably a 283B6-Iike sequence; and in particular 283B6 or (preferably) a humanized variant of 283 B6; wherein each of these constructs, proteins or polypeptides optionally have been provided with an increased half-life (as described herein), and may for example for this purpose further comprise a serum-albumin binding peptide or binding domain capable of increasing the half- life of the construct (compared to the same construct without the serum-albumin binding peptide or binding domain), also as described herein.
  • the constructs, proteins or polypeptides preferably comprise two ISV's against CXC - 4 (at least one of which is an ISV of the invention, i.e., so as to form a bivalent or biparatopic construct of the invention), and preferably two ISV's of the invention, and are optionally half-life extended (e.g., in that they further contain a half-life extending moiety as described herein).
  • half-life extended e.g., in that they further contain a half-life extending moiety as described herein.
  • polypeptides of the invention comprising a first ISV and a second ISV (at least one and preferably both of which are an ISV of the invention) may represented as follows (with the N-terminus of the polypeptide towards the right and the C-terminus towards the left):
  • first ISV of the invention y-iinker- " econd ISV of the invention ], which construct may optionally be pegylated for increased half-life in circulation;
  • first ISV of the invention -[serum albumin binding peptide (monovalent or in tandem)];
  • polypeptides and/or immunoglobulin single variable domains may optionally be tagged with tags known to the skilled person such as 3xFlag-His6.
  • proteins or polypeptides may comprise one or more further binding domains or binding units directed against CXCR-4 (and in particular, against human CXCR-4) preferably one or more other ISV's directed against CXCR-4 (and in particular, against human CXCR-4).
  • ISV's against human CXCR-4 described in WO 09/138519, US 61/358,495, US 61/358,495 and PCT/EP2010/064766 and/or the amino acid sequences of SEQ ID NO's: 371. to 458 (or humanized and/or sequence-optimized variants thereof).
  • the invention relates to an ISV (as defined herein) that has an IC 5 o in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM).
  • ISV may be as further described herein, and may for example be a domain antibody, single domain antibody or dAb, but is preferably a Nanobody® (e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH).
  • Such an ISV is preferably an ISV of the invention (as defined herein), and according to a specific aspect of the invention is a 10E9-type sequence, a 281E10-type sequence, a 10E12- type sequence, a lOAlO-type sequence, a lOGlO-type sequence, a 14A2-type sequence, a 15Al-type sequence, a 15H3-type sequence or a 283B6-type sequence; and preferably a
  • the invention further relates to a compound, construct, protein or polypeptide that comprises or essentially consists of an ISV (as defined herein) that has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM).
  • an ISV may be as further described herein, and may for example be a domain antibody, single domain antibody or dAb, but is preferably a Nanobody® (e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH); and again such an ISV is preferably an ISV of the invention (as defined herein), and according to a specific aspect of the invention may in particular be a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a 10A10-type sequence, a lOGlO-type sequence, a 14A2-type sequence, a 15Al-type sequence, a 1.5H3- type sequence or a 283B6-type sequence; and is preferably a 10E9-like sequence, a 281E10- like sequence, a 10E12-like sequence, a 10A10-like sequence, a lOGlO-like sequence, a
  • .Another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers (for example, in the constructs of SEQ ID NO's: 279 to 359, the ISV's of the invention are linked to each other via a 20GS linker and linked to the half-life extending Nanobody ⁇ Alb-8 via a 35GS linker.
  • the ISV's of the invention have been provided with a short C-terminal GGGC sequence to which for example a PEG-group can be linked).
  • the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain: an illustrative and non-limiting example of such a construct is the construct of SEQ ID NO:369).
  • the invention further generally relates to a compound, construct, protein or
  • polypeptide comprising or essentially consisting of a first ISV that is directed against CXCR- 4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), in which the first and second ISV may be the same or different, and in which at least one of the ISV's present therein have an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM) when measured in the corresponding monovalent format.
  • both (or all) ISV's present therein have an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM) when measured in the corresponding monovalent format.
  • both the first and second ISV may be as further described, herein, and may for example (each or both) be a domain antibody, single domain antibody or dAb, but is preferably a Nanobody® ⁇ e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH), and are preferably both ISV's of the invention (as defined herein), and according to a specific aspect of the invention may in particular be a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a 10A10-type sequence, a l OGlO-type sequence, a 14A2-type sequence, a 15Al-type sequence, a 15H3-type sequence or a 283B6-type sequence; and is preferably a 10E9-like sequence, a 281E10-like sequence, a 10E12-like sequence, a 10A10-like sequence, a lOGlO-
  • Another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers.
  • the two or more (and preferably two) ISV s of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain).
  • the ISV's may be the same (so as to provide a bi- or multivalent protein or polypeptide of the invention) or may be different, and when they are different they may be directed against the same epitope, domain or (extracellular) loops on CXCR-4 or against different epitopes, domains or (extracellular) loops on CXCR-4.
  • the invention further generally relates to a compound, construct, protein or
  • polypeptide comprising or essentially consisting of a first ISV that is directed against CXCR- 4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), in which the first and second ISV may be the same or different, and in which the first and second ISV may each independently be a 10E9-type sequence (as defined herein); a 281E10-type sequence (as defined herein); a 10E12-type sequence (as defined herein): a 10A10-type sequence (as defined herein); a lOGlO-type sequence (as defined herein); a 14A2-type sequence (as defined herein); a 15A1- type sequence (as defined herein); a 15H3-type sequence (as defined herein); and or a 283B6- type sequence (as defined herein).
  • another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half- life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers.
  • the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain).
  • the invention generally relates to a compound, construct, protein or polypeptide, comprising or essentially consisting of a first ISV that is directed against CXCR-4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), wherein the first and second ISV may be the same or different, and in which, the first and second ISV may each independently be a 10E9-like sequence (as defined herein); a 281E10- like sequence (as defined herein); a 10E12-like sequence (as defined herein); a 10A10-like sequence (as defined herein); a lOGlO-like sequence (as defined herein); a 14A2-like sequence (as defined herein); a 15Al-like sequence (as defined herein); a 15H3-like sequence (as defined herein); and/or a 283B6-like sequence (a
  • another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers, in the above constructs, the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain).
  • the invention generally relates to a compound, construct, protein or polypeptide, comprising or essentially consisting of a first ISV that is directed against CXCR-4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), wherein the first and second ISV may be the same or different, and in which the first and second ISV may each independently be 10E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 281E10 or (preferably) a humanized (and/or sequence optimized) variant of 281E10; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 1 OA 10 or (preferably) a humanized (and/or sequence optimized) variant of 1 OA 10; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of ⁇ 0G10; 14A2 or (preferably)
  • another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers.
  • the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain).
  • All the compounds, construct, protein or polypeptide are preferably such that they have IC 5 o in the Jurkat assay of Example 1, C-4; of better than 20.0 nM, preferably better than ⁇ , more preferably better than 7.0 nM, even more preferably better than 5.0 nM, such as better than 3.0 nM.
  • bivalent or biparatopic constructs of the invention will generally bind with higher avidity to CXCR-4 than the corresponding monovalent ISV(s) and thus will generally be expected to perform better in the Jurkat assay than the corresponding monovalent ISV(s) (and also show better potency in other suitable assays)
  • half-life extended constructs may, depending on the half-life extension technique used, have a lower potency in the Jurkat assay (and/or in other assays) than the corresponding construct without the half-life extension and even compared to some or all of the building blocks present in the construct.
  • the bulky PEG groups may interfere with target binding and thus reduce affinity/avidity and/or potency.
  • the use of the ISV's of the invention will be of (even) great(er) advantage, as it allows the constructs to be made starting from building blocks with higher potency which may compensate to some extent for any loss of potency due to half-life extension.
  • the invention further relates to the use of a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a lOAlO-type sequence, a lOGlO-type sequence, a 14A2- type sequence, a 15Al-type sequence, a 15H3-type sequence or a 283B6-type sequence as a binding domain or binding unit in a compound, construct, protein or polypeptide that is directed against (as defined herein) CXCR-4, and in particular (at least) against human CXCR-4.
  • the invention also further relates to the use of a 10E9-like sequence, a 281E10-like sequence, a 10E12-like sequence, a 10A10-like sequence, a 10G10-like sequence, a 14A2- like sequence, a 15Al-like sequence, a 15H3-like sequence or a 283B6-like sequence as a binding domain or binding unit in a compound, construct, protein or polypeptide that is directed against (as defined herein) CXCR-4, and in particular (at least) against human CXCR-4.
  • the invention also further relates to the use of 10E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 281E10 or (preferably) a humanized (and/or sequence optimized) variant of 281E10; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 1 OA 10 or (preferably) a humanized (and/or sequence optimized) variant of 10A10; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of 10G10; 14A2 or (preferably) a humanized (and/or sequence optimized) variant of 14A2; 15A1 or (preferably) a humanized (and/or sequence optimized) variant of 15A1 ; 15H3 or (preferably) a humanized (and/or sequence optimized) variant of 15H3;
  • 283B6 or (preferably) a humanized (and/or sequence optimized) variant of 283B6 as a binding domain or binding unit in a compound, construct, protein or polypeptide that is directed against (as defined herein) CXCR-4, and in particular (at least) against human CXCR-4.
  • the (use of and/or constructs or polypeptides comprising the) following ISV's are preferred: a 10E9-type sequence, 10E12-type sequence, lOGlO-type sequence, 15H3-type sequence, 283B6-type sequence, with the following ISV's of the invention being more preferred: a 10E9-Hke sequence, 10E12-like sequence, 10G10-like sequence, 15H3-like sequence or 283B6-like sequence; and the following ISV's of the invention being particularly preferred: 3.0E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of 10G10; 15H3 or (preferably) a humanized (and/or sequence optimized) variant of 15H3; and/or 283B6 or (preferably) a humanized (and/
  • amino acid sequences, compounds, constructs and polypeptides of the invention are directed against human CXCR4.
  • they can be used for the same purposes, uses and applications as described in WO 09/138519 and the non-prepublished US application 61 /358,495, for example to inhibit signaling that is mediated by human CXCR4 and/or its ligand(s); and/or i the prevention or treatment of diseases associated with an increased signaling of CXCR4 and/or with an undesired proliferation of cells that express or over-express CXCR-4, such as the various diseases in the group of cancer such as hematopoietic cancers like CLL, AML, ALL, MM, Non-Hodgkin lymphoma, (other) solid tumors such as breast cancer, lung cancer (such as SCLC or non-SCLC NSCLC lung cancer), liver cancer (such as HCC), brain tumors (such as glioblastoma), ovarian cancer, stromal
  • the amino acid sequences of the invention can be used for stem cell mobilization in various patients in need of stem cells after X-ray radiation such as e.g., cancer patients after radiation treatment to replenish the stem cell pool after radiation in cancer patients, or in patients in need of more stem cells, e.g. , in patients with ischemic diseases such as myocardial infarction (MI), stroke and/or diabetes (i.e., patients in need of tissue repair) wherein more stem cell would be re-transfused (after mobilization, screening, selection for lineage in need (e.g., cardiac, vascular lineages) and ex- vivo expansion of patient's own stem cells).
  • ischemic diseases such as myocardial infarction (MI), stroke and/or diabetes
  • MI myocardial infarction
  • diabetes i.e., patients in need of tissue repair
  • mobilization, screening, selection for lineage in need e.g., cardiac, vascular lineages
  • ex- vivo expansion of patient's own stem cells ex-
  • EP 1 061 944 for other possible indications for therapeutics against CXCR-4, reference is for example made to EP 1 072 273 and WO99/50461.
  • amino acid sequences of the invention are very potent (i.e., EC 50 values as measured, e.g., in the experimental part in the pM range) antagonists of human CXCR4 and some may be inverse agonists in certain continuously active human CXCR4 mutants (see e.g., Example 5 of WO 09/138519).
  • Such multispecific constructs are preferably proteins and polypeptides (i.e., encoded by a nucleotide sequence and/or capable of being expressed by a host or host cell), as also generally described in WO 09/138519.
  • the efficacy of the amino acid sequences and polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved.
  • the in vivo activity of the ISV's and constructs of the invention can be tested in any suitable animal model of the cancer to be treated, for example in a chemically induced model, a xenograft model and/or a transgenic model, and suitable models will be clear to the skilled person, depending on the cancer to be treated.
  • suitable animal model of the cancer to be treated for example in a chemically induced model, a xenograft model and/or a transgenic model, and suitable models will be clear to the skilled person, depending on the cancer to be treated.
  • the ability of the ISV's or constructs of the invention to mobilize stem cells can for example be determined according to the methods described in Example 10 of US 61/293, 279.
  • the ISV's and constructs of the invention may be agonists or antagonists in respect of the (desired or undesired) activity of CXCR-4 (i.e., the activity to be obtained/increased or decreased/inhibited).
  • the invention is also not limited to any particular hypothesis, mechanism or explanation as to how the ISV's or constructs of the invention achieve their (desired) biological effect, and may for example do so by competing with the ligand for binding to the receptor, preventing or inhibiting ligand binding, by steric hindrance, by preventing or inhibiting conformational changes that need to occur in order for the ligand to bind to the receptor and/or for receptor-mediated signaling to occur upon binding of the ligand, by preventing, decreasing or conversely increasing receptor internalization or recycling, by promoting or decreasing receptor dimerisation (where required for signaling) or by allosteric interaction; or any combination of one or more of the foregoing, depending on the specific ISV and/or construct, the epitope(s), loop(s) or domains, or confonnation(s) to which it binds, and the interaction of the ISV or construct with the receptor.
  • amino acid sequences and polypeptides that are directed against CXCR-4 from a first species of warm-blooded animal may or may not show cross-reactivity with CXCR-4 from one or more other species of warm-blooded animal.
  • amino acid sequences and polypeptides directed against human CXCR-4 may or may not show cross reactivity with CXCR-4 from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys ⁇ Macaca fascicuiaris) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus)) and/or with CXCR-4 from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with CXCR-4 (such as the species and animal models mentioned herein).
  • primates such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys ⁇ Macaca fascicuiaris) and/or rhesus monkeys (Macaca mulatto
  • amino acid sequences and polypeptides of the invention that are cross-reactive with CXCR-4 from multiple species of mammal will usually be advantageous for use in veterinary applications, since it will allow the same amino acid sequence or polypeptide to be used across multiple species.
  • amino acid sequences and polypeptides directed against CXCR-4 from one species of animal can be used in the treatment of another species of animal, as long as the use of the amino acid sequences and/or polypeptides provide the desired effects in the species to be treated.
  • the ISV's and constructs of the invention are preferably (at least) cross-reactive with CXCR-4 from cynomolgus monkeys ⁇ Macaca fascicularis).
  • the present invention is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of CXCR-4 against which the amino acid sequences and polypeptides of the invention are directed (and as shown herein, the invention advantageously provides a number of ISV's binding to different epitopes, which may be an advantage whe the ISV's are to be used as building blocks for making - for example - biparatopic constructs).
  • amino acid sequences and polypeptides may or may not be directed against an "interaction site” (as defined herein).However, it is generally assumed and preferred that the amino acid sequences and polypeptides of the invention are preferably directed against an interaction site (as defined herein).
  • a polypeptide of the invention may contain two or more amino acid sequences of the invention that are directed against CXCR-4. Generally, such polypeptides will bind to CXCR-4 with increased avidity compared to a single amino acid sequence of the invention.
  • Such a polypeptide may for example comprise two amino acid sequences of the invention that are directed against the same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of CXCR-4 (which may or may not be an interaction site); or comprise at least one "first" amino acid sequence of the invention that is directed against a first same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of CXCR-4 (which may or may not be an interaction site); and at least one "second” amino acid sequence of the invention that is directed against a second antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) different from the first (and which again may or may not be an interaction site).
  • at least one amino acid sequence of the invention is directed against an interaction site (as defined herein), although the invention in its broadest sense is not limited thereto.
  • amino acid sequences and polypeptides may be such that they compete with the cognate binding partner (e.g., the ligand, receptor or other binding partner, as applicable) for binding to the target. It is also within the scope of the invention that, where applicable, an amino acid sequence of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or confimiations of CXCR-4.
  • the antigenic determinants, epitopes, parts, domains or subunits of CXCR-4 to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if CXCR-4 contains repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the invention may bind to such different antigenic determinants, epitopes, parts, domains, subunits of CXCR-4 with an affinity and/or specificity which may be the same or different).
  • the amino acid sequences and polypeptides of the invention may bind to either one of these confirmation, or may bind to both these confirmations (i.e., with an affinity and/or specificity which may be the same or different).
  • the amino acid sequences and polypeptides of the invention may bind to a conformation of CXCR-4 in which it is bound to a pertinent ligand, may bind to a conformation of CXCR-4 in which it is not bound to a pertinent ligand, or may bind to both such conformations (again with an affinity and/or specificity which may be the same or different).
  • amino acid sequences and polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of CXCR-4; or at least to those analogs, variants, mutants, alleles, parts and fragments of CXCR-4 that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the amino acid sequences and polypeptides of the invention bind in CXCR-4 (e.g., in wild-type CXCR-4).
  • the amino acid sequences and polypeptides of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that are the same as, or that are different from (i.e., higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to (wild- type) CXCR-4. It is also included within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of CXCR-4, but not to others.
  • proteins or polypeptides that contain two or more amino acid sequences directed against CXCR-4 may bind with higher avidity to CXCR-4 than the corresponding monomeric amino acid sequence(s).
  • proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of CXCR-4 may (and usually will) bind with higher avidity than each of the different monomers, and proteins or polypeptides that contain two or more amino acid sequences directed against CXCR-4 may (and usually will) bind also with higher avidity to a multimer of CXCR-4.
  • an ISV as described and used herein 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 a suitable fragment thereof.
  • the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a VH sequence that is derived from a human antibody) or be a so-called VHH-sequence (as defined herein) that is derived from a so-called “heavy chain antibody” (as defined herein).
  • any ISV described herein may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb” (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® (as defined herein, and including but not limited to a VHH sequence); other single variable domains, or any suitable fragment of any one thereof.
  • dAb or an amino acid sequence that is suitable for use as a dAb
  • Nanobody® as defined herein, and including but not limited to a VHH sequence
  • the invention is not limited as to the origin of the amino acid sequence of the invention (or of the nucleotide sequence of the invention used to express it), nor as to the way that the amino acid sequence or nucleotide sequence of the invention is (or has been) generated or obtained.
  • the amino acid sequences of the invention may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences.
  • the amino acid sequence is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to "humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VRH sequences or Nanobodies ⁇ ), "camelized” (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences.
  • nucleotide sequences of the invention may be naturally occurring nucleotide sequences or synthetic or semi- synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequences that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.
  • a suitable naturally occurring template e.g., DNA or RNA isolated from a cell
  • nucleotide sequences that have been isolated from a library and in particular, an expression library
  • nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence using any suitable
  • the ISV's used herein are preferably Nanobodies® and may as such for example be naturally occurring VHH's, humanized VHH's or camelized VH's (such as camelized human VH's).
  • suitable Nanobodies® may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring VHH sequences (i.e., from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to "humanized” (as defined herein) Nanobodies®, “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies® that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PGR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well
  • Nanobodies® of the invention comprises a synthetic or semi -synthetic sequence (such as a partially humanized sequence)
  • said Nanobody® may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized Nanobodies® of the invention.
  • humanized Nanobodies® may be amino acid sequences that are as generally defined for Nanobodies® 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 (as defined herein).
  • a humanizing substitution as defined herein.
  • Nanobody® may be partially humanized or fully humanized.
  • the invention relates to a compound or construct, and in particular a protein or polypeptide (also referred to herein as a "compound of (he invention” or
  • polypeptide of the invention that comprises or essentially consists of one or more amino acid sequences of the invention (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units.
  • such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence of the invention (and/or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence of the invention.
  • such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the compound or construct is a (fusion) protein or (fusion) polypeptide.
  • said one or more other groups, residues, moieties or binding units are immunoglobulin sequences.
  • said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain. antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb's", amino acid sequences that are suitable for use as a dAb, or Nanobodies®.
  • such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active.
  • such groups may be linked to the one or more amino acid sequences of the invention so as to provide a
  • compounds or constructs that comprises or essentially consists of one or more derivatives as described herein, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers.
  • said one or more other groups, residues, moieties or binding units are amino acid sequences.
  • the one or more amino acid sequences of the invention 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 compound or construct is a fusion (protein) or fusion (polypeptide).
  • amino acid sequences of the invention can be used as "building blocks" to form polypeptides of the invention, i.e., by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or constructs as described herein (such as, without limitations, the biparatopic. bi/multivalent and bi/multispecific polypeptides of the invention described herein) which combine within one molecule one or more desired properties or biological functions.
  • the compounds or polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the one or more amino acid sequences of the invention to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the invention.
  • Polypeptides of the invention can also 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.
  • a compound of the invention or a polypeptide of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention.
  • Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half- life of the amino acid sequence of the invention.
  • polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more amino acid sequences of the invention 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 acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb's", amino acid sequences 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, or transferrine; reference is made to the further description and.
  • serum proteins or fragments thereof such as (human) serum albumin or suitable fragments thereof
  • binding units that can bind to serum
  • polypeptides in which an amino acid sequence of the invention is linked to an Fc portion such as a human Fc
  • polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins such as, witliout limitation, the proteins and peptides described in WO 2008/068280, WO 2009/127691 or the non-prepublished US application 61/301,819 by applicant, or a serum-albumin binding ISV.
  • the half-life extending moiety is an albumin-binding TSianobody® (see for example WO 2004/041863), and in particular Alb-1 or a humanized version of Alb- 1 such as Alb-8 (for which reference is for example made to WO 06/122787).
  • the compounds or 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 amino acid sequence of the invention er se.
  • the compounds or polypeptides of the invention with increased half-life may 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 corresponding amino acid sequence of the invention per se.
  • such compounds or polypeptides of the invention have a serum 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 corresponding amino acid sequence of the invention per se.
  • such compounds or 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.
  • compounds or 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 1 1 days (such as about 1 1 to 16 days), more preferably at least about 12 days (such as about 12 to 1.8 days or more), or more than 14 days (such as about 14 to 19 days).
  • the invention in another aspect, relates to a nucleic acid that encodes an amino acid sequence of the invention or a polypeptide of the invention (or a suitable fragment thereof).
  • a nucleic acid will also be referred to herein as a "nucleic acid of the invention” and may for example be in the form of a genetic construct, as further described herein.
  • the invention in another aspect, relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention.
  • a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention.
  • Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.
  • the invention further relates to a product or composition containing or comprising at least one amino acid sequen ce of the invention, at least one polypeptide of the invention (or a suitable fragment thereof) and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e., depending on the intended use of the composition.
  • Such 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).
  • a pharmaceutical composition as described herein
  • a veterinary composition or a product or composition for diagnostic use (as also described herein).
  • the invention also relates to the use of an amino acid sequence, Nanobody ⁇ or polypeptide of the invention, or of a composition comprising the sam e, in (methods or compositions for) modulating CXCR-4, 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 CXCR-4 related disease or disorder (such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention) or in need of stem cell mobilisation).
  • a CXCR-4 related disease or disorder such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention
  • the invention also relates to methods for modulating CXCR-4, either in vitro (e.g., in an in vitro or cellular assay) or in vivo (e.g., in an 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 CXCR-4 related disease or disorder (such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention) or in need of stem cell mobilisation), which method comprises at least the step of contacting CXCR-4 with at least one amino acid sequence, Nanobody® or polypeptide of the invention, or with a composition comprising the same, in a manner and in an amount suitable to modulate CXCR-4, with at least one amino acid sequence, Nanobody® or polypeptide of the invention.
  • a CXCR-4 related disease or disorder such as one of the disease mentioned herein, for example a cancer that can
  • the invention also relates to the use of an one amino acid sequence, Nanobody® or polypeptide of the invention in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating CXCR-4, either in vitro (e.g.. in an in vitro or cellular assay) or in vivo (e.g., in an 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 CXCR-4 related disease or disorder (such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention) or in need of stem cell mobilisation).
  • a composition such as, without limitation, a pharmaceutical composition or preparation as further described herein
  • CXCR-4 related disease or disorder such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention
  • modulating or “to modulate” generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, CXCR-4, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein).
  • modulating or “to modulate” may mean either reducing or inhibiting the activity of, or alternatively increasing the activity of CXCR-4, as measured in vitro using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of CXCR-4 in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.
  • modulating may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of CXCR-4 for one or more of its targets, ligands or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of CXCR-4 for one or more conditions in the medium or surroundings in which CXCR-4 is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.
  • this may again be determined, in any suitable manner and/or using any suitable assay known per se, such as the assays described herein or in the prior art cited herein.
  • Modulating may also mean effecting a change ⁇ i.e., an activity as an agonist or as an antagonist, respectively) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which CXCR-4 (or in which its substrate(s), Iigand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved.
  • a change ⁇ i.e., an activity as an agonist or as an antagonist, respectively
  • CXCR-4 or in which its substrate(s), Iigand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects
  • an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.
  • Modulating may for example involve reducing or inhibiting the binding of CXCR-4 to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to CXCR-4. Modulating may also involve activating CXCR-4 or the mechanism or pathway in which it is involved. Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.
  • the invention further relates to appli cations and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host ceils, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with CXCR-4. Some preferred but non-limiting applications and uses will become clear from the further description herein.
  • the invention also relates to the amino acid, sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy.
  • the invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy 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 amino acid sequence, compound, construct or polypeptide as described herein.
  • the invention relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of various forms of cancer (as listed above) and/or in stem cell mobilisation.
  • Nanobodies® generally offer certain advantages (outlined herein) compared to "dAb's" or similar (single) domain antibodies or immunoglobulin sequences, which advantages are also provided by the
  • the Nanobodies® of the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more Nanobodies® of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers).
  • the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or
  • polypeptide which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e., against one or more other targets than CXCR-4), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein.
  • a protein or polypeptide may comprise or essentially consist of one or more Nanobodies® of the invention and optionally one or more (other) Nanobodies® (i.e., directed against other targets than CXCR-4), all optionally linked via one or more suitable linkers, so as to provide a monovalent, multivalent or multispecific
  • Nanobody® construct respectively, as further described herein.
  • polypeptides may also be in essentially isolated form (as defined herein).
  • the binding site for binding against CXCR-4 is preferably formed by the CDR sequences.
  • a Nanobody® of the invention may also, and in addition to the at least one binding site for binding against CXCR-4, contain one or more further binding sites for binding against other antigens, proteins or targets.
  • Nanobody® of the invention (or a polypeptide of the invention comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably directed against human CXCR-4; whereas for veterinary purposes, it is preferably directed against CXCR-4 from the. species to be treated. Also, as with the amino acid sequences of the invention, a Nanobody® of the invention may or may not be cross-reactive (i.e., directed against CXCR-4 from, two or more species of mammal, such as against human CXCR-4 and CXCR-4 from at least one of the species of mammal mentioned herein).
  • the amino acid sequence and structure of a Nanobody® can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's" (or sometimes also referred to as “FW's"), which are referred to in the art and herein as “Framework region 1" or “FR1”; as “Framework region 2" or “FR2”; as “Framework region 3" or “FR3”; and as “Framework region 4" or “FR4", respectively; which framework regions are interrupted by three complementary detenriining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region For "CDR1”; as “Complementarity Determining Region 2" or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”, respectively.
  • Nanobodies® of the invention are as described herein.
  • Other suitable CDR sequences can be obtained by the methods described herein.
  • the CDR sequences present in) the Nanobodies® of the invention are such that:
  • the Nanobodies® can bind to CXCR-4 with a dissociation constant (K D ) of 10 "5 to 10 " 12 moles/liter or less, and preferably 30 " 7 to 10 * 1 ⁇ " moles/hter or less and more preferably 10 "8 to 10 ⁇ 12 moles/liter (i.e., with an association constant ( A) of 10 ⁇ to 10 12 liter/ moles or more, and preferably 10 to 10 liter/moles or more and more preferably 10 to 10° liter/moles);
  • K D dissociation constant
  • A association constant
  • the Nanobodies® can bind to CXCR-4 with a k on -rate of between 10 2 IvfV 1 to about 10 7 MV, preferably between 10 3 M 1 and 10 7 M ⁇ V 1 , more preferably between 10 M ' V and 10 7 IvlV, such as between 10 5 M ' V 1 and 10 7 VP ' s "1 :
  • the CDR sequences present in) the Nanobodies® of the invention are such that: a monovalent Nanobody® of the invention (or a polypeptide that contains only one Nanobody® of the invention) is preferably such that it will bind to CXCR-4 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
  • the affinity of the Nanobody® of the invention against CXCR-4 can be determined in a manner known per se for example using the general techniques for measuring KD. KA, f or kocut mentioned herein, as well as some of the specific assays described herein.
  • Nanobodies® of the invention and of polypeptides comprising the same
  • CXCR-4 Some preferred IC50 values for binding of the Nanobodies® of the invention (and of polypeptides comprising the same) to CXCR-4 will become clear from the further description and examples herein.
  • a polypeptide of the invention comprises one or more (such as two or preferably one) ISV's of the invention linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the invention to cross the blood brain barrier.
  • said one or more amino acid sequences that allow the resulting polypeptides of the invention to cross the blood brain barrier may be one or more (such as two and preferably one) Nanobodies®, such as the Nanobodies® described in WO
  • FC44 SEQ ID NO: 1 89 of WO 06/040153
  • FC5 SEQ ID NO: 190 of WO 06/040154
  • polypeptides comprising one or more ISV's of the invention are preferably such that they:
  • KD dissociation constant
  • K A association constant
  • a polypeptide that contains only one amino acid sequence of the invention is preferably such that it will bind to CXCR-4 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
  • an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
  • Nanobodies® of the invention may bind to CXCR-4 with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the invention.
  • nucleic acid that encodes an amino acid sequence of the invention (such as a Nanobody® of the invention) or a polypeptide of the invention comprising the same.
  • an amino acid sequence of the invention such as a Nanobody® of the invention
  • a polypeptide of the invention comprising the same.
  • such a nucleic acid may be in the form of a genetic construct, as defined herein.
  • the invention relates to host or host cell that expresses or that is capable of expressing an amino acid sequence (such as a Nanobody® of the invention) and/or a polypeptide of the invention compri sing the same; and/or that contains a nucleic acid of the invention.
  • an amino acid sequence such as a Nanobody® of the invention
  • a polypeptide of the invention compri sing the same; and/or that contains a nucleic acid of the invention.
  • Another aspect of the invention relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention and/or at least one nucleic acid of the invention, 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).
  • the invention further relates to methods for preparing or generating the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and.
  • compositions described herein are preferred but non-limiting examples of such methods will become clear from the further description herein.
  • the invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with CXCR-4.
  • an ISV of the invention is preferably a Nanobody®, e.g., a V H3 ⁇ 4 a humanized VHH o a camelized V H (such as a cameiized human VH). Accordingly, herein, the invention is described with particular reference to such Nanobodies® (which are also referred to herein as "Nanobodies® of the invention”. It will however be clear to the skilled person that the teaching herein can also be applied to other ISV's of the invention.
  • a Nanobody® in its broadest sense can be generally defined as a polypeptide comprising:
  • amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E;
  • amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.
  • a Nanobody® of the invention may have the structure
  • FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q;
  • the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid or a cysteine and the amino acid residue at position 44 according to the Kabat numbering is preferably E;
  • amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S. and is in. particular chosen from the group consisting of R and S;
  • CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • Nanobody® in its broadest sense can be generally defined as a polypeptide comprising:
  • amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting ofR and S.
  • a Nanobody® of the invention may have the structure
  • FR1 - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4. respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q;
  • amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;
  • CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • Nanobody® against CXCR-4 may have the structure:
  • FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q;
  • amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;
  • a Nanobody® can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which;
  • amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and
  • amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L. R or C; and is preferably chosen from the group consisting of L or R; and
  • the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W;
  • amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q;
  • the amino acid residue at position 45 according to the Kabat numbering is R; and b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W;
  • amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q;
  • amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E. D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and
  • amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and
  • amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q;
  • CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • a Nanobody® of the invention may have the structure FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D. G, Q, R, S, L; and is preferably chosen from the group consisting of G. E or Q;
  • amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R;
  • the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W;
  • CD l , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • a Nanobody® of the invention may have the structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4. respectively, and in which CDR1 to CDR3 refer to the complementaiity determining regions 1 to 3, respectively, and in which: b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q;
  • the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W;
  • amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q;
  • CDR1 , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • Nanobody® of the invention may have the structure
  • FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: 1. the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G. E and Q;
  • amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R;
  • amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S;
  • amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q;
  • CDR1 , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • Nanobodies® of the invention Two particularly preferred, but non-limiting groups of the Nanobodies® of the invention are those according to a) above; according to (a-1) to (a-4) above; according to b) above; according to (b-1 ) to (b-4) above; according to (c) above; and/or according to (c-1) to (c-4) above, in which either:
  • amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-iike sequence as described) and the amino acid residue at position 108 is Q or L, and is preferably Q.
  • a Nanobody® of the invention may have the structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q;
  • CD 1 , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • Nanobody® of the invention may have the structure
  • FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) the amino acid residues at positions 43-46 according to the abat numbering form the sequence KERE or QRE (or a KERE-Hke sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q;
  • CDR1 , CDR2 and CDR3 are as defined herein, and. are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • the amino acid residue at position 37 is most preferably F.
  • the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably V.
  • the Nanobodies® of the invention can generally be classified on the basis of the following three groups:
  • the "GLE -group” Nanobodies® with the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering and Q at position 108 according to the Kabat numbering.
  • Nanobodies® within this group usually have a V at position 37, and can have a W, P, R or S at position 103, and preferably have a W at position 103.
  • the GLEW group also comprises some GLEW-like sequences such as those mentioned in Table B-2 below. More generally, and without limitation,
  • Nanobodies® belonging to the GLEW-group can be defined as Nanobodies® with a G at position 44 and/or with a W at position 47, in which position 46 is usually E and in which preferably position 45 is not a charged amino acid residue and not cysteine; ii)
  • the u KERE-group Nanobodies® with the amino acid sequence KERB or KQRE (or another KERE-like sequence) at positions 43-46 according to the abat numbering and Q or L at position 108 according to the Kabat numbering.
  • Nanobodies® within this group usually have a F at position 37, an L or F at position 47; and caii have a W, P.
  • Nanobodies® belonging to the KERE-group can be defined as Nanobodies® with a K, Q or R at position 44 (usually K) in which position 45 is a charged amino acid residue or cysteine, and position 47 is as further defined herein;
  • Nanobodies® can have either the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering or the amino acid sequence KERB or KQRE at positions 43-46 according to the Kabat numbering, the latter most preferably in combination with an F at position 37 and an L or an F at position 47 (as defined for the KERE-group); and can have Q or L at position 108 according to the Kabat numbering, and preferably have Q.
  • Nanobodies® may belong to ⁇ i.e., have characteristics of) two or more of these classes.
  • one specifically preferred group of Nanobodies® has GLEW or a GLEW- like sequence at positions 44-47; P,R. or S (and in particular R) at position 103; and Q at position 108 (which may be humanized to L).
  • Nanobodies® in the form of a native (i.e., non-humanized) VHH sequence
  • humanized variants of these Nanobodies® may contain other amino acid residues than those indicated above (i.e., one or more humanizing substitutions as defined herein).
  • humanized Nanobodies® of the GLEW-group or the 103 P, R, S-group, Q at position 108 may be humanized to 108L.
  • other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein.
  • a potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human V H 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 combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework, regions of) a Nanobody® may be partially humanized or fully humanized.
  • a Nanobody® of the invention may be a Nanobody® belonging to the GLEW-group (as defined herein), and in which CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • a Nanobody® of the invention may be a Nanobody® belonging to the KERE-group (as defined herein), and CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • a Nanobody® of the invention may be a Nanobody® belonging to the 103 P, R, S-group (as defined herein), and in which CDRl , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • the Nanobodies® of the invention can contain, at one or more positions that in a conventional VR domain wou!d form (part of) the VH/VL interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring V H sequence, and in particular one or more charged amino acid residues (as mentioned in Table A-2 on page 48 of the international application WO 08/020079).
  • Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table B-2 below; as well as the substitutions that are described in the International Application WO 00/29004 for so-called “microbodies”, e.g., so as to obtain a Nanobody® with Q at position 108 in combination with KLEW at positions 44-47, Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein.
  • the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.
  • the amino acid residue at position 83 is chosen from the group consisting of R, , N, E, G, I, T and Q; and is most preferably either or E (for Nanobodies® corresponding to naturally occurring V H H domains) or R (for "humanized” Nanobodies®, as described herein).
  • the amino acid residue at position 84 is chosen from the group consisting of P, A, R, S, D T, and V in one aspect, and is most preferably P (for Nanobodies® corresponding to naturally occurring V H H domains) or R (for "humanized” Nanobodies®; as described herein).
  • the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G.
  • the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 which, in the Nanobodies® are as mentioned above, will also be referred to herein as the "Hallmark Residues".
  • the Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human VH domain, VH3, are summarized in Table B-2.
  • Table B-3 Some preferred but non-limiting combinations of Hallmark Residues in naturally occurring Nanobodies®.
  • each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the abat numbering) of a naturally occurring VHH domain.
  • Tables B-4 - B-7 also contain data on the VHH entropy (" VHH Ent”) and VHH variability ("VHH Var ”) at each amino acid position for a representative sample of 7732 VHH sequences (including data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University).
  • the values for the V HH entropy and the V H H variability provide a measure for the variability and degree of conservation of amino acid, residues between the 7732 V H H sequences analyzed: low values (i.e., ⁇ 1, such as ⁇ 0.5) indicate that an amino acid residue is highly conserved between the V HH sequences ( . e., little variability).
  • the G at position 9 and the W at position 36 have values for the VH H entropy of 0.01 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 36 is W in all 7732 sequences analysed), whereas for residues that form part of the CDR's generally values of 1.5 or more are found (data not shown).
  • the data represented below support the hypothesis that the amino acid residues at positions 27-30 and maybe even also at positions 93 and 94 already form part of the CDR's (although the invention is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used).
  • Table B-5 Non-limiting examples of amino acid residues in FR2 (for the footnotes, see the footnotes to Table B-2)
  • Table B-7 Non-limiting examples of amino acid residues in FR4 (for the footnotes, see the footnotes to Table B-2)
  • a Nanobody® of the invention can be defined as an amino acid sequence with the (general) structure FR1 - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
  • one or more of the amino acid residues at positions 1 1, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2;
  • CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • the above Nanobodies® may for example be VHH sequences or may be humanized Nanobodies®.
  • the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein.
  • the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.
  • a Nanobody® of the invention can be an amino acid sequence with the (general) structure FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
  • said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are disregarded;
  • CDR1 , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
  • the above Nanobodies® may for example be V H H sequences or may be humanized Nanobodies®.
  • the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein.
  • the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.
  • Table B-8 Representative amino acid sequences for Nanobodtes® of the KERE, GLEW and P,R,S 103 group.
  • KJERE sequence no. 3 SEQ ID NO:3 AVQLVDSGGGLVQAGDSLKLSCALTGGAETXXXXXWFRQTPGREREFVAXXXXXRFn
  • KERE sequence no. 4 SEQ ID NO:4 QVQEVESGGGEVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTIS
  • KERE sequence no. 6 SEQ ID NO:6 DVKI ' V ESGC;GLVQAGGSI,RI.,SCVASGFNFDXXXXX FRQAPGKEREEVAXXXXXRFTI
  • KERE sequence no. 7 SEQ ID NO:7 QVREAESGC;GLVQSGGSLRI,SCVASGS'I Y I'XXXXXWYRQYPGKQRALVAXXXXXRFTI
  • KERE sequence tio. 8 SEQ ID NO:8 EVQLVESGGGLVQAGGSLRESCAASGFTSDXXXXWFRQAPGKPREGVSXXXXXRFTIS
  • KERE sequence no. 10 SEQ ID NO: 10 QVPVVESGGGLVQAGDSERi.,FCAVPSF ' rSTXXXXXWFRQAPGKEREFVAXXXXXRFTIS

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Abstract

La présente invention concerne des séquences d'acides aminés qui sont des domaines variables simples d'immunoglobuline (tels que définis ici) dirigés contre le CXCR4. En particulier, la présente invention concerne des domaines variables simples d'immunoglobuline dirigés contre le CXCR4 doués d'une meilleure activité thérapeutique et des produits de recombinaison les comprenant. Ainsi, l'invention concerne des composés, des produits de recombinaison, des protéines ou des polypeptides comprenant ou consistant essentiellement en l'une ou plusieurs de telles séquences d'acides aminés.
PCT/EP2011/050156 2010-01-08 2011-01-07 Domaines variables simples d'immunoglobuline dirigés contre le cxcr4 doués d'une meilleure activité thérapeutique et produits de recombinaison les comprenant WO2011083140A1 (fr)

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