WO2010066835A2

WO2010066835A2 - Eph receptor and ephrin ligand interaction

Info

Publication number: WO2010066835A2
Application number: PCT/EP2009/066821
Authority: WO
Inventors: Christoph Blanchetot; Michael John Scott Saunders; Johannes Joseph Wilhelmus De Haard; Peter Vanlandschoot
Original assignee: Ablynx Nv
Priority date: 2008-12-10
Filing date: 2009-12-10
Publication date: 2010-06-17
Also published as: WO2010066835A3

Abstract

The present invention relates to amino acid sequences that are directed against proteins from the group of the Eph receptor family such as EphA1 to EphA10, EphB1 to EphB6 and Ephrin ligands such as EphrinA1 to EphrinA6 and EprhinB1 to EphrinB3 as well as to constructs that comprise or essentially consist of one or more of such amino acid sequences.

Description

Eph receptor and ephrin ligand interaction

The present invention relates to amino acid sequences that are directed against proteins from the group of the Eph receptor family such as EphAl to EpIiAlO, EphB 1 to EphB6 and Ephrin ligands, also Ephrins and/or Eph, such as EphrinAl to EphrinAό and EphrinBl to EphrinBS, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more of such amino acid sequences.

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.

Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein. The Eph receptors comprise the largest group of receptor tyrosine kinases. Their ligands are the Ephrins. a family of membrane-bound proteins. Both play important roles in the establishment of neuronal and vascular networks during embryonic development, adult and (neuronal and vascular) pathological processes (Elena B. Pasquale, "Eph receptor signalling casts a wide net on cell behaviour", Nature Rev MoI Cell Biol (June 2005) 462-475) through their participative role in cellular repulsion, migration and adhesion. Eph/Ephrin signalling has the peculiar properties to be bi-directional.

There are 16 known Eph receptors (15 found in mammals) - (see e.g. the website of the Human Genome Organisation (HUGO) Gene Nomenclature Committee, http://www.genenames.org/data/hgnc_data.php, for sequences).

EphAl , EphA2, EpliA3, EphA4, EphAS. EphAό, EphA7, EphA8, EphA9, EpIiAlO (hereinafter EphA receptor sub-family or EphA subfamily) • EphBl, EphB2, EphB3, EphB4, EρhB5. EρhB6 (hereinafter EphB receptor sub-family or EphB subfamily)

There are 9 known ephrin ligands (8 found in mammals) - (see e.g. the website of the Human Genome Organisation (HUGO) Gene Nomenclature Committee, http://www.genenames.org/data/hgnc_data.php, for sequences, use EFN instead of Ephrin):

• EphrinAl, EphrmA2, EphrinA3, EphrinA4, EphiinA5 (hereinafter EphrinA ligand sub-family or EphrinA subfamily)

• EphrinBl, EphrinB2, EphrinB3 (hereinafter EphrinB ligand sub-family or EphrinB subfamily)

The Eph family is composed of at least 16 known members in vertebrate (EphAl to EphAlO and EphBl to EphB6). All have an ectodomain comprising a ligand (Ephrin-) binding domain followed by a cystein-rich region and two fibronectin III (FN3). After a single membrane-spanning helix, the intracellular domain is divided in a juxtamembrane segment, a tyrosine kinase domain and finally a C-terminal SAM domain. Only the kinase domain of EphAlO and EphB6 are inactive. The Eph family is subdivided in 2 classes, the EphA and EphB, based on sequences homology in the ectodomain which appears to correspond to their binding preference. EphrinA or EphrinB respectively.

The Ephrins are subdivided into the EphrinA classes which contains 6 members (EphrinAl to EphrinAό) and are characterized by their GPI-anchoring into the membrane in contrast to the EphrinB classe (EphrinBl to EphrinB3) which are anchored to the membrane by their transmembrane domain. (Pasquale, 2005, supra).

Eph receptor tyrosine kinase and Ephrins are key players in cellular repulsion, migration and adhesion. Deregulation of any to this function can lead to tumorigenesis and/or metastasis. Indeed, Eph and Ephrin overexrpression can result in tumorigenesis either directly (proliferation and tumor survival), or through their role in angiogenesis and metastasis (see Hanna Surawska, Patrick C. Ma, Ravi Salgia, The role of Ephrins and Eph receptors in cancer, Cytokine & Growth Factor Reviews 15 (2004) 419-433). The role of Eph/Ephrin is suggested by their significant differential expression in lung cancer (upregulation of EphA6, EphA7, EphB3, EphrinA3, EphrinB3 and downregulation of EphB4, EphB6, EphrinAl .

Interestingly, EphrinA2 is expressed in lung tumors but not in healthy lung tissue. High level

1297188 1 of EphA2 correlates with more advanced stage of NSCLC. EρhA2 was proposed to be a marker of aggressive behavior in brain metastasis. In breast cancer, upregulation of EphA2, EphA7, EphAl O₅ EphrinA2. Ephrin B3 and down regulation of several Eph and Ephrin are likely involved in tumorigenesis and/or invasiveness. EphBό (a kinase dead receptor) is downregulated in most aggressive cell line suggesting its potential role as a prognostic indicator. Other example of cancer where Eph/Ephrin have been shown to be involved in are Prostate cancer, brain cancer, melanoma and leukaemia (Surawska, 2004, supra: and references therein). However, the role of EphB4 is not clear yet. The EphB4 has been shown to have tumor-promoting and tumor-suppressing activities in breast cancer (see review Nicole K. Noren and Elena B. Pasquale, Paradoxes of the EphB4 Receptor in Cancer Cancer Research 67, (May 2007) 3994-3997). The dual role of EphB4 is probably linked to its angiogenic activity. The role of EphA2 is more clear as a tumor-promotor (Brantley-Sieders, DM, Fang, WB, Hicks. DJ, Zhuang. G, Shyr, Y, Chen, J Impaired tumor micro environment in EphA2-deficient mice inhibits tumor angiogenesis and metastatic progression. FASEB J., 19(13), (2005) 1884-6). Antibodies targeting EphA2 have been shown to reduce tumor growth of several cancer. The antibodies induce internalisation and therefore inactivation of the EphA2 oncogenic effect. Interestingly, specific EphA2 epitope were identified in normal versus malignant cells.

In addition to their role during development, the regulated expression of Eph and Ephrin in adult CNS suggest a role in regeneration after injury. Several evidence suggests that the Eph-Ephrin signaling complexes inhibit axonal regeneration and are key regulator of astrocyte reactivity and glial scar formation. Blocking Eph/Ephrin following CNS injury may therefore be of therapeutic benefit to enhance axonal regenaration after CNS injury (see Goldshmit, Y., McLenachan, S. and Turnley, A, Roles of Eph receptors and Ephrins and/or Eph in the normal and damaged adult CNS, Volume: 52, Issue: 2 (September 2006), 327-

345). In Multiple sclerosis, Eph and Ephrins have been shown to be up regulated in activated astrocytes in demyelinated areas.

Moreover, the EphBl/EphrinB2 pathway was recently shown to be involved in neuropathic pain and that EphrinB2 expression lowering agents (e.g. EphrinB2 siRNA) or EphrinB2 activity lowering agents may a potential therapeutic for neuropatic pain (H. Kobayashi et al, Involvement of EphBl receptor/EphrinB2 Ligand in Neuropathic Pain, SPINE Volume 32, Number 15, pp 1592-1598. 2007). Kitamυra and colleagues also have recently shown that high expression of ephrinBl might be closely associated with rheumatoid arthritis pathogensis and thus lowering ephrinBl levels or masking ephrinBl activity in patients or animals in need thereof (i.e. have RA or are in the progress of developing RA due to high epthrinBl levels or activities) by e.g. the antibodies of this invention may be beneficial to treat RA in humans or animals (Kitamura, T., Enhancement of lymphocyte migration and cytokine production by ephrinBl system in rheumatoid arthritis, Am J Physio log Cell Physiol., October 2007).

Furthermore, Eph receptor tyrosine kinase and Ephiins are suggested to be related to fibrosis-related diseases (see patent MEDIMMUNE WO2006/047638) and to infectious diseases (see patent MEDIMMUNE WO2006/047637).

Also, as will be clear from the further disclosure herein, and depending on the Eph against which they are directed and their desired (therapeutic) effect, the amino acid sequences, Nanobodies and polypeptides of the invention may act as (full or partial) agonists, (full or partial, and competitive or non-competitive) antagonists or as inverse agonists of a Eph and/or of the biological function, pathway, mechanism, effect, signalling or response associated therewith. They may do so in an irreversible but preferably reversible manner.

The polypeptides and compositions of the present invention can generally be used to modulate, and in particular inhibit and/or prevent, binding of Ephrins and/or Eph receptors, and thus to modulate, and in particular inhibit or prevent, the signalling that is mediated by Ephrins and/or Eph receptors, to modulate the biological pathways in which Ephrins and/or Eph receptors are involved, and/or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways.

As such, the polypeptides and compositions of the present invention can be used for the prevention and treatment (as defined herein) of cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases. Generally, "cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases" can be defined as diseases and disorders that can be prevented and/or treated, respectively, by suitably administering to a subject in need thereof (i.e. having the disease or disorder or at least one symptom thereof and/or at risk of attracting or developing the disease or disorder) of either a polypeptide or composition of the invention (and in particular, of a pharmaceutically active amount thereof) and/or of a known active principle active against Ephrins and/or Eph or a biological pathway or mechanism in which Ephrins and/or Eph is involved (and in particular, of a pharmaceutically active amount thereof). Examples of such cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases will be clear to the skilled person based on the disclosure herein.

In particular, the polypeptides and compositions of the present invention can be used for the prevention and treatment of cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases which are characterized by excessive and/or unwanted signalling mediated by Ephrins and/or Eph or by the pathway(s) in which Ephrins and/or Eph is involved. Examples of such cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases will again be clear to the skilled person based on the disclosure herein. Thus, without being limited thereto, the amino acid sequences and polypeptides of the invention can for example be used to prevent and/or to treat all diseases and disorders that are currently being prevented or treated with active principles that can modulate Ephrins and/or Eph-mediated signalling, such as those mentioned in the prior art cited above. It is also envisaged that the polypeptides of the invention can be used to prevent and/or to treat all diseases and disorders for which treatment with such active principles is currently being developed, has been proposed, or will be proposed or developed in future. In addition, it is envisaged that, because of their favourable properties as further described herein, the polypeptides of the present invention may be used for the prevention and treatment of other diseases and disorders than those for which these known active principles are being used or will be proposed or developed; and/or that the polypeptides of the present invention may provide new methods and regimens for treating the diseases and disorders described herein.

Other applications and uses of the amino acid sequences and polypeptides of the invention will become clear to the skilled person from the further disclosure herein.

Generally, it is an object of the invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the diagnosis, prevention and/or treatment of cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or use of such agents and compositions.

In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods that have certain advantages compared to the agents, compositions and/or methods that are currently used and/or known in the art. These advantages will become clear from the further description below.

More in particular, it is an object of the invention to provide therapeutic proteins that can be used as pharmacologically active agents, as well as compositions comprising the same, for the diagnosis, prevention and/or treatment of cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibro sis-related diseases, and infectious diseases and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or the use of such therapeutic proteins and compositions. Accordingly, it is a specific object of the present invention to provide amino acid sequences that are directed against (as defined herein) Ephrins and/or Eph. in particular against Ephrins and/or Eph from a warm-blooded animal, more in particular against Ephrins and/or Eph from a mammal, and especially against human Ephrins and/or Eph; and to provide proteins and polypeptides comprising or essentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that are suitable for prophylactic, therapeutic and/or diagnostic use in a warm-blooded animal, and in particular in a mammal, and more in particular in a human being. More in particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used for the prevention, treatment, alleviation and/or diagnosis of one or more diseases, disorders or conditions associated with Ephrins and/or Eph and/or mediated by Ephrins and/or Eph (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used in the preparation of pharmaceutical or veterinary compositions for the prevention and/or treatment of one or more diseases, disorders or conditions associated with and/or mediated by Ephrins and/or Eph (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being. In the invention, generally, these objects are achieved by the use of the amino acid sequences, proteins, polypeptides and compositions that are described herein.

In general, the invention provides amino acid sequences that are directed against (as defined herein) and/or can specifically bind (as defined herein) to Ephrins and/or Eph; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

More in particular, the invention provides amino acid sequences that can bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a Kp-value (actual or apparent), a K_A-value (actual or apparent), a k_on-rate and/or a k_Ofr rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the invention are preferably such that they: bind to Ephrins and/or Eph with a dissociation constant (K_D) of 10^"5 to 10^'12 moles/liter or less, and preferably 10^"7 to 10^"1 moles/liter or less and more preferably 10^"8 to 10^"12 moles/liter (i.e. with an association constant (K_A) of 10⁵ to 10¹² liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸ to 10¹² liter/moles); and/or such that they: - bind to Ephrins and/or Eph with a k_on-rate of between 10² M^"V^! to about 10⁷ M^"!s^"'. preferably between 10^"' M^-1S^"1 and 10⁷ M^-1S^'1, more preferably between 10⁴ M^" V¹ and 10⁷ IvT¹S^'1, such as between 10⁵ M^'V¹ and 10⁷ MV ; and/or such that they: bind to Ephrins and/or Eph with a k_off rate between Is^"1 (ti_/2-0.69 s) and 10^"6 s^"1 (providing a near irreversible complex with a Ua of multiple days), preferably between

10^~2 s^"! and 10^"6 s^"1, more preferably between 10^"3 s^"1 and 10^"6 s^"1, such as between 10^"4 s^" ' and 10^"6 S^"1. Preferably, a monovalent amino acid sequence of the invention (or a polypeptide that contains only one amino acid sequence of the invention) is preferably such that It will bind to Ephrins and/or Eph with an affinity less than 500 nM, preferably less than 200 iiM, more preferably less than 10 nM, such as less than 500 pM. Some preferred IC50 values for binding of the amino acid sequences or polypeptides of the invention to Ephrins and/or Eph will become clear from the further description and examples herein.

For binding to Ephrins and/or Eph, an amino acid sequence of the invention will usually contain within its amino acid sequence one or more amino acid residues or one or more stretches of amino acid residues (i.e. with each "stretch'' comprising two or amino acid residues that are adjacent to each other or in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence) via which the amino acid sequence of the invention can bind to Ephrins and/or Eph. which amino acid residues or stretches of amino acid residues thus form the "site'^" for binding to Ephrins and/or Eph (also referred to herein as the ''antigen binding site").

The amino acid sequences provided by the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than Ephrins and/or Eph), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. Such a protein or polypeptide may also be in essentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as such preferably essentially consist of a single amino acid chain that is not linked via disulphide bridges to any other amino acid sequence or chain (but that may or may not contain one or more intramolecular disulphide bridges. For example, it is known that Nanobodies - as described herein - may sometimes contain a disulphide bridge between CDR3 and CDRl or FR2). However, it should be noted that one or more amino acid sequences of the invention may be linked to each other and/or to other amino acid sequences (e.g. via disulphide bridges) to provide peptide constructs that may also be useful in the invention (for example Fab' fragments, F(ab')₂ fragments, ScFv constructs, "diabodies" and other multispecific constructs. Reference is for example made to the review by Holliger and Hudson, Nat Biotechnol. 2005 Sep;23(9): l 126-36).

Generally, when an amino acid sequence of the invention (or a compound, construct or polypeptide comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably either an amino acid sequence that does not occur naturally in said subject; or, when it does occur naturally in said subject, in essentially isolated form (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use, the amino acid sequences of the invention (as well as compounds, constructs and polypeptides comprising the same) are preferably directed against human Ephrins and/or Eph; whereas for veterinary purposes, the amino acid sequences and polypeptides of the invention are preferably directed against Ephrins and/or Eph from the species to be treated, or at at least cross-reactive with Ephrins and/or Eph from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, and in addition to the at least one binding site for binding against Ephrins and/or Eph, contain one or more further binding sites for binding against other antigens, proteins or targets. 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. Suitable assays and animal models will be clear to the skilled person, and for example include the following i) in vitro models: Biacore, FLIPR, ELISA and/or Competitive ELISA; ii) cell-based models: adhesion assay, Eph activation (e.g. by phosphorylation), induction of EC sprouting in a 3D spheroid-based assay, induction of EC chord-like structures on matrigel matrix; iii) in vivo animal models: mouse model for neuronal regeneration, anti-tumorigenic and/or anti-angiogenic mouse models (e.g. xenograft experiments), as well as the assays and animal models used in the experimental part below and in the prior art cited herein.

Also, according to the invention, amino acid sequences and polypeptides that are directed against Ephrins and/or Eph from a first species of warm-blooded animal may or may not show cross-reactivity with Ephrins and/or Eph from one or more other species of warmblooded animal. For example, amino acid sequences and polypeptides directed against human Ephrins and/or Eph may or may not show cross reactivity with Ephrins and/or Eph from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys (Macaca fascicularϊs) and/or rhesus monkeys (Macaca mulatto,)) and baboon (Papio ursinus)) and/or with Ephrins and/or Eph 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 Ephrins and/or Eph (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross- reactivity, when present, may have advantages from a drug development point of view, since it allows the amino acid sequences and polypeptides against human Ephrins and/or Eph to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the invention that are cross-reactive with Ephrins and/or Eph 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. Thus, it is also encompassed within the scope of the invention that amino acid sequences and polypeptides directed against Ephrins and/or Eph from one species of animal (such as amino acid sequences and polypeptides against human Ephrins and/or Eph) 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 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 Ephrins and/or Eph against which the amino acid sequences and polypeptides of the invention are directed. However, it is generally assumed and preferred that the amino acid sequences and polypeptides of the invention are preferably directed against EphrinAl, EphrinB2. EphA2, EphB4, or EphBό, more preferably against the mouse and/or human version, most preferred against human EphrinAl, EphrinB2, EphA2, EphB4. or EphBό. Thus, in one preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are directed against mouse and/or human EphrinAl (GeneBank ref: NMJ 82685), human EphrinB2 (GeneBank ref: NM_004093), human EρhA2 (GeneBank ref: NM_004431), human EphB4 (GeneBank ref: BC004264), or human EphBό (GeneBank ref: NM_004445), and are as further defined herein.

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 confirmations of Ephrins and/or Eph. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of Ephrins and/or Eph to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if Ephrins and/or Eph 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 Ephrins and/or Eph with an affinity and/or specificity which may be the same or different). Also, for example, when Ephrins and/or Eph exists in an activated conformation and in an inactive conformation, 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). Also, for example, the amino acid sequences and polypeptides of the invention may bind to a conformation of Ephrins and/or Eph in which it is bound to a pertinent ligand, may bind to a conformation of Ephrins and/or Eph in which it 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). It is also expected that the 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 Ephrins and/or Eph; or at least to those analogs, variants, mutants, alleles, parts and fragments of Ephrins and/or Eph 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

Ephrins and/or Eph (e.g. in wild-type Ephrins and/or Eph). Again, in such a case, 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) Ephrins and/or Eph. 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 Ephrins and/or Eph, but not to others.

When Ephrins and/or Eph exists in a monomelic form and in one or more multimeric forms, it is within the scope of the invention that the amino acid sequences and polypeptides of the invention only bind to Ephrins and/or Eph in monomelic form, only bind to Ephrins and/or Eph in multimeric form, or bind to both the monomeric and the multimeric form. Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to the monomeric form 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 the multimeric form.

Also, when Ephrins and/or Eph can associate with other proteins or polypeptides to form protein complexes (e.g. with multiple subunits), it is within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to Ephrins and/or Eph in its non-associated state, bind to Ephrins and/or Eph in its associated state, or bind to both. In all these cases, the amino acid sequences and polypeptides of the invention may bind to such multimers or associated protein complexes with an affinity and/or specificity that may be the same as or different from (i.e. higher than or lower than) the affinity and/or specificity with which the amino acid sequences and polypeptides of the invention bind to Ephrins and/or Eph in its monomeric and non-associated state. Also, as will be clear to the skilled person, proteins or polypeptides that contain two or more amino acid sequences directed against Ephrins and/or Eph may bind with higher avidity to Ephrins and/or Eph than the corresponding monomeric amino acid sequence(s). For example, and without limitation, proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of Ephrins and/or Eph 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 Ephrins and/or Eph may (and usually will) bind also with higher avidity to a multimer of Ephrins and/or Eph.

Generally, amino acid sequences and polypeptides of the invention will at least bind to those forms of Ephrins and/or Eph (including monomeric, multimeric and associated forms) that are the most relevant from a biological and/or therapeutic point of view, as will be clear to the skilled person. It is also within the scope of the invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the amino acid sequences and polypeptides of the invention, and/or to use proteins or polypeptides comprising or essentially consisting of one or more of such parts, fragments, analogs, mutants, variants, alleles and/or derivatives, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs, mutants, variants, alleles and/or derivatives will usually contain (at least part of) a functional antigen-binding site for binding against Ephrins and/or Eph; and more preferabh will be capable of specific binding to Ephrins and/or Eph, and even more preferably capable of binding to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a K_D- value (actual or apparent), a K_A-value (actual or apparent), a k_OJ1-rate and/or a karate, or alternatively as an IC^o value, as further described herein) that is as defined herein. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and/or polypeptides will become clear from the further description herein. Additional fragments or polypeptides of the invention may also be provided by suitably combining (i.e. by linking or genetic fusion) one or more (smaller) parts or fragments as described herein.

In one specific, but non-limiting aspect of the invention, which will be further described herein, such analogs, mutants, variants, alleles, derivatives have an increased half- life in serum (as further described herein) compared to the amino acid sequence from which they have been derived. For example, an amino acid sequence of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide a derivative of an amino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that 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). Reference is inter alia made to the review by Halaby et al.. J. (1999) Protein Eng. 12, 563-71. Preferably, when properly folded so as to form an immunoglobulin fold, such an amino acid sequence is capable of specific binding (as defined herein) to Ephrins and/or Eph; and more preferably capable of binding to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a K_ϋ-value (actual or apparent), a KA-value (actual or apparent), a k_on-rate and/or a k₀j_f-rate₅ or alternatively as an ICso value, as further described herein) that is as defined herein. Also, parts, fragments, analogs, mutants, variants, alleles and/or derivatives of such amino acid sequences are preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold. In particular, but without limitation, the amino acid sequences of the invention may be amino acid sequences that essentially consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR 3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR' s, as further described herein).

The amino acid sequences of the invention may in particular be an immunoglobulin sequence or a suitable fragment thereof, and more in particular be an immunoglobulin variable domain sequence or a suitable fragment thereof, such as light chain variable domain sequence (e.g. a V_L-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a V_H -sequence) or a suitable fragment thereof. When 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 Vn sequence that is derived from a human antibody) or be a so-called Vnπ-sequence (as defined herein) that is derived from a so-called "heavy chain antibody^'' (as defined herein).

However, it should be noted that 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. Thus, 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. In a specific but non-limiting aspect of the invention, 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 V_HSI 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, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing. Reference is for example made to the standard handbooks, as well as to the further description and prior art mentioned herein.

Similarly, the 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 sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular a "single variable domain" or "single variable domains" (hereinafter "single variable domains"). The single variable domains of the invention are any variable domain that forms a single antigen binding unit. Generally, such single variable domains will be amino acid sequences that essentially consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's, as further described herein). Such single variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold. As such, the single variable domain may for example comprise 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 Vπ-sequence or V_HH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e. a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies and ScFv fragments that need to interact with another variable domain - e.g. through a V_H/V_L interaction - to form a functional antigen binding domain). For example, the single variable domain 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 V_HH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term "dAb's", reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544- 6), to Holt et al, Trends Biotechnol., 2003, 21(11):484-490; as well as to for example WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so- called "IgNAR domains", see for example WO 05/18629).

In particular, the amino acid sequence of the invention may be a Nanobody™ or a suitable fragment thereof. [Note: Nanobody™, Nanobodies™ and Nanoclone™ are trademarks ofAblynx N. VJ. For a further description of VH_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 Brussels; 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. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO

04/041865, WO 04/041863, WO 04/062551 , WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153, which list and references are incorporated herein by reference. As described in these references, generally, Nanobodies (in particular V_HII sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more " ^''Hallmark residues" in one or more of the framework sequences. Such Nanobodies directed against Ephrins and/or Eph will also be referred to herein as "Nanobodies of the invention".

For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described Nanobodies of the so-called "V_H3 class" (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V_H3 class such as DP-47, DP-51 or DP-29), which Nanobodies form a preferred aspect of this invention. It should however be noted that the invention in its broadest sense generally covers any type of Nanobody directed against Ephrins and/or Eph, and for example also covers the Nanobodies belonging to the so-called "V_H4 class" (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the Vπ4 class such as DP-78), as for example described in the US provisional application 60/792,279 by Ablynx N, V, entitled "DP-78-like Nanobodies" filed on April 14, 2006. Generally, Nanobodies (in particular V_HH sequences and partially humanized

Nanobodies) can in particular be characterized by the presence of one or more ''Hallmark residues" (as described herein) in one or more of the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as 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 one or more of the Hallmark residues are as further defined herein.

In particular, a Nanobody 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 the framework sequences are as further defined herein.

More in particular, a Nanobody can be an amino acid sequence with the (general) 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: i) preferably one or more of the amino acid residues at positions 11. 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A- 3 below; and in which: ii) 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.

In these Nanobodies, the CDR sequences are generally as further defined herein. Thus, the invention also relates to such Nanobodies that can bind to (as defined herein) and/or are directed against Ephrins and/or Eph, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such Nanobodies and/or suitable fragments.

SEQ ID NO's 714 to 797 give the amino acid sequences of a number of V_ΠH sequences that have been raised against Ephrins and/or Eph. Accordingly, some particularly preferred Nanobodies of the invention are Nanobodies which can bind (as further defined herein) to and/or are directed against to Ephrins and/or Eph and which: i) have 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 , in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded. In this respect, reference is also made to Table A-I. which lists the framework 1 sequences (SEQ ID NO's: 126 to 209), framework 2 sequences (SEQ ID NO's: 294 to 377), framework 3 sequences (SEQ ID NO's: 462 to 545) and framework 4 sequences (SEQ ID NO's: 630 to 713) of the Nanobodϊes of SEQ ID NO's: 714 to 797 (with respect to the amino acid residues at positions 1 to 4 and 27 to 30 of the framework 1 sequences, reference is also made to the comments made below. Thus, for determining the degree of amino acid identity, these residues are preferably disregarded); and in which: ii) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A- 3 below. In these Nanobodies, the CDR sequences are generally as further defined herein.

Again, such Nanobodies may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring V_HH 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, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when a Nanobody comprises a V_HH sequence, said Nanobody may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized Nanobodies of the invention. Similarly, when a Nanobody 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. In particular, 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). Some preferred, but non-limiting humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_HH sequence with the corresponding framework sequence of one or more closely related human Vn sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V_HH sequence (in any manner known per se, as further described herein) and the resulting humanized V_HH 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.

Some particularly preferred humanized Nanobodies of the invention are humanized variants of the Nanobodies of SEQ ID NO's: 714 to 797 . Thus, some other preferred Nanobodies of the invention are Nanobodies which can bind (as further defined herein) to Ephrins and/or Eph and which: i) are ahumanized variant of one of the amino acid sequences of SEQ ID NO's: 714 to

797 ; and/or ii) have 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 . in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded: and in which: i) preferably one or more of the amino acid residues at positions 11, 37, 44. 45, 47, 83,

84. 103. 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 below.

According to another specific aspect of the invention, the invention provides a number of stretches of amino acid residues (i.e. small peptides) that are particularly suited for binding to Ephrins and/or Eph. These stretches of amino acid residues may be present in, and/or may be incorporated into, an amino acid sequence of the invention, in particular in such a way that they form (part of) the antigen binding site of an amino acid sequence of the invention. As these stretches of amino acid residues were first generated as CDR sequences of heavy chain antibodies or V_HII sequences that were raised against Ephrins and/or Eph (or may be based on and/or derived from such CDR sequences, as further described herein), they will also generally be referred to herein as "CDR sequences"¹ (i.e. as CDRl sequences, CDR2 sequences and CDR3 sequences, respectively). It should however be noted that the invention in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in an amino acid sequence of the invention, as long as these stretches of amino acid residues allow the amino acid sequence of the invention to bind to Ephrins and/or Eph. Thus, generally, the invention in its broadest sense comprises any amino acid sequence that is capable of binding to Ephrins and/or Eph and that comprises one or more CDR sequences as described herein, and in particular a suitable combination of two or more such CDR sequences, that are suitably linked to each other via one or more further amino acid sequences, such that the entire amino acid sequence forms a binding domain and/or binding unit that is capable of binding to Ephrins and/or Eph. It should however also be noted that the presence of only one such CDR sequence in an amino acid sequence of the invention may by itself already be sufficient to provide an amino acid sequence of the invention that is capable of binding to Ephrins and/or Eph; reference is for example again made to the so-called "Expedite fragments^"' described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises at least one amino acid sequence that is chosen from the group consisting of the CDRl sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof). In particular, an amino acid sequence of the invention may be an amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least one amino acid sequence that is chosen from the group consisting of the CDRl sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof). Generally, in this aspect of the invention, the amino acid sequence of the invention may be any amino acid sequence that comprises at least one stretch of amino acid residues, in which said stretch of amino acid residues has an amino acid sequence that corresponds to the sequence of at least one of the CDR sequences described herein. Such an amino acid sequence may or may not comprise an immunoglobulin fold. For example, and without limitation, such an amino acid sequence may be a suitable fragment of an immunoglobulin sequence that comprises at least one such CDR sequence, but that is not large enough to form a (complete) immunoglobulin fold (reference is for example again made to the "'Expedite fragments'^' described in WO 03/050531). Alternatively, such an amino acid sequence may be a suitable "protein scaffold" that comprises least one stretch of amino acid residues that corresponds to such a CDR sequence (i.e. as part of its antigen binding site). Suitable scaffolds for presenting amino acid sequences will be clear to the skilled person, and for example comprise, without limitation, to binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as Affϊbodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz el al., Nat. Biotech 2005, VoI 23:1257). and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al._^Comb Chem High Throughput Screen 2006 9(8):619-32).

Again, any amino acid sequence of the invention that comprises one or more of these CDR sequences is preferably such that it can specifically bind (as defined herein) to Ephrins and/or Eph, and more in particular such that it can bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a Kp-value (actual or apparent), a K._A-value (actual or apparent), a k_on-rate and/or a k_otr-rate. or alternatively as an IC₅₀ value, as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect of the invention may be any amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least two amino acid sequences that are chosen from the group consisting of the CDRl sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that (i) when the first amino acid sequence is chosen from the CDRl sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein or the CDR3 sequences described herein; (ii) when the first amino acid sequence is chosen from the CDR2 sequences described herein, the second amino acid sequence is chosen from the CDRl sequences described herein or the CDR3 sequences described herein; or (iii) when the first amino acid sequence is chosen from the CDR.3 sequences described herein, the second amino acid sequence is chosen from the CDRl sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention may be amino acid 5 sequences that comprise at least one antigen binding site, wherein said antigen binding site comprises at least three amino acid sequences that are chosen from the group consisting of the CDRl sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that the first amino acid sequence is chosen from the CDRl sequences described herein, the second amino acid sequence is chosen from the CDR2

] O sequences described herein, and the third amino acid sequence is chosen from the CDR3 sequences described herein. Preferred combinations of CDRl, CDR2 and CDR3 sequences will become clear from the further description herein. As will be clear to the skilled person, such an amino acid sequence is preferably an immunoglobulin sequence (as further described herein), but it may for example also be any other amino acid sequence that comprises a

15 suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against Ephrins and/or Eph, that comprises one or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 210 to 293;

20 b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; d) the amino acid sequences of SEQ ID NO's: 378 to 461 ;

25 e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; g) the amino acid sequences of SEQ ID NO's: 546 to 629;

30 h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c): i) any amino acid substitution in such an amino acid sequence according to b) and/or c) is preferably, and compared to the corresponding amino acid sequence according to a), a conservative amino acid substitution, (as defined herein); and/or ii) the amino acid sequence according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to a); and/or iii) the amino acid sequence according to b) and/or c) may be an amino acid sequence that is derived from an amino acid sequence according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f): i) any amino acid substitution in such an amino acid sequence according to e) and/or f) is preferably, and compared to the corresponding amino acid sequence according to d), a conservative amino acid substitution, (as defined herein); and/or ii) the amino acid sequence according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to d); and/or iii) the amino acid sequence according to e) and/or f) may be an amino acid sequence that is derived from an amino acid sequence according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se. Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i): i) any amino acid substitution in such an amino acid sequence according to h) and/or i) is preferably, and compared to the corresponding amino acid sequence according to g), a conservative amino acid substitution, (as defined herein); and/or ii) the amino acid sequence according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to g); and/or iii) the amino acid sequence according to h) and/or i) may be an amino acid sequence that is derived from an amino acid sequence according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively. In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of: i) the amino acid sequences of SEQ ID NO's: 210 to 293; ii) the amino acid sequences of SEQ ID NO's: 378 to 461; and iii) the amino acid sequences of SEQ ID NO's: 546 to 629; or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against Ephrins and/or Eph.

In a more specific, but again non- limiting aspect, the invention relates to an amino acid sequence directed against Ephrins and/or Eph, that comprises two or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 210 to 293; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; d) the amino acid sequences of SEQ ID NO's: 378 to 461 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461; g) the amino acid sequences of SEQ ID NO's: 546 to 629; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g), h) or i); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), g), h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g), h) or i), the second stretch of amino acid residues corresponds to om of the amino acid sequences according to a), b), c), d). e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of: i) the amino acid sequences of SEQ ID NO's: 210 to 293; ii) the amino acid sequences of SEQ ID NO's: 378 to 461; and iii) the amino acid sequences of SEQ ID NO's: 546 to 629; such that, (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 210 to 293, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 378 to 461 or of SEQ ID NO's: 546 to 629; (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 378 to 461, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 210 to 293 or of SEQ ID NO's: 546 to 629; or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 546 to 629. the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 210 to 293 or of SEQ ID NO's: 378 to 461.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against Ephrins and/or Eph. In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against Ephrins and/or Eph, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 210 to 293; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; the second stretch of amino acid residues is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 378 to 461; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; and the third stretch of amino acid residues is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO^*s: 546 to 629; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629.

Preferably, in this specifc aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 210 to 293; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 378 to 461 ; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 546 to 629. Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against Ephrins and/or Eph.

Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 . This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO^'s: 714 to 797 , in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein. Also, such amino acid sequences are preferably such that they can specifically bind

(as defined herein) to Ephrins and/or Eph; and more in particular bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a K_A- value (actual or apparent), a Ic_0n -rate and/or a k_oir rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein. When the amino acid sequence of the invention essentially consists of 4 framework regions (FRl to FR4, respectively) and 3 complementarity determining regions (CDRl to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 210 to 293 ; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 210 to 293; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; and/or - CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 378 to 461 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461; and/or

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 546 to 629; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 546 to 629; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629.

In particular, such an amino acid sequence of the invention may be such that CDRl is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 210 to 293; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 378 to 461; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 546 to 629.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FRl to FR4, respectively) and 3 complementarity determining regions (CDRl to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 210 to 293; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; c) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; and

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 378 to 461; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; and

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 546 to 629; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be such that CDRl is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 210 to 293; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 378 to 461 ; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 546 to 629.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to Ephrins and/or Eph; and more in particular bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a K_D- value (actual or apparent), a Revalue (actual or apparent), a k_on-rate and/or a k_Oβ-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FRl to FR4, respectively) and 3 complementarity determining regions (CDRl to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 . This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 714 to 797 , in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In such an amino acid sequence of the invention, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

The framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a V_L-sequence) and/or from a heavy chain variable domain (e.g. a V_H- sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a V_RI r sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional V_H sequences that have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequence of the invention is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); is a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody); is a "dAb" (or an amino acid sequence that is suitable for use as a dAb); or is aNanobody™ (including but not limited to Vim sequence). Again, suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acid sequences of the invention may contain one or more of Hallmark residues (as defined herein), such that the amino acid sequence of the invention is a Nanobody™. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

Again, as generally described herein for the amino acid sequences of the invention, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR/ s and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived). Such fragments may also again be such that they comprise or can form an immunoglobulin fold, or alternatively be such that they do not comprise or cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequence as described herein (and in particular a CDR3 sequence), that is flanked on each side by (part of) a framework sequence (and in particular, part of the framework sequence(s) that, in the immunoglobulin sequence from which the fragment is derived, are adjacent to said CDR sequence. For example, a CDR3 sequence may be preceded by (part of) a FR3 sequence and followed by (part of) a FR4 sequence). Such a fragment may also contain a disulphide bridge, and in particular a disulphide bridge that links the two framework regions that precede and follow the CDR sequence, respectively (for the purpose of forming such a disulphide bridge, cysteine residues that naturally occur in said framework regions may be used, or alternatively cysteine residues may be synthetically added to or introduced into said framework regions). For a further description of these "Expedite fragments", reference is again made to WO 03/050531 , as well as to the US provisional application of Ablynx N. V. entitled "Peptides capable of binding to serum proteins" of Ablynx N. V, (inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; and Hoogenboom, Hendricus Renerus Jacobus Mattheus) filed on December 5, 2006.

In another aspect, the invention relates to a construct, and in particular a protein or polypeptide (also referred to herein as a "compound of the invention'^* or ''polypeptide of the invention", respectively) 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. As will become clear to the skilled person from the further disclosure herein, 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 construct in which it is present) and may or may not modify the properties of the amino acid sequence of the invention.

For example, such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, 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, ^MdAb"^"s, amino acid sequences that are suitable for use as a dAb, or Nanobodies.

Alternatively, 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. For example, and without limitation, such groups may be linked to the one or more amino acid sequences of the invention so as to provide a "derivative" of an amino acid sequence or polypeptide of the invention, as further described herein. Also within the scope of the present invention are 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. Preferably, said one or more other groups, residues, moieties or binding units are amino acid sequences. In the compounds or constructs described above, 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. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting construct is a fusion (protein) or fusion (polypeptide).

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.

The process of designing/selecting and/or preparing a compound or polypeptide of the invention, starting from an amino acid sequence of the invention, is also referred to herein as ''formatting" said amino acid sequence of the invention; and an amino acid of the invention that is made part of a compound or polypeptide of the invention is said to be "f" ormatted" or to be '"in the formal of said compound or polypeptide of the invention. Examples of ways in which an amino acid sequence of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted amino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, 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. Examples of 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 references mentioned herein): polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or 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, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to the US provisional application of Ablynx N. V. entitled "Peptides capable of binding to serum proteins" of Ablynx N. V. filed on December 5. 2006.

Generally, 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 per se. For example, 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.

In a preferred, but non-limiting aspect of the invention, 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.

In another preferred, but non-limiting aspect of the invention, 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. For example, 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 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days). In another aspect, the invention 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). Such 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.

In another aspect, the invention 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. 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 sequence 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). Some preferred but non-limiting examples of such products or compositions will become clear from the further description 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 same, in (methods or compositions for) modulating Ephrins and/or Eph, 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 cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases).

The invention also relates to methods for modulating Ephrins and/or Eph, 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 cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases), which method comprises at least the step of contacting Ephrins and/or Eph 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 Ephrins and/or Eph, with at least one amino acid sequence, Nanobody or polypeptide of the invention.

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 Ephrins and/or Eph. 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 cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis- related diseases, and infectious diseases). In the context of the present invention, ''modulating" or "to modulate" generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, Ephrins and/or Eph, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). In particular, "modulating" or "'to modulate" may mean either reducing or inhibiting the activity of, or alternatively increasing the activity of Ephrins and/or Eph, as measured 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 ofEphrins and/or Eph in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody or polypeptide of the invention. As will be clear to the skilled person, "modulating" may also involve effecting a change (which may either be an increase or a descrease) in affinity, avidity, specificity and/or selectivity of Ephrins and/or Eph 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 Ephrins and/or Eph for one or more conditions in the medium or surroundings in which Ephrins and/or Eph 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. As will be clear to the skilled person, 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 Ephrins and/or Eph (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, 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 Ephrins and/or Eph to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to Ephrins and/or Eph. Modulating may also involve activating Ephrins and/or Eph 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 methods for preparing or generating the amino acid sequences, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

Generally, these methods may comprise the steps of: providing a set, collection or library of amino acid sequences; and screening said set, collection or library of amino acid sequences for amino acid sequences that can bind to and/or have affinity for Ephrins and/or Eph; and isolating the amino acid sequence(s) that can bind to and/or have affinity for Ephrins and/or Eph.

In such a method, the set, collection or library of amino acid sequences may be any suitable set, collection or library of amino acid sequences. For example, the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin sequences (as described herein), such as a naϊve set. collection or library of immunoglobulin sequences; a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences: and/or a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation. Also, in such a method, the set, collection or library of amino acid sequences may be a set, collection or library of heavy chain variable domains (such as V_H domains or V_HH domains) or of light chain variable domains. For example, the set. collection or library of amino acicl sequences may be a set collection or library of domain antibodies or single domain antibodies, or may be a set, collection or library of amino acici sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with Ephrins and/or Eph or with a suitable antigenic determinant based thereon or derived there from, such as an antigenic part, fragment, region, domain, loop or other epitope thereof, In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequences comprises at least the steps of: a) providing a collection or sample of cells expressing amino acid sequences; b) screening said collection or sample of cells for cells that express an amino acid sequence that can bind to and/or have affinity for Ephrins and/or Eph; and c) either (i) isolating said amino acid sequence; or (U) isolating from said cell a nucleic acid sequence that encodes said amino acid sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulin sequence. the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a mammal that has been suitably immunized with Ephrins and/or Eph or with a suitable antigenic determinant based thereon or derived there from, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s). The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824. WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001 ).

In another aspect, the method for generating an amino acid sequence directed against Eplirins and/or Eph may comprise at least the steps of: a) providing a set. collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for Ephrins and/or Eph; and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence. In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naive set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi -synthetic set. collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as Vn domains or V_HH domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set. collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody,

In a preferred aspect of this method, the set. collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with Ephrins and/or Eph or with a suitable antigenic determinant based thereon or derived there from, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_HH sequences.

In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology. 23, 9. 1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se. such as by expression in a suitable host cell or host organism or by chemical synthesis. Also, following the steps above, one or more amino acid sequences of the invention may be suitably humanized (or alternatively camelized); and/or the amino acid sequence(s) thus obtained may be linked to each other or to one or more other suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the invention. Also, a nucleic acid sequence encoding an amino acid sequence of the invention may be suitably humanized (or alternatively camelized) and suitably expressed; and/or one or more nucleic acid sequences encoding an amino acid sequence of the invention may be linked to each other or to one or more nucleic acid sequences that encode other suitable amino acid sequences (optionally via nucleotide sequences that encode one or more suitable linkers), after which the nucleotide sequence thus obtained may be suitably expressed so as to provide a polypeptide of the invention.

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 Ephrins and/or Eph. 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.

In particular, 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.

More in particular, 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 cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description herein, in which the invention will be described and discussed in more detail with reference to the Nanobodies of the invention and polypeptides of the invention comprising the same, which form some of the preferred aspects of the invention .

As will become clear from the further description herein, 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 Nanobodies of the invention. However, it will be clear to the skilled person that the more general aspects of the teaching below can also be applied (either directly or analogously) to other amino acid sequences of the invention.

In the present description, examples and claims: a) Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory

Manual" ( 2nd.Ed.), VoIs. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, "Genes II". John Wilev & Sons, New York, N.Y., (1985); Old et a!.. "Principles of Gene Manipulation: An Introduction to Genetic Engineering". 2nd edition, University of California Press, Berkeley, CA (1981), Roilt et al., "Immunology" (6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitf s Essential Immunology, 10^th Ed. Blackwell Publishing, UK (2001); and

Janeway et al., "'Imrnunobiology" (6th Ed.), Garland Science Publishing/Churchill Livingstone, New York (2005), as well as to the general background art cited herein; b) Unless indicated otherwise, the term "immunoglobulin sequence" - whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as Vj _HI domains or V_H/V_L domains, respectively). In addition, the term '"sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence^*', "variable domain sequence", "V_HH sequence'" or '"protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation. Also, the term "nucleotide sequence'^" as used herein also encompasses a nucleic acid molecule with said nucleotide sequence, so that the terms "'nucleotide sequence" and '"nucleic acid'^" should be considered equivalent and are used interchangeably herein; c) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se. as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta. Adv. Drug Deliv. Rev. 2006. 58 (5-6): 640-56; Levin and Weiss. MoL Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2). 31-45: Schmitz et al., Placenta. 2000. 21 Suppl. A, S106-12. Gonzales et al., Tumour Biol., 2005, 26(1). 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins. d) Amino acid residues will be indicated according to the standard three-letter or one- letter amino acid code, as mentioned in Table A-2;

e) For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity^'" between a first nucleotide sequence and a second nucleotide sequence may be calculated by dividing [the number of nucleotides in the fust nucleotide sequence that are identical Io the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI Blast v2.0, using standard settings.

Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0 967 284, EP 1 085 089. WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.

Usually, for the purpose of determining the percentage of '"sequence identity" between two nucleotide sequences in accordance with the calculation method outlined hereinabove, the nucleotide sequence with the greatest number of nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the '"second" nucleotide sequence; f) For the purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence (also referred to herein as "amino acid identity ") may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e. as an "amino acid difference" as defined herein.

Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.

Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences in accordance with the calculation method outlined hereinabove, the amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.

Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein. Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala. Ser, Thr. Pro and GIy; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, GIu and GIn; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, VaI and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Ala into G3y or into Ser; Arg into Lys; Asn into GIn or into His; Asp into GIu; Cys into Ser: GIn into Asn; GIu into Asp; GIy into Ala or into Pro; His into Asn or into GIn; He into Leu or into VaI; Leu into He or into VaI; Lys into Arg, into GIn or into GIu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr;

Tyr into Trp; and/or Phe into VaI, into He or into Leu. Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al, Principles of Protein Structure, Springer-Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol, 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Nad. Acad ScL USA 81 : 140-144. 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132, 198 1, and Goldman et al.. Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a V_HH domain from a llama is for example given by Desmyter et al., Nature Structural Biology. Vol. 3, 9, 803 (1996); SpinelK et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information about some of the amino acid residues that in conventional Vn domains form the V_H/V_L interface and potential cameϊizing substitutions on these positions can be found in the prior art cited above. g) Amino acid sequences and nucleic acid sequences are said to be '"exactly the same'^'' if they have 100% sequence identity (as defined herein) over their entire length; h) When comparing two amino acid sequences, the term ''amino acid difference" refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences; i) When a nucleotide sequence or amino acid sequence is said to '"comprise" another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein). By means of a non-limiting example, when a Nanobody of the invention is said to comprise a CDR sequence, this may mean that said CDR sequence has been incorporated into the Nanobody of the invention, but more usually this generally means that the Nanobody of the invention contains within its sequence a stretch of amino acid residues with the same amino acid sequence as said CDR sequence, irrespective of how said Nanobody of the invention has been generated or obtained. It should also be noted that when the latter amino acid sequence has a specific biological or structural function, it preferably has essentially the same, a similar or an equivalent biological or structural function in the first mentioned amino acid sequence

(in other words, the first mentioned amino acid sequence is preferably such that the latter sequence is capable of performing essentially the same, a similar or an equivalent biological or structural function). For example, when a Nanobody of the invention is said to comprise a CDR sequence or framework sequence, respectively, the CDR sequence and framework are preferably capable, in said Nanobody, of functioning as a

CDR sequence or framework sequence, respectively. Also, when a nucleotide sequence is said to comprise another nucleotide sequence, the first mentioned nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g. a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the first mentioned, larger nucleotide sequence), j) A nucleic acid sequence or amino acid sequence is considered to be ''(in) essentially isolated (form/' - for example, compared to its native biological source and/or the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeplide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a nucleic acid sequence or amino acid sequence is considered "essentially isolated" when it has been purified at least 2 -fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more. A nucleic acid sequence or amino acid sequence that is "in essentially isolated form" is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as poly acrylamide- gel electrophoresis; k) The term "domain'' as used herein generally refers to a globular region of an amino acid sequence (such as an antibody chain, and in particular to a globular region of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region.

Usually, such a domain will comprise peptide loops (for example 3 or 4 peptide loops) stabilized, for example, as a sheet or by disulfide bonds. The term ''binding domain" refers to such a domain that is directed against an antigenic determinant (as defined herein); 1) The term "antigenic determinant^"' refers to the epitope on the antigen recognized by the antigen-binding molecule (such as a Nanobody or a polypeptide of the invention) and more in particular by the antigen-binding site of said molecule. The terms ''antigenic determinant" and "epitope" may also be used interchangeably herein, m) An amino acid sequence (such as a Nanobody, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be ''against" or "directed againsf said antigenic determinant, epitope, antigen or protein. n) The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as a Nanobody or a polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (K_D), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the K_D, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (K_A), which is 1/K_D). AS will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a Nanobody or polypeptide of the invention) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as the amino acid sequences, Nanobodies and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (K_D) of 10^" to 10^"1 moles/liter or less, and preferably 1(T⁷ to 10^"12 moles/liter or less and more preferably 10^"8 to 10^"12 moles/liter (i.e. with an association constant (K_A) of IO⁵ to 10¹ liter/ moles or more, and preferably 10⁷ to 10ⁱ² liter/moles or more and more preferably 10⁸ to 10¹² liter/moles). Any K_D value greater than 10⁴ mol/liter (or any K_A value lower than 10⁴ M^"') liters/mol is generally considered to indicate non-specific binding. Preferably, a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM. more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example. Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein. The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more then 10^"4 moles/liter or 10^~3 moles/liter (e,g, of 10^"2 moles/liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant (K_A), by means of the relationship [K_D = 1/K_A]. The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the K_D, or dissociation constant, which has units of mol/liter (or M). The affinity can also be expressed as an association constant, K_A, which equals

1/K_D and has units of (mol/liter)^"1 (or M^"1). In the present specification, the stability of the interaction between two molecules (such as an amino acid sequence, Nanobody or polypeptide of the invention and its intended target) will mainly be expressed in terms of the K_D value of their interaction; it being clear to the skilled person that in view of the relation K_A =1/K_D, specifying the strength of molecular interaction by its K_D value can also be used to calculate the corresponding K_A value. The Ko-value characterizes the strength of a molecular interaction also in a thermodynamic sense as it is related to the free energy (DG) of binding by the well known relation DG=RTJn(Ko) (equivalently DG-RT-In(KO). where R equals the gas constant, T equals the absolute temperature and In denotes the natural logarithm. The K_D for biological interactions which are considered meaningful (e.g. specific) are typically in the range of 10^'10M (0.1 nM) to 10^"5M (10000 nM). The stronger an interaction is, the lower is its K_D.

The K_D can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as k_Off, to the rate of its association, denoted k_on (so that K_D =k_off/k_on and K_A = Wk_off). The off-rate Ic_0Jf has units s⁴ (where s is the SI unit notation of second). The on-rate k_on has units M^-1S^"1. The on-rate may vary between 10² M^'V¹ to about 10⁷ M^"'s^"

', approaching the diffusion-limited association rate constant for bimolecular interactions. The off-rate is related to the half-life of a given molecular interaction by the relation ti_/2=ln(2)/k_Off . The off-rate may vary between 10^' s^" (near irreversible complex with a tm of multiple days) to 1 s^"1 (Iv₂=O.69 s). The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al, Intern. Immunology, 13. 1551- 1559. 2001) where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k_ϋn, k_off measurements and hence K_D (or K_Λ) values. This can for example be performed using the well-known BIACORE instruments.

It will also be clear to the skilled person that the measured K_D may correspond to the apparent K_D if the measuring process somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent K_D may be measured if one molecule contains more than one recognition sites for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules.

Another approach that may be used to assess affinity is the 2-steρ ELISA (Enzyme- Linked Immunosorbent Assay) procedure of Friguet et a (J. Immunol. Methods, 77. 305-] 9. 1985). This method establishes a solution phase binding equilibrium measurement and avoids possible artefacts relating to adsorption of one of the molecules on a support such as plastic.

However, the accurate measurement of K_D may be quite labour-intensive and as consequence, often apparent K_D values are determined to assess the binding strength of two molecules. It should be noted that as long all measurements are made in a consistent way (e.g. keeping the assay conditions unchanged) apparent K_D measurements can be used as an approximation of the true K_D and hence in the present document K_D and apparent K_D should be treated with equal importance or relevance. Finally, it should be noted that in many situations the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule.

For example, to assess the binding strength between molecules A and B₅ one may e.g. use a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B. As a result an IC₅₀ value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved. Provided K_{D re}f- the K_D of the reference molecule, is known, as well as the total concentration c_ref of the reference molecule, the apparent K_D for the interaction A-B can be obtained from following formula: KD ^~lCso/(l⁺c_Kf/ Koref)- Note that if c_ref « K_{D ref}, K_D ~ IC50. Provided the measurement of the IC50 is performed in a consistent way (e.g. keeping c_ref fixed) for the binders that are compared, the strength or stability of a molecular interaction can be assessed by the IC₅₀ and this measurement is judged as equivalent to K_D or to apparent K_D throughout this text. o) The half-life of an amino acid sequence, compound or polypeptide of the invention can generally be defined as the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half- life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. and may for example generally involve the steps of suitably administering to a warm-blooded animal (i.e. to a human or to another suitable mammal, such as a mouse, rabbit, rat, pig, dog or a primate, for example monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys {Macaca mulatto)) and baboon (Papio nr sinus)) a suitable dose of the amino acid sequence, compound or polypeptide of the invention; collecting blood samples or other samples from said animal; determining the level or concentration of the amino acid sequence, compound or polypeptide of the invention in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence, compound or polypeptide of the invention has been reduced by 50% compared to the initial level upon dosing. Reference is for example made to the Experimental Part below, as well as to Dennis et al., J. Biol. Chem 277:35035-42 (2002), and to the standard handbooks, such as

Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). As will also be clear to the skilled person (see for example pages 6 and 7 of WO

04/003019 and in the further references cited therein), the half-life can be expressed using parameters such as the 11/2 -alpha, tl/2-beta and the area under the curve (AUC). In the present specification, an "increase in half-life^"' refers to an increase in any one of these parameters, such as any two of these parameters, or essentially all three these parameters. As used herein "increase in half-life" or "increased half-life" in particular refers to an increase in the tl/2-beta, either with or without an increase in the 11/2 -alpha and/or the AUC or both. For example, the half-life of an amino acid sequence or polypeptide of the invention may be determined by means of a pharmacokinetic study, performed in a rodent or non- human primate model, as follows. Groups of animals (n=2-10) are given an intravenous bolus injection of lmg/kg or 10 mg/kg 2D3-17D12 fusion protein. Plasma samples are obtained via a vein at different timepoints after dosing (eg. 1, 2, 4, 6, 8, 12, 24, 48,144,

192, 240, 288 and 336 h after dosing) and analyzed for the presence of the 2D3-17D12 fusion protein by ELlSA. Plasma concentration versus time are fitted to a two- compartment elimination model. The pharmacokinetic parameters of clearance, Vl, steady state volume (V ss), T¹A, AUC, and AUC corrected for actual dose administered (AUC/dose) are averaged for each treatment group. Differences between groups are determined by analysis of variance. p) In the context of the present invention, "modulating^"' or ""to modulate" generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay. In particular, "modulating" or "to modulate^*' may mean either reducing or inhibiting the activity of. or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), 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 the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention.

As will be clear to the skilled person, "'modulating'^* may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ϋgands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, depending on the target or antigen involved.

"Modulating" may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s). ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, depending on the target or antigen involved. In particular, 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 construct of the invention. Modulating may for example also involve allosteric modulation of the target or antigen; and/or reducing or inhibiting the binding of the target or antigen to one of its substrates or ligands and/or competing with a natural ligand. substrate for binding to the target or antigen. Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or confirmation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its confirmation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating may for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds (such as ions). Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner; o) The invention also provides amino acid sequences that cross-block the binding of one of the amino acid sequences described in this application and/or are cross-blocked from binding Eph and/or ephrin by one of the amino acid sequences described in this application. The terms "cross-block", "cross-blocked" and '"cross-blocking" are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents to interfere with the binding of other amino acid sequences or binding agents of the invention to Eph and/or ephrin. The extend to which an amino acid sequence or other binding agents of the invention is able to interfere with the binding of another to Eph and/or ephrin. and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequence or another binding agents in terms of their binding to Eph and/or ephrin. Other preferred amino acid sequences of the invention are amino acid sequences comprising at least one single variable domain that cross-block at least one of the amino acid sequences with SEQ ID NOs 714 to 797 or are cross-blocked by at least one of the amino acid sequences with SEQ ID NOs 714 to 797. The following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the invention. It will be appreciated that the assay can be used with any of the Eph and/or ephrin binding agents described herein. The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations. Thus in one cross- blocking assay, the Eph and/or ephrin protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a Eph and/or ephrin- coated surface.

Typically 200- 800 resonance units of Eph and/or ephrin would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used). Two test amino acid sequences (termed A* and B*) to be assessed for their ability to cross- block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture.

When calculating the concentrations on a binding site basis the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of Eph and/or ephrin binding sites on that amino acid sequence. The concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the Eph and/or ephrin molecules captured on the Biacore chip. The amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix. The test mixture is passed over the Eph and/or ephrm-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound Eph and/or ephrin. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of A* alone is then passed over the Eph and/or ephrin-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound Eph and/or ephrin. The solution of B* alone is then passed over the Eph and/or ephrin-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each amino acid sequence when passed over the Eph and/or ephrin surface alone. If the actual recorded binding of the mixture is less than this theoretical maximum then the two amino acid sequences are cross- blocking each other. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to Eph and/or ephrin in the above Biacore cross-blocking assay such that during the assay and in the presence of a second amino acid sequence or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination. The Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to Eph and/or ephrin coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on Eph and/or ephrin is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of Eph and/or ephrin. for example N-terminal His-tagged Eph and/or ephrin (R & D Systems, Minneapolis, MN, USA; 2005 cat# 1406-ST-025). In this particular format, an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged Eph and/or ephrin would be passed over the surface of the chip and captured by the anti-His amino acid sequence. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged Eph and/or ephrin would be loaded back onto the anti-His amino acid sequence coated surface. In addition to the example given using N-terminal His-tagged Eph and/or ephrin, C-terminal His-tagged Eph and/or ephrin could alternatively be used. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross- blocking analysis (e.g. HA tag with anti-HA antibodies: FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin). The following generally describes an ELISA assay for determining whether an anti-Eph and/or ephrin amino acid sequence or other Eph and/or ephrin binding agent cross-blocks or is capable of cross- blocking according to the invention. It will be appreciated that the assay can be used with any of the Eph and/or ephrin binding agents described herein. The general principal of the assay is to have an anti-Eph and/or ephrin amino acid sequence coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking. anti-Eph and/or ephrin amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of Eph and/or ephrin is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of Eph and/or ephrin molecules. The plate is washed to remove Eph and/or ephrin that has not been bound by the coated amino acid sequence of the invention and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and Eph and/or ephrin. The amount of bound Eph and/or ephrin is then measured using an appropriate Eph and/or ephrin detection reagent. An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of Eph and/or ephrin molecules that the coated amino acid sequence can bind relative to the number of Eph and/or ephrin molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence. In the instance where the first amino acid sequence, e.g. a nanobody-X, is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of the second amino acid sequence, i.e. Nanobody-Y, is then added to the ELISA plate such that the moles of Nanobody-Y Eph and/or ephrin binding sites per well are at least 10 fold higher than the moles of Nanobody-X Eph and/or ephrin binding sites that were used, per well, during the coating of the ELISA plate. Eph and/or ephrin is then added such that the moles of Eph and/or ephrin added per well are at least 25 -fold lower than the moles of Nanobody-X Eph and/or ephrin binding sites that were used for coating each well. Following a suitable incubation period the ELISA plate is washed and a Eph and/or ephrin detection reagent is added to measure the amount of Eph and/or ephrin specifically bound by the coated anti-Eph and/or ephrin amino acid sequence (in this case Nanobody-X). The background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Nanobody-X), second solution phase amino acid sequence (in this case Nanobody-Y), Eph and/or ephrin buffer only (i.e. no Eph and/or ephrin) and Eph and/or ephrin detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Nanobody-X), second solution phase amino acid sequence buffer only (i.e. no second solution phase amino acid sequence), Eph and/or ephrin and Eph and/or ephrin detection reagents. The ELISA assay may be run in such a manner so as to have the positive control signal be at least 6 times the background signal. To avoid any artefacts (e.g. significantly different affinities between Nanobody-X and Nanobody-Y for Eph and/or ephrin) resulting from the choice of which amino acid sequence to use as the coating amino acid sequence and which to use as the second (competitor) amino acid sequence, the cross-blocking assay may to be run in two formats: 1) format 1 is where Nanobody-X is the amino acid sequence that is coated onto the ELISA plate and Nanobody-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Nanobody-Y is the amino acid sequence that is coated onto the ELISA plate and Nanobody-X is the competitor amino acid sequence that is in solution. Nanobody-X and Nanobody-Y are defined as cross-blocking if. either in format 1 or in format 2, the solution phase anti-Eph and/or ephrin amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the Eph and/or ephrin detection signal {i.e. the amount of Eph and/or ephrin bound by the coated amino acid sequence) as compared to the Eph and/or ephrin detection signal obtained in the absence of the solution phase anti- Eph and/or ephrin amino acid sequence (i.e. the positive control wells); p) As further described herein, the total number of amino acid residues in a Nanobody can be in the region of 110- 120, is preferably 112-115, and is most preferably 1 13. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a Nanobody are not particularlv limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein; q) The amino acid residues of a Nanobody are numbered according to the general numbering for V_H domains given by Kabat et al. (''Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda. MD, Publication No. 91), as applied to V_HH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23: 240 (1-2): 185-195 (see for example Figure 2 of this publication); or referred to herein. According to this numbering, FRl 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, and FR4 of a Nanobody comprises the amino acid residues at positions 103-113. [In this respect, it should be noted that - as is well known in the art for V_H domains and for V_HH domains - 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). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDR's, position 1 according to the Kabat numbering corresponds to the start of FRl 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, and position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.]. Alternative methods for numbering the amino acid residues of V_H domains, which methods can also be applied in an analogous manner to V_HH domains from Camelids and to Nanobodies, are the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, claims and figures, the numbering according to Kabat as applied to V_HH domains by Riechmami and Muyldermans will be followed, unless indicated otherwise; r) In respect of a target or antigen, the term "interaction site" on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerization or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generally, an "interaction site" can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention can bind such that the target or antigen (and/or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein); and s) The Figures, Sequence Listing and the Experimental Part/Examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or of the appended claims in any way, unless explicitly indicated otherwise herein.

For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the prior art cited herein, 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 Brussels; 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. and Ablynx N. V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867. WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858. WO 06/40153. WO 06/079372, WO 06/122786. WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153. which list and references are incorporated herein by reference.

In accordance with the terminology used in the art (see the above references), the variable domains present in naturally occurring heavy chain antibodies will also be referred to as "V _HH domains'^'', in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein below as 'Υ_H domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein below as '"Vi domains^'1'). As mentioned in the prior art referred to above, V_HH domains have a number of unique structural characteristics and functional properties which make isolated V_HH domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring V_HH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, V_HH domains (which have been '"designed" by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small. functional antigen-binding structural unit, domain or protein. This distinguishes the V_HH domains from the V_H and V_L domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv' s fragments, which consist of a V_H domain covalently linked to a V_L domain).

Because of these unique properties, the use of V_HH domains and Nanobodies as single antigen -binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional V_H and V_L domains, scFv's or conventional antibody fragments (such as Fab- or F(ab')₂-fragments): only a single domain is required to bind an antigen with high affinity and with high selectivity, so that there is no need to have two separate domains present, nor to assure that these two domains are present in the right conformation and configuration (i.e. through the use of especially designed linkers, as with scFv's);

V_HH domains and Nanobodies can be expressed from a single gene and require no post- translational folding or modifications;

V_HH domains and Nanobodies can easily be engineered into multivalent and multispeciilc formats (as further discussed herein); - V_HH domains and Nanobodies are highly soluble and do not have a tendency to aggregate (as with the mouse-derived "dAb^'s" described by Ward et al.₅ Nature, Vol. 341 , 1989, p. 544);

V_HH domains and Nanobodies are highly stable to heat, pH, proteases and other denaturing agents or conditions (see for example Ewert et al, supra); - V_HH domains and Nanobodies are easy and relatively cheap Io prepare, even on a scale required for production. For example, V_HH domains. Nanobodies and proteins/polypeptides containing the same can be produced using microbial fermentation (e.g. as further described below) and do not require the use of mammalian expression systems, as with for example conventional antibody fragments; - V_HH domains and Nanobodies are relatively small (approximately 15 IcDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigen-binding fragments thereof, and therefore show high(er) penetration into tissues (including but not limited to solid tumours and other dense tissues) than such conventional 4-chain antibodies and antigen-binding fragments thereof; V_HΠ domains and Nanobodies can show so-called cavity-binding properties (inter alia due to their extended CDR3 loop, compared to conventional V_H domains) and can therefore also access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding fragments thereof. For example, it has been shown that V_HH domains and Nanobodies can inhibit enzymes (see for example WO 97/49805; Transue et aL, Proteins 1998 Sep 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998 JuI 1 ; 17(13): 3512-20). In a specific and preferred aspect, the invention provides Nanobodies against Ephrins and/or Eph, and in particular Nanobodies against Ephrins and/or Eph from a warm-blooded animal, and more in particular Nanobodies against Ephrins and/or Eph from a mammal, and especially Nanobodies against human Ephrins and/or Eph; as well as proteins and/or polypeptides comprising at least one such Nanobody, In particular, the invention provides Nanobodies against Ephrins and/or Eph, and proteins and/or polypeptides comprising the same, that have improved therapeutic and/or pharmacological properties and/or other advantageous properties (such as, for example, improved ease of preparation and/or reduced costs of goods), compared to conventional antibodies against Ephrins and/or Eph or fragments thereof, compared to constructs that could be based on such conventional antibodies or antibody fragments (such as Fab' fragments, F(ab^")2 fragments, ScFv constructs, "diabodies" and other multispecific constructs (see for example the review by Holliger and Hudson, Nat Biolechnol. 2005 Sep;23(9):1126- 36)), and also compared to the so-called '"dAb's" or similar (single) domain antibodies that may be derived from variable domains of conventional antibodies. These improved and advantageous properties will become clear from the further description herein, and for example include, without limitation, one or more of: increased affinity and/or avidity for Ephrins and/or Eph, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described herein below); - better suitability for formatting in a multivalent format (for example in a bivalent format); better suitability for formatting in a mullispecific format (for example one of the multispecific formats described herein below); improved suitability or susceptibility for "humanizing" substitutions (as defined herein); - less immunogenicity, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described herein below); increased stability, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described herein below); increased specificity towards Ephrins and/or Eph, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described herein below); decreased or where desired increased cross -reactivity with Ephrins and/or Eph from different species; and/or one or more other improved properties desirable for pharmaceutical use (including prophylactic use and/or therapeutic use) and/or for diagnostic use (including but not limited to use for imaging purposes), either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described herein below).

As generally described herein for the amino acid sequences of the invention, 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). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than Ephrins and/or Eph), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. In particular, such 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 Ephrins and/or Eph), 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. Such proteins or polypeptides may also be in essentially isolated form (as defined herein). In a Nanobody of the invention, the binding site for binding against Ephrins and/or Eph is preferably formed by the CDR sequences. Optionally, a Nanobody of the invention may also, and in addition to the at least one binding site for binding against Ephrins and/or Eph, contain one or more further binding sites for binding against other antigens, proteins or targets. For methods and positions for introducing such second binding sites, reference is for example made to Keck and Huston, Biophysical Journal, 71, October 1996, 2002-2011 ; EP 0 640 130; WO 06/07260 and the US provisional application by Ablynx N. V. entitled "Immunoglobulin domains with multiple binding sites" filed on November 27, 2006.

As generally described herein for the amino acid sequences of the invention, when a 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 Ephrins and/or Eph; whereas for veterinary purposes, it is preferably directed against Ephrins and/or Eph 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 Ephrins and/or Eph from two or more species of mammal, such as against human Ephrins and/or Eph and Ephrins and/or Eph from at least one of the species of mammal mentioned herein).

Also, again as generally described herein for the amino acid sequences of the invention, the Nanobodies of the invention may generally be directed against any antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of Ephrins and/or Eph. However, it is generally assumed and preferred that the Nanobodies of the invention (and polypeptides comprising the same) are directed against EphrinAl, EphrinB2. EphA2, EphB4, or EphB6, more preferably against the mouse and/or human version, most preferred against human EphrinAl , EphrinB2. EphA2, EphB4, or EphB6. As already described 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 "PR's" (or sometimes also referred to as "FWs"), which are referred to in the ait and herein as "Framework region !^"' or "FRl"; as "Framework region 2" or "FR2"; as -'Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR' s". which are referred to in the art as "Complementarity Determining Region F'or "CDRl"; as "Complementarity Determining Region 2" or "CDR2"; and as '"Complementarity Determining Region 3" or "CDR3'^", respectively. Some preferred framework sequences and CDR^" s (and combinations thereof) that are present in the Nanobodies of the invention are as described herein. Other suitable CDR sequences can be obtained by the methods described herein, According to a non-limiting but preferred aspect of the invention, (the CDR sequences present in) the Nanobodies of the invention are such that: the Nanobodies can bind to Ephrins and/or Eph with a dissociation constant (K_D) of 10^" ⁵ to 10^"1 moles/liter or less, and preferably 10^"7 to 10^"12 moles/liter or less and more preferably 10^" to 10^" moles/liter (i.e. with an association constant (K_A) of 10 to 10 liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably

10^s to 10¹² liter/moles); and/or such that: the Nanobodies can bind to Ephrins and/or Eph with a k_on-rate of between IG² M^" V to about 10⁷ M^-1S^"1, preferably between 10³ M^-1S^"1 and 10⁷ M^'V¹. more preferably between 10⁴ M^"V^S and 10⁷ M^-1S^"1, such as between 10⁵ M^"V^] and 10⁷ VT¹S^"1; and/or such that they : the Nanobodies can bind to Ephrins and/or Eph with a k_Off rate between Is^"1 (tj_/2=0.69 s) and 10^" s^"! (providing a near irreversible complex with a ti_/2 of multiple days), preferably between 10^"2 s^"! and 10^"6 s^"!. more preferably between 10^"3 s^"1 and 10^"6 s^"1, such as between 10^"4 s^"1 and 10^"6 s^"1.

Preferably, (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 Ephrins and/or Eph 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 Ephrins and/or Eph can be determined in a manner known per se, for example using the general techniques for measuring K_D. K_Af W or k_on mentioned herein, as well as some of the specific assays described herein.

Some preferred 1C50 values for binding of the Nanobodies of the invention (and of polypeptides comprising the same) to Ephrins and/or Eph will become clear from the further description and examples herein.

In a preferred but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against Ephrins and/or Eph, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), in which: - CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 210 to 293; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 210 to 293; c) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; and/or

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 378 to 461; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; and/or

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 546 to 629; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^"s: 546 to 629; or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against Ephrins and/or Eph, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), in which:

CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 210 to 293; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 210 to 293; and - CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO' s: 378 to 461 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 378 to 461; and

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 546 to 629; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 546 to 629; or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of the invention, when a Nanobody of the invention contains one or more CDRl sequences according to b) and/or c): i) any amino acid substitution in such a CDR according to b) and/or c) is preferably, and compared to the corresponding CDR according to a), a conservative amino acid substitution (as defined herein); and/or ϋ) the CDR according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to a); and/or iii) the CDR according to b) and/or c) may be a CDR that is derived from a CDR according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se. Similarly, when a Nanobody of the invention contains one or more CDR2 sequences according to e) and/or f): i) any amino acid substitution in such a CDR according to e) and/or f) is preferably, and compared to the corresponding CDR according to d), a conservative amino acid substitution (as defined herein); and/or ii) the CDR according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to d); and/or iii) the CDR according to e) and/or f) may be a CDR that is derived from a CDR according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.

Also, similarly, when a Nanobody of the invention contains one or more CDR3 sequences according to h) and/or i): i) any amino acid substitution in such a CDR according to h) and/or i) is preferably, and compared to the corresponding CDR according to g), a conservative amino acid substitution (as defined herein); and/or ii) the CDR according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to g); and/or iii) the CDR according to h) and/or i) may be a CDR that is derived from a CDR according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se. Il should be understood that the last three paragraphs generally apply to any Nanobody of the invention that comprises one or more CDRl sequences, CDR2 sequences and/or CDR3 sequences according to b), c)₅ e), f), h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more of the CDR's explicitly listed, above are particularly preferred; Nanobodies comprising two or more of the CDR's explicitly listed above are more particularly preferred; and Nanobodies comprising three of the CDR's explicitly listed above are most particularly preferred.

Some particularly preferred, but non- limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table A-I below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies of the invention. As will be clear to the skilled person, a combination of CDRl, CDR2 and CDR3 sequences that occur in the same clone (i.e. CDRl, CDR2 and CDR3 sequences that are mentioned on the same line in Table A-I) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table A-I). Also, a combination of CDR sequences and framework sequences that occur in the same clone (i.e. CDR sequences and framework sequences that are mentioned on the same line in Table A-I) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table A-I , as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinations of CDR's mentioned in Table A-I, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR's; in which: i) any amino acid substitution in such a CDR is preferably, and compared to the corresponding CDR sequence mentioned in Table A-I, a conservative amino acid substitution (as defined herein); and/or ii) any such CDR sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR sequence mentioned in Table A-I; and/or iii) any such CDR sequence is a CDR that is derived by means of a technique for affinity maturation known per se, and in particular starting from the corresponding CDR sequence mentioned in Table A-I.

However, as will be clear to the skilled person, the (combinations of) CDR sequences, as well as (the combinations of) CDR sequences and framework sequences mentioned in Table A-I will generally be preferred.

Thus, in the NanobodJes of the invention, at least one of the CDRl, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I ; or from the group of CDRl, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%. more preferably at least 95%, even more preferably at least 99% "sequence identity" (as defined herein) with at least one of the CDR3 , CDR2 and CDR3 sequences, respectively, listed in Table A-I; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I.

In this context, by "suitably chosen" is meant that, as applicable, a CDRl sequence is chosen from suitable CDRl sequences (i.e. as defined herein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively. More in particular, the CDR sequences are preferably chosen such that the Nanobodies of the invention bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a K_D- value (actual or apparent), a K_A-value (actual or apparent), a k_on-rate and/or a k_orrrate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-I or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-I ; and/or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table A-L

Preferably, in the Nanobodies of the invention, at least two of the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I or from the group consisting of CDRl, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 ''amino acid difference(s)" with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I .

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-I or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-I₅ respectively; and at least one of the CDRl and CDR2 sequences present is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table A-I or from the group of CDRl and CDR2 sequences, respectively, that have at least 80%. preferably at least 90%. more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl and CDR2 sequences, respectively, listed in Table A-I; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table A-I .

Most preferably, in the Nanobodies of the invention, all three CDRl. CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I or from the group of CDRl, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRi, CDR2 and CDR3 sequences, respectively, listed in Table A-I. Even more preferably, in the Nanobodies of the invention, at least one of the CDRl.

CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-L Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%. even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences, respectively, listed in Table A-I; and/or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table A-] .

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table A-I. Preferably, in this aspect, at least one and preferably both of the CDRl and CDR2 sequences present are suitably chosen from the groups of CDRl and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%. more preferably at least 95%, even more preferably at least 99% sequence identity with the CDRl and CDR2 sequences, respectively, listed in Table A-! ; and/or from the group consisting of the CDRl and CDR2 sequences, respectively. that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table A-I .

Even more preferably, in the Nanobodies of the invention, at least two of the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-I ; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table A-I.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-I. and either the CDRl sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table A-I. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-I; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table A-L Even more preferably, in the Nanobodies of the invention, all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I .

Also, generally, the combinations of CDR's listed in Table A-I (i.e. those mentioned on the same line in Table A-I) are preferred. Thus, it is generally preferred that, when a CDR in aNanobody of the invention is a CDR sequence mentioned in Table A-I or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table A- 1 ; and/or from the group consisting of CDR sequences that have 3. 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table A-I, that at least one and preferably both of the other CDR's are suitably chosen from the CDR sequences that belong to the same combination in Table A-I (i.e. mentioned on the same line in Table A-I) or are suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%. more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR sequence(s) belonging to the same combination and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the CDR sequence(s) belonging to the same combination. The other preferences indicated in the above paragraphs also apply to the combinations of CDR's mentioned in Table A-L Thus, by means of non-limiting examples, aNanobody of the invention can for example comprise a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table A-I, a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-I (but belonging to a different combination), and a CDR3 sequence. Some preferred Nanobodies of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table A-I; a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-I (but belonging to a different combination); and a CDR3 sequence that has more than 80 % sequence identity with one of the CDR3 sequences mentioned in Table A-I (but belonging to a different combination); or (2) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table A-I; a CDR2 sequence, and one of the CDR3 sequences listed in Table A-I; or (3) a CDRl sequence; a CDR2 sequence that has more than 80% sequence identity with one of the CDR2 sequence listed in Table A-I; and a CDR3 sequence that has 3, 2 or 1 amino acid differences with the CDR3 sequence mentioned in Table A-I that belongs to the same combination as the CDR2 sequence. Some particularly preferred Nanobodies of the invention may for example comprise:

(1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table A-I; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table A-I that belongs to the same combination; and a CDR3 sequence that has more than 80 % sequence identity with the CDR3 sequence mentioned in Table A-I that belongs to the same combination; (2) a CDRl sequence; a CDR 2 listed in Table A-I and a CDR3 sequence listed in Table A-I (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).

Some even more preferred Nanobodies of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table A-I; the CDR2 sequence listed in Table A-I that belongs to the same combination; and a CDR3 sequence mentioned in Table A-I that belongs to a different combination; or (2) a CDRl sequence mentioned in Table A-I; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table A-I that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence listed in Table A-I that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for example comprise a CDRl sequence mentioned in Table A- 1, a CDR2 sequence that has more than 80 % sequence identity with the CDR2 sequence mentioned in Table A-I that belongs to the same combination; and the CDR3 sequence mentioned in Table A-I that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDRl, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDRl, CDR2 and CDR3 sequences, respectively, listed in Table A-I. According to another preferred, but non-limiting aspect of the invention (a) CDRl has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues; and/or (c) CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues. In another preferred, but non-limiting aspect, the invention relates to a Nanobody in which the CDR sequences (as defined herein) have more than 80%. preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 . Generally. Nanobodies with the above CDR sequences may be as further described herein, and preferably have framework sequences that are also as further described herein. Thus, for example and as mentioned herein, such Nanobodies may be naturally occurring Nanobodies (from any suitable species), naturally occurring V_HH sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences or Nanobodies, including but not limited to partially humanized Nanobodies or V_HH sequences, fully humanized Nanobodies or V_HH sequences, camelized heavy chain variable domain sequences, as well as Nanobodies that have been obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates to a humanized Nanobody, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), in which CDRl to CDR3 are as defined herein and in which said humanized Nanobody comprises at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein). In another preferred, but non-limiting aspect, the invention relates to a Nanobody in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO^'s: 714 to 797 . This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 714 to 797 , in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies can be as further described herein.

In another preferred, but non-limiting aspect, the invention relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 714 to 797 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%. more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 .

Another preferred, but non-limiting aspect of the invention relates to humanized variants of the Nanobodies of SEQ ID NO's: 714 to 797 , that comprise, compared to the corresponding native V_HH sequence, at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

The polypeptides of the invention comprise or essentially consist of at least one Nanobody of the invention. Some preferred, but non-limiting examples of polypeptides of the invention are given in SEQ ID NO's: 714 to 797 .

It will be clear to the skilled person that the Nanobodies that are mentioned herein as "preferred'^" (or '"more preferred^"', "even more preferred'^*, etc.) are also preferred (or more preferred, or even more preferred, etc.) for use in the polypeptides described herein. Thus, polypeptides that comprise or essentially consist of one or more "'preferred" Nanobodies of the invention will generally be preferred, and polypeptides that comprise or essentially consist of one or more "'more preferred^"' Nanobodies of the invention will generally be more preferred, etc.

Generally, proteins or polypeptides that comprise or essentially consist of a single Nanobody (such as a single Nanobody of the invention) will be referred to herein as ''monovalent" proteins or polypeptides or as "monovalent constructs^". Proteins and polypeptides that comprise or essentially consist of two or more Nanobodies (such as at least two Nanobodies of the invention or at least one Nanobody of the invention and at least one other Nanobody) will be referred to herein as "multivalent" proteins or polypeptides or as "multivalent constructs", and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention. Some non-limiting examples of such multivalent constructs will become clear from the further description herein. According to one specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least two Nanobodies of the invention, such as two or three Nanobodies of the invention. As further described herein, such multivalent constructs can provide certain advantages compared to a protein or polypeptide comprising or essentially consisting of a single Nanobody of the invention, such as a much improved avidity for Ephrins and/or Eph. Such multivalent constructs will be clear to the skilled person based on the disclosure herein.

According to another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one Nanobody of the invention and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is preferably also a Nanobody. Such proteins or polypeptides are also referred to herein as "multispecific" proteins or polypeptides or as 'multi specific constructs", and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention (as will become clear from the further discussion herein of some preferred, but-noniimiting multispecific constructs). Such multispecific constructs will be clear to the skilled person based on the disclosure herein.

According to yet another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one Nanobody of the invention, optionally one or more further Nanobodies, and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the Nanobody of the invention and/or to the resulting fusion protein. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention. Some non-limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein. It is also possible to combine two or more of the above aspects, for example to provide a trivalent bispecific construct comprising two Nanobodies of the invention and one other Nanobody, and optionally one or more other amino acid sequences. Further non- limiting examples of such constructs, as well as some constructs that are particularly preferred within the context of the present invention, will become clear from the further description herein.

In the above constructs, the one or more Nanobodies and/or other amino acid sequences may be directly linked to each other and/or suitably linked to each other via one or more linker sequences. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.

In one specific aspect of the invention, a Nanobody of the invention or a compound, construct or polypeptide of the invention comprising at least one Nanobody 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 Nanobodies, compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise Nanobodies 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. Reference is for example made to the US provisional application by Abiynx N. V. entitled "Immunoglobulin domains with multiple binding sites" filed on November 27, 2006); or polypeptides of the invention that comprise at least one Nanobody 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 Nanobody of the invention. Examples of 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 Nanobodies of the invention are suitable linked to one or more serum proteins or fragments thereof (such as serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, Nanobodies or (single) domain antibodies that can bind to serum proteins such as serum albumin, serum immunoglobulins such as IgG, or trans ferrine); polypeptides in which a Nanobody of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more Nanobodies of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to the US provisional application of Abiynx N. V. entitled "Peptides capable of binding to serum proteins" of Abiynx N.V. filed on December 5, 2006. Again, as will be clear to the skilled person, such Nanobodies, compounds, constructs or polypeptides may contain one or more additional groups, residues, moieties or binding units, such as one or more further amino acid sequences and in particular one or more additional Nanobodies (i.e. not directed against Ephrins and/or Eph). so as to provide a tri- of multispecific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs or polypeptides comprising the same) 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 per se. For example, the Nanobodies, compounds, constructs 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.

In a preferred, but non-limiting aspect of the invention, such Nanobodies, compound, constructs 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. For example, 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 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days). In another one aspect of the invention, a polypeptide of the invention comprises one or more (such as two or preferably one) Nanobodies 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. In particular, 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 02/057445, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of the invention are preferably such that they: bind to Ephrins and/or Eph with a dissociation constant (K_D) of 10° to 10^"12 moles/liter or less, and preferably 10^"7 to 10^"ϊ2 moles/liter or less and more preferably 10^"8 to 10^"12 moles/liter (i.e. with an association constant (K_A) of 10⁵ to 10 liter/ moles or more, and preferably 10 to 10 liter/moles or more and more preferably 10 to 10 liter/moles); and/or such that they: - bind to Ephrins and/or Eph with a k_on-rate of between 10² M⁴S^"1 to about 10⁷ M^-1S⁴. preferably between 10³ M⁴S^"1 and 10⁷ M⁴S⁴. more preferably between 10 M⁴S⁴ and 10⁷ M⁴S⁴, such as between 10⁵ M⁴S⁴ and 10⁷ M⁴S⁴; and/or such that they: bind to Ephrins and/or Eph with a k_Ofτ rate between Is⁴ (ti_/2=0.69 s) and 10^"6 s⁴ (providing a near irreversible complex with a t]/₂ of multiple days), preferably between

10^'2 s⁴ and 10^"6 s⁴. more preferably between 10^'3 s⁴ and 10^"6 s⁴ _; such as between 10^"4 s^" ¹ and 10^"6 s⁴.

Preferably, a polypeptide that contains only one amino acid sequence of the invention is preferably such that it will bind to Ephrins and/or Eph with an affinity less than 500 nM, preferably less than 200 nM. more preferably less than 10 nM, such as less than 500 pM. In this respect, it will be clear to the skilled person that a polypeptide that contains two or more Nanobodies of the invention may bind to Ephrins and/or Eph with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the invention.

Some preferred IC₅₀ values for binding of the amino acid sequences or polypeptides of the invention to Ephrins and/or Eph will become clear from the further description and examples herein.

Other polypeptides according to this preferred aspect of the invention may for example be chosen from the group consisting of amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more '"sequence identity" (as defined herein) with one or more of the amino acid sequences of SEQ ID NO's: 714 to 797 , in which the Nanobodies comprised within said amino acid sequences are preferably as further defined herein.

Another aspect of this invention relates to a nucleic acid that encodes a Nanobody of the invention or a polypeptide of the invention comprising the same. Again, as generally described herein for the nucleic acids of the invention, such a nucleic acid may be in the form of a genetic construct, as defined herein. In another aspect, the invention relates to host or host cell that expresses or that is capable of expressing a Nanobody of the invention and/or a polypeptide of the invention comprising the same; and/or that contains a nucleic acid of the invention. Some preferred but non-limiling examples of such hosts or host cells will become clear from the further description herein.

Another aspect of the invention relates to a product or composition containing or comprising at least one Nanobody 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. 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). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention further relates to methods for preparing or generating the Nanobodies, polypeptides, nucleic acids, host cells, products and compositions described herein. Some 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 Nanobodies, 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 Ephrins and/or Eph. Some preferred but non-limiting applications and uses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description herein below. Generally, it should be noted that the term Nanobody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the Nanobodies of the invention can generally be obtained: (1 ) by isolating the V_HH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring V_HH domain; (3) by "humanizalion" (as described herein) of a naturally occurring V_HΠ domain or by expression of a nucleic acid encoding a such humanized V_HH domain: (4) by "camelization" (as described herein) of a naturally occurring V_H domain from any animal species, and in particular a from species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized V_H domain; (5) by "camelization^"' of a "domain antibody^"' or "Dab^" as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized V_H domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and for example include the methods and techniques described in more detail herein.

One preferred class of Nanobodies corresponds to the V_HH domains of naturally occurring heavy chain antibodies directed against Ephrins and/or Eph. As further described herein, such V_HH sequences can generally be generated or obtained by suitably immunizing a species of Camelid with Ephrins and/or Eph (i.e. so as to raise an immune response and/or heavy chain antibodies directed against Ephrins and/or Eph). by obtaining a suitable biological sample from said Camelid (such as a blood sample, serum sample or sample of B- cells), and by generating V_HH sequences directed against Ephrins and/or Eph, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person and/or are further described herein. Alternatively, such naturally occurring V_HH domains against Ephrins and/or Eph, can be obtained from naϊve libraries of Camelid Vm r sequences, for example by screening such a library using Ephrins and/or Eph, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681. WO 01/90190. WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naϊve V_HH libraries may be used, such as V_HH libraries obtained from naϊve V_HH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.

Thus, in another aspect, the invention relates to a method for generating Nanobodies, that are directed against Ephrins and/or Eph. In one aspect, said method at least comprises the steps of: a) providing a set, collection or library of Nanobody sequences; and b) screening said set, collection or library of Nanobody sequences for Nanobody sequences that can bind to and/or have affinity for Ephrins and/or Eph; and c) isolating the amino acid sequence(s) that can bind to and/or have affinity for Ephrins and/or Eph.

In such a method, the set, collection or library of Nanobody sequences may be a naϊve set, collection or library of Nanobody sequences; a synthetic or semi -synthetic set, collection or library of Nanobody sequences; and/or a set, collection or library of Nanobody sequences that have been subjected to affinity maturation. In a preferred aspect of this method, the set, collection or library of Nanobody sequences may be an immune set. collection or library of Nanobody sequences, and in particular an immune set. collection or library of V_HH sequences, that have been derived from a species of Camelid that has been suitably immunized with Ephrins and/or Eph or with a suitable antigenic determinant based thereon or derived there from, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set. collection or library of Nanobody or V_HH sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) Nanobody sequences will be clear to the person skilled in the art. for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23. 9, 1105-1116 (2005). In another aspect, the method for generating Nanobody sequences comprises at least the steps of; a) providing a collection or sample of cells derived from a species of Camelid that express immunoglobulin sequences; b) screening said collection or sample of cells for (i) cells that express an immunoglobulin sequence that can bind to and/or have affinity for Ephrins and/or Eph: and (ii) cells that express heavy chain antibodies, in which substeps (i) and (ii) can be performed essentially as a single screening step or in any suitable order as two separate screening steps, so as to provide at least one cell that expresses a heavy chain antibody that can bind to and/or has affinity for Ephrins and/or Eph; and c) either (i) isolating from said cell the V_HH sequence present in said heavy chain antibody; or (ii) isolating from said cell a nucleic acid sequence that encodes the V_HH sequence present in said heavy chain antibody, followed by expressing said V_HH domain,

In the method according to this aspect, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a Camelid that has been suitably immunized with Ephrins and/or Eph or a suitable antigenic determinant based thereon or derived there from, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s). The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is made to the so-called "Nanoclone™" technique described in International application WO 06/079372 by Ablynx N. V.

In another aspect, the method for generating an amino acid sequence directed against Ephrins and/or Eph may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody sequences; b) screening said set collection or library of nucleic acid sequences for nucleic acid sequences that encode a heavy chain antibody or a Nanobody sequence that can bind to and/or has affinity for Ephrins and/or Eph; and c) isolating said nucleic acid sequence, followed by expressing the V_HH sequence present in said heavy chain antibody or by expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody sequences may for example be a set collection or library of nucleic acid sequences encoding a naϊve set, collection or library of heavy chain antibodies or V_HH sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of Nanobody sequences; and/or a set, collection or library of nucleic acid sequences encoding a set. collection or library of Nanobody sequences that have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences encoding heavy chain antibodies or V_HH sequences derived from a Camelid that has been suitably immunized with Ephrins and/or Eph or with a suitable antigenic determinant based thereon or derived there from, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set. collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set. collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23. 9, 1105-1116 (2005). As will be clear to the skilled person, the screening step of the methods described herein can also be performed as a selection step. Accordingly the term "screening" as used in the present description can comprise selection, screening or any suitable combination of selection and/or screening techniques. Also, when a set, collection or library of sequences is used, it may contain any suitable number of sequences, such as 1. 2, 3 or about 5, 10, 50, 100, 500, 1000. 5000. 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collection or library of amino acid sequences may be obtained or defined by rational, or semi-empirical approaches such as computer modelling techniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two or more sequences that are variants from one another (e.g. with designed point mutations or with randomized positions), compromise multiple sequences derived from a diverse set of naturally diversified sequences (e.g. an immune library)), or any other source of diverse sequences (as described for example in Hoogenboom et al, Nat Biotechnol 23:1105. 2005 and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection or library of sequences can be displayed on the surface of a phage particle, a ribosome. a bacterium, a yeast cell, a mammalian cell, and linked to the nucleotide sequence encoding the amino acid sequence within these carriers. This makes such set, collection or library amenable to selection procedures to isolate the desired amino acid sequences of the invention. More generally, when a sequence is displayed on a suitable host or host cell, it is also possible (and customary) to first isolate from said host or host cell a nucleotide sequence that encodes the desired sequence, and then to obtain the desired sequence by suitably expressing said nucleotide sequence in a suitable host organism, Again, this can be performed in any suitable manner known per se, as will be clear to the skilled person.

Yet another technique for obtaining V_HH sequences or Nanobody sequences directed against Ephrins and/or Eph involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against Ephrins and/or Eph), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said V_HH sequences or Nanobody sequences (such as a blood sample, serum sample or sample of B-cells). and then generating V_JJH sequences directed against Ephrins and/or Eph, starting from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens et al., Proc. Natl. Acad. Sci .USA. 2006 Oct 10; 103 (41): 15130-5 can be used. For example, such heavy chain antibody expressing mice can express heavy chain antibodies with any suitable (single) variable domain, such as (single) variable domains from natural sources (e.g. human (single) variable domains, Camelid (single) variable domains or shark (single) variable domains), as well as for example synthetic or semi-synthetic (single) variable domains.

The invention also relates to the V_HH sequences or Nanobody sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said V_HH sequence or Nanobody sequence; and of expressing or synthesizing said V_HH sequence or Nanobody sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of the invention comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_HH domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V_HH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a V_H domain from a conventional 4-chaiii antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanization referred to herein. Again, it should be noted that such humanized Nanobodies of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1) — (8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_HΠ domain as a starting material.

Another particularly preferred class of Nanobodies of the invention comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_H domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V_H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V_HH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and/or are present at the V_H-V_L interface, and/or at the so- called Camelidae hallmark residues, as defined herein (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the V_H sequence that is used as a starting material or starting point for generating or designing the camelized Nanobody is preferably a V_H sequence from a mammal, more preferably the V_H sequence of a human being, such as a Vip sequence. However, it should be noted that such camelized Nanobodies of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1) - (8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_H domain as a starting material.

For example, again as further described herein, both "'humanization'^" and "camelization" can be performed by providing a nucleotide sequence that encodes a naturally occurring Vim domain or V_H domain, respectively, and then changing, in a manner known per se. one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" Nanobody of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to provide the desired Nanobody of the invention. Alternatively, based on the amino acid sequence of a naturally occurring Vππ domain or V_H domain, respectively, the amino acid sequence of the desired humanized or camelized Nanobody of the invention, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring V_HH domain or Vn domain, respectively, a nucleotide sequence encoding the desired humanized or camelized Nanobody of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies of the invention and/or nucleic acids encoding the same, starting from naturally occurring V_H sequences or preferably V_HH sequences, will be clear from the skilled person, and may for example comprise combining one or more parts of one or more naturally occurring V_H sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring V_HH sequences (such as one or more FR sequences or CDR sequences), and/or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide a Nanobody of the invention or a nucleotide sequence or nucleic acid encoding the same (which may then be suitably expressed). Nucleotide sequences encoding framework sequences of V_HH sequences or Nanobodies will be clear to the skilled person based on the disclosure herein and/or the further prior art cited herein (and/or may alternatively be obtained by PCR starting from the nucleotide sequences obtained using the methods described herein) and may be suitably combined with nucleotide sequences that encode the desired CDR' s (for example, by PCR assembly using overlapping primers), so as to provide a nucleic acid encoding a Nanobody of the invention.

As mentioned herein, Nanobodies may in particular be characterized by the presence of one or more " 'Hallmark residues" (as described herein) in one or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of the invention, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising: a) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q; and/or: b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the 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; and/or: c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the 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.

Thus, in a first preferred, but non-limiting aspect, 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) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which: b) 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; and/or in which: c) the 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 in which: d) 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.

In particular, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising: a) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q; and/or: b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or: c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the 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.

Thus, according to a preferred, but non-limiting aspect, 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 CDRJ 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; and/or in which: b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which: c) the 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 in which: d) 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. In particular, a Nanobody against Ephrins and/or Eph according to 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) the amino acid residue at position 108 according to the Kabat numbering is Q: and/or in which: b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which: c) the 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 in which: d) 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.

In particular, according to one preferred, but non-limiting aspect of the invention, 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; 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; and a-2) the 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 a-3) 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; a-4) the amino acid residue at position 108 according to the Kabat numbering is Q: or 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; and b-2) 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; b-4) the 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: or in which: c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A₅ G, E, D, Q. R, S and L; and is preferably chosen from the group consisting of G. E and Q; and c-2) the 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 c-3) the 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; and in which d) 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. Thus, in another preferred, but non-limiting aspect, 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, D₅ G, Q, R, S. L; and is preferably chosen from the group consisting of G, E or Q; and in which: a-2) the 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 in which: a-3) 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; and in which a-4) the amino acid residue at position 108 according to the Kabat numbering is Q; and in which: d) 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.

In another preferred, but non-limiting aspect, 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: b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and in which: b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and in which: 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; and in which: b-4) the 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; and in which: d) 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 OΏS of the more preferred aspects herein. In another preferred, but non-limiting aspect, 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: c-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, R₅ S and L; and is preferably chosen from the group consisting of G, E and Q; and in which: c-2) the 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 in which: c-3) the 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 in which: 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; and in which: d) 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.

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: i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-Hke sequence as described herein) and the amino acid residue at position 108 is Q; or in which: ii) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence as described) and the amino acid residue at position 108 is Q or L, and is preferably Q. Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may- have the structure FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FR] Io 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: 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: and in which: ii) 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.

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: i) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence) and the amino acid residue at position 108 is Q or L. and is preferably Q; and in which: ii) 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.

In the Nanobodies of the invention in which the amino acid residues at positions 43- 46 according to the Kabat numbering form the sequence KERE or KQRE. the amino acid residue at position 37 is most preferably F. In the Nanobodies of the invention in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW. 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. Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the Nanobodies of the invention can generally be classified on the basis of the following three groups: i) The "GLEW-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. As further described herein, 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 A-3 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 KERE or KQRE (or another KERE-like sequence) at positions 43-46 according to the Kabat numbering and Q or L at position 108 according to the Kabat numbering. As further described herein,

Nanobodies within this group usually have a F at position 37, an L or F at position 47; and can have a W. P. R or S at position 103, and preferably have a W at position 103. More generally, and without limitation. 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; iii) The "103 P, R, S-group": Nanobodies with a P, R or S at position 103. These Nanobodies can have either the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering or the amino acid sequence KERE 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.

Also, where appropriate, Nanobodies may belong to (i.e. have characteristics of) two or more of these classes. For example, 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). More generally, it should be noted that the definitions referred to above describe and apply to Nanobodies in the form of a native (i.e. non-humanized) V_HH sequence, and that 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). For example, and without limitation, in some humanized Nanobodies of the GLEW-group or the 103 P, R, S-group, Q at position 108 may be humanized to 108L, As already mentioned herein, other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_HH 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 Vm _Ϊ sequence (in any manner known per se, as further described herein) and the resulting humanized V_HH 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. Thus, in another preferred, but non-limiting aspect, 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. In another preferred, but non-limiting aspect, 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. Thus, in another preferred, but non-limiting aspect, 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.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,S residues mentioned above, the Nanobodies of the invention can contain, at one or more positions that in a conventional V₁] domain would form (part of) the V_H/V_L 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). Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table A-3 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.

In one aspect of the Nanobodies of the invention, the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.

Also, in one aspect of the Nanobodies of the invention, the amino acid residue at position 83 is chosen from the group consisting of R, K, N, E, G, I, T and Q; and is most preferably either K or E (for Nanobodies corresponding to naturally occurring V_HH 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_HΠ domains) or R (for "humanized" Nanobodies, as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G. Collectively, 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 V_H domain, V_H3, are summarized in Table A-3. Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring V_HH domains are mentioned in Table A-4. For comparison, the corresponding amino acid residues of the human V_H3 called DP-47 have been indicated in italics.

In the 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 Kabat numbering) of a naturally occurring V_HΠ domain.

Such amino acid residues will be clear to the skilled person. Tables A- 5 to A-8 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FRl, FR2, FR3 and FR4 of naturally occurring V_HH domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring V_HH domain (and which is the most preferred amino acid residue for said position in a Nanobody) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring V_HH domains supports the hypothesis underlying the numbering by Chothia (supra) that the residues at these positions already form part of CDRl.)

In Tables A-5 - A-8, some of the non-limiting residues that can be present at each position of a human V_H3 domain have also been mentioned. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human V_H3 domain is indicated in bold; and other preferred amino acid residues have been underlined.

For reference only, Tables A-5-A-8 also contain data on the V_HH entropy ("V Ent ") and V_HH variability ("V_HH Var ") at each amino acid position for a representative sample of 1118 V_HH sequences (data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The values for the V_HΠ entropy and the V_ΠH variability provide a measure for the variability and degree of conservation of amino acid residues between the 1118 V_HH 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 (i.e. little variability). For example, the G at position 8 and the G at position 9 have values for the V_ΠH entropy of 0.1 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 9 is G in all 1118 sequences analysed), whereas for residues that form part of the CDR' s generally values of 1.5 or more are found (data not shown). Note that (1) the amino acid residues listed in the second column of Tables A-5-A-8 are based on a bigger sample than the 1118 V_HH sequences that were analysed for determining the V_HH entropy and V_HH variability referred to in the last two columns; and (2) 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). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et al., PROTEINS: Structure. Function and Genetics, 52: 544-552 (2003).

Thus, in another preferred, but not limiting aspect. aNanobody of the invention can be defined as 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: i) one or more of the amino acid residues at positions 11. 37, 44. 45, 47. 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 ; and in which: ii) 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 V_HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies. they may optionally be further suitably humanized, again as described herein. In particular, 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: i) (preferably) one or more of the amino acid residues at positions 11, 37, 44.45, 47, 83, 84, 103. 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 (it being understood that Vππ sequences will contain one or more Hallmark residues; and that partially humanized Nanobodies will usually, and preferably, [still] contain one or more Hallmark, residues [although it is also within the scope of the invention to provide - where suitable in accordance with the invention - partially humanized Nanobodies in which all Hallmark residues, but not one or more of the other amino acid residues, have been humanized]; and that in fully humanized Nanobodies, where suitable in accordance with the invention, all amino acid residues at the positions of the Hallmark residues will be amino acid residues that occur in a human Vjp sequence. As will be clear to the skilled person based on the disclosure herein that such V_HH sequences, such partially humanized Nanobodies with at least one Hallmark residue, such partially humanized Nanobodies without Hallmark residues and such fully humanized Nanobodies all form aspects of this invention); and in which: ii) 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; and in which: iii) 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 V_HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

In particular, a Naπobody of the invention of the KERE group can be an amino acid sequence with the (general) structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which: i) the 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; and in which: ii) FRl is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: vi) 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.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized

Nanobodies, they may optionally be further suitably humanized, again as described herein.

With regard to framework 1. it will be clear to the skilled person that when an amino acid sequence as outlined above is generated by expression of a nucleotide sequence, the first four amino acid sequences (i.e. amino acid residues 1 -4 according to the Kabat numbering) may often be determined by the primer(s) that have been used to generate said nucleic acid. Thus, for determining the degree of amino acid identity, the first four amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering considered to be part of the framework regions (and not the CDR' s), it has been found by analysis of a database of more than 1000 V_HH sequences that the positions 27 to 30 have a variability (expressed in terms of V_JΠI entropy and V_HH variability - see Tables A-5 to A-8) that is much greater than the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino acid residues at positions 27 to 30 are preferably also disregarded. In view of this, a Nanobody of the KERE class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which: i) the 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; and in which: ii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies of the KERE-class; and in which: iv) 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 GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which i) preferably, when the Nanobody of the GLEW-class is a non-humanized Nanobody, the amino acid residue in position 108 is Q; ii) FRl is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: vi) 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. In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies).

With regard to framework 1 , it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which: i) preferably, when the Nanobody of the GLEW-class is a non-humanized Nanobody, the amino acid residue in position 108 is Q; and in which: ii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iii) FR2, FR3 and FR4 are as mentioned herein for FR2. FR3 and FR4 of Nanobodies of the GLEW-class; and in which: iv) 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 V_HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein. In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which i) the amino acid residue at position 103 according to the Kabat numbering is different from W; and in which: ii) preferably the amino acid residue at position 103 according to the Kabat numbering is

P, R or S, and more preferably R; and in which: iii) FRl is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which iv) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: v) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table A-22: Representative FR3 sequences for Nanobodies of the P,R,S 103-group.

and in which: vi) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: vii) 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.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies). With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, aNanobody of the P.R.S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which: i) the amino acid residue at position 103 according to the Kabat numbering is different from W; and in which: ii) preferably the amino acid residue at position 103 according to the Kabat numbering is

P, R or S, and more preferably R; and in which: iii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies of the P₅R₅S 103 class; and in which: v) 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.

In another preferred, but non-limiting aspect, the invention relates to a Nanobody as described above, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 . This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 714 to 797 , in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies may be as further described herein.

As already mentioned herein, another preferred but non-limiting aspect of the invention relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 714 to 797 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 714 to 797 . Also, in the above Nanobodies: i) any amino acid substitution (when it is not a humanizing substitution as defined herein) is preferably, and compared to the corresponding amino acid sequence of SEQ ID NO's: 714 to 797 . a conservative amino acid substitution, (as defined herein); and/or: ii) its amino acid sequence preferably contains either only amino acid substitutions, or otherwise preferably no more than 5, preferably no more than 3, and more preferably only 1 or 2 amino acid deletions or insertions, compared to the corresponding amino acid sequence of SEQ ID NO's: 714 to 797 ; and/or iii) the CDR' s may be CDR's that are derived by means of affinity maturation, for example starting from the CDR's of to the corresponding amino acid sequence of SEQ ID NO's: 714 to 797 . Preferably, the CDR sequences and FR sequences in the Nanobodies of the invention are such that the Nanobodies of the invention (and polypeptides of the invention comprising the same): bind to Ephrins and/or Eph with a dissociation constant (K_D) of 1 CT⁵ to 10^{"i 2} moles/liter or less, and preferably 10^" to 10^" moles/liter or less and more preferably 10^" to 10^{" "} moles/liter (i.e. with an association constant (K_A) of IO⁵ to 10¹² liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸ to 10¹² liter/moles); and/or such that they: - bind to Ephrins and/or Eph with a k_on-rate of between 10² IVfV¹ to about 10⁷ M^-1S^"1. preferably between 10³ M^-1S^"1 and 10⁷ M^-1S^"1, more preferably between 10⁴ VT¹S^"1 and 10⁷ M-¹S^"1, such as between 10⁵ M^'V¹ and 10⁷ MV; and/or such that they: bind to Ephrins and/or Eph with a Ic_0Jf rate between Is^-1 (ti_/2=0.69 s) and 10^"6 s^"1 (providing a near irreversible complex with a tj_/2 of multiple days), preferably between

10^"2 s^"1 and 10^"6 s^"1, more preferably between 10^"3 s^'[ and 1 O^-6 s^"1, such as between 10^"4 s^" ' and 10^"6 S^"1.

Preferably, CDR sequences and FR sequences present in the Nanobodies of the invention are such that the Nanobodies of the invention will bind to Ephrins and/or Eph with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

According to one non-limiting aspect of the invention, a Nanobody may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring human V_H domain, and in particular compared to the corresponding framework region of DP-47. More specifically, according to one non-limiting aspect of the invention, a Nanobody may be as defined herein, but with the proviso that it has at least "'one amino acid difference" (as defined herein) at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring human V_H domain, and in particular compared to the corresponding framework region of DP-47. Usually, a Nanobody will have at least one such amino acid difference with a naturally occurring V_H domain in at least one of FR2 and/or FR4, and in particular at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

Also, a humanized N anobody of the invention may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring V_HH domain. More specifically, according to one non- limiting aspect of the invention, a humanized Nanobody may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring V_HH domain. Usually, a humanized Nanobody will have at least one such amino acid difference with a naturally occurring V_HH domain in -at least one of FR2 and/or FR4, and in particular at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs") of the Nanobodies of the invention as defined herein, and in particular analogs of the Nanobodies of SEQ ID NCs 714 to 797 . Thus, according to one aspect of the invention, the term "Nanobody of the invention" in its broadest sense also covers such analogs. Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the Nanobodies of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDR' s. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and/or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another V_1-1H domain (see Tables A- 5 to A- 8 for some non-limiting examples of such substitutions), although the invention is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Nanobody of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody of the invention (i.e. to the extent that the Nanobody is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the Nanobodies thus obtained. For example, and depending on the host organism used to express the Nanobody or polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein).

As can be seen from the data on the V_HH entropy and VHH variability given in Tables A-5 to A- 8 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a K_Λ-value (actual or apparent), a k_on-rate and/or a karate, or alternatively as an IC₅0 value, as further described herein) that is as defined herein for the Nanobodies of the invention.

The analogs are preferably also such that they retain the favourable properties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree of sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95% or 99% or more; and/or preferably have at most 20, preferably at most 10, even more preferably at most 5, such as 4, 3, 2 or only 1 amino acid difference (as defined herein), with one of the Nanobodies of SEQ ID NOs: 714 to 797 .

Also, the framework sequences and CDR' s of the analogs are preferably such that they are in accordance with the preferred aspects defined herein. More generally, as described herein, the analogs will have (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103.

One preferred class of analogs of the Nanobodies of the invention comprise Nanobodies that have been humanized (i.e. compared to the sequence of a naturally occurring Nanobody of the invention). As mentioned in the background art cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V_HH with the amino acid residues that occur at the same position in a human V_H domain, such as a human V_R3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing substitutions mentioned in the background art cited herein, and/or from a comparisons between the sequence of a Nanobody and the sequence of a naturally occurring human V_H domain.

The humanizing substitutions should be chosen such that the resulting humanized Nanobodies still retain the favourable properties of Nanobodies as defined herein, and more preferably such that they are as described for analogs in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the invention may become more "human-like", while still retaining the favourable properties of the Nanobodies of the invention as described herein. As a result, such humanized Nanobodies may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring V_HH domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring V_HH domains on the other hand.

The Nanobodies of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined herein) or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. One preferred humanizing substitution for Nanobodies of the "P₅R₅S-103 group" or the "KERE group" is Q108 into L108. Nanobodies of the "GLEW class" may also be humanized by a Q108 into L108 substitution, provided at least one of the other Hallmark residues contains a Camelid (camelizing) substitution (as defined herein). For example, as mentioned above, one particularly preferred class of humanized Nanobodies has GLEW or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding the same, can be provided in any manner known per se. For example, the analogs can be obtained by providing a nucleic acid that encodes a naturally occurring V_HH domain, changing the codons for the one or more amino acid residues that are to be substituted into the codons for the corresponding desired amino acid residues (e.g. by site-directed mutagenesis or by PCR using suitable mismatch primers), expressing the nucleic acid/nucleotide sequence thus obtained in a suitable host or expression system; and optionally isolating and/or purifying the analog thus obtained to provide said analog in essentially isolated form (e.g. as further described herein). This can generally be performed using methods and techniques known per se. which will be clear to the skilled person, for example from the handbooks and references cited herein, the background art cited herein and/or from the further description herein. Alternatively, a nucleic acid encoding the desired analog can be synthesized in a manner known per se (for example using an automated apparatus for synthesizing nucleic acid sequences with a predefined amino acid sequence) and can then be expressed as described herein. Yet another technique may involve combining one or more naturally occurring and/or synthetic nucleic acid sequences each encoding a part of the desired analog, and then expressing the combined nucleic acid sequence as described herein. Also, the analogs can be provided using chemical synthesis of the pertinent amino acid sequence using techniques for peptide synthesis known per se. such as those mentioned herein. In this respect, it will be also be clear to the skilled person that the Nanobodies of the invention (including their analogs) can be designed and/or prepared starting from human V_H sequences (i.e. amino acid sequences or the corresponding nucleotide sequences), such as for example from human V_H 3 sequences such as DP-47, DP-51 or DP-29. i.e. by introducing one or more camelizing substitutions (i.e. changing one or more amino acid residues in the amino acid sequence of said human Vn domain into the amino acid residues that occur at the corresponding position in a V_HH domain), so as to provide the sequence of aNanobody of the invention and/or so as to confer the favourable properties of a Nanobody to the sequence thus obtained. Again, this can generally be performed using the various methods and techniques referred to in the previous paragraph, using an amino acid sequence and/or nucleotide sequence for a human V_H domain as a starting point.

Some preferred, but non-limiting camelizing substitutions can be derived from Tables A-5 - A-8. It will also be clear that camelizing substitutions at one or more of the Hallmark residues will generally have a greater influence on the desired properties than substitutions at one or more of the other amino acid positions, although both and any suitable combination thereof are included within the scope of the invention. For example, it is possible to introduce one or more camelizing substitutions that already confer at least some the desired properties, and then to introduce further camelizing substitutions that either further improve said properties and/or confer additional favourable properties. Again, the skilled person will generally be able to determine and select suitable camelizing substitutions or suitable combinations of camelizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible camelizing substitutions and determining whether the favourable properties of Nanobodies are obtained or improved (i.e. compared to the original V_H domain). Generally, however, such camelizing substitutions are preferably such that the resulting an amino acid sequence at least contains (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably also an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P₅ R or S at position 103; and optionally one or more further camelizing substitutions. More preferably, the camelizing substitutions are such that they result in a Nanobody of the invention and/or in an analog thereof (as defined herein), such as in a humanized analog and/or preferably in an analog that is as defined in the preceding paragraphs. As will also be clear from the disclosure herein, it is also within the scope of the invention to use parts or fragments, or combinations of two or more parts or fragments, of the Nanobodies of the invention as defined herein, and in particular parts or fragments of the Nanobodies of SEQ ID NO's: 714 to 797 . Thus, according to one aspect of the invention, the term "Nanobody of the invention" in its broadest sense also covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention (including analogs thereof) have amino acid sequences in which, compared to the amino acid sequence of the corresponding full length Nanobody of the invention (or analog thereof), one or more of the amino acid residues at the N-terminal end, one or more amino acid residues at the C- terminal end, one or more contiguous internal amino acid residues, or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a Kπ-value (actual or apparent), a K_A- value (actual or apparent), a k_on-rate and/or a k_off rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues, of the amino acid sequence of the corresponding full length Nanobody of the invention. Also, any part or fragment is such preferably that it comprises at least one of CDRl,

CDR2 and/or CDR3 or at least part thereof (and in particular at least CDR3 or at least part thereof). More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least one other CDR (i.e. CDRl or CDR2) or at least part thereof, preferably connected by suitable framework sequence(s) or at least part thereof. More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least part of the two remaining CDR's, again preferably connected by suitable framework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect, such a part or fragment comprises at least CDR3, such as FR3, CDR3 and FR4 of the corresponding full length Nanobody of the invention, i.e. as for example described in the International application WO 03/050531 (Lasters et al). As already mentioned above, it is also possible to combine two or more of such parts or fragments (i.e. from the same or different Nanobodies of the invention), i.e. to provide an analog (as defined herein) and/or to provide further parts or fragments (as defined herein) of a Nanobody of the invention. It is for example also possible to combine one or more parts or fragments of a Nanobody of the invention with one or more parts or fragments of a human V_H domain.

According to one preferred aspect, the parts or fragments have a degree of sequence identity of at least 50%. preferably at least 60%, more preferably at least 70%. even more preferably at least 80%, such as at least 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs 714 to 797 .

The parts and fragments, and nucleic acid sequences encoding the same, can be provided and optionally combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codon in a nucleic acid that encodes a full-sized Nanobody of the invention, and then expressing the nucleic acid thus obtained in a manner known per se (e.g. as described herein). Alternatively, nucleic acids encoding such parts or fragments can be obtained by suitably restricting a nucleic acid that encodes a full-sized Nanobody of the invention or by synthesizing such a nucleic acid in a manner known per se. Parts or fragments may also be provided using techniques for peptide synthesis known per se.

The invention in its broadest sense also comprises derivatives of the Nanobodies of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g. enzymatical) modification, of the Nanobodies of the invention and/or of one or more of the amino acid residues that form the Nanobodies of the invention.

Examples of such modifications, as well as examples of amino acid residues within the Nanobody sequence that can be modified in such a manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. by covalent linking or in an other suitable manner) of one or more functional groups, residues or moieties into or onto the Nanobody of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the Nanobody of the invention. Example of such functional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or the absorption of the Nanobody of the invention, that reduce the immunogenicity and/or the toxicity of the Nanobody of the invention, that eliminate or attenuate any undesirable side effects of the Nanobody of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the Nanobodies and/or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv' s and single domain antibodies), for which reference is for example made to Remington's

Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton. PA (1980). Such functional groups may for example be linked directly (for example covalently) to a Nanobody of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person, One of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypo]y(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv' s); reference is made to for example Chapman, Nat. Biotechnol., 54, 531 -545 (2002); by Veronese and Harris, Adv. Drag Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA. Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a Nanobody of the invention, a Nanobody of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG. or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of a Nanobody of the invention, all using techniques of protein engineering known per se to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000.

Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co~translational and/or post-translational modification, depending on the host cell used for expressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled Nanobody. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allopbycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as ¹⁵²Eu or others metals from the lanthanide series), phosphorescent labels, chemi luminescent labels or bioluminescent labels (such as luminal, isoluminol. theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵1, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr₅ ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates or metallic cations (for example metallic cations such as ^99mTc. ¹²³I, ¹¹¹In, ¹³¹L ⁹⁷Ru, ⁶⁷Cu₅ ⁶⁷Ga, and ⁶⁸Ga or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labelled Nanobodies and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label. As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethyl- enetriaminepentaacetic acid (DTPA) or ethyl enediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin~(strept)avidin binding pair. Such a functional group may be used to link the Nanobody of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a Nanobody of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated Nanobody may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the Nanobody of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh. Journal of Drug Targetting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the Nanobody of the invention.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a cell, the Nanobodies of the invention may also be linked to a toxin or to a toxic residue or moiety. Examples of toxic moieties, compounds or residues which can be linked to a Nanobody of the invention to provide - for example - a cytotoxic compound will be clear Io the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g. to study function- activity relationships). Reference is for example made to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to Ephrins and/or Eph with an affinity (suitably measured and/or expressed as a Ko-vaiue (actual or apparent), a K_A-value (actual or apparent), a k_OT1-rate and/or a k_0frrate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of or comprise at least one Nanobody of the invention. By "essentially consist of is meant that the amino acid sequence of the polypeptide of the invention either is exactly the same as the amino acid sequence of a Nanobody of the invention or corresponds to the amino acid sequence of a Nanobody of the invention which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1 -6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwise influence the (biological) properties of the Nanobody and may or may not add further functionality to the Nanobody. For example, such amino acid residues: can comprise an N-terminal Met residue, for example as result of expression in a heterologous host cell or host organism. may form a signal sequence or leader sequence that directs secretion of the Nanobody from a host cell upon synthesis. Suitable secretory leader peptides will be clear to the skilled person, and may be as further described herein. Usually, such a leader sequence will be linked to the N-terrm^'nus of the Nanobody, although the invention in its broadest sense is not limited thereto; may form a sequence or signal that allows the Nanobody to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumours, or the blood-brain-barrier. Examples of such amino acid sequences will be clear to the skilled person. Some non-limiting examples are the small peptide vectors ("Pep-trans vectors") described in WO 03/026700 and in Temsamani et al.. Expert Opin. Biol. Ther., I₅ 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296. 124-131 (2001), and the membrane translocator sequence described by Zhao et al., Apoptosis, 8, 631-637 (2003). C- termina] and N-terminal amino acid sequences for intracellular targeting of antibody fragments are for example described by Cardinale et al., Methods, 34, 171 (2004).

Other suitable techniques for intracellular targeting involve the expression and/or use of so-called "intrabodies" comprising a Nanobody of the invention, as mentioned below: may form a "'tag", for example an. amino acid sequence or residue that allows or facilitates the purification of the Nanobody, for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g. by chemical or enzymatical cleavage) to provide the Nanobody sequence (for this purpose, the tag may optionally be United to the Nanobody sequence via a cleavable linker sequence or contain a cleavable motif). Some preferred, but non- limiting examples of such residues are multiple histidine residues, glutatione residues and a myc-tag (see for example SEQ ID NO:31 of WO 06/12282). may be one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups will be clear to the skilled person and include, but are not limited to. the amino acid residues and functional groups mentioned herein for the derivatives of the Nanobodies of the invention.

According to another aspect, a polypeptide of the invention comprises a Nanobody of the invention, which is fused at its amino terminal end. at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein comprising said Nanobody of the invention and the one or more further amino acid sequences. Such a fusion will also be referred to herein as a "Nanobody fusion".

The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the Nanobody, and may or may not add further functionality to the Nanobody or the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody or the polypeptide of the invention. For example, the further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the Nanobody of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv' s and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the Nanobody of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).

In particular, it has been described in the art that linking fragments of immunoglobulins (such as V_H domains) to serum albumin or to fragments thereof can be used to increase the half-life. Reference is for made to WO 00/27435 and WO 01/077137). According to the invention, the Nanobody of the invention is preferably either directly linked to serum albumin (or to a suitable fragment thereof) or via a suitable linker, and in particular via a suitable peptide linked so that the polypeptide of the invention can be expressed as a genetic fusion (protein). According to one specific aspect, the Nanobody of the invention may be linked to a fragment of serum albumin that at least comprises the domain III of serum albumin or part thereof. Reference is for example made to the US provisional application 60/788,256 of Ablynx N.V. entitled "Albumin derived amino acid sequence, use thereof for increasing the half-life of therapeutic proteins and of other therapeutic proteins and entities, and constructs comprising the same" filed on March 31, 2006.

Alternatively, the further amino acid sequence may provide a second binding site or binding unit that is directed against a serum protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Such amino acid sequences for example include the Nanobodies described below, as well as the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO 04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as to EP 0 368 684, as well as to the following the US provisional applications 60/843,349, 60/850.774, 60/850,775 by Ablynx N.V. mentioned herein and US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins" filed on December 5, 2006 (also mentioned herein). Such amino acid sequences may in particular be directed against serum albumin (and more in particular human serum albumin) and/or against IgG (and more in particular human IgG). For example, such amino acid sequences may be amino acid sequences that are directed against (human) serum albumin and amino acid sequences that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787) and/or amino acid sequences that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see again for example WO 06/0122787); amino acid sequences that have or can provide an increased half-life (see for example the US provisional application 60/843,349 by Ablynx N.V, entitled "Serum albumin binding proteins with long half-lives" filed on September 8, 2006); amino acid sequences against human serum albumin that are cross- reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such, as, and in particular, cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ur sinus), reference is again made to the US provisional application 60/843,349); amino acid sequences that can bind to serum albumin in a pH independent manner (see for example the US provisional application 60/850,774 by Ablynx N.V. entitled ''Amino acid sequences that bind to serum proteins in a manner thai is essentially independent ofthepH, compounds comprising the same, and uses thereof, filed on October 11, 2006) and/or amino acid sequences that are conditional binders (see for example the US provisional application 60/850,775 by Ablynx N.V. entitled "Amino acid sequences thai bind to a desired molecule in a conditional manner', filed on October 11, 2006). According to another aspect, the one or more further amino acid sequences may comprise one or more parts, fragments or domains of conventional 4-chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a Nanobody of the invention may be linked to a conventional (preferably human) V_H or V_L domain or to a natural or synthetic analog of a V_H or V_L domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et a!.).

The at least one Nanobody may also be linked to one or more (preferably human) C_HI , C_H2 and/or C_H3 domains, optionally via a linker sequence. For instance, a Nanobody linked to a suitable CnI domain could for example be used - together with suitable light chains - to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab')₂ fragments, but in which OΪΪQ or (in case of an F(ab')₂ fragment) one or both of the conventional Vn domains have been replaced by a Nanobody of the invention. Also, two Nanobodies could be linked to a C_H3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, one or more Nanobodies of the invention may be linked to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more C_H2 and/or C_H3 domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) and Fc region, for example from IgG, from IgE or from another human Ig, For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid V_HH domain or a humanized derivative thereof (i.e. a Nanobody), in which the

Camelidae C_H2 and/or C_H3 domain have been replaced by human C_H2 and C_H3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a Nanobody and human C_H2 and C_H3 domains (but no C_HI domain), which immunoglobulin has the effector function provided by the Cj {I and C_H3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the Nanobodies of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04/058820, WO 99/42077 and WO 05/017148. as well as the review by Holliger and Hudson, supra. Coupling of a Nanobody of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding Nanobody of the invention. For some applications, the use of an Fc portion and/or of constant domains (i.e. C_R2 and/or C_H3 domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more Nanobodies and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two Nanobodies linked to a C_H3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD. the cut-off value for renal absorption.

The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the Nanobody or the polypeptide of the invention from a host cell upon synthesis (for example to provide a pre-, pro- or prepro- form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention).

The further amino acid sequence may also form a sequence or signal that allows the Nanobody or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody or polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumours, or the blood-brain-barrier. Suitable examples of such amino acid sequences wiϊl be clear to the skilled person, and for example include, but are not limited to, the "Peptrans" vectors mentioned above, the sequences described by Cardinale et al. and the amino acid sequences and antibody fragments known per se that can be used to express or produce the Nanobodies and polypeptides of the invention as so-called '"intrabodies", for example as described in WO 94/02610. WO 95/22618, US-A-7004940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo, A. & Biocca. S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer- Verlag; and in Kontermann, Methods 34, (2004). 163-170, and the further references described therein. For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation of such a cell, the Nanobodies of the invention may also be linked to a (cylo)toxic protein or polypeptide. Examples of such toxic proteins and polypeptides which can be linked to a Nanobody of the invention to provide - for example - a cytotoxic polypeptide of the invention will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or more further amino acid sequences comprise at least one further Nanobody, so as to provide a polypeptide of the invention that comprises at least two. such as three, four, five or more Nanobodies. in which said Nanobodies may optionally be linked via one or more linker sequences (as defined herein). Polypeptides of the invention that comprise two or more Nanobodies. of which at least one is a Nanobody of the invention, will also be referred to herein as "multivalent" polypeptides of the invention, and the Nanobodies present in such polypeptides will also be referred to herein as being in a "multivalent format". For example a "bivalent" polypeptide of the invention comprises two Nanobodies, optionally linked via a linker sequence, whereas a "trivalent" polypeptide of the invention comprises three Nanobodies, optionally linked via two linker sequences; etc.: in which at least one of the Nanobodies present in the polypeptide, and up to all of the Nanobodies present in the polypeptide, is/are a Nanobody of the invention. In a multivalent polypeptide of the invention, the two or more Nanobodies may be the same or different, and may be directed against the same antigen or antigenic determinant (for example against the same ρart(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. For example, a bivalent polypeptide of the invention may comprise (a) two identical Nanobodies; (b) a first Nanobody directed against a first antigenic determinant of a protein or antigen and a second Nanobody directed against the same antigenic determinant of said protein or antigen which is different from the first Nanobody; (c) a first Nanobody directed against a first antigenic determinant of a protein or antigen and a second Nanobody directed against another antigenic determinant of said protein or antigen; or (d) a first Nanobody directed against a first protein or antigen and a second Nanobody directed against a second protein or antigen (i.e. different from said first antigen). Similarly, a trivalent polypeptide of the invention may, for example and without being limited thereto, comprise (a) three identical Nanobodies; (b) two identical Nanobody against a first antigenic determinant of an antigen and a third Nanobody directed against a different antigenic determinant of the same antigen; (c) two identical Nanobody against a first antigenic determinant of an antigen and a third Nanobody directed against a second antigen different from said first antigen; (d) a first Nanobody directed against a first antigenic determinant of a first antigen, a second Nanobody directed against a second antigenic determinant of said first antigen and a third Nanobody directed against a second antigen different from said first antigen; or (e) a first Nanobody directed against a first antigen, a second Nanobody directed against a second antigen different from said first antigen, and a third Nanobody directed against a third antigen different from said first and second antigen.

Polypeptides of the invention that contain at least two Nanobodies, in which at least one Nanobody is directed against a first antigen (i.e. against Ephrins and/or Eph,) and at least one Nanobody is directed against a second antigen (i.e. different from Ephrins and/or Eph,), will also be referred to as "multispecific" polypeptides of the invention, and the Nanobodies present in such polypeptides will also be referred to herein as being in a "multispecific format". Thus, for example, a "bispecific'^" polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigen (i.e. Ephrins and/or Eph,) and at least one further Nanobody directed against a second antigen (i.e. different from Ephrins and/or Eph,), whereas a '"tri specific" polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigen (i.e. Ephrins and/or Eph,), at least one further Nanobody directed against a second antigen (i.e. different from Ephrins and/or Eph,) and at least one further Nanobody directed against a third antigen (i.e. different from both Ephrins and/or Eph, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against Ephrins and/or Eph. and a second Nanobody directed against a second antigen, in which said first and second Nanobody may optionally be linked via a linker sequence (as defined herein); whereas a trispecific polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against Ephrins and/or Eph, a second Nanobody directed against a second antigen and a third Nanobody directed against a third antigen, in which said first, second and third Nanobody may optionally be linked via one or more, and in particular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multispecific polypeptide of the invention may comprise at least one Nanobody against Ephrins and/or Eph, and any number of Nanobodies directed against one or more antigens different from Ephrins and/or Eph.

Furthermore, although it is encompassed within the scope of the invention that the specific order or arrangement of the various Nanobodies in the polypeptides of the invention may have some influence on the properties of the final polypeptide of the invention (including but not limited to the affinity, specificity or avidity for Ephrins and/or Eph, or against the one or more other antigens), said order or arrangement is usually not critical and may be suitably chosen by the skilled person, optionally after some limited routine experiments based on the disclosure herein. Thus, when reference is made to a specific multivalent or multispecific polypeptide of the invention, it should be noted that this encompasses any order or arrangements of the relevant Nanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that the polypeptides of the invention contain two or more Nanobodies and one or more further amino acid sequences (as mentioned herein). For multivalent and multispecific polypeptides containing one or more V_HH domains and their preparation, reference is also made to Conrath et ai, J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (200I)₅ 277-302; as well as to for example WO 96/34103 and WO 99/23221. Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that provides for an increased half-life. Such Nanobodies may for example be Nanobodies that are directed against a serum protein, and in particular a human serum protein, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one of the serum proteins listed in WO 04/003019. Of these, Nanobodies that can bind to serum albumin (and in particular human serum albumin) or to IgG (and in particular human IgG, see for example Nanobody VH-I described in the review by Muyldermans. supra) are particularly preferred (although for example, for experiments in mice or primates, Nanobodies against or cross-reactive with mouse serum albumin (MSA) or serum albumin from said primate, respectively, can be used. However, for pharmaceutical use, Nanobodies against human serum albumin or human IgG will usually be preferred). Nanobodies that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies directed against serum albumin that are described in WO 04/041865, in WO 06/122787 and in the further patent applications by Ablynx N.V,, such as those mentioned above. For example, the some preferred Nanobodies that provide for increased half-life for use in the present invention include Nanobodies that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787); Nanobodies that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see for example WO 06/0122787); Nanobodies that have or can provide an increased half-life (see for example the US provisional application 60/843,349 by Ablynx N.V mentioned herein); Nanobodies against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ur sinus)) (see for example the US provisional application 60/843,349 by Ablynx N.V); Nanobodies that can bind to serum albumin in a pH independent manner (see for example the US provisional application 60/850,774 by Ablynx N.V. mentioned herein) and/or Nanobodies that are conditional binders (see for example the US provisional application 60/850,775 by Ablynx N /V.).

Some particularly preferred Nanobodies that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies ALB-I to ALB-IO disclosed in WO 06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly preferred. According to a specific, but non-limiting aspect of the invention, the polypeptides of the invention contain, besides the one or more Nanobodies of the invention, at least one Nanobody against human serum albumin. Generally, any polypeptides of the invention with increased half-life thai contain one or more Nanobodies of the invention, and any derivatives of Nanobodies of the invention or of such polypeptides that have an increased half-life, preferably have a half-life that is at least 1.5 limes, 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 Nanobody of the invention per se. For example, such a derivative or polypeptides 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 Nanobody of the invention per se. In a preferred, but non-limiting aspect of the invention, such derivatives or polypeptides may 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. For example, such derivatives or polypeptides may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable of binding to one or more molecules which can increase the half-life of the polypeptide in vivo. The polypeptides of the invention are stabilised in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.

Another preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such Nanobodies include Nanobodies that are directed towards specific cell-surface proteins, markers or epitopes of the desired organ, tissue or cell (for example cell-surface markers associated with tumor cells), and the single- domain brain targeting antibody fragments described in WO 02/057445 and WO 06/040153, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies and the one or more polypeptides may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecifϊc polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers mat are used in the ait to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent V_H and V₁, domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each Nanobody by itself forms a complete antigen -binding site).

For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly_xser_y)_z, such as (for example (gly₄ser)₃ or (giysse^, as described in WO 99/42077 and the GS30. GS 15, GS9 and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825). as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678 ).

Some other particularly preferred linkers are poly-alanine (such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825). Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, poly(ethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026. It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for Ephrins and/or Eph, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

For example, in multivalent polypeptides of the invention that comprise Nanobodies directed against a multimeric antigen (such as a multimeric receptor or other protein), the length and flexibility of the linker are preferably such that it allows each Nanobody of the invention present in the polypeptide to bind to the antigenic determinant on each of the subunits of the multimer. Similarly, in a multispecific polypeptide of the invention that comprises Nanobodies directed against two or more different antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor, channel or protein), the length and flexibility of the linker are preferably such that it allows each Nanobody to bind to Its intended antigenic determinant. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g. as described herein for the derivatives of the Nanobodies of the invention). For example. linkers containing one or more charged amino acid residues (see Table A- 2 above) can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a polypeptide of the invention comprises three of more Nanobodies, it is possible to link them by use of a linker with three or more '"arms", which each "'arm" being linked to a Nanobody, so as to provide a '"star-shaped" construct. It is also possible, although usually less preferred, to use circular constructs.

The invention also comprises derivatives of the polypeptides of the invention, which may be essentially analogous to the derivatives of the Nanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that "essentially consist" of a polypeptide of the invention (in which the wording "essentially consist of^* has essentially the same meaning as indicated hereinabove).

According to one aspect of the invention, the polypeptide of the invention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids of the invention can be prepared in a manner known per sc, as will be clear to the skilled person from the further description herein. For example, the Nanobodies and polypetides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the amino acid sequences, Nanobodies, polypeptides and nucleic acids include the methods and techniques described herein.

As will be clear to the skilled person, one particularly useful method for preparing an amino acid sequence, Nanobody and/or a polypeptide of the invention generally comprises the steps of: i) the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said amino acid sequence, Nanobody or polypeptide of the invention (also referred to herein as a "nucleic acid of the invention"), optionally followed by: ii) isolating and/or purifying the amino acid sequence, Nanobody or polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of: i) cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one amino acid sequence. Nanobody and/or polypeptide of the invention; optionally followed by: ii) isolating and/or purifying the amino acid sequence, Nanobody or polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).

According to one aspect of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein. The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. To provide analogs, nucleotide sequences encoding naturally occurring V_HH domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least one nucleotide sequence encoding a Nanobody and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner. Techniques for generating the nucleic acids of the invention will be clear Io the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site- directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers, using for example a sequence of a naturally occurring form of Ephrins and/or Eph as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al.. mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of. be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention'^'. The genetic constructs of the invention may be DNA or RNA, and are preferably double- stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting aspect, a genetic construct of the invention comprises i) at least one nucleic acid of the invention; operably connected to ii) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also iii) one or more further elements of genetic constructs known per se; in which the terms "regulatory element", "promoter", "terminator" and "operably connected" have their usual meaning in the art (as further described herein); and in which said "further elements" present in the genetic constructs may for example be 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to the skilled person, and may for instance depend upon the type of construct used, the intended host cell or host organism; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g. via constitutive, transient or inducible expression); and/or the transformation technique to be used. For example, regulatory requences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in an essentially analogous manner.

Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the invention and said regulatory elements, and. optionally said one or more further elements, are "operably linked" to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered "operably linked" to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of said promotor). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.

Preferably, the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism.

For instance, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant that (for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked (as defined herein).

Some particularly preferred promoters include, but are not limited to, promoters known per se for the expression in the host cells mentioned herein; and in particular promoters for the expression in the bacterial cells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows - i.e. under appropriate selection conditions - host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.

A leader sequence should be such that - in the intended host cell or host organism - it allows for the desired post-translational modifications and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism. Leader sequences may not be required for expression in a bacterial cell. For example, leader sequences known per se for the expression and production of antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments) may be used in an essentially analogous manner. An expression marker or reporter gene should be such that - in the host cell or host organism - it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also allow for the localisation of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP. Some preferred, but non-limiting examples of suitable promoters, terminator and further elements include those that can be used for the expression in the host cells mentioned herein; and in particular those that are suitable for expression in bacterial cells, such as those mentioned herein and/or those used in the Examples below. For some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention - such as terminators, transcriptional and/or translational enhancers and/or integration factors - reference is made to the general handbooks such as Sambrook et al. and Ausubel et al. mentioned above, as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191 , WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, US-A-7,207,410, US-A- 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Reference is also made to the general background art cited above and the further references cited herein.

The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the Examples below, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the amino acid sequence, Nanobody or polypeptide of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, for example: a bacterial strain, including but not limited to gram-negative strains such as strains of Escherichia coli; of Proteus, for example of Proteus mirabilis; of Pseudomonas, for example of Pseudomonas fluorescens; and gram-positive strains such as strains of

Bacillus, for example of Bacillus subtilis or of Bacillus brevis; of Streptomyces, for example of ^' Streptomyces lividans; of Staphylococcus, for example of Staphylococcus carnosus; and of Lactococcus, for example of Lactococcus lactis; a fungal cell, including but not limited to cells from species of Trichoderma, for example from Trichoderma reesei; of Neurospora, for example from Neυrospora crassa; of Sordaria, for example from Sordaria macrospora; of Aspergillus, for example from Aspergillus niger or from Aspergillus sojae; or from other filamentous fungi; a yeast cell, including but not limited to cells from species of Saccharomyces. for example of Saccharomyces cerevisiae; of Schizosaccharomyces_^ for example of Schi∑osaccharomyces pombe; of Pichia, for example of Pichia pastoris or of Pichia methanolica: of Hansenula, for example of Hansenula polymorpha\ of Kluyveromyces, for example of Kluyveromyces lactis; of Arxula, for example of Arxula adeninivorans; of Yarrowia. for example of Yarrowia lipolytica; an amphibian cell or cell line, such as Xenopus oocytes; - an insect-derived cell or cell line, such as cells/cell lines derived from lepidoptera, including but not limited to Spodoptera SF9 and Sf21 cells or cells/cell lines derived from Drosophila, such as Schneider and Kc cells; a plant or plant cell, for example in tobacco plants; and/or a mammalian cell or cell line, for example a cell or cell line derived from a human, a cell or a cell line from mammals including but not limited to CHO-cells, BHK-cells (for example BHK-21 cells) and human cells or cell lines such as HeLa, COS (for example

COS-7) and PER.C6 cells; as well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the skilled person. Reference is also made to the general background art cited hereinabove, as well as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et al., (1998), supra; Riechmann and Muyldermans, (1999), supra; van der Linden, (2000), supra; Thomassen et al., (2002), supra; Joosten et al., (2003), supra: Joosten et al., (2005), supra; and the further references cited herein. The amino acid sequences, Nanobodies and polypeptides of the invention can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g. as a gene therapy). For this purpose, the nucleotide sequences of the invention may be introduced into the cells or tissues in any suitable way, for example as such (e.g. using liposomes) or after they have been inserted into a suitable gene therapy vector (for example derived from retroviruses such as adenovirus, or parvoviruses such as adeno-associated virus). As will also be clear to the skilled person, such gene therapy may be performed in vivo and/or in situ in the body of a patient by administering a nucleic acid of the invention or a suitable gene therapy vector encoding the same to the patient or to specific cells or a specific tissue or organ of the patient; or suitable cells (often taken from the body of the patient to be treated, such as explanted lymphocytes, bone marrow aspirates or tissue biopsies) may be treated in vitro with a nucleotide sequence of the invention and then be suitably (re-)introduced into the body of the patient. All this can be performed using gene therapy vectors, techniques and delivery systems which are well known to the skilled person, and for example described in Culver, K. W., "Gene Therapy", 1994. p. xii, Mary Ann Liebert, Inc., Publishers, New York, N. Y); Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911 -919; Anderson, Science 256 (1992),808-813: Verma, Nature 389 (1994),239; Isner, Lancet 348 (1996),370-374; Muhlhauser, Circ. Res. 77 (1995),1077-1086; Onodera, Blood 91; (1998),30- 36; Verma, Gene Ther. 5 (1998),692-699; Nabel, Ann. N. Y. Acad. Sci. : 81 1 (1997), 289-292: Verzeletti, Hum. Gene Ther. 9 (1998). 2243-51 ; Wang, Nature Medicine 2 (1996),714-716; WO 94/29469; WO 97/00957, US 5,580,859; US 5,5895466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. For example, in situ expression of ScFv fragments (Afanasieva et al, Gene Ther., 10, 1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has been described in the art.

For expression of the Nanobodies in a cell, they may also be expressed as so-called "intrabodies", as for example described in WO 94/02610, WO 95/22618 and US-A-7004940; WO 03/014960; in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer- Ver lag; and in Kontermann, Methods 34, (2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the invention can for example also be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep (see for example US-A-6,741,957. US-A-6,304,489 and US-A- 6,849,992 for general techniques for introducing transgenes into mammals), in plants or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or turbers (for example in tobacco, maize, soybean or alfalfa) or in for example pupae of the silkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Some preferred, but non-limiting examples include expression in the wheat germ system: in rabbit reticulocyte lysates; or in the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies is that the polypeptides based thereon can be prepared through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, host cells, regulatory elements, etc.. will be clear to the skilled person, for example from the references cited above. It should however be noted that the invention in its broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention in a form that is suitable for pharmaceutical use, and such expression systems will again be clear to the skilled person. As also will be clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the (industrial) production of Nanobodies or Nanobody-containing protein therapeutics include strains of E. coli, Pichia pastoris, S. cerevisiae that are suitable for large scale expression/production/fermentation, and in particular for large scale pharmaceutical (i.e. GMP grade) expression/production/fermentation. Suitable examples of such strains will be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above. The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a Nanobody-containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e. leading to a protein that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mimics human glycosylation. Generally, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast usually leads to a glycosylation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cells and expression systems can be used in the invention, depending on the desired amino acid sequence, Nanobody or polypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the amino acid sequence. Nanobody or polypeptide of the invention is glycosylated. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is non-glyco sy lated .

According to one preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above.

According to another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acid sequences, Nanobodies and the polypeptides of the invention, the amino acid sequences. Nanobodies and polypeptides of the invention can be produced either intracellularly (e.g. in the cytosol. in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracelhilarly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. When eukaryotic host cells are used, extracellular production is usually preferred since this considerably facilitates the further isolation and downstream processing of the Nanobodies and proteins obtained. Bacterial cells such as the strains of E. coli mentioned above normally do not secrete proteins extracellularly, except for a few classes of proteins such as toxins and hemolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane to the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product can be identical to the natural gene product after cleavage of the secretion signal sequence by a specific signal peptidase. Also, there appeal's to be much less protease activity in the periplasm than in the cytoplasm. In addition, protein purification is simpler due to fewer contaminating proteins in the periplasm. Another advantage is that correct disulfide bonds may form because the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, so-called inclusion bodies. These inclusion bodies may be located in the cytosol or in the periplasm; the recovery of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. Many recombinant proteins, including therapeutic proteins, are recovered from inclusion bodies. Alternatively, as will be clear to the skilled person, recombinant strains of bacteria that have been genetically modified so as to secrete a desired protein, and in particular an amino acid sequence, Nanobody or a polypeptide of the invention, can be used. Thus, according to one non-limiting aspect of the invention, the amino acid sequence,

Nanobody or polypeptide of the invention is an amino acid sequence. Nanobody or polypeptide that has been produced intracellularly and that has been isolated from the host cell, and in particular from a bacterial cell or from an inclusion body in a bacterial cell. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is an amino acid sequence, Nanobody or polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host cell is cultivated. Some preferred, but non-limiting promoters for use with these host cells include, for expression in E. colt lac promoter (and derivatives thereof such as the lacUV5 promoter); arabinose promoter; left- (PL) and rightward (PR) promoter of phage lambda; promoter of the trp operon; hybrid lac/trp promoters (tac and trc); T7-promoter (more specifically that of T7-phage gene 10) and other T-phage promoters; promoter of the TnIO tetracycline resistance gene; engineered variants of the above promoters that include one or more copies of an extraneous regulatory operator sequence; for expression in S. cerevisiae: constitutive: ADHl (alcohol dehydrogenase 1), ENO (enolase), CYCl (cytochrome c iso-1). GAPDH (glyceraldehydes-3 -phosphate dehydrogenase), PGKl (phosphoglycerate kinase). PYKl (pyruvate kinase); regulated:

GAL L 10,7 (galactose metabolic enzymes), ADH2 (alcohol dehydrogenase 2), PHO5 (acid phosphatase), CUPl (copper metallothionein): heterologous: CaMV (cauliflower mosaic virus 35S promoter); for expression in Pichia pastoris: the AOXl promoter (alcohol oxidase I); for expression in mammalian cells: human cytomegalovirus (hCMV) immediate early enhancer/promoter: human cytomegalovirus (hCMV) immediate early promoter variant that contains two tetracycline operator sequences such that the promoter can be regulated by the Tet repressor; Herpes Simplex Virus thymidine kinase (TK) promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR) enhancer/promoter; elongation factor lα (hEF-lα) promoter from human, chimpanzee, mouse or rat: the SV40 early promoter; HIV-I long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cells include: vectors for expression in mammalian cells: pMAMneo (Clontech), pcDNAS (Invitrogen), pMClneo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146). pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565), as well as viral-based expression systems, such as those based on adenovirus; vectors for expression in bacterial cells: pET vectors (Novagen) and pQE vectors (Qiagen); vectors for expression in yeast or other fungal cells: pYES2 (Invitrogen) and Pichia expression vectors (Invitrogen); vectors for expression in insect cells: pBlueBacII (ϊnvitrogen) and other baculovirus vectors vectors for expression in plants or plant cells: for example vectors based on cauliflower mosaic virus or tobacco mosaic virus, suitable strains of Agrobacterium, or Ti-plasmid based vectors.

Some preferred, but non-limiting secretory sequences for use with these host cells include: for use in bacterial cells such as E. colt PeIB. BIa, OmpA, OmpC, OmpF, OmpT, StIL PhoA. PhoE. MaIE, Lpp, LamB, and the like; TAT signal peptide, hemolysin C- terminal secretion signal; for use in yeast: α-mating factor prepro- sequence, phosphatase (phol). invertase (Sue), etc.; for use in mammalian cells: indigenous signal in case the target protein is of eukaryotic origin; murine Ig ic-chain V-J2-C signal peptide; etc. Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.

After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g. using specific antibodies.

The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions), an amino acid sequence, Nanobody or polypeptide of the invention (and in case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention that may for instance be obtained by cell division or by sexual or asexual reproduction. To produce/obtain expression of the amino acid sequences of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) amino acid sequence, Nanobody or polypeptide of the invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the amino acid sequences of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequence, Nanobody or polypeptide of the invention may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell/host organism used. Also, the amino acid sequence, Nanobody or polypeptide of the invention may be glycosylated, again depending on the host cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention may then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using a specific, cleavable amino acid sequence fused with the amino acid sequence, Nanobody or polypeptide of the invention) and/or preparative immunological techniques (i.e. using antibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation or compositions comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation maj be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein. Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one amino acid of the invention, at least one Nanobody of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances. Generally, the amino acid sequences, Nanobodies and polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865 and WO 04/041867) as well as to the standard handbooks, such as Remington^'s Pharmaceutical Sciences, 18^th Ed., Mack Publishing Company, USA (1990) or Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005).

For example, the amino acid sequences, Nanobodies and polypeptides of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv' s and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra- arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, sterile water and aqueous buffers and solutions such as physiological phosphate-buffered saline. Ringer's solutions, dextrose solution, and Hank's solution; water oils; glycerol; ethanol; glycols such as propylene glycol or as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof. Usually, aqueous solutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Patent No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene encoding an amino acid sequence, Nanobody or polypeptide of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumours, or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression. Thus, the amino acid sequences, Nanobodies and polypeptides of the invention may be systemicaliy administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient^'s diet. For oral therapeutic administration, the amino acid sequences. Nanobodies and polypeptides of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the amino acid sequence, Nanobody or polypeptide of the invention. Their percentage in the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the amino acid sequence, Nanobody or polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen. or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the amino acid sequences, Nanobodies and polypeptides of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the amino acid sequences. Nanobodies and polypeptides of the invention may be incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also be provided with an enteric coating that will allow the constructs of the invention to resist the gastric environment and pass into the intestines. More generally, preparations and formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the invention may also be administered intravenously or intraperitoncally by infusion or injection. Solutions of the amino acid sequences, Nanobodies and polypeptides of the invention or their salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use. these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars. buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example. aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the amino acid sequences, Nanobodies and polypeptides of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the amino acid sequences. Nanobodies and polypeptides of the invention may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water- alcohol/glycol blends, in which the amino acid sequences. Nanobodies and polypeptides of the invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the amino acid sequences, Nanobodies and polypeptides of the invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992.478). Smith et al. (U.S. Pat. No. 4,559, 157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptides of the invention can be determined by comparing their in vitro activity, and in vivo activity in • animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, Nanobodies and polypeptides of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt- %.

The amount of the amino acid sequences, Nanobodies and polypeptides of the invention required for use in treatment will vary not only with the particular amino acid sequence, Nanobody or polypeptide selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the amino acid sequences, Nanobodies and polypeptides of the invention varies depending on the target cell, tumor, tissue, graft, or organ. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye. An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E. W.. ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication.

In another aspect, the invention relates to a method for the prevention and/or treatment of at least one cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In the context of the present invention, the term "prevention and/or treatment'^" not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated. The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention relates to a method for the prevention and/or treatment of at least one disease or disorder that is associated with Ephrins and/or Eph, with its biological or pharmacological activity, and/or with the biological pathways or signalling in which Ephrins and/or Eph is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be treated by modulating Ephrins and/or Eph, its biological or pharmacological activity, and/or the biological pathways or signalling in which Ephrins and/or Eph is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, said pharmaceutically effective amount may be an amount that is sufficient to modulate Ephrins and/or Eph, its biological or pharmacological activity, and/or the biological pathways or signalling in which Ephrins and/or Eph is involved; and/or an amount that provides a level of the amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention in the circulation that is sufficient to modulate Ephrins and/or Eph, its biological or pharmacological activity, and/or the biological pathways or signalling in which Ephrins and/or Eph is involved.

The invention furthermore relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence of the invention, a Nanobody of the invention or a polypeptide of the invention to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. More in particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder chosen from the group consisting of the diseases and disorders listed herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same, In the above methods, the amino acid sequences, Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, the amino acid sequences. Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can for example be administered orally, intraperitoneally (e.g. intravenously, subcutaneously. intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), intranasally, transdermally, topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. The clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the clinician. The amino acid sequences. Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific amino acid sequence. Nanobody or polypeptide of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician. Generally, the treatment regimen will comprise the administration of one or more amino acid sequences, Nanobodies and/or polypeptides of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to administered can be determined by the clinician, again based on the factors cited above. Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency of the specific amino acid sequence. Nanobody and polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the amino acid sequences, Nanobodies and polypeptides of the invention will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1, 10. 100 or 1000 microgram per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, talcing into account however differences in affinity/avidity, efficacy, biodistribution. half-life and similar factors well known to the skilled person. Usually, in the above method, a single amino acid sequence, Nanobody or polypeptide of the invention will be used. It is however within the scope of the invention to use two or more amino acid sequences, Nanobodies and/or polypeptides of the invention in combination. The Nanobodies, amino acid sequences and polypeptides of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e. as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement. In particular, the amino acid sequences, Nanobodies and polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.

When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side- effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved. as will be ciear to the clinician. The clinician will also be able, where appropriate and on a case -by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side -effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand. Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

In another aspect, the invention relates to the use of an amino acid sequence,

Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one cancers such as lung, breast, and brain cancer,

CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases; and/or for use m one or more of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention also relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence, Nanobody or polypeptide of the invention to a patient.

More in particular, the invention relates to the use of an amino acid sequence,

Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of cancers such as lung, breast, and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, and infectious diseases, and in particular for the prevention and treatment of one or more of the diseases and disorders listed herein. Again, in such a pharmaceutical composition, the one or more amino acid sequences, Nanobodies or polypeptides of the invention may also be suitably combined with one or more other active principles, such as those mentioned herein.

Finally, although the use of the Nanobodies of the invention (as defined herein) and of the polypeptides of the invention is much preferred, it will be clear that on the basis of the description herein, the skilled person will also be able to design and/or generate, in an analogous manner, other amino acid sequences and in particular (single) domain antibodies against Ephrins and/or Eph. as well as polypeptides comprising such (single) domain antibodies. For example, it will also be clear to the skilled person that it may be possible to

"graft^'' one or more of the CDR' s mentioned above for the Nanobodies of the invention onto such (single) domain antibodies or other protein scaffolds, including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be clear to the skilled person and are well known in the art, see for example US- A-7,180,370, WO 01/27160, EP 0 605 522, EP 0 460 167, US-A-7,054,297, Nicaise et al, Protein Science (2004), 13: 1882-1891; Ewert et al.. Methods. 2004 OcI; 34(2): 184-199; Kettleborough et al., Protein Eng. 1991 Oct; 4(7): 773-783; O'Brien and Jones, Methods MoI. Biol. 2003: 207: 81-100; Skerra, J. MoI. Recognit. 2000: 13: 167-187, and Saerens et al.. J. MoI. Biol. 2005 Sep 23;352(3):597-607, and the further references cited therein. For example, techniques known per se for grafting mouse or rat CDR' s onto human frameworks and scaffolds can be used in an analogous manner to provide chimeric proteins comprising one or more of the CDR' s of the Nanobodies of the invention and one or more human framework regions or sequences.

It should also be noted that, when the Nanobodies of the inventions contain one or more other CDR sequences than the preferred CDR sequences mentioned above, these CDR sequences can be obtained in any manner known per se, for example from Nanobodies (preferred), V_H domains from conventional antibodies (and in particular from human antibodies), heavy chain antibodies, conventional 4-chain antibodies (such as conventional human 4-chain antibodies) or other immunoglobulin sequences directed against Ephrins and/or Eph. Such immunoglobulin sequences directed against Ephrins and/or Eph can be generated in any manner known per se, as will be clear to the skilled person, i.e. by immunization with Ephrins and/or Eph or by screening a suitable library of immunoglobulin sequences with Ephrins and/or Eph, or any suitable combination thereof. Optionally, this may be followed by techniques such as random or site-directed mutagenesis and/or other techniques for affinity maturation known per se. Suitable techniques for generating such immunoglobulin sequences will be clear to the skilled person, and for example include the screening techniques reviewed by Hoogenboom, Nature Biotechnology, 23, 9, 1105-1116 (2005) Other techniques for generating immunoglobulins against a specified target include for example the Nanoclone technology (as for example described in the published US patent application 2006-021 1088). so-called SLAM technology (as for example described in the European patent application 0 542 810), the use of transgenic mice expressing human immunoglobulins or the well-known hybridoma techniques (see for example Larrick el al. Biotechnology, Vol.7, 1989, p. 934). All these techniques can be used to generate immunoglobulins against Ephrins and/or Eph, and the CDR' s of such immunoglobulins can be used in the Nanobodies of the invention, i.e. as outlined above. For example, the sequence of such a CDR can be determined, synthesized and/or isolated, and inserted into the sequence of a Nanobody of the invention (e.g. so as to replace the corresponding native CDR), all using techniques known per se such as those described herein, or Nanobodies of the invention containing such CDR' s (or nucleic acids encoding the same) can be synthesized de novo. again using the techniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, polypeptides, nucleic acids, genetic constructs and hosts and host cells of the invention will be clear to the skilled person based on the disclosure herein. For example, and without limitation, the amino acid sequences of the invention can be linked to a suitable carrier or solid support so as to provide a medium than can be used in a manner known per se to purify Ephrins and/or Eph from compositions and preparations comprising the same. Derivatives of the amino acid sequences of the invention that comprise a suitable detectable label can also be used as markers to determine (qualitatively or quantitatively) the presence of Ephrins and/or Eph in a composition or preparation or as a marker to selectively detect the presence of Ephrins and/or Eph on the surface of a cell or tissue (for example, in combination with suitable cell sorting techniques).

The invention will now be further described by means of the following non-limiting Figure 1. Representative result of a competitive binding ELISA showing the neutralizing activity of nanobodies (in periplasm fraction) for mEphA2. niEphrinAl-Fc is coated on maxisorp (0.5ug/ml in lOOul PBS) and binding of biotynilated mEphA2 (0. lug/ml in 10 OuI l%skimmed milk/PBS) is detected with Streptavidine-HRPO. Nanobodies are selected as in example 3b. In dark background color there is the periplasm fraction containing EphA2 competing nanobodies (low signal, less than 0.1 arbitrary unit). In light background color there is the nanobodies partially competing (value less than 0.12 arbitrary unit).

The invention will now be further described by means of the following non-limiting experimental part.

Experimental Part:

Example 1: Immunizations

Two llamas were immunized according to standard protocols with 6 boosts of a cocktail (2x40ug + 4x20ug, containing mouse EphrinAl and EphrinB2 (llama 100 and 101) or mouse EphA2 and EphB4 (llama 164 and 165) or human EphBό (llama 171 and 172)). Blood was collected from these animals at 6 and 10 days after Die 6^th boost.

All proteins were recombinant proteins fused to human Fc and purchased from R&D systems: mEphrhiAl -Fc-HIS (Cat. No 602-A1 ) mEphrinB2-Fc-HIS (Cat. No 496-EB) mEphA2/Fc-HIS (Cat. No 639-A2) niEphB4-Fc-HIS (Cat. No 446-B4) hEPHB6-Fc (Cat. No3384-B6)

Example 2; Library construction

Peripheral blood mononuclear cells were prepared from blood samples using Ficoll- Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells as well as from the lymph node bow cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored at 4°C for further use, making phage libraries 100b, 101b, 164, 165, 173 and 174 (according to the immunized Llama). pAX50: An expression vector was used derived from pUCl 19 which contained the LacZ promoter, a coliphage pill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector coded for a C -terminal c-myc tag and a (His)6 tag.

Example 3a: Selections

To identify nanobodies recognizing Eph and Ephrin, corresponding phage libraries were used for selections on the immunized proteins. The proteins were immobilized independently at 5 μg/ml, 0.5 ug/ml or 0ug/ml (control) on Nunc Maxisorp ELISA plates (10OuI per wells). Phages were added in a PBS solution containing 4% skimmed milk and total human IgG (250ug/ml). After incubation and extensive washing, the bound phages were eluted using trypsine (lmg/ml) as in standard protocols.

Output of both Ri selections were analyzed for enrichment factor (phage present in eluate relative to controls). Based on these parameters the best selections were chosen for further analysis. When enrichment was not satisfactory, a second round of selection was done using the same condition as the first round, giving R2 output. In both cases, the polyclonal outputs were then recloned in pAX51 and individual

TGl colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for nanobody expression. Periplasmic extracts (volume: ~ 100 ul) were prepared according to standard methods (see for example the prior ait and applications filed by applicant cited herein). pAX51 : An expression vector was used derived from pUCl 19 which contained the LacZ promoter, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector codes for a C-terminal c-myc tag and a (His)6 tag.

Example 3b: Selections of neutralizing nanobodies. To identify nanobodies specifically neutralizing the Eph-Ephrin interaction, specific competitive elution were done during R2 (phage rescued from Rl are used): After coating of 0.5ug/ml (lOOul/well) and phage binding as in Selection 3a, the bound phages were eluted with 50ug/ml (100ul/well) of:

Ephrin Al when EphA2 was coated

Ephrin B2 when EphB4 or EphB6 was coated.

EphB2 when Ephrin Bl was coated.

EphA2 when Ephrin Al was coated.

Output of R2 selections were rescued individual colonies containing expressing nanobodies are picked, grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for nanobody expression. Periplasmic extracts (volume: - 100 ul) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 4: Screening for binding

In order to determine binding specificity to the Eph/Ephrin, the clones were tested in an ELISA binding assay setup:

In short, 2ug/ml of protein or 20ug of total human IgG (control) were immobilized directly on Maxisorp microliter plates (Nunc). Free binding sites were blocked using 200ul of 4% Marvel (skimmed milk) in PBS. Next, 1 OOul of 2% Marvel PBS containing 7.5 ul of periplasmic extract within the nanobody of the different clones were allowed to bind to the immobilized antigens. After incubation and washing, nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with an AP- conjugated goat- anti-mouse antibody and a pNPP solution. Binding specificity was determined based on OD values compared to controls (as shown in Table B-I).

Example 5: Screening for neutralizing activity

In order to determine neutralizing activity of the nanobodies, the clones were tested in a receptor/ligand binding assay (Competitive ELISA). Briefly, the Eph (or Ephrin) was coated in 96 wells (Maxisorp, Nunc). After washing and blocking as usual, the coated Eph (or Ephrin) was incubated with 15ul periplasmic fraction prepared from above. Finally, specific biotinylated ligand (or receptor) was added. After washing, the presence of bound biotinylated-ligand (or receptor) was detected using streptavidine-HRPO antibody. In the case where a nanobody present in the periplasm neutralizes the receptor (i.e. competes for ligand binding), no ligand bound was detected. In all cases, the concentration of protein tested was used to get sub-optimal response (see Table B-7).

For mEphA2, biotinylated-mEphrinAl was used.

For mEphiϊnAL biotinylated-mEphA2 was used.

For mEphB4, biotinylated-mEphrinB2 was used.

Above Nanobodies with partial or full blocking properties are considered EpMnAl blocker or neutralizing antibodies of EphrinAl and in particular of human and/or mouse EphrinAl . Further functional assay to confirm this function can be found later in this experimental part.

Above Nanobodies with partial or full blocking properties are considered antagonists of mEphA2 and in particular of human and/or mouse EphA2. Further functional assay to confirm this function can be found later in this experimental part.

Above Nanobodies with partial or full blocking properties are considered antagonists of mEphB4 and in particular of human and/or mouse EphB4. Further functional assay to confirm this function can be found later in this experimental part. Example 6: List of generat in vitro, celt-based or in vivo assays: In vitro

- Biacore, - FLIPR

- ELISA and Competitive ELISA

-In vitro binding assays: ELISA, FLIPR, competitive ELISA, Biacore

-In vivo binding assay: Flow cytometry

Cell based:

- Adhesion assay

- Eph activation (phsophorylation). - Induction of EC sprouting in a 3D spheroid-based assay

- Induction of EC chord-like structures on Matrigel matrix

- neurite outgrowth assay

Animal models: - Mouse model for neuronal regeneration (model for spinal cord injury, stroke disease)

- Anti-tumorigenic and anti-angio genie mouse model (xenograft experiment) - model for cancer

Frey test for mechanical allodynia. i.e. sensitivity to non-noxious mechanical stimuli, see e.g. Kobayashi et al, supra, 2007. - Collagen- antibody induced arthritis (CAIA), animal model of human rheumatoid arthritis (RA), see e.g. Kitamura et al, supra. 2007.

Example 7: List of particularly preferred embodiments of amino acid sequences of the invention:

• Amino acid sequence comprising e.g. 2 Nanobodies with antagonistic effect for the same target, e.g. EphAl to EphAl 0, such as e.g. EphA2; EphBl to EphB6 such as e.g. EphB4 or EphB6, either being directed against two different epitopes, or being against the same epitope.

♦ Amino acid sequence comprising a Nanobody against an Eph receptor, e.g. EphAl to EphAlO, such as e.g. EphA2; EphBl to EphB6 such as e.g. EphB4 or EphB6, and a Nanobody against an Ephrin, e.g. EphrinAl to EphrinAό or EphrinBl to EphrinB3 such as e.g. EphrinAl or EphrinB2.

• Amino acid sequence comprising a Nanobody against the EphA2 receptor and a Nanobody against EphrinAl ,

• Amino acid sequence comprising a Nanobody against the EphB4 receptor and a Nanobody against EphrinB2.

• Amino acid sequence comprising a cytotoxic compound (e.g. peptidic toxin, e.g. immunotoxήi) and a Nanobody wherein the said Nanobody is able to bind to any Eph or Ephrin, preferably a nanobody which may disrupt at least one of the Eph/Ephrin interactions, e.g. EphA2/Ephrinl or EphB4/EphrinB2 interactions. The amino acid sequences of the invention such as those presented e.g. in SEQ ID NOs: 714 to 797 may be used for targeting specific types of cancers.

• Amino acid sequences comprising a Nanbody which is directed against an "oncogenic" epitope of an Ephrin or Eph, e.g. EphA2, i.e. an epitope of an Eph. e.g. EphA2, which is only present or accessable on cancer cells (see Coffmann, supra. 2007).

Example 9: HOMOLOGIES of selected target proteins between mouse and human form:

m/h EPHA2 92% HOMOLOGY m/h EPHB4 92% HOMOLOGY m/h EPHB6 90% HOMOLOGY hEPHB4/hEPHB6 42% HOMOLOGY

m/h Ephrin-Al 85% HOMOLOGY m/h Ephrin-B2 95% HOMOLOGY

Example 10: Testing of ephrinAl fligand) / EphA2 (receptor) Nanobodies in a functional cellular (spreading) assay.

Reference:

Carter N, Nakamoto T. Hirai H, Hunter T. EphrinAl -induced cytoskeletal re-organization requires FAK and p!30(cas). Nat Cell Biol. 2002, 4, 565-73.

The coordinated migration of cells during development is a process that requires reorganization of the cell cytoskeleton through a series of signaling systems.

The Eph family of receptor tyrosine kinases (RTKs) and their ephrin ligands are important in cell segregation and axon targeting in the developing nervous system. Although there are differences in affinity between A-type ephrins and various A-type Eph RTKs, most A~type ephrins activate most EphA RTKs in vitro.

Carter et al. (Nat Cell Biol. 2002. 4, 565.) have shown that in immortalized mouse fibroblastic cell lines, primary (mouse embryonic fibroblasts) MEFs or MEF cell lines, the ephrinAl -EphA2 system promotes adhesion and cell spreading on an ephrinAl through a unique type of actin cytoskeletal re-organization at the cell periphery. EphA2 is the main RTK expressed in NIH3T3 cells and primary MEFs. Signaling through A- type Eph receptors positively regulates re-organization of the actin cytoskeleton and promotes cell spreading in murine fibroblasts.

Cells expressing Eph receptors in vivo are likely to encounter membrane-bound ephrins in a pseudo-two-dimensional array on the plasma membrane of a neighboring cell. Hence we will investigate the interaction between NIH3T3 cells in a two-dimensional array of ephrinAl.

NIH 3T3 fibroblasts (expressing EphA2) will be plated on ephrinAl -coated or poly D-Lysine (PDL) coverslips in presence of EphA2 or ephrinAl nanobodies vs. control nanbodies. EphrinAl is expected to promote cell adherence and spreading of the NIH 3T3 fibroblasts (expressing EphA2). as compared to control.

To determine whether the adherence of NIH3T3 cells to the ephrinAl -coated surface is caused by ephrinAl and/or its cognate receptor, EphA2, NIH3T3 cells will be plated in the presence of EphA2 nanobodies or ephrinAl nanobodies (respectively at 0.8, 4, 20, 100 and 500 nM). In both cases we expect adherence of the cells to ephrinAl to be reduced to control level, whereas inclusion of EphA2 nanobodies or ephrinAl nanobodies in the medium will most likely have no effect on NIH3T3 cell adherence to PDL.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in their entirety for the purpose and information indicated in the specification. Preferred embodiments:

1. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of EphAl , EphA2, EphA3, EphA4, EphA5. EphA6. EphA7, EphA8, EphΛ9, EphAl O₅

EphBl, EphB2, EphB3, EphB4, EphB5, EphB6. EphrinAl, EphrinA2, EphrinA3, EphrinA4, EphrinAS, EphrinAό. EphrinBl, EphrinB2, and EphrinB3.

2. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of EphAl, EphA2, EphA3, EphA4. EphA5, EphAό. EphA7. EphA8, EphA9, EphAl 0, EphBl, EphB2, EphB3, EphB4. EphB5, and EphB6.

3. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of EphrinAl , EphrinA2, EphrinA3. EphrinA4, EphrinAS, EphrinAό. EphrinBl, EphrinB2, and EphrinB3.

4. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of EphAl, EphA2, EphA3, EphA4, EphA5, EphAό, EphA7. EphA8. EphA9. and EphAl 0.

5. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting ofEphBl, EphB2, EphB3, EphB4, EphB5, and EphB6.

6. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of EphrinAl, EphrinA2, EphrinAS, EphrinA4. EphrinAS. and EphrinAβ. 7. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of EphrinBl, EphrinB2, and EphrinB3.

8. Amino acid sequence according to embodiment Ho 7 that is in essentially isolated form.

9. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of hEphΛl, hEphA2. hEphA3. hEphA4, hEphA5, hEphA6, hEphA7₅ hEphA8, hEphA9, hEphAlO, hEphBl, hEphB2, hEphB3, hEphB4, hEphBS, hEphBό. hEphrinAl, hEphrinA2, hEphrinA3, hEphrinA4, hEphrmAS, hEphrinAδ, hEphrinBl, hEphrinB2, and hEphrinBS.

10. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of hEphAl, hEphA2, hEphA3, hEphA4, hEphAS, hEpfiAό, hEphA7, hEphA8. hEphA9, hEphAlO, hEphBl, hEphB2, hEphB3, hEphB4. hEphBS, and hEphB6.

1 1. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of hEphrinAl, hEphrinA2. hEphrinA3, hEphrinA4. hEphrinAS. hEphrinAό, hEphrinBl, hEphrinB2, and hEphririB3.

12. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of hEphAl, hEphA2, hEphA3, hEphA4, hEphAS, hEphAό, hEphA7, hEphAS, hEphA9, and hEphAlO.

13. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting ofhEphBl. hEphB2, hEphBS, hEphB4. hEphBS, and hEphBό. 14. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of hEphrinAl, hEphrinA2, hEphririA3, hEphrinA4, hEphrinA5₅ and hEphrinAό.

15. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of hEphrinBl , hEphrinB2, and hEphrmB3.

16. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of the EphA2, EphB4, EphBό, EphrinAl, and EρhrinB2.

17. Amino acid sequence comprising at least one single variable domain that is directed against and/or that specifically binds to a protein selected from the group consisting of the human and/or mouse EphA2, EphB4. EphB6, EphrinAl , and EphrinB2,

18. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Eph, e.g. human and/or mouse Eph; and ii) blocks the interaction between said Eph, e.g. human and/or mouse Eph, and an Ephrin. e.g. human and/or mouse Ephrin.

19. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Eph, e.g. human and/or mouse Eph; and ii) blocks the interaction between said at least one Eph, e.g. human and/or mouse Eph, and at least one Ephrin. e.g. human and/or mouse Ephrin; and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis. c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis. whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein. 19a. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Eph. e.g. human and/or mouse Eph, e.g. EphA2 and/or EphB6; and ii) blocks the interaction between said at least one Eph, e.g. human and/or mouse Eph, and at least one Ephrin, e.g. human and/or mouse Ephrin; and iii) wherein said amino acid sequence has an inhibitory effect on angiogenesis, whereas said biological effect or effects may e.g. be measured in in vitro cell based or animal assays, e.g. in a xenograft experiment.

19b. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Eph, e.g. human and/or mouse Eph, e.g. EphA4 and/or EphA5; and ii) blocks the interaction between said at least one Eph, e.g. human and/or mouse Eph. and at least one Ephrin, e.g. human and/or mouse Ephrin; and iii) wherein said amino acid sequence has a growth promoting effect on neurite outgrowth, whereas said biological effect or effects may e.g. be measured in in vitro cell based or animal assays, e.g. in a neurite outgrowth assay.

19c. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Eph, e.g. human and/or mouse Eph; and ii) blocks the interaction between said at least one Eph, e.g. human and/or mouse Eph, and at least one Ephrin, e.g. human and/or mouse Ephrin, e.g. EphrinB2; and iii) wherein said amino acid sequence has an inhibitory effect on neuropatic pain, whereas said biological effect or effects may e.g. be measured in in vitro cell based or animal assays, e.g. in a Frey test for mechanical allodynia.

19d. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Eph, e.g. human and/or mouse Eph: and ii) blocks the interaction between said at least one Eph, e.g. human and/or mouse Eph, and at least one Ephrin, e.g. human and/or mouse EpMn, e.g.

EphrinBl; and iii) wherein said amino acid sequence has an inhibitory effect on pathogenesis of rheumatoid arthritis, whereas said biological effect or effects may e.g. be measured in in vitro cell based or animal assays, e.g. in CAIA model for RA. 20. Amino acid sequence according to any any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to at least two Eph, e.g. human and/or mouse Eph; and ii) blocks the interaction between said at least two Eph, e.g. human and/or mouse Eph, and at least one Ephrin, e.g. human and/or mouse Ephrin; and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

21. Amino acid sequence according to any any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to at least one Eph, e.g. human and/or mouse Eph; and ii) blocks the interaction between said Eph, e.g. human and/or mouse Eph, and at least two Ephrin, e.g. human and/or mouse Ephrin; and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis., and/or b) inhibitory effect on tumorgensis. c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

22. Amino acid sequence according to any any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to at least one Eph, e.g. human and/or mouse Eph; and ii) does not block the interaction between said Eph, e.g. human and/or mouse Eph, and any Ephrin, e.g. human and/or mouse Ephrin; and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein. 23. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Ephrin, e.g. human and/or mouse Ephrin; and ii) blocks the interaction between said Ephrin, e.g. human and/or mouse Ephrin, and an Eph, e.g. human and/or mouse Eph.

24. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to an Ephrin, e.g. human and/or mouse Ephrin; and ii) blocks the interaction between said at least one Ephrin, e.g. human and/or mouse Ephrin, and at least one Eph, e.g. human and/or mouse Eph; and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

25. Amino acid sequence according to any any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to at least two Ephrin. e.g. human and/or mouse Ephrin; and ii) blocks the interaction between said at least two Ephrin, e.g. human and/or mouse Ephrin, and at least one Eph, e.g. human and/or mouse Eph; and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

26. Amino acid sequence according to any any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to at least one Ephrin, e.g. human and/or mouse Ephrin; and ii) blocks the interaction between said Ephrin, e.g. human and/or mouse Ephrin, and at least two Eph, e.g. human and/or mouse Eph: and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis. c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

27. Amino acid sequence according to any any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to at least one Ephrin, e.g. human and/or mouse Ephrin; and ii) does not block the interaction between said Ephrin, e.g. human and/or mouse Ephrin. and any Eph, e.g. human and/or mouse Eph; and iii) wherein said amino acid sequence has at least one of the following biological effects, a) an inhibitory effect on angiogenesis. and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

28. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to human EphA2; and ii) blocks the interaction between human EphA2 and human EphrinAl .

29. Amino acid sequence according to any previous or following embodiments, wherein the variable domain wherein the variable domain i) is directed against and/or specifically binds to human EphrinAl; and ii) blocks the interaction between human EphrinAl and human EphA2.

30. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to the human form of an Eph; and ii) blocks the interaction between said human Eph and an human Ephrin. 31. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to human and/or mouse EphrinBl; and ii) blocks the interaction between human and/or mouse EphrinBl and mouse and/or human EphB2.

32. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to the human and/or mouse form of EphB4; and ii) blocks the interaction between human and/or mouse EphB4 and human and/or mouse EphrinB2.

33. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to human and/or mouse EphrinB 1 ; and ii) blocks the interaction between human and/or mouse EphrinB 1 and mouse and/or human EphB2.

34. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to human EphA2; and ii) blocks the interaction between human EphA2 and human EphrinAl.

35. Amino acid sequence according to any previous or following embodiments, wherein the variable domain i) is directed against and/or specifically binds to human EphB4; and ii) blocks the interaction between human EphB4 and human EphrinB2.

36. Amino acid sequence according to any previous or following embodiments, wherein said amino acid sequence has at least one of the following biological effects: a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein. 37. Amino acid sequence according to any previous or following embodiments, wherein said amino acid sequence has an inhibitory effect on angiogenesis.

38. Amino acid sequence according to any previous or following embodiments, wherein said amino acid sequence has an inhibitory effect on tumorgensis.

39. Amino acid sequence according to any previous or following embodiments, wherein said amino acid sequence has a growth promoting effect on central nervous system neurite outgrowth.

39a. Amino acid sequence according to any previous or following embodiments, wherein said amino acid sequence has an inhibitory effect on neuropatic pain.

39b. Amino acid sequence according to any previous or following embodiments, wherein said amino acid sequence has a inhibitoy effect on RA initiation and/or progression.

40. Amino acid sequence according to any previous or following embodiments, wherein the variable domain has an antagonistic effect to at least one member of the group of proteins consisting of EphAl, EphA2, EphA3, EphA4, EphAS, EphA6, EphA7, EphA8, EphA9, EpIiAl 0. EpIiB 1. EphB2, EρhB3. EphB4, EphB5. EpliBβ.

41. Amino acid sequence according to any previous or following embodiments, wherein the variable domain has an agonistic effect to at least one member of the group of proteins consisting of EphAl, EphA2, EphA3, EphA4, EphAS, EphAό. EphA7, EphA8. EphA9. EphAlO, EphBl. EphB2, EphB3, EphB4, EphB5, EphB6.

42. Amino acid sequence according to any previous or following embodiments, wherein the variable domain has an antagonistic effect to human and/or mouse EphA2 or EphB4. 43. Amino acid sequence according to any previous or following embodiments, wherein the variable domain has an agonistic effect to human and/or mouse EphA2 or EphB4.

44. Amino acid sequence according to any previous or following embodiments, wherein the variable domain is able to inhibit the assembly of an Eph homodimerε, e.g. such as human and/or mouse EphA2 or EphB4 homodimers; or an amino acid sequence according to any of the preceding embodiments, wherein the variable domain is able to enhance the assembly of an Eph homodimers, e.g. such as human and/or mouse EphA2 or EphB4 homodimers.

45. Amino acid sequence according to any previous or following embodiments that can specifically bind to at least one member of the group of proteins consisting of EphAl, EphA2, EphA3, EphA4, EρhA5, EphA6, EphA7, EphA8, EphA9. EpIiAlO, EphBl, EphB2, EphB3, EphB4, EphB5, EphB6, EpMnAl, EphrinA2, EphrinA3, EρhrinA4,

EphrinA5, EpMnAo, EphrinBl, EphrinB2, EpMnB3 with a dissociation constant (K_D) of 10^" ⁵ to 10^"i2 moles/liter or less, and preferably 10^~7 to 10^"1 moles/liter or less and more preferably 10^" to 10^" moles/liter.

46. Amino acid sequence according to any previous or following embodiments, that can specifically bind to at least one member of the group of proteins consisting of EphAl, EphA2, EphA3, EphA4, EρhA5_? EphAό, EphA7, EphA8, EphA9, EphAlO, EphBl, EphB2. EphB3, EphB4. EphB5, EphBό, EpMnAl, EplirinA2, EpMnA3, EphrinA4, EpMnAS₅ EpMiiAό, EphrinBl, EphrinB2, EphrinB3 with a rate of association (k_on~rate) of between 10² M^-1S^"1 to about 10⁷ M⁴S^'1, preferably between 10³ M^'V¹ and 10⁷ M^ls ^l, more preferably between 10⁴ M^" V^] and 10⁷ M^~V^], such as between 10⁵ M^'V¹ and 10⁷ M^-1S^"1.

47. Amino acid sequence according to any previous or following embodiments, that can specifically bind to at least one member of the group of proteins consisting of EphAl , EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphAlO, EphBl , EphB2, EpliB3, BphB4. EphB5. EρhB6_; EphrinAi, EpMnA2, EρhrinA3, EpMnA4, EpMnAS, EphrinAό. EphrinBl, EphrinB2. EphrinB3 with a rate of dissociation (k_off rate) between 1 s^"1 and 10^~6 s^"!, preferably between 10^'2 s^'1 and 10^"6 s^'1, more preferably between 10^"3 s^"1 and 10^"6 s \ such as between 10^"4 s^"1 and 10^"6 s^"1.

48. Amino acid sequence according to any previous or following embodiments, that can specifically bind to at least one member of the group of proteins consisting of

EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphAlO, EphBL EphB2. EphB3, EphB4, EphB5, EphBό, EphrinAl , EphrinA2, EphrinA3, EphrinA4, EphrinA5, EphrinAό, EphrinBl, EphrinB2. EphrinB3 with an affinity less than 500 nM, preferably less than 200 nM. more preferably less than 10 nM, such as less than 500 pM.

49. Amino acid sequence according to any previous or following embodiments, that is a naturally occurring amino acid sequence (from any suitable species) or a synthetic or semi-synthetic amino acid sequence.

50. Amino acid sequence according to any previous or following embodiments, that comprises an immunoglobulin fold or that under suitable conditions is capable of forming an immunoglobulin fold.

51. Amino acid sequence according to any previous or following embodiments, that essentially consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively).

52. Amino acid sequence according to any previous or following embodiments, that is an immunoglobulin sequence.

53. Amino acid sequence according to any of the preceding embodiments, that is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence.

54, Amino acid sequence according to any of the preceding embodiments that is a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation. 55. Amino acid sequence according to any previous or following embodiments, that essentially consists of a light chain variable domain sequence (e.g. a V_L-sequence); or of a heavy chain variable domain sequence (e.g. a V_H~sequence).

56. Amino acid sequence according to any previous or following embodiments, that essentially consists of a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or that essentially consist of a heavy chain variable domain sequence that is derived from heavy chain antibody.

57. Amino acid sequence according to any previous or following embodiments, that essentially consists of a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody™ (including but not limited to a Vm i sequence).

58. Amino acid sequence according to any previous or following embodiments, that essentially consists of a Nanobody™.

59. Amino acid sequence according to any previous or following embodiments, that essentially consists of a Nanobody™ that i) has 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 foπn the CDR sequences are disregarded; and in which: ii) preferably 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 A-3.

60. Amino acid sequence according to any previous or following embodiments, that essentially consists of a Nanobody™ that i) has 80% amino acid identity with at least one of the amino acid sequences of SEQ ID N(Ts: 714 to 797, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 11 , 37, 44, 45, 47. 83,

84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3.

61a. An amino acid sequence according to any previous or following embodiments comprising at least one variable domain that cross-blocks the binding of at least one of the amino acid sequences with SEQ ID NOs 714 to 797 to an Eph and/or an Ephrin.

61b. An amino acid sequence according to any previous or following embodiments comprising at least one variable domain that is cross-blocked by at least one of the amino acid sequences with SEQ ID NOs 714 to 797 to an Eph and/or an Ephrin.

61c. An amino acid sequence according to embodiments 61a or 61b wherein the ability of said amino acid sequence to cross-block oi to be cross-blocked h delected in a Biacorε assa} .

61 d. An amino acid sequence according to embodiments 61 a or 61 b wherein the ability of said amino acid sequence to cross-block or to be cross-blocked is detected in an ELISA assay.

61. Amino acid sequence according to any previous or following embodiments, that essentially consists of a humanized Nanobody™.

62. Construct, that comprises or essentially consists of one or more amino acid sequences according to any of embodiments 1 to 6 L and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers.

63. Construct, that comprises or essentially consists of one or more amino acid sequences according to any of embodiments 1 to 61 , and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers; and wherein said construct has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, and/or c) has a growth promoting effect on central nervous system neurite outgrowth, whereas the biological effect or effects a), b) or c) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

64. Construct according to embodiments 62 or 63, in which said one or more other groups, residues, moieties or binding units are amino acid sequences.

65. Construct according to embodiments 62 to 64 in which said one or more linkers, if present, are one or more amino acid sequences.

66. Construct according to any of embodiments 62 to 65, in which said one or more other groups, residues, moieties or binding units are immunoglobulins.

67. Construct according to any of embodiments 62 to 66, in which 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.

68. Construct according to any of embodiments 62 to 67, in which said one or more amino acid sequences of the invention are immunoglobulin sequences.

69. Construct according to any of embodiments 62 to 68, in which said one or more amino acid sequences of the invention 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. 70. Construct, that comprises or essentially consists of one or more Nanobodies according to any of previous embodiments and in which said one or more other groups, residues, moieties or binding units are Nanobodies.

71. Construct according to any of embodiments 62 to 70, which is a multivalent construct.

72. Construct according to any of embodiments 62 to 71, which is a multispecific construct.

73. Construct according to any of embodiments 62 to 72, which has an increased half-life, compared to the corresponding amino acid sequence according to any of embodiments 1 to 61 per se.

74. Construct according to embodiment 73. in which said one or more other groups, residues, moieties or binding units provide the construct with increased half-life, compared to the corresponding amino acid sequence according to any of embodiments 1 to 61 per se.

75. Construct according to embodiment 74, in which said one or more other groups, residues, moieties or binding units that provide the construct with increased half-life is chosen from the group consisting of serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

76. Construct according to embodiment 75, in which said one or more other groups, residues, moieties or binding units that provide the construct with increased half-life is chosen from the group consisting of human serum albumin or fragments thereof.

77. Construct according to embodiment 76, in which said one or more other groups, residues, moieties or binding units that provides the construct with increased half-life are chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG). 78. Construct according to embodiment 77, in which said one or more other groups, residues, moieties or binding units that provides the construct with increased half-life 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 that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

79. Construct according to embodiment 78, in which said one or more other groups, residues, moieties or binding units that provides the construct with increased half-life is a Nanobody that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

80. Construct according to any of embodiments 73 to 79, that has a serum 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 according to any of embodiments 1 to 21 per se.

81. Construct according to any of embodiments 73 to 80, that has 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 according to any of embodiments 1 to 21 per se.

82. Construct according to any of embodiments 73 to 81 , that has 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; for example, 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 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days). 83. Construct according to any of embodiments 62 to 82 that comprises or essentially consists of two amino acid sequences according to any of embodiments 1 to 61.

84. Construct according to embodiment 83, wherein said two amino acid sequences are directed against and/or specifically bind to same target protein which is selected from the group consisting of EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphAS, EphA9. EphAlO, EphBl, EphB2, EphB3, EphB4, EphB5, EphB6, EplirinAl, EphrinA2, EphrinA3, EphrinA4_! EphrinA5, EphrinA6, EphrinBl, EphrinB2. EphiinB3, said binding either being directed against two different epitopes or being against the same epitope.

85. Construct according to embodiments 83 or 84, wherein said target protein is selected from the group consisting of hEphAl, hEphA2, hEphA3, hEphA4, hEphA5, hEphAβ, hEphA7, hEphAS, hEphA9, hEphAlO, hEphBl, hEphB2, hEphB3, hEphB4, hEphB5, hEphB6, liEphrinA] , hEphrinA2, hEphrinA3, hEphrinA4, hEphrinAS. hEphrinAό, hEphrinBl, hEphrinB2, hEphrinB3.

86. Construct according to embodiment 85, wherein said first amino acid sequence is directed against and/or specifically binds to human EpnA2, EphB4, EphB6, EphrinAl, and EphrinB2 and wherein said second amino acid sequence is directed against and/or specifically binds to human EphA2, EphB4. EphB6, EphrinAl, and EphrinB2.

87. Construct according to embodiment 85, wherein said first amino acid sequence is directed against and/or specifically binds to human EphA2 or EphB4 and wherein said second amino acid sequence is directed against and/or specifically binds to human EphrinAl or EphrinB2.

88. Construct according to embodiment 85, wherein said first amino acid sequence is directed against and/or specifically binds to human EphA2 and wherein said second amino acid sequence is directed against and/or specifically binds to a human Ephrin. 89. Construct according to embodiment 85. wherein said first amino acid sequence is directed against and/or specifically binds to human EphB4 and wherein said second amino acid sequence is directed against and/or specifically binds to human Ephrin.

90. Construct according to any embodiments 62 to 89; wherein said construct has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

90a. Construct according to any embodiments 62 to 89; wherein said construct comprises two Nanobodies which are bispecific and designed in order that no clustering of of Eph and/or Ephrins occurs, and wherein said construct has at least one of the following biological effects, a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tumorgensis, c) has a growth promoting effect on central nervous system neurite outgrowth, d) inhibitory effect on neuropathic pain, and/or e) inhibitory effect on rheumatoid arthritis pathogensis, whereas the biological effect or effects a), b) c), d) or e) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

91. Construct according to embodiments 62 to 90, that comprises or essentially consists of one or more amino acid sequences according to any of embodiments 1 to 61, and optionally further comprises one or more toxic groups, toxic residues, toxic moieties or toxic binding units, optionally linked via one or more linkers.

92. Construct according to embodiment 91 , wherein the toxic group is selected from the group of immunotoxins.

93. Monovalent construct, comprising or essentially consisting of one amino acid sequence according to any of embodiments 1 to 61. 94. Monovalent construct according to embodiment 93, in which said amino acid sequence of the invention is 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, "dAbⁿ's. amino acid sequences that are suitable for use as a dAb, or Nanobodies.

95. Monovalent construct, comprising or essentially consisting of one Nanobody according to any of embodiments 1 to 61.

96, Nucleic acid or nucleotide sequence, that encodes an amino acid sequence according to any of embodiments 1 to 61 , a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 to 95.

97. Nucleic acid or nucleotide sequence according to embodiment 96, that is in the form of a genetic construct.

98. Host or host cell that expresses, or that under suitable circumstances is capable of expressing, an amino acid sequence according to any of embodiments 1 to 61, a construct according to any of embodiments 62 to 92. or a monovalent construct according to any of embodiments 93 to 95; and/or that comprises a nucleic acid or nucleotide sequence according to embodiment 96 or 97.

99. Method for producing an amino acid sequence according to any of embodiments 1 to 61, a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 to 95, said method at least comprising the steps of: a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid or nucleotide sequence according to embodiment 96. or a genetic construct according to embodiment 97; optionally followed by: b) isolating and/or purifying the amino acid sequence according to any of embodiments 1 to 61 , a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 to 95. 100. Method for producing an amino acid sequence according to any of embodiments 1 to 61. a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 to 95. said method at least comprising the steps of: a) cultivating and/or maintaining a host or host cell according to embodiment 98 under conditions that are such that said host or host cell expresses and/or produces at least one amino acid sequence according to any of embodiments 1 to 61, a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 to 95; optionally followed by: b) isolating and/or purifying the amino acid sequence according to any of embodiments 1 to 61, a construct according Io any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 to 95.

101. Composition, comprising at least one amino acid sequence according to any of embodiments 1 to 61, a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 to 95, or nucleic acid or nucleotide sequence according to embodiment 96 or 97.

102. Composition according to embodiment 101, which is a pharmaceutical composition

103. Composition according to embodiment 102, which is a pharmaceutical composition, that further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and that optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.

104. Method for the prevention and/or treatment of at least one disease or disorder related to excessive or insufficient angiogenesis, cancers such as lung, breast, prostate and brain cancer, CNS injury such as spinal cord injury and/or stroke, neuropathic pain, rheumatoid arthritis, fibrosis-related diseases, and infectious diseases, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one amino acid sequence according to any of embodiments 1 to 61, a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 or 95, or composition according to any of embodiments 101 to 103.

105. Method for the prevention and/or treatment of at least one disease or disorder that is associated with a protein selected from the group consisting of EpIiAl. EphA2, EphA3. EphA4, EphA5, EphA6, EphA7, EphAS, EphA9, EphAlO. EphBl , EphB2. EphB3. EphB4, EphB5, EphB6, EphrinAl, EphrinA2, EphrinA3, EphrinA4, EphrinAS, EphrinAό, EphrinBl. EphrinB2, EphrinB3, with its biological or pharmacological activity, and/or with the biological pathways or signalling in which said protein is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one amino acid sequence according to any of embodiments 1 to 61 , a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 or 95, or composition according to any of embodiments 101 to 103.

106. Method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering, to a subject in need thereof, an amino acid sequence according to any embodiments 1 to 61, a construct according to any of embodiments 62 to 92. or a monovalent construct according to any of embodiments 93 or 95, or composition according to any of embodiments 101 to 103, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one amino acid sequence according to any embodiments 1 to 61, a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 or 95, or composition according to any of embodiments 101 to 103.

107. Use of an amino acid sequence according to any embodiments 1 to 61, a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 or 95 in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one disease or disorder related to excessive or insufficient angiogenesis, cancers such as lung, breast, prostate and brain cancer, neuropathic pain, rheumatoid arthritis, CMS injury such as spinal cord injury and/or stroke, fibrosis- related diseases, and infectious diseases. 108. Use of an amino acid sequence according to any embodiments 1 to 61, a construct according to any of embodiments 62 to 92, or a monovalent construct according to any of embodiments 93 or 95 for the prevention and/or treatment of at least one disease or disorder related to excessive or insufficient angiogenesis, cancers such as lung, breast, prostate and brain cancer, CNS injury such as spinal cord injury and/or stroke, neuropathic pain, rheumatoid arthritis, fibro sis-related diseases, and infectious diseases.

Claims

Claims:

1. Amino acid sequence comprising at least one single variable domain that is directed against a protein selected from the group consisting of EphAl, EphA2, EphA3, EphA4_; EphA5, EphAό, EphA7, EphA8, EphA9, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB5, EphB6.

2. Amino acid sequence according to claim 1 , which is in essentially isolated form.

3. Amino acid sequence according to claim 1 , wherein the variable domain is directed against a protein selected from the group consisting of the human and/or mouse form of EphAl, EphA2, EphA3, EphA4, EphA5, EphAό, EphA7, EphA8, EphA9, EphAlO, EphBl , EphB2, EphB3, EphB4, EphB5, EphB6, preferably human form.

4. Amino acid sequence according to claim 1 , wherein the variable domain has an antagonistic effect to human form of EphAl, EphA2, EpliA3, EphA4, EphA5, EphAό, EρhA7, EρhA8_; EphA9, EphAl 0.

5. Construct comprising at least two amino acid sequences of claims 1 to 4.

6. Amino acid sequence accoding to claim 1 or construct of claim 5, wherein said amino acid sequence or construct has at least one of the following biological effects: a) an inhibitory effect on angiogenesis, and/or b) inhibitory effect on tirmorgensis, and/or c) has a growth promoting effect on central nervous system neurite outgrowth, whereas the biological effect or effects a), b) or c) may e.g. be measured in in vitro cell based or animal assays as disclosed herein.

7. Composition comprising at least one amino acid sequence according to claims 1 to 4 or claim 6 or a construct of claim 5 or 6; and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

8. Method for the prevention and/or treatment of at least one disease or disorder related to excessive or insufficient angiogenesis, cancers such as lung, breast, prostate and brain cancer, CNS injury such as spinal cord injury and/or stroke, fibrosis-related diseases, neuropathic pain, rheumatoid arthritis- and infectious diseases, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one amino acid sequence according to claim 1 or construct of claim 5.

9. Use of an amino acid sequence according to claim 1 for prevention and/or treatment of at least one disease or disorder related to excessive or insufficient angiogenesis; or use of an amino acid sequence according to claim 1 or construct of claim 5 in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one disease or disorder related to excessive or insufficient angiogenesis, cancers such as lung, breast, prostate and brain cancer, CNS injury such as spinal cord injury and/or stroke, neuropathic pain, rheumatoid arthritis, fϊbrosis-related diseases, and infectious diseases.

10. Method for producing an amino acid sequence according to claim 1 or construct of claim 5, said method at least comprising the steps of: i. cultivating and/or maintaining a suitable host or host cell under conditions that are such that said host or host cell expresses and/or produces at least one amino acid sequence according to claim 1 or construct of claim 5; optionally followed by: ii. isolating and/or purifying the amino acid sequence according to claim 1 or construct of claim 5.