NZ614249B2 - Bispecific binding molecules binding to vegf and ang2 - Google Patents

Abstract

Discloses a bispecific binding molecule comprising - at least one VEGF-binding component, at least one serum albumin binding component, and at least one Ang2-binding component and, wherein said Ang2-binding component binds to Ang2 with a potency at least 5,000 times higher than to Ang1 or to Ang4, and wherein said VEGF-, serum albumin and Ang2-binding components are immunoglobulin single variable domains, each immunoglobulin single variable domain consisting of four framework regions and three complementarity determining regions (CDRs), and wherein said bispecific binding molecule is selected from the group consisting of bispecific binding molecules having (i) the CDR sequences as present in VEGFANGBII00022 (SEQ ID NO: 207), (ii) the CDR sequences as present in VEGFANGBII00025 (SEQ ID NO: 210), and (iii) the CDR sequences as present in VEGFANGBII00028 (SEQ ID NO: 213). nd wherein said VEGF-, serum albumin and Ang2-binding components are immunoglobulin single variable domains, each immunoglobulin single variable domain consisting of four framework regions and three complementarity determining regions (CDRs), and wherein said bispecific binding molecule is selected from the group consisting of bispecific binding molecules having (i) the CDR sequences as present in VEGFANGBII00022 (SEQ ID NO: 207), (ii) the CDR sequences as present in VEGFANGBII00025 (SEQ ID NO: 210), and (iii) the CDR sequences as present in VEGFANGBII00028 (SEQ ID NO: 213).

Description

/055901 Bispecific binding molecules binding to VEGF and AngZ FIELD OF THE INVENTION The invention relates to the field of human therapy, in particular cancer therapy and agents and compositions useful in such therapy.

BACKGROUND OF THE INVENTION When tumors reach a critical size of imately 1 mm3 they become dependent on angiogenesis for maintaining blood supply with oxygen and nutritients to allow for further growth. Anti-angiogenesis therapies have become an important treatment option for several types of tumors. These therapies have focused on blocking the VEGF pathway ra et al., Nat Rev Drug Discov. 2004 May;3(5):391-400.) by neutralizing VEGF (Avastin) or its receptors (Sutent and Sorafinib). Recent studies in mice have shown, that oietin2 (Ang2), a ligand of the Tie2 receptor, ls vascular remodeling by ng the functions of other angiogenic factors, such as VEGF. Ang2 is primarily sed by endothelial cells, strongly induced by hypoxia and other angiogenic factors and has been demonstrated to regulate tumor vessel plasticity, allowing vessels to respond to VEGF and FGF2 (Augustin et al., Nat Rev Mol Cell Biol. 2009 Mar;10(3):165-77.).

Consistent with this role, the deletion or inhibition of Ang2 results in reduced angiogenesis (Gale et al., Dev Cell. 2002 3):302—4.) (Falcon et al., Am J Pathol. 2009 Nov;175(5):2159—70.). Elevated Ang2 serum concentrations have been reported for patients with colorectal cancer, NSCLC and melanoma (Goede et al., Br J Cancer. 2010 Oct (9):1407-14),(Park et al., Chest. 2007 Jul;132(1): 200-6.),(Helfrich et al., Clin Cancer Res. 2009 Feb 15;15(4):1384-92.). ln CRC cancer Ang2 serum levels correlate with therapeutic response to anti-VEGF therapy.

The Ang—Tie system consists of 2 receptors (Tie1 and Tie2) and 3 ligands (Ang1, Ang2 and Ang4) (Augustin et al., Nat Rev Mol Cell Biol. 2009 (3):165-77.). Tie2, Ang1 and Ang2 are the best studied members of this family, Tie1 is an orphan receptor and the role of Ang4 for vascular remodelling still needs to be defined. Ang2 and Angl mediate opposing functions upon Tie2 binding and activation. Ang2-mediated Tie2 activation results in endothelial cell activation, pericyte dissociation, vessel leakage and induction of vessel sprouting. In contrast to Ang2, Ang1 signaling maintains vessel ity by recruitment of pericytes, thereby maintaining endothelial cell quiescence.

Angiopoietin 2 (Ang2) is a secreted, 66 kDa ligand for the Tie2 receptor tyrosine kinase (Augustin et al., Nat Rev Mol Cell Biol. 2009 Mar;10(3):165-77.). Ang2 consists of an N- terminal —coil domain and a inal fibrinogen-like domain, the latter is ed for Tie2 interaction. Ang2 is primarily expressed by endothelial cells and strongly induced by hypoxia and other angiogenic factors, including VEGF. Tie2 is found on elial cells, haematopoietic stem cells and tumor cells. Ang2-Tie2 has been trated to regulate tumor vessel plasticity, ng vessels to respond to VEGF and FGF2.

In vitro Ang2 has been shown to act as a modest mitogen, chemoattractant and inducer of tube formation in human umbilical vein endothelial cells (HUVEC). Ang2 induces tyrosine phosphorylation of ectopically expressed Tie2 in fibroblasts and promotes downstream signaling events, such as phosphorylation of ERK-MAPK, AKT and FAK in HUVEC. An antagonistic role of Ang2 in Ang1-induced endothelial cell responses has been described.

AngZ -deficiency has been shown to result in a profound tic ning defect in mice. Although the loss of Ang2 is dispensable for embryonic vascular development, Ang2 -deficient mice have persistent ar s in the retina and kidney. Together with the dynamic pattern of Ang2 expression at sites of angiogenesis (for example ovary), these findings indicate that Ang2 controls vascular remodeling by enabling the functions of other angiogenic factors, such as VEGF.

The Ang2-Tie2 system exerts crucial roles during the angiogenic switch and later stages of tumor angiogenesis. Ang2 sion is strongly up-regulated in the tumor-associated endothelium. Reduced growth of tumors has been observed when implanted into Ang2 - deficient mice, especially during early stages of tumor . Therapeutic blocking of Ang2 with Ang2 mAbs has shown broad efficacy in a variety of tumor xenograft models.

Additive effects of Ang2 mAbs with inhibitors of VEGFR2 (mAbs and small molecular weight tors) have been described.

As described in e.g. U82008/OO14196 and W02008/101985, angiogenesis is implicated in the pathogenesis of a number of disorders, including solid tumors and metastasis as well as eye diseases. One of the most important pro—angiogenic factors is vascular endothelial growth factor (VEGF), also termed VEGF-A or vascular permeability factor (VPF). VEGF belongs to a gene family that includes ta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E and VEGF—F. Alternative splicing of mRNA of a single gene of human VEGF s in at least six isoforms (VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206), VEGF165 being the most abundant Two VEGF ne kinase receptors (VEGFR) have been identified that interact with VEGF, i.e. VEGFR-1 (also known as Flt-1) and VEGFR-2 (also known as KDR or FlK-1). VEGFR-1 has the highest affinity for VEGF, while VEGFR- 2 has a somewhat lower affinity for VEGF. Ferrara rine Rev. 2004, 25: 1) provide a ed description of VEGF, the interaction with its receptors and its on in normal and pathological processes can be found in Hoeben etal. Pharmacol. Rev. 2004, 56: 549—580.

VEGF has been reported to be a pivotal regulator of both normal and abnormal angiogenesis (Ferrara and Davis-Smyth, Endocrine Rev. 1997, 18: 4-25; Ferrara J. MoL Med. 1999, 77: 527-543). Compared to other growth factors that contribute to the processes of vascularformation, VEGF is unique in its high specificity for endothelial cells within the vascular system.

VEGF mRNA is overexpressed by the majority of human tumors. In the case of tumor growth, enesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment for the growth and metastasis of the tumor (Folkman et a/., 1989, Nature 339-58), which allows the tumor cells to acquire a growth advantage compared to the normal cells. Therefore, anti-angiogenesis therapies have become an important treatment option for several types of tumors. These therapies have focused on blocking the VEGF pathway (Ferrara et al., Nat Rev Drug Discov. 2004 May; 3(5): 391-400.

VEGF is also involved in eye diseases. The concentration of VEGF in eye fluids is highly ated with the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies. Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by age-related macular ration (AMD). Up-regulation of VEGF has also been obsen/ed in various inflammatory disorders. VEGF has been implicated in the pathogenesis of rheutatoid tis, an inflammatory disease in which angiogenesis plays a significant role.

The elucidation of VEGF and its role in angiogenesis and different processes has provided a potential new target of therapeutic ention. The function of VEGF has been inhibited by small les that block or prevent activation of VEGF receptor tyrosine kinases (Schlaeppi and Wood, 1999, Cancer Metastasis Rev, 18: 473-481) and consequently interfere with the VEGF receptor signal transduction pathway. Cytotoxic conjugates containing bacterial or plant toxins can inhibit the stimulating effect of VEGF on tumor angiogenesis. VEGF-DT385 toxin conjugates (diphtheria toxin domains fused or chemically conjugated to VEGF165), for example, efficiently inhibit tumor growth in vivo. Tumor growth inhibition could also be achieved by ring a Flk-1 mutant or soluble VEGF ors by a irus.

VEGF-neutralizing antibodies, such as A4.6.l and MV833, have been developed to block VEGF from binding to its receptors and have shown preclinical antitumor activity (Kim et al. Nature 1993, 362: 841-844; Folkman Nat. Med. 1995, 1: 27-31; Presta etal. Cancer Res. 1997, 57: 4593-4599; Kanai et al. Int. J. Cancer 1998, 77: 933-936; Ferrara and Alitalo Nat. Med. 1999, 5: 364; 320, 340. For a review of therapeutic anti-VEGF approaches trials, see Campochiaro and Hackett (Oncogene 2003, 22: 6537-6548).

Most al ence has been obtained with A4.6.1, also called bevacizumab (Avastin®; Genentech, San Francisco, CA).

WO2008/101985 describes immunoglobulin single variable domains from des (VHHs or “Nanobodies, as defined ) that bind to VEGF, and their use in the treatment of conditions and diseases characterized by excessive and/or ogical angiogenesis or neovascularization.

It has been an object of the present invention to provide novel anti-angiogenic g molecules for human therapy.

It has been a further object of the invention to provide methods for the prevention, treatment, alleviation and/or diagnosis of such diseases, disorders or conditions, involving the use and/or stration of such binding molecules and compositions comprising them. In particular, it is has been an object of the invention to provide such pharmacologically active g molecules, compositions and/or methods that provide ages compared to the , compositions and/or methods currently used and/or known in the art. These advantages e improved therapeutic and/or pharmacological properties and/or other advantageous properties, e.g. for manufacturing purposes, especially as compared to conventional antibodies as those described above, or fragments thereof.

BRIEF SUMMARY OF THE INVENTION Herein disclosed are bispecific binding molecules, such as bispecific immunoglobulins, such as globulin single variable domains like VHHs and domain antibodies, which comprise at least one VEGF-binding component and at least one Ang2-binding component in a single molecule. Said bispecific g molecules may r comprise a serum albumin binding component.

More specifically, herein disclosed is a bispecific binding molecule of the invention which essentially comprises (i) a Ang2-binding component ically g to at least one epitope of Ang2 and (ii) a VEGF-binding ent specifically binding to at least an epitope of VEGF, wherein the components are linked to each other in such a way that they simultaneously bind to Ang2 and VEGF or that they bind to either Ang2 or VEGF at a time.

The two components may comprise one or more immunoglobulin single variable domains that may be, independently of each other, VHHs or domain antibodies, and/or any other sort of immunoglobulin single variable domains, such as VL domains, as defined herein, provided that each of these immunoglobulin single variable domains will bind the antigen, i.e. Ang2 or VEGF, respectively.

According to an embodiment of the invention, there is provided a bispecific binding molecule comprising - at least one VEGF-binding component, - at least one serum albumin binding component, and - at least one Ang2-binding component and, wherein said Ang2-binding component binds to Ang2 with a potency at least 5,000 times higher than to Ang1 or to Ang4, and n said VEGF-, serum albumin and Ang2-binding components are immunoglobulin single variable domains, each immunoglobulin single le domain consisting of four framework regions and three complementarity determining regions (CDRs), and wherein said ific binding molecule is ed from the group consisting of ific binding molecules having (i) the CDR sequences as present in GBII00022 (SEQ ID NO: 207), (ii) the CDR sequences as t in VEGFANGBII00025 (SEQ ID NO: 210), and (iii) the CDR sequences as present in VEGFANGBII00028 (SEQ ID NO: 213).

According to a preferred embodiment, the immunoglobulin single variable domains are of the same type, in particular, all immunoglobulin single variable domains are VHHs or domain dies.

According to a particularly preferred embodiment, all immunoglobulin single le domains are VHHs, preferably humanized (or “sequence-optimized”, as defined herein) VHHs. Accordingly, the invention relates to bispecific binding molecules comprising an (optionally humanized or sequence-optimized) anti-Ang2 VHH and an (optionally humanized or sequence-optimized) anti-VEGF VHH.

However, it will be clear to the d person that the teaching herein may be applied analogously to bispecific binding molecules ing other anti-Ang2 or anti-VEGF immunoglobulin single variable domains, such as domain antibodies.

In another aspect, the invention relates to nucleic acids encoding the bispecific binding molecules of the invention as well as vectors and host cells containing same.

The invention further relates to a product or composition containing or comprising at least one bispecific binding molecule of the invention and optionally one or more further components of such compositions.

The invention further relates to methods for preparing or ting the bispecific binding molecules, nucleic acids, host cells, products and compositions described herein.

The invention further relates to applications and uses of the bispecific binding molecules, nucleic acids, host cells, products and compositions bed , as well as to methods for the prevention and/or treatment for diseases and disorders that can be modulated by inhibition of Ang2.

It has been found that the Ang2-binding component of the bispecific g molecules according to the present invention binds to and antagonizes Ang2 with a potency at least 5,000 times higher, preferably 10,000 times higher than to Ang1 or Ang4. This will largely avoid blocking activation of Ang1-mediated signalling, which would counter the intended anti-angiogenetic effect.

It has further been found that the VEGF-binding component of the bi-specific binding molecules according to the present invention binds to VEGF-A with an affinity of at least 1,000 times , preferebly at least 5,000 times higher, more preferably at least ,000 times higher than to VEGF-B, VEGF-C, VEGF-D or PLGF. Due to the highly preferential binding to VEGF-A the signaling of VEGFR3, which modulates of lymph angiogenesis, is not interfered with.

In a preferred embodiment the bispecific binding les of the present invention are provided as linked VHH domains. Such les are significantly smaller than conventional antibodies and have thus the potential for penetrating into a tumor deeper than such conventional antibodies. This benefit is further accentuated by the specific sequences disclosed herein after being free of glycosylation sites. r, due to the bispecific nature (VEGF- and Ang2-binding components in one molecule) the tumor ation of both functionalities will be necessarily equal, which will ensure that the beneficial effects of the combined antagonism of VEGF and Ang2 will be provided within the whole depth of penetration of the tumor. This is an advantage over the combination of dual antagonists against these targets, since the depth of penetration of individual antagonists will always vary to some degree.

Another advantage of a preferred bispecific binding molecules of the present invention is their increased serum ike due to a serum albumin g component such as a serum albumin binding le as described herein.

These and other aspects, ments, advantages and applications of the invention will become clear from the further ption hereinbelow.

DEFINITIONS 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.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, , and Roitt et a/., "Immunology" (2nd Ed), Gower Medical Publishing, London, New York (1989), as well as to the general background art cited herein; rmore, unless indicated othenivise, 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, to the general background art ed to above and to the further references cited therein.

The term “bispecific binding molecule” refers to a molecule comprising at least one AngZ-binding le (or “Ang2-binding component”) and at least one inding le (or “VEGF-binding component”). A bispecific g molecule may contain more than one Ang2-binding molecule and/or more than one VEGF-binding molecule, i.e. in the case that the bispecific binding molecule contains a biparatopic (as defined below) Ang2-binding molecule and/or a biparatopic VEGF-binding molecule, in the part of the molecule that binds to Ang2 or to VEGF, i.e. in its “Ang2-binding component” (or anti-Ang2 component) or “VEGF-binding component” (or anti-VEGF component), respectively. The word “bispecific” in this context is however not to be ued as to exclude further g components with g specificity to molecules other than VEGF and Ang2 from the bispecific binding molecule. Non-limiting examples of such further binding components are binding components binding to serum albumin.

Unless ted otherwise, the terms "immunog/obulin" and “immunog/obu/in sequence" - whether used herein to refer to a heavy chain dy or to a conventional n antibody - are used as general terms to include both the ize antibody, the individual chains thereof, as well as all parts, domains orfragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VHNL domains, respectively). In addition, the term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "(single) variable domain sequence", "VHH sequence" or in sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.

The term n" (of a polypeptide or protein) as used herein refers to a folded protein structure which has the ability to retain its tertiary structure ndently of the rest of the protein. Generally, domains are responsible for discrete functional ties of proteins, and in many cases may be added, d or transferred to other ns without loss of function of the remainder of the protein and/or of the domain.

The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (such as e.g. a chain of a conventional 4-chain antibody or of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region. lmmunoglobulin domains are characterized in that they retain the globulin fold characteristic of antibody molecules, which consists of a 2—layer sandwich of about 7 antiparallel beta-strands ed in two beta-sheets, optionally stabilized by a conserved disulphide bond. An immunoglobulin domain comprises (a) variable domain(s), i.e., one or more immunoglobulin variable s.

The term "immunoglobulin van'ab/e domain" as used herein means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and hereinbelow as work region 1" or "FR1"; as "framework region 2" or"FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are ed to in the art and hereinbelow as ementarity determining region 1"or "CDR1"; as "complementarity ining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. In the context of the present invention immunoglobulin single variable domains like VHHs and domain antibodies are preferred.

The term "immunoglobulin single variable domain" as used herein means an immunoglobulin variable domain which is capable of specifically binding to an epitope of the antigen without pairing with an additional variable globulin domain. One example of immunoglobulin single variable domains in the meaning of the present invention are "domain antibodies", such as the immunoglobulin single le s VH and VL (VH domains and VL domains). Another example of immunoglobulin single variable s are "VHH domains" (or simply "VHHs") from camelids, as defined hereinafter.

In view of the above definition, the antigen-binding domain of a tional 4-chain dy (such as an lgG, lgM, lgA, lgD or lgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a hide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e. by a VH- VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective anfigen.

"VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (variable) domain of "heavy chain antibodies" (i.e. of "antibodies devoid of light chains"; Hamers-Casterman C, Atarhouch T, Muyldermans 8, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are t in tional 4-chain antibodies (which are ed to herein as "VH domains" or ”VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are ed to herein as "VL domains" or "VL domains"). VHH domains can specifically bind to an e without an additional antigen binding domain (as opposed to VH or VL s in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain together with a VH domain). VHH domains are small, robust and efficient n recognition units formed by a single immunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH, VHH domain, VHH dy fragment, VHH antibody, as well as "Nanobody®" and "Nanobody® domain" ("Nanobody" being a trademark of the y Ablynx N.V.; Ghent; Belgium) are used interchangeably and are representatives of globulin single variable domains (having the structure FR1-CDR1-FR2—CDR2—FR3—CDR3-FR4 and specifically binding to an epitope without requiring the presence of a second immunoglobulin variable domain), and which are distinguished from VH domains by the so-called "hallmark residues", as defined in e.g. /109635, Fig. 1.

The amino acid residues of a immunoglobulin single variable domain, e.g. a VHH, are numbered according to the general numbering for VH domains given by Kabat et al.

("Sequence of proteins of immunological st", US Public Health Services, NIH da, MD, Publication No. 91), as d to VHH domains from Camelids, as shown e.g. in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, —38 . According to this numbering - FR1 comprises the amino acid residues at positions 1-30, - CDR1 comprises the amino acid residues at positions 31-35, - FR2 comprises the amino acids at ons 36-49, - CDR2 comprises the amino acid residues at positions 50-65, - FR3 comprises the amino acid residues at positions 66-94, - CDR3 comprises the amino acid residues at positions 95-102, and - FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering ing to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the present description, claims and figures, the numbering according to Kabat and applied to VHH s as described above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer ces may also be suitable for the purposes described herein. lmmunoglobulin single variable domains, e.g. VHHs and domain antibodies, according to the preferred embodiments of the invention, have a number of unique structural characteristics and functional properties which makes them highly advantageous for use in y as functional antigen-binding molecules. In particular, and without being limited thereto, VHH domains (which have been "designed" by nature to onally bind to an antigen without pairing with a light chain le domain) can function as single, vely small, functional antigen-binding structural units.

Due to their unique properties, immunoglobulin single variable domains, as defined herein, like VHHs or VHs (or VLs) - either alone or as part of a larger ptide, e.g. a biparatopic molecule - offer a number of significant advantages: . only a single domain is required to bind an antigen with high affinity and with high ivity, so that there is no need to have two separate domains present, nor to assure that these two domains are t in the right spacial conformation and uration (i.e. through the use of especially designed linkers, as with scFv's); . globulin single variable domains can be expressed from a single nucleic acid molecule and do not require any post-translational modification (like glycosylation; immunoglobulin single variable domains can easily be ered into alent and multispecific formats (as further discussed ); immunoglobulin single variable domains have high specificity and affinity for their target, low inherent toxicity and can be administered via alternative routes than infusion or injection; immunoglobulin single variable domains are highly stable to heat, pH, proteases and other denaturing agents or conditions and, thus, may be ed, stored or transported without the use of refrigeration equipments; immunoglobulin single variable domains are easy and relatively inexpensive to prepare, both on small scale and on a manufacturing scale. For example, immunoglobulin single variable domains can be produced using microbial fermentation (e.g. as further described below) and do not e the use of mammalian expression systems, as with for example conventional antibodies; immunoglobulin single variable domains are relatively small (approximately 15 kDa, or 10 times smaller than a conventional lgG) compared to conventional 4-chain antibodies and antigen—binding fragments thereof, and therefore show r) penetration into tissues ding but not limited to solid tumors and other dense tissues) and can be administered in higher doses than such tional 4-chain antibodies and antigen-binding nts thereof; VHHs have specific so-called “cavity-binding properties” (inter alia due to their extended CDR3 loop, compared to VH domains from 4-chain antibodies) and can therefore also access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding nts thereof; VHHs have the particular advantage that they are highly soluble and very stable and do not have a tendency to ate (as with the mouse-derived antigen- binding domains described by Ward et al., Nature 341: 544-546 (1989)).

The immunoglobulin single le domains of the invention are not limited with respect to a specific biological source from which they have been ed or to a specific method of preparation. For example, obtaining VHHs may include the following steps: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; or screening a library sing heavy chain antibodies or VHHs and isolating VHHs therefrom; (2) sing a c acid molecule encoding a VHH with the naturally occurring sequence; (3) "humanizing" (as described herein) a VHH, optionally after affinity maturation, with a naturally occurring sequence or expressing a nucleic acid encoding such zed VHH" (4) "camelizing" (as described below) a immunoglobulin single variable heavy domain from a naturally occurring antibody from an animal species, in particular a species of mammal, such as from a human being, or expressing a nucleic acid molecule encoding such camelized domain; (5) izing" a VH, or expressing a c acid molecule encoding such a camelized VH; (6) using techniques for preparing synthetically or semi-synthetically proteins, polypeptides or other amino acid sequences; (7) preparing a c acid molecule encoding a VHH domain using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; (8) subjecting heavy chain antibodies or VHHs to affinity maturation, to mutagenesis (e.g. random mutagenesis or site-directed mutagenesis) and/or any other technique(s) in order to increase the affinity and/or specificity of the VHH; and/or (9) combinations or ions of the foregoing steps.

Suitable methods and techniques for performing the above-described steps are known in the art and will be clear to the skilled person. By way of example, methods of obtaining VHH domains binding to a specific antigen or epitope have been described in W02006/O40153 and W02006/122786.

According to specific embodiments, the immunoglobulin single variable s of the invention or present in the polypeptides of the invention are VHH domains with an amino acid sequence that essentially corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized" or “sequence-optimized” (optionally after affinity-maturation), i.e. by replacing one or more amino acid residues in the amino acid ce of said naturally occurring VHH sequence by one or more of the amino acid residues that occur at the corresponding on(s) in a le heavy domain of a conventional 4-chain dy from a human being. This can be performed using methods known in the art, which can by routinely used by the skilled person.

A humanized VHH domain may contain one or more fully human framework region sequences, and, in an even more specific embodiment, may contain human framework region sequences derived from the human germline Vh3 sequences DP-29, DP-47, DP-51, or parts f, or be highly homologous o, optionally combined with JH sequences, such as JH5. Thus, a humanization protocol may comprise the replacement of any of the VHH residues with the corresponding framework 1, 2 and 3 (FRI, FR2 and FR3) residues of germline VH genes such as DP 47, DP 29 and DP 51) either alone or in combination. Suitable framework regions (FR) of the immunoglobulin single variable s of the invention can be selected from those as set out e.g. in W02006/OO4678 and specifically, include the so-called "KERE" and "GLEW" classes. Examples are immunoglobulin single variable domains having the amino acid sequence W at about positions 44 to 47, and their respective humanized counterparts. A humanized VHH domain may contain one or more fully human framework region sequences.

By way of example, a humanizing substitution for VHHs belonging to the 103 P,R,S— group and/or the GLEW-group (as defined below) is 108Q to 108L. Methods for humanizing immunoglobulin single variable domains are known in the art.

Binding immunoglobulin single le s with ed properties in view of therapeutic application, e.g. enhanced affinity or decreased immunogenicity, may be obtained from individual binding les by techniques known in the art, such as 2012/055901 affinity maturation (for e, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, humanizing, 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, also termed “sequence optimization”, as described herein. Reference is, for example, made to standard handbooks, as well as to the further description and Examples.

If appropriate, a binding molecule with increased affinity may be obtained by affinity- maturation of r binding molecule, the latter representing, with respect to the affinity-matured molecule, the “parent” binding molecule.

Methods of obtaining VHHs that bind to a specific antigen or epitope have been described earlier, e.g. in W02006/O40153 and W02006/122786. As also described therein in detail, VHH domains derived from camelids can be "humanized" (also termed nce-optimized” herein, “sequence-optimizing” may, in addition to humanization, encompass an additional modification of the sequence by one or more mutations that h the VHH with improved properties, such as the l of potential post translational modification sites) by replacing one or more amino acid es in the amino acid sequence of the al VHH ce by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional n antibody from a human being. A humanized VHH domain can contain one or more fully human framework region sequences, and, in an even more specific embodiment, can contain human framework region sequences derived from DP-29, DP-47, DP-51, or parts thereof, optionally combined with JH sequences, such as JH5.

Domain antibodies, also known as "Dab"s and "dAbs" (the terms "Domain Antibodies" and "dAbs" being used as trademarks by the GlaxoSmithKline group of ies) have been described in e.g. Ward, E.S., et a/.: "Binding ties of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli"; Nature 341: 544-546 (1989); Holt, L.J. et a/.: "Domain antibodies: proteins for therapy"; TRENDS in Biotechnology 21(11): 484-490 ; and W02003/002609.

WO 31078 Domain antibodies essentially correspond to the VH or VL domains of antibodies from non—camelid mammals, in particular human n antibodies. In order to bind an e as a single antigen binding domain, i.e. without being paired with a VL or VH domain, respectively, ic selection for such n binding properties is ed, e.g. by using libraries of human single VH or VL domain sequences.

Domain antibodies have, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not e humanization for e.g. therapeutical use in humans. As in the case of VHH domains, they are well expressed also in prokaryotic expression systems, providing a significant reduction in overall manufacturing cost.

Furthermore, it will also be clear to the skilled person that it is possible to "graf " one or more of the CDR's mentioned above onto other "scaffolds", including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting are known in the art.

The terms "epitope" and "antigenic determinant“, which can be used interchangeably, refer to the part of a macromolecule, such as a polypeptide, that is recognized by antigen-binding molecules, such as tional antibodies or the ptides of the invention, and more particularly by the antigen-binding site of said molecules. Epitopes define the m binding site for an immunoglobulin, and thus ent the target of specificity of an immunoglobulin.

A polypeptide (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain of the invention, or generally an antigen-binding molecule or a fragment thereof) that can "bind to" or "specifically bind to", that "has affinity fof' and/or that "has specificity fof' a certain epitope, antigen or protein (orfor at least one part, fragment or epitope thereof) is said to be "against“ or "directed t" said epitope, antigen or protein or is a "binding" molecule with respect to such epitope, antigen or protein. In this t, a VEGF-binding component may also be referred to as “VEGF-neutralizing”.

Generally, the term "specificity' refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein (such as an immunoglobulin single variable domain of the invention) molecule can bind. The specificity of an antigen-binding molecule can be ined based on its ty and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a measure for the binding strength between an e and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an epitope and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding molecule (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain or a ptides ning it and the pertinent antigen. Avidity is related to both the affinity between an epitope and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule.

The part of an antigen-binding molecule that recognizes the epitope is called a paratope.

Unless indicated othen/vise, the term “VEGF-binding molecule” or “Ang2—binding molecule” includes anti-VEGF or anti-Ang2 dies, anti-VEGF antibody or anti-Ang2 antibody fragments, “anti-VEGF antibody-like molecules” or “anti-Ang2 antibody-like molecules”, as defined herein, and conjugates with any of these. Antibodies include, but are not limited to, monoclonal and chimerized monoclonal antibodies. The term ,,antibody“ encompasses complete immunoglobulins, like monoclonal antibodies produced by recombinant expression in host cells, as well as antibody fragments or ody-like molecules”, including single-chain antibodies and linear dies, so- called ” (“Small Modular lmmunopharmaceuticals”), as e.g described in W02002/O56910; Antibody-like molecules include immunoglobulin single variable s, as defined herein. Other es for antibody-like molecules are immunoglobulin amily antibodies , or afted molecules.

“Ang2-binding molecule” or binding le" respective/y, refers to both monovalent target-binding molecules (i.e. molecules that bind to one epitope of the respective target) as well as to bi- or multivalent binding molecules (i.e. binding les that bind to more than one epitope, e.g. ”biparatopic” molecules as defined hereinbelow). Ang2(or VEGF)-binding molecules containing more than one Ang2(or binding immunoglobulin single variable domain are also termed “formatted” binding molecules, they may, within the target-binding component, in addition to the immunoglobulin single variable domains, comprise linkers and/or moieties with effector functions, e.g. half-life-extending moieties like albumin-binding globulin single variable s, and/or a fusion partner like serum albumin and/or an attached polymer like PEG.

The term "biparatopic An92(or binding molecule” or "biparatopic immunoglobulin single variable domain” as used herein shall mean a binding molecule comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as herein defined, wherein the two molecules bind to two non-overlapping epitopes of the respective antigen. The topic binding molecules are composed of immunoglobulin single variable s which have different specificities with respect to the epitope. The part of an antigen-binding molecule (such as an antibody or an globulin single variable domain of the invention) that recognizes the epitope is called a pe.

A formatted binding molecule may, albeit less preferred, also comprise two identical immunoglobulin single variable domains or two different immunoglobulin single variable domains that recognize the same or overlapping epitopes or their respective n. In this case, with respect to VEGF, the two immunoglobulin single variable domains may bind to the same or an overlapping epitope in each of the two rs that form the VEGF dimer.

Typically, the binding molecules of the invention will bind with a dissociation constant (KB) of 10E-5 to “ICE-14 liter (M) or less, and preferably 10E-7 to 10E-14 moles/liter (M) or less, more preferably 10E-8 to 10E-14 moles/liter, and even more preferably 10E-11 to 1OE-13, as measured e.g. in a Biacore or in a Kinexa assay), and/or with an association constant (KA) of at least ‘IOE7 ME-1, preferably at least 10E8 ME—1, more preferably at least 10E9 ME-1, such as at least 10E11 ME-1. Any KD value greater than 10E-4 M is generally considered to indicate non—specific binding.

WO 31078 Preferably, a polypeptide of the invention will bind to the desired n, i.e. VEGF or Ang2, respectively, with a KD 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 epitope can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or itive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and ch competition assays, and the different variants thereof known per se in the art.

Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code, as generally known and agreed upon in the art. When ing two amino acid sequences, the term "amino acid difference" refers to insertions, deletions or tutions of the indicated number of amino acid residues at a position of the reference sequence, compared to a second sequence. In case of substitution(s), such substitution(s) will preferably be conservative amino acid tution(s), which means that an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no nce on the function, activity or other biological properties of the polypeptide. Such conservative amino acid tutions are well known in the art, for example from WO1998/49185, wherein conservative amino acid substitutions preferably are substitutions in which one amino acid within the following groups (i) - (v) is tuted by another amino acid residue within the same group: (i) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar, negatively d residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar, positively charged residues: His, Arg and Lys; (iv) large aliphatic, nonpolar residues: Met, Leu, lle, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu;Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; lle into Leu or into Val; Leu into lie or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into lle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser;Trp into Tyr; Tyr into Trp or into Phe; Val into lie or into Leu.

A polypeptide or nucleic acid molecule is considered to be "(in) essentially isolated (form)" - for example, when ed 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 protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor ent. In ular, a polypeptide or nucleic acid molecule 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 polypeptide or nucleic acid molecule that is "in essentially isolated form" is preferably essentially homogeneous, as ined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide gel ophoresis. nce identity" between two VEGF-binding molecule sequences or n two AngZ—binding molecule sequences indicates the percentage of amino acids that are identical between the sequences. It may be calculated or determined as described in paragraph f) on pages 49 and 50 of W02008/020079. "Sequence similarity" indicates the percentage of amino acids that either are cal or that represent conservative amino acid substitutions.

Alternative s for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the present description, claims and figures, the numbering ing to Kabat and applied to VHH domains as described above will be followed, unless indicated otherwise.

An "affinity-matured” VEGF-binding molecule or inding molecule, in particular a VHH or a domain antibody, has one or more alterations in one or more CDRs which result in an ed affinity for VEGF or Ang2, as compared to the respective parent VEGF-binding molecule or Ang2—binding molecule. Afffinity-matured VEGF-binding molecules or Ang2—binding molecules of the invention may be prepared by methods known in the art, for example, as described by Marks et a/., 1992, Biotechnology 10: 3, or Barbas, eta/., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier eta/., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins etal., 1992, J. Mol. Biol. 226(3): 889 896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

Forthe t invention, an “amino acid ces of SEQ ID NO: x”: includes, if not otherwise stated, an amino acid sequence that is 100% identical with the ce shown in the respective SEQ ID NO: x; a) amino acid sequences that have at least 80% amino acid identity with the sequence shown in the respective SEQ ID NO: x; b) amino acid sequences that have 3, 2, or 1 amino acid differences with the sequence shown in the respective SEQ ID NO: x.

The terms "cancer“ and "cancerous” refer to or describe the physiological condition in s that is typically characterized by unregulated cell growth/proliferation.

Examples of cancer to be treated with a bispecific binding molecule of the ion, include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

More particular es of such s, as suggested for treatment with VEGF antagonists in US 2008/0014196, include squamous cell cancer, small-cell lung , non—small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval , thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and s types of head and neck cancer. Dysregulation of angiogenesis can lead to many disorders that can be d by compositions and methods of the invention. These disorders include both non-neoplastic and neoplastic conditions. sties include but are not limited those described above.

Non—neoplastic disorders include, but are not limited to, as ted fortreatment with VEGF antagonists in U82008/0014196, undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, sclerotic plaques, diabetic and other proliferative retinopathies including retinopathy of prematurity, ental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal/choroidal neovascularization, neovascularization of the angle (rubeosis), ocular neovascular disease, vascular restenosis, ovenous malformations (AVM), meningioma, hemangioma, ibroma, thyroid lasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, acute lung injury/ ARDS, sepsis, primary pulmonary ension, malignant pulmonary effusions, al edema (e.g., associated with acute stroke/ closed head injury/ trauma), synovial inflammation, pannus formation in RA, myositis ossificans, hypertropic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, 3rd spacing of fluid diseases eatitis, compartment syndrome, burns, bowel e), uterine fibroids, premature labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, nephrotic syndrome, undesired or aberrant tissue mass growth (non-cancer), hemophilicjoints, hypertrophic scars, inhibition of hair growth, Osier—Weber syndrome, pyogenic granuloma retrolental fibroplasias, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.

DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the t invention relates to a bispecific binding molecule comprising at least one Ang2—binding component and at least one VEGF-binding component.

In a preferred embodiment, the present ion relates to a bispecific g molecule comprising at least one VEGF-binding component and at least one Ang2—binding component which further comprises at least a further binding component, ably a serum albumin binding component (serum albumin binding molecule).

In a red embodiment, the serum albumin binding ent of the binding molecule of the t invention is an isolated immunoglobulin single variable domain or a ptide ning one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four ork regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 257, 260, 263, 266, 269, 272, or 275.

More preferably, said one or more immunoglobulin single variable domain of the serum n binding component contain a. a CDR3 with an amino acid sequence ed from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs N03: 257, 260, 263, 266, 269, 272, or275; b. a CDR1 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs:255, 258, 261, 264, 267, 270, or 273; c. a CDR2 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs:256, 259, 262, 265, 268, 271, or 274.

In a more preferred embodiment, said one or more immunoglobulin single variable domains of the serum albumin g component are VHHs, preferably having an amino acid sequence shown in SEQ ID NOs: 98 or 254.

According to preferred embodiments, said inding component and said VEGF— binding component comprise at least one Ang2-binding immunoglobulin single variable domain and at least one VEGF-binding immunoglobulin single variable domain, tively.

In a preferred aspect, said Ang2-binding component and said VEGF-binding component each comprise at least one VEGF-binding immunoglobulin single variable domain and at least one Ang2—binding immunoglobulin single variable domain, respectively, wherein each of said immunoglobulin single variable domains has four framework s and three complementarity determining regions CDR1, CDR2 and CDR3, respectively.

Thus, the ng2 and/or the anti-VEGF component contained in the ific binding molecules of the invention may include two (or more) anti-Ang2 (or anti-VEGF, respectively) immunoglobulin single variable s, wherein the immunoglobulin single variable domains are directed against ent epitopes within the Ang2 (or VEGF) target. Thus, the two immunoglobulin single le domains in a ific binding le will have different antigen specificity and therefore different CDR sequences.

Such bivalent binding molecules are also named "biparatopic single domain antibody constructs" (if the immunoglobulin single le domains consist or essentially consist of single domain antibodies), or "biparatopic VHH constructs" (if the immunoglobulin single variable domains consist or essentially consist of VHHs), respectively, as the two immunoglobulin single variable domains will include two different paratopes.

In the bispecific binding le of the invention, one or both of the binding molecules may be bivalent; e.g. the VEGF-binding component may be biparatopic and the Ang2- g component may be one immunoglobulin single le domain, or the VEGF- binding component may be one immunoglobulin single variable domain and the AngZ- binding component may be biparatopic. ln bispecific binding molecules of the invention, it is preferably the VEGF-binding component that ns a bivalent VEGF-binding immunoglobulin single variable domain, e.g. a biparatopic VHH.

Such VEGF-binding immunoglobulin single variable domain may be two or more VEGF- binding VHHs, which are a. identical VHHs that are e of blocking the interaction between inant human VEGF and the recombinant human VEGFR-2 with an inhibition rate of 2 60% or b. different VHHs that bind to non-overlapping epitopes of VEGF, wherein at least one VHH is capable of blocking the interaction between recombinant human VEGF and the recombinant human VEGFR-2 with an inhibition rate of 2 60% and wherein at least one VHH is capable of blocking said interaction with an inhibition rate of s 60 %.

WO 31078 The VEGF-binding component sing at least a variable domain with four framework regions and three complementarity determining s CDR1, CDR2 and CDRB, respectively, wherein said CDR3 has the amino acid sequence Ser Arg Ala Tyr Xaa Ser Xaa Arg Leu Arg Leu Xaa Xaa Thr Tyr Xaa Tyr as shown in SEQ ID NO: 1, wherein Xaa at position 5 is Gly or Ala; Xaa at position 7 is Ser or Gly; Xaa at position 12 is Gly, Ala or Pro; Xaa at position 13 is Asp or Gly; Xaa at position 16 is Asp or Glu; and wherein said VEGF-binding component is capable of blocking the interaction of human recombinant VEGF165 with the human recombinant VEGFR—2 with an inhibition rate of 260%.

According to red embodiments, Xaa at position 5 is Gly, Xaa at position 7 is Ser, Xaa at position 12 is Ala, and Xaa at position 13 is Asp.

In particular, said CDR3 has a sequence selected from SEQ ID N012 SRAYGSSRLRLGDTYDY, SEQ ID NO: 3 SRAYGSSRLRLADTYDY; SEQ ID NO: 4 SRAYGSSRLRLADTYEY; SEQ ID NO: 5 GRLRLADTYDY; SEQ ID NO: 6 SRAYASSRLRLADTYDY; SEQ ID NO: 7 SRAYGSSRLRLPDTYDY; SEQ ID NO: 8 SRAYGSSRLRLPGTYDY.

According to certain embodiments, a VEGF-binding component comprises one or more immunoglobulin single variable domains each containing a. a CDR3 with an amino acid sequence ed from a first group of sequences shown in SEQ ID NO: 2 to 8; b. a CDR1 and a CDR2 with an amino acid sequences that is contained, as indicated in Table 3, in a sequence selected from a second group of amino acid sequences shown in SEQ ID NOs: 9 to 46, wherein said second sequence contains the respective CDR3 selected according to a).

According to preferred embodiments, the immunoglobulin single variable domains are VHHs.

According to ic ments, the VHHs have amino acid sequences selected from sequences shown in SEQ ID NOs: 9 - 46.

According to another specific embodiment, the VHHs have amino acid sequences selected from SEQ ID N08: 15, SEQ ID NO: 18 and SEQ ID NO: 25.

The invention also relates to VEGF-binding component that have been obtained by affinity maturation and/or sequence optimization of an above-defined VHH, e.g. to a VHH that has been ed by sequence optimization of a VHH having an amino acid sequence shown in SEQ ID NO: 18. Examples are VHHs having amino acid sequences selected from sequences shown in SEQ ID N03: 47 — 57.

According to certain embodiments, a VEGF-binding domain of the invention may be formatted, as herein defined, e.g. it may be biparatopic or se two identical immunoglobulin single variable s. Such VEGF-binding components may comprise two or more VHHs, which are a) identical VHHs that are capable of blocking the interaction between recombinant human VEGF and the recombinant human VEGFR-2 with an inhibition rate of 2 60% or b) ent VHHs that bind to non-overlapping epitopes of VEGF, wherein at least one VHH is capable of blocking the interaction between inant human VEGF and the recombinant human VEGFR-2 with an inhibition rate of 2 60% and wherein at least one VHH binds is e of blocking said interaction with an inhibition rate of s 60 %.

The percentage of blocking said interaction at an inhibition rate of 2 60% or s 60 %, respectively, refers to an inhibition rate as determined by an Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen®), a competition ELISA, a plasmon resonance (SPR) based assay (Biacore®) as used in the Examples.

In the following, the ability of VHHs according to a) is also termed “receptor-blocking”, while the ability of VHHs according to b) is also termed “non-receptor-blocking”.

Preferably, the receptor-blocking VHHs have an inhibition rate of 2 80%, more preferably 2 90%; the most preferred VHHs being te receptor blockers, i.e. have an inhibition rate of 100 %.

A VEGF-binding ent may contain two or more identical VHHs a) selected from VHHs having amino acid sequences shown in SEQ ID NOs: 9 - 46 or VHHs that have been obtained by affinity maturation and/or sequence optimization of such VHH. The VHH may be selected from VHHs having the amino acid shown in SEQ ID NO: 18 or SEQ ID NO: 47 — 57. ing to red embodiments, a formatted VEGF-binding component comprises two VHHs each having the amino acid sequence shown in SEQ ID NO: 57.

In formatted VEGF-binding components comprising two different VHHs a) said one or more VHHs with an inhibition rate of 2 60% are selected from i. VHHs having an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 9 — 46 or ii. VHHs that have been obtained by affinity maturation and/or sequence optimization of such VHHs, and wherein b) said one or more VHHs with an inhibition rate of s 60 % are ed from i. SEQ ID NOs: 58 — 124 or ii. VHHs that have been obtained by affinity maturation and/or sequence optimization of such VHH.

According to red embodiments, two VHHs are contained in polypeptides with amino acid sequences shown in SEQ ID NOs: 128 — 168, separated by linker sequences as indicated in Table 15.

In a preferred VEGF-binding component VHH a) i. has an amino acid sequence shown in SEQ ID NO: 18 and VHH b) i. has an amino acid sequence shown in SEQ ID NO: 64.

In other preferred VEGF-binding components VHHs according to a) ii. are selected from VHHs having an amino acid sequence shown in SEQ ID N03: 47 — 57 and VHHs according to b) ii. are selected from VHHs having an amino acid sequence shown in SEQ ID NOs: 125— 127.

Particularly preferred is a biparatopic VEGF-binding component sing two VHHs, one of them having the amino acid shown in SEQ ID NO: 57 and one of them having the amino acid shown in SEQ ID NO: 127.

The Ang2-binding component comprises at least a variable domain with four framework regions and three mentarity ining regions CDR1, CDR2 and CDR3, tively, wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID N03: 226, 229, 232, 235, 238, 241, 244, 247, 250, or 253.

In a second aspect, said Ang2-binding component is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single le domains, wherein said immunoglobulin single variable domain consists of four ork regions and three complementarity determining s CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 226, 229, 232, 235, 238, 241, 244, 247, 250, or 253.

In a further aspect, said immunoglobulin single variable domain of the AngZ-binding component contains a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 226, 229, 232, 235, 238, 241, 244, 247, 250, or 253 (see also Table 49); WO 31078 b. a CDR1 with an amino acid sequences that is contained, as indicated in Table 36-A, 38-A, 41-A, or 45-A, as l sequence in a sequence selected from a second group of amino acid ces shown SEQ ID N03: 224, 227, 230, 233, 236, 239, 242, 245, 248, or 251 (see also Table 49); c. a CDR2 with an amino acid sequences that is contained, as indicated in Table 36-A, 38—A, 41-A, or 45-A, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 225, 228, 231, 234, 237, 240, 243, 246, 249, or 252 (see also Table 49).

Preferably, the immunoglobulin single variable domain of the Ang2-binding component is a VHH, preferably having amino acid sequence selected from amino acid sequences shown in SEQ ID N03: 214, 215, 216, 217, 218, 219, 220, 221, 222, or 223.

In another preferred embodiment, the immunoglobulin single variable domain of the inding component has been obtained by affinity maturation or humanization of an immunoglobulin single variable domain as described herein.

Similarly, the present invention also relates to a VHH which has been obtained by affinity maturation or humanization of a VHH of the Ang2-binding component as described herein.

The present invention thus also relates to an Ang2-binding VHH with an amino acid sequence selected from acid ces shown in SEQ ID NOs: 214, 215, 216, 217, 218, 219, 220, 221, 222, or 223.

Suitable parent Ang2-binding components for affinity maturation are, by way of example, the above-described VHHs with amino acid sequences shown in SEQ ID NOs:214, 215, 216, 217, 21i8, or 219.

Accordingly, the invention also s to inding molecules that have been obtained by ty maturation and/or sequence optimization of an above-defined VHH, eg. to a VHH that has been obtained by sequence optimization of a VHH having an amino acid sequence shown as SEQ ID NOs:217, 218, 219,220, 221, 222, or 223. The “source” amino acid sequences that were used to generate the latter VHHs are shown in SEQ ID NOs: 214, 215, or 216. Also these amino acid sequences are le Ang2- binding components that can be applied in the binding molecules of the present invenfion.

As described herein, the binding molecule of the present invention preferably comprises at least one serum albumin binding component. Particularly preferred binding molecules thus have at least one VEGF-binding component, at least one inding component and at least one serum albumin binding component. The order of these three binding components could be any possible order such as the order set out in Table 36-8, 38—8, 40, 41-8, 42, 43, 45-8, 46-A, or 47-A; or in Figure 20, 23, 27, or 30, e.g. the VEGF-, Ang2—or serum albumin binding component can be N-terminal or C-terminal. y, “1DO1” (SEQ ID No: 214), “11807”, “00027” (SEQ ID No:216), “00908”, “7G08” (SEQ ID No:215), “00919”, “00921” (SEQ ID No: 220), “00928” (SEQ ID No:221), “00932”, “00933”, “00934”, “00935”, “00936”, “00937”, “00938” (SEQ ID No:222), or ” (SEQ ID No:223) as referred to in the legend of the aforementioned Tables and Figures stand for inding components, while “00038” stands for a VEGF-binding ent and “ALB1 1” stands for a serum albumin binding component. None of them is to be construed to a specific sequence, but stands for a Ang2-, VEGF- and serum albumin binding ent in general when used in the context of possible set-ups of binding molecules of the t invention.

However, it is preferred that the serum albumin binding component is in between the VEGF- and inding component (or vice , while it is particularly preferred that at least one VEGF-binding component is N-terminal, followed by at least one serum albumin binding component, followed by at least one Ang2-binding component at the C-Terminus. This set-up is shown to be specifically useful.

The present invention relates thus in a preferred aspect to binding molecules comprising at least one VEGF-binding ent, at least one Ang2-binding component and at least one serum albumin binding component having an amino acid sequence ed from the amino acid ces shown in SEQ ID NOs: 180-213, “At least one” binding component (VEGF, Ang2 or serum albumin) when used herein includes that a binding molecule of the present invention may contain one, two, three, four orfive VEGF-, Ang2-, and/or serum albumin binding components (i.e., entities/units) which are preferably represented by an immunoglobulin singly variable domain as described herein.

The VEGF- and/or Ang2-binding components with improved properties in view of therapeutic application, e.g. enhanced affinity or decreased immunogenicity, may be obtained from individual VEGF-or AngZ-binding components of the invention by techniques known in the art, such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin ces), CDR grafting, humanizing, combining fragments derived from ent immunoglobulin sequences, PCR assembly using overlapping s, and similar techniques for ering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing, also termed “sequence optimization”, as described herein.

Reference is, for example, made to standard oks, as well as to the further description and Examples.

If appropriate, a VEGF-or inding component of the invention with increased affinity may be obtained by affinity-maturation of another VEGF-or Ang2—binding ent, the latter representing, with respect to the affinity—matured le, the “parent” VEGF-binding ent.

In VEGF or Ang2 VHHs of the invention that start with EVQ, the N-terminal E may be replaced by a D (which is often a result of sequence—optimization) or it may be missing (as for expression of the VHH in Eco/i). Forformatted VEGF—binding components, this usually applies only to the VHH that is situated N-terminally.

A preferred, but non-limiting humanizing substitution for VEGF VHH domains belonging to the 103 P,R,S-group and/or the GLEW-group (as defined below) is 1080 to 108L.

Methods for zing immunoglobulin single variable domains are known in the art.

According to another embodiment, the immunoglobulin single variable domain is a domain dy, as defined herein.

In yet another embodiment, the representatives of the class of VEGF-and/or Ang2- binding immunoglobulin single variable domains of the invention have amino acid sequences that correspond to the amino acid sequence of a naturally occurring VH domain that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a lly occurring le heavy chain from a conventional n antibody by one or more amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the d person, and reference is additionally be made to WO1994/O4678. Such camelization may entially occur at amino acid positions which are present at the VH-VL interface and at the so-called Camelidae Hallmark residues (see for example also WO1994/O4678). A detailled description of such "humanization" and "camelization" techniques and preferred framework region sequences consistent therewith can additionally be taken from e.g. pp. 46 and pp. 98 of W02006/040153 and pp. 107 of W02006/122786.

The VEGF-binding components of the invention, e.g. immunoglobulin single le domains, have specificity for VEGF in that they comprise one or more immunoglobulin single variable domains specifically binding to one or more epitopes within the VEGF molecule. The same is true for Ang2-binding components of the invention.

Specific binding of an VEGF-binding ent to its antigen VEGF can be ined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA and ELISA) and ch competition assays, and the ent variants thereof known per se in the art. The same is true for an Ang2—binding component when binding to its antigen.

With regard to the antigen VEGF, a VEGF-binding ent of the invention, e.g. an immunoglobulin single variable domain, is not limited with regard to the species. Thus, the immunoglobulin single variable domains of the invention preferably bind to human VEGF, if intended for eutic purposes in humans. However, immunoglobulin single variable domains that bind to VEGF from another mammalian species are also within the scope of the invention. An immunoglobulin single variable domain of the invention binding to one species form of VEGF may cross-react with VEGF, which has a different sequence than the human one, from one or more other species. For example, immunoglobulin single variable domains of the invention binding to human VEGF may exhibit cross reactivity with VEGF from one or more other species of primates and/or with VEGF from one or more species of animals that are used in animal models for diseases, for example monkey, mouse, rat, rabbit, pig, dog, and in particular in animal models for diseases and ers associated with VEGF-mediated s on angiogenesis (such as the species and animal models mentioned herein). globulin single variable domains of the invention that show such cross-reactivity are advantageous in a research and/or drug development, since it allows the immunoglobulin single variable domains of the ion to be tested in ledged disease models such as monkeys, in particular Cynomolgus or Rhesus, or mice and rats.

Preferably, in view of cross-reactivity with one or more VEGF molecules from s other than human that is/are intended for use as an animal model during development of a therapeutic VEGF antagonist, a inding component recognizes an epitope in a region of the VEGF of interest that has a high degree of identity with human VEGF.

An immunoglobulin single variable domain of the invention recognizes an epitope which is, totally or in part, located in a region of VEGF that is relevant for binding to its receptor, in particular to VEGFR-2, which has been shown to be the receptor whose activation is causally involved in the neovascularisation of tumors. According to preferred aspects, globulin single variable domains of the invention block VEGF receptor tion, in particular 2 activation, at least partially, preferably substantially and most preferably totally.

As described above, the ability of a VEGF-binding component to block the ction between VEGF and its receptors, in particular the VEGFR-2, can be determined by an Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen®), a competition ELISA, or a plasmon resonance (SPR) based assay (Biacore®), as described in the Examples.

Preferably, an immunoglobulin single variable domain of the invention binds to VEGF with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than nM, such as less than 500 pM (as determined by Surface Plasmon Resonance analysis, as described in Example 5.7). The same is true for an immunoglobulin single le domain of the invention binds to angiopoietin.

Preferably, the immunoglobulin single le domains of the invention have IC50 values, as measured in a competition ELISA assay as bed in Example 5.1. in the range of 10'6 to ‘IO'10 moles/litre or less, more preferably in the range of 10'8 to 10'10 moles/litre or less and even more preferably in the range of 10'9 to 10'10 moles/litre or less.

According to a non-limiting but red embodiment of the invention, VEGF-binding immunoglobulin single variable domains of the ion bind to VEGF with an dissociation constant (KB) of 10'5 to 10‘12 moles/liter (M) or less, and preferably 10'7 to '12 moles/liter (M) or less and more preferably 10'8 to 10'12 moles/liter (M), and/or with an association constant (KA) of at least 107 M1, preferably at least 108 M", more preferably at least 109 M1, such as at least 1012 M1; and in particular with a KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The KD and KA values of the immunoglobulin single variable domain of the invention against VEGF can be determined. The same is true for an Ang2—binding immunoglobulin single variable domain of the invention. topic inding components comprising two or more immunoglobulin single variable domains essentially consist of or comprise (i) a first immunoglobulin single le domain specifically binding to a first epitope of VEGF and (ii) a second immunoglobulin single variable domain specifically binding to a second epitope of VEGF, wherein the first epitope of VEGF and the second epitope of VEGF are not identical es. In other words, such polypeptide of the invention comprises or essentially consist of two or more immunoglobulin single variable domains that are ed t at least two non-overlapping epitopes present in VEGF, wherein said immunoglobulin single variable domains are linked to each other in such a way that they are capable of simultaneously binding VEGF. In this sense, the polypeptide of the invention can also be regarded as a “bivalent” or "multivalent" immunoglobulin construct, and especially as a "multivalent immunoglobulin single variable domain construct", in that the polypeptide ns at least two binding sites for VEGF. (Such constructs are also termed “formatted” VEGF binding molecules, e.g. “formatted” VHHs). The same is true for biparatopic Ang2-binding components, mutatis mutandis.

Such VEGF-or Ang2-binding ent of the invention includes (at least) two anti— VEGF or Ang2 immunoglobulin single variable domains, respectively, wherein (the) two immunoglobulin single variable domains are preferably directed against non-overlapping epitopes within the VEGF le or angiopoietin molecule, respectively. Thus, these two immunoglobulin single variable domains will have a ent antigen specificity and therefore different CDR sequences. For this reason, such polypeptides of the invention will herein also be named "biparatopic ptides", or "biparatopic domain dy constructs" (if the immunoglobulin single variable domains consist or essentially consist of domain antibodies), or "biparatopic VHH ucts" (if the immunoglobulin single variable domains t or essentially consist of VHHs), respectively, as the two immunoglobulin single variable domains will e two different paratopes.

If a polypeptide of the invention is a biparatopic molecule as defined herein, at least one of the immunoglobulin single variable domain components binds to an epitope such that the interaction between recombinant human VEGF and recombinant humen VEGFR-2 is blocked at an inhibition rate of 280%. As has been shown in experiments of the invention, n formatted molecules contain two VHHs that both block the VEGFR2 receptor at an inhibition rate of 280%. Certain VHHs of the invention block the VEGFR-2 at an inhibition rate of 100%, i.e. they are complete blockers.

In both cases, onal sequences and moieties may be present within the VEGF- binding components of the invention, e.g. N-terminally, inally, or located between the two immunoglobulin single le domains, e.g. linker sequences and sequences providing for effector ons, as set out in more detail herein.

According to another, albeit less preferred embodiment, a VEGF-binding component of the invention may include more than two anti-VEGF immunoglobulin single variable domains, i.e. three, four or even more anti-VEGF VHHs. In this case, at least two of the anti—VEGF immunoglobulin single variable domains are directed against non-overlapping epitopes within the VEGF molecule, wherein any further immunoglobulin single variable domain may bind to any of the two non-overlapping es and/or a further epitope present in the VEGF molecule.

According to the invention, the two or more immunoglobulin single variable domains can be, ndently of each other, VHHs or domain antibodies, and/or any other sort of immunoglobulin single variable domains, such as VL domains, as d herein, provided that these immunoglobulin single variable domains will bind the antigen, i.e.

VEGF or angiopoietin, respectively.

The detailed description of the g components is primarily provided for the VEGF- binding component. However, all features and options outlined herein forthe VEGF- binding component also apply equivalently for the Ang2—binding component, mutatis mutandis.

According to preferred embodiments, the binding molecules present in the bispecific binding molecules (the Ang2—binding molecules within the Ang2-binding component or the VEGF-binding molecules within the VEGF-binding ent or the two adjacent Ang2— and VEGF-binding components) may be connected with each other directly (i.e. without use of a linker) or via a linker. The linker is preferably a linker peptide and will be selected so as to allow binding of the two ent binding molecules to each of non- pping epitopes of the targets, either within one and the same target molecule, or within two different molecules.

In the case of topic binding molecules, selection of s within the Ang2— or the VEGF-binding component will inter alia depend on the es and, specifically, the distance between the epitopes on the target to which the immunoglobulin single variable domains bind, and will be clear to the skilled person based on the disclosure , optionally after some limited degree of routine mentation.

Two g molecules (two VHHs or domain antibodies or VHH and a domain antibody), or two binding components, may be linked to each other via an additional VHH or domain antibody, respectively (in such binding molecules, the two or more immunoglobulin single variable domains may be linked directly to said additional WO 31078 immunoglobulin single variable domain or via suitable linkers). Such an additional VHH or domain antibody may for example be a VHH or domain antibody that provides for an increased half-life. For example, the latter VHH or domain antibody may be one that is capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin.

Alternatively, the two or more immunoglobulin single variable domains that bind to the respective target may be linked in series (either directly or via a suitable linker) and the additional VHH or domain antibody (which may provide for increased half-life) may be connected directly or via a linker to one of these two or more aforementioned immunoglobulin sequences.

Suitable linkers are described herein in connection with specific ptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 9 or more amino acids, more preferably at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides.

The linker ce may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is preferably munogenic in the subject to which the ific binding molecule of the invention is stered.

One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO1996/34103 and W01994/O4678.

Other examples are lanine linker sequences such as Ala- Ala- Ala.

Further preferred examples of linker sequences are Gly/Ser s of different length such as (glyxsery)z linkers, including er)3, (gly4ser)4, (gly4ser), er), glyg, and (gly3ser2)3.

Some non-limiting examples of linkers are contained in ific binding molecules of the invention shown in Table 15 (SEQ ID NOs 128 — 168), e.g. the linkers GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS; SEQ ID NO: 169); GGGGSGGGS (9G8; SEQ ID NO: 170); GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (4OGS; SEQ ID NO: 171).

If a ted bispecific binding molecule of the invention is modified by the attachment of a polymer, for e of a polyethylene glycol PEG (polyethylene glycol) moiety, the linker sequence preferably includes an amino acid residue, such as a cysteine or a lysine, allowing such modification, e.g. tion, in the linker region.

Examples of linkers useful forfor PEGylation are: GGGGCGGGS (“GSQ,C5”, SEQ ID NO: 172); GGGGCGGGGSGGGGSGGGGSGGGGS (“6825,C5, SEQ ID NO:173) GGGSGGGGSGGGGCGGGGSGGGGSGGG ,C14", SEQ ID NO:174), GGGGSGGGGSGGGGCGGGGSGGGGSGGGGSGGGGS ("GSB5,C15", SEQ ID NO:175), and GGGGCGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (“GS35,C5”, SEQ ID NO:176).

Furthermore, the linker may also be a poly(ethylene glycol) moiety, as shown in e.g.

W02004/081026.

In r embodiment, the immunoglobulin single variable domains are linked to each other via r moiety (optionally via one or two linkers), such as another polypeptide which, in a preferred but miting embodiment, may be a further immunoglobulin single variable domain as described above. Such moiety may either be essentially inactive or may have a biological effect such as improving the desired properties of the polypeptide or may confer one or more additional d properties to the polypeptide.

For example, and without limitation, the moiety may improve the half-life of the protein or polypeptide, and/or may reduce its immunogenicity or improve any other desired property.

WO 31078 According to a preferred embodiment, a bispecific binding le of the invention includes, especially when intended for use or used as a therapeutic agent, a moiety which extends the half-life of the polypeptide of the invention in serum or other body fluids of a patient. The term life" is defined as the time it takes for the serum concentration of the (modified) polypeptide to reduce by 50%, in vivo, for example due to degradation of the polypeptide and/or clearance and/or sequestration by natural isms.

More specifically, such half-life extending moiety can be covalently linked to or fused to an immunoglobulin single variable domain and may be, without limitation, an Fc portion, an albumin , a fragment of an albumin moiety, an albumin binding moiety, such as an lbumin immunoglobulin single variable domain, a transferrin binding moiety, such as an anti—transferrin immunoglobulin single variable domain, a polyoxyalkylene molecule, such as a polyethylene glycol molecule, an albumin binding peptide or a hydroxyethyl starch (HES) derivative.

In another embodiment, the bispecific binding molecule of the invention comprises a moiety which binds to an antigen found in blood, such as serum albumin, serum immunoglobulins, thyroxine-binding protein, fibrinogen or transferrin, thereby conferring an increased half-life in vivo to the resulting polypeptide of the invention. According to a specifically preferred ment, such moiety is an albumin-binding immunoglobulin and, ally preferred, an albumin-binding immunoglobulin single variable domain such as an albumin-binding VHH domain.

If intended for use in humans, such albumin-binding immunoglobulin single variable domain preferably binds to human serum albumin and preferably is a humanized albumin-binding VHH . lmmunoglobulin single variable domains binding to human serum n are known in the art and are described in further detail in e.g. W02006/122786. ically, useful albumin binding VHHs are ALB 1 and its humanized counterpart, ALB 8 (W02009/095489). Other albumin g VHH s mentioned in the above patent publication may, however, be used as well.

A specifically useful albumin binding VHH domain is ALB8 which consists of or ns the amino acid sequence shown in SEQ ID NO: 98 or 254.

According to a further embodiment of the invention, the two immunoglobulin single variable domains, in preferably VHHs, may be fused to a serum albumin molecule, such as described e.g. in WO2001/79271 and WO2003/59934. As e.g. described in WO2001/79271, the fusion protein may be obtained by conventional recombinant technology: a DNA le coding for serum albumin, or a fragment thereof, is joined to the DNA coding for the bispecific binding molecule, the obtained construct is inserted into a plasmid suitable for expression in the selected host cell, e.g. a yeast cell like Pichia pastoris or a ial cell, and the host cell is then transfected with the fused nucleotide sequence and grown under suitable conditions. The sequence of a useful HSA is shown in SEQ ID NO: 99.

According to another embodiment, a half-life extending modification of a polypeptide of the ion (such modification also reducing genicity of the polypeptide) comprises attachment of a suitable pharmacologically acceptable polymer, such as straight or branched chain thylene glycol) (PEG) or derivatives thereof (such as methoxypoly(ethylene ) or mPEG). Generally, any suitable form of PEGylation can be used, such as the tion used in the art for antibodies and dy fragments (including but not limited to domain antibodies and scFv's); reference is made, for example, to: Chapman, Nat. Biotechnol, 54, 531-545 (2002); Veronese and Harris, Adv.

Drug Deliv. Rev. 54, 453-456 ; Harris and Chess, Nat. Rev. Drug. Discov. 2 (2003); and WO2004/060965.

Various reagents for PEGylation of polypeptides are also commercially available, for example from Nektar Therapeutics, USA, or NOF Corporation, Japan, such as the Sunbright® EA Series, SH Series, MA Series, CA Series, and ME Series, such as Sunbright® ME—100MA, Sunbright® ME-200MA, and Sunbright® ME-400MA.

Preferably, site—directed tion is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering 16, 761-770 (2003)). For example, for this e, PEG may be attached to a cysteine residue that naturally occurs in a ptide of the invention, a polypeptide 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 sing one or more ne residues for attachment of PEG may be fused to the N- and/or C-terminus of a polypeptide of the invention, all using techniques of protein engineering known per se to the skilled person.

Preferably, for the polypeptides of the ion, a PEG is used with a molecular weight of more than 5 kDa, such as more than 10 kDa and less than 200 kDa, such as less than 100 kDa; for example in the range of 20 kDa to 80 kDa.

With regard to tion, its should be noted that generally, the invention also encompasses any bispecific binding molecule that has been PEGylated at one or more amino acid positions, ably in such a way that said PEGylation either (1) ses the half-life in vivo; (2) reduces immunogenicity; (3) provides one or more further cial properties known per se for PEGylation; (4) does not essentially affect the affinity of the polypeptide for its target (e.g. does not reduce said ty by more than 50 %, and more preferably not by more than 10%, as determined by a suitable assay bed in the art); and/or (4) does not affect any of the other desired properties of the bispecific binding molecules of the invention. Suitable PEG-groups and methods for attaching them, either ically or non-specifically, will be clear to the skilled person.

Various reagents for PEGylation of polypeptides are also commercially available, for example from Nektar Therapeutics, USA, or NOF ation, Japan, such as the Sunbright® EA Series, SH Series, MA Series, CA Series, and ME Series, such as Sunbright® ME—1OOMA, Sunbright® ME-2OOMA, and Sunbright® ME-4OOMA.

According to an especially preferred embodiment of the invention, a PEGylated polypeptide of the invention includes one PEG moiety of linear PEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEG moiety is attached to the polypeptide in a linker region and, specifially, at a Cys residue at position 5 of a GSQ-Iinker peptide as shown in SEQ ID NO:93, at position 14 of a GSZ7—Iinker peptide as shown in SEQ ID NO:95, or at position 15 of a GSB5-linker peptide as shown in SEQ ID NO:96, or at position 5 of a 35GS-Iinker peptide as shown in SEQ ID NO:97. 2012/055901 A bispecific binding molecule of the invention may be PEGylated with one of the PEG reagents as mentioned above, such as "Sunbright® ME-4OOMA", as shown in the following chemical a: Bispecific binding molecules that contain s and/or half-life extending functional groups are shown in SEQ ID NO: 81 and in Figure 48.

According to another ment, the immunoglobulin single variable domains are domain antibodies, as defined herein. globulin single variable domains present in the bispecific binding molecules of the invention may also have sequences that correspond to the amino acid sequence of a naturally occurring VH domain that has been "camelized", i.e. by ing one or more amino acid es in the amino acid sequence of a naturally occurring variable heavy chain from a conventional n antibody by one or more amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody.

This can be performed in a manner known per se, which will be clear to the skilled person, and reference is additionally be made to WO1994/O4678. Such camelization may preferentially occur at amino acid positions which are present at the VH-VL interface and at the so-called dae Hallmark residues (see for example also W01994/O4678). A led description of such "humanization" and "camelization" techniques and preferred framework region sequences consistent therewith can additionally be taken from eg. pp. 46 and pp. 98 of W02006/040153 and pp. 107 of W02006/122786.

The binding components have specificity for Ang2 or VEGF, respectively, in that they comprise in a preferred embodiment one or more immunoglobulin single variable domains specifically binding to one or more epitopes within the Ang2 molecule or within the VEGF molecule, respectively.

Specific binding of a binding component to its antigen Ang2 or VEGF can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatohard analysis and/or competitive g assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA and ELISA) and sandwich competition assays, and the different variants thereof known per se in the art.

With regard to the antigen Ang2 or VEGF, respectively, an immunoglobulin single variable domain is not limited with regard to the species. Thus, the immunoglobulin single variable domains preferably bind to human AngZ or to human VEGF, respectively, if intended rapeutic purposes in humans. r, globulin single variable domains that bind to Ang2 or VEGF, respectively, from another ian species, or polypeptides containing them, are also within the scope of the invention. An immunoglobulin single variable domain binding to one species form of Ang2 or VEGF may cross—react with the respective antigen from one or more other species. For example, immunoglobulin single le domains binding to the human antigen may exhibit cross reactivity with the respective n from one or more other s of primates and/or with the antigen from one or more species of animals that are used in animal models for diseases, for example monkey (in particular Cynomolgus or Rhesus), mouse, rat, rabbit, pig, dog or) and in particular in animal models for diseases and disorders that can be modulated by inhibition of Ang2 (such as the species and animal models mentioned herein). lmmunoglobulin single variable domains of the invention that show such cross-reactivity are advantageous in a research and/or drug development, since it allows the immunoglobulin single variable domains of the invention to be tested in acknowledged disease models such as monkeys, in particular Cynomolgus or Rhesus, or mice and rats.

Also, the binding components are not limited to or defined by a specific domain or an nic determinant of the antigen against which they are directed. Preferably, in view of cross-reactivity with one or more antigen molecules from species other than human that is/are intended for use as an animal model during development of a therapeutic GF nist, a binding component recognizes an epitope in a region of the the respective n that has a high degree of identity with the human antigen. By way of example, in view of using a mouse model, an anti-Ang2 immunoglobulin single variable domain contained in the ific binding molecules of the invention izes an epitope which is, totally or in part, located within the FLD domain of AngZ, which shows a high identity between human and mouse.

Preferably, the VEGF-binding component binds to the VEGF isoforms VEGF165 and/or VEGF121.

In another aspect, the invention relates to nucleic acid molecules that encode bispecific g molecules of the invention. Such nucleic acid molecules will also be referred to herein as "nucleic acids of the invention" and may also be in the form of a genetic uct, as defined herein. A c acid 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 ed host cell or host organism). According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated form, as defined hereabove.

The nucleic acid of the invention may also be in the form of, may be present in and/or may be part of a vector, such as for example a plasmid, cosmid or YAC. The vector may especially be an expression vector, i.e. a vector that can provide for sion of the bispecific binding le in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression ). Such expression vector generally comprises at least one nucleic acid of the invention that is operably linked to one or more suitable regulatory elements, such as er(s), enhancer(s), terminator(s), and the like. Such elements and their selection in view of expression of a specific ce in a specific host are common knowledge of the skilled person. Specific examples of regulatory elements and other ts useful or necessary for expressing bispecific binding molecules of the invention, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes, and the like, are disclosed e.g. on pp. 131 to 133 of W02006/O40153.

The nucleic acids of the invention may be prepared or obtained in a manner known per se (e.g. by automated DNA synthesis and/or recombinant DNA technology), based on the ation on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source.

In another aspect, the invention relates to host cells that express or that are capable of expressing one or more bispecific binding molecules of the ion; and/or that contain a nucleic acid of the invention. According to a particularly red ment, said host cells are bacterial cells; other useful cells are yeast cells, fungal cells or mammalian cells.

Suitable bacterial cells include cells from gram-negative ial s such as strains of Eschen'chia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cell include cells from species of Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of Saccharomyces (for example Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizosaccharomyces pombe), Pichia (for example Pichia pastoris and Pichia methanolica), and Hansenula.

Suitable mammalian cells include for example CHO cells, BHK cells, HeLa cells, COS cells, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as well.

The ion further provides methods of manufacturing a bispecific binding molecule of the invention, such methods generally comprising the steps of: - culturing host cells comprising a nucleic acid capable of encoding a bispecific binding le under conditions that allow expression of the bispecific binding molecule of the on;and - recovering or ing the polypeptide expressed by the host cells from the culture; - optionally further purifying and/or modifying and/or formulating the bispecific binding molecule of the invention.

For production on an industrial scale, preferred host organisms include strains of E. coli, Pichia paston‘s, and S. cerevisiae that are suitable for large scale expression, production and tation, and in ular for large scale ceutical expression, production and fermentation.

The choice of the specific expression system depends in part on the requirement for certain post-translational modifications, more specifically glycosylation. The tion of a bispecific binding molecule of the invention for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the sed 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.

Bispecific binding molecules of the invention may be produced either in a cell as set out above intracellullarly (e.g. in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further ed; or they can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the e medium and optionally r purified.

Methods and reagents used for the recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods of induction of protein expression, culture ions, and the like, are known in the art. Similarly, protein isolation and purification techniques useful in a method of manufacture of a polypeptide of the invention are well known to the skilled In a further , the invention relates to a peptide having an amino acid sequence of a CDR3 contained in an anti-VEGF-VHH having an amino acid sequence selected from sequences shown in SEQ ID NOs: 9 to 57 or SEQ ID NOs: 58 - 127, respectively, and a nucleic acid molecule encoding same.

These peptides correspond to CDR3s d from the VHHs of the invention. They, in particular the nucleic acid molecules encoding them, are useful for CDR grafting in order to replace a CDR3 in an immunoglobulin chain, or for ion into a non- immunoglobulin scaffold, e.g. a protease inhibitor, nding protein, cytochrome b562, a helix-bundle protein, a disulfide-bridged peptide, a lipocalin or an anticalin, thus conferring target-binding ties to such scaffold. The method of CDR-grafting is well known in the art and has been widely used, e.g. for humanizing antibodies (which WO 31078 usually comprises grafting the CDRs from a rodent antibody onto the Fv frameworks of a human antibody).

In order to obtain an immunoglobulin or a non-immunoglobulin scaffold containing a CDR3 of the invention, the DNA encoding such molecule may be obtained according to standard methods of molecular biology, e.g. by gene synthesis, by oligonucleotide annealing or by means of overlapping PCR fragments, as e.g. bed by Daugherty et al., 1991, Nucleic Acids Research, Vol. 19, 9, 2471 — 2476. A method for ing a VHH CDR3 into a non-immunoglobulin scaffold has been described by Nicaise et al., 2004, Protein Science, 13, 1882 — 1891.

The invention further relates to a product or composition containing or comprising at least one ific binding molecule of the invention and ally one or more r components of such compositions known perse, i.e. depending on the intended use of the ition.

For pharmaceutical use, a bispecific binding molecule of the invention may be formulated as a pharmaceutical preparation or composition comprising at least one bispecific binding molecule of the ion 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- ng examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by enous, 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 f, 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 bispecific binding molecule, in particular one immunoglobulin single variable domain, of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for ceutical use), and optionally one or more further active substances.

The bispecific binding molecules of the invention may be formulated and administered in any suitable manner known per se: Reference, in particularfor the immunoglobulin single variable domains, is for example made to W02004/041862, /041863, W02004/041865, W02004/041867 and W02008/020079, as well as to the standard handbooks, such as Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, an immunoglobulin single variable domain 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 ations 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 ons, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable rs or diluents for such preparations for example include, without limitation, e water and pharmaceutically able aqueous buffers and ons such as logical phosphate-buffered saline, Ringer*s solutions, dextrose solution, and Hank's solution; water oils; ol; ethanol; s 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 es thereof. Usually, aqueous solutions or suspensions will be preferred.

Thus, the bispecific binding molecule of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. For oral therapeutic stration, the bispecific binding molecule of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, es, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the binding molecule 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 bispecific binding molecule of the ion in such therapeutically useful compositions is such that an ive dosage level will be obtained.

The tablets, pills, capsules, and the like may also contain binders, excipients, disintegrating agents, lubricants and sweetening or flavouring agents, for example those mentioned on pages 143-144 of W02008/020079. 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 ble oil or a polyethylene . Various other materials may be present as coatings or to othen/vise 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 binding molecules 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 bispecific g molecules of the invention may be incorporated into sustained-release preparations and s.

Preparations and ations for oral administration may also be provided with an c 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 ific binding molecules of the invention may also be administered intravenously or intraperitoneally by infusion or injection, as r described on pages 144 and 145 of W02008/020079.

Fortopical administration of the bispecific binding molecules of the invention, 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, as further described on page 145 of W02008/020079.

Generally, the concentration of the bispecific g molecules of the invention in a liquid composition, such as a , will be from about 01-25 wt-%, preferably from about 05-10 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 bispecific binding molecules of the invention required for use in treatment will vary not only with the particular binding molecule selected, but also with the route of administration, the nature of the condition being treated and the age and condition of the t and will be ultimately at the discretion of the attendant physician or clinician. Also, the dosage of the binding les of the ion 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 ses 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 stration regimen may include long-term, daily treatment. By “long-term” is meant at least two weeks and preferably, several weeks, months, or years of duration. ary 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 n, 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.

According to a further embodiment, the invention relates to the use of bispecific binding molecules, e.g. immunoglobulin single variable s, for therapeutic purposes, such - for the prevention, treatment and/or alleviation of a disorder, disease or condition, especially in a human being, that is associated with VEGF- and / or AngZ-mediated effects on angiogenesis or that can be prevented, treated or alleviated by modulating the Notch signaling pathway and/or the Tie2 signalling y with a bispecific binding molecule according to the invention, - in a method of treatment of a patient in need of such y, such method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one bispecific binding molecule of the ion, e.g. an immunoglobulin single variable domain, or a pharmaceutical composition containing same; - for the preparation of a medicament for the prevention, treatment or alleviation of disorders, diseases or conditions associated with VEGF- and/or Ang2-mediated effects on angiogenesis; - as an active ingredient in a pharmaceutical composition or medicament used for the above purposes.

According to a specific aspect, said disorder disorder, disease or condition is a cancer or cancerous disease, as defined .

According to another aspect, the disease is an eye disease associated with VEGF- and/or Ang2—mediated effects on angiogenesis or which can be treated or alleviated by modulating the Notch signaling pathway with a bispecific binding molecule. ing on the cancerous disease to be treated, a bispecific g molecule of the ion may be used on its own or in combination with one or more additional therapeutic agents, in particular selected from chemotherapeutic agents like DNA damaging agents or therapeutically active compounds that inhibit angiogenesis, signal uction pathways or mitotic checkpoints in cancer cells.

The onal therapeutic agent may be administered simultaneously with, optionally as a component of the same pharmaceutical ation, or before or after administration of the binding molecule.

In certain embodiments, the additional therapeutic agent may be, without limitation (and in the case of the receptors, including the respective ligands), one or more tors selected from the group of tors of EGFR, VEGFR, HER2—neu, Her3, AuroraA, AuroraB, PLK and Pl3 , FGFR, PDGFR, Raf, Ras, KSP, PDK1, PTK2, lGF-R or Further examples of additional therapeutic agents are inhibitors of CDK, Akt, srC/bcr abl, CKit, cMet/HGF, c-Myc, Flt3, HSP90, hedgehog antagonists, inhibitors of JAK/STAT, MEK, mTor, aB, the proteasome, Rho, an inhibitor of wnt signaling or an inhibitor of the ubiquitination pathway or another inhibitor of the Notch signaling pathway.

Examples for Aurora inhibitors are, without tion, PHA-739358, AZD-1152, AT 9283, CYC-116, R-763, VX-680, VX-667, MLN-8045, PF-3814735.

An example for a PLK tor is GSK-461364.

Examples for rat inhibitors are BAY-734506 (also a VEGFR inhibitor), PLX 4032, RAF-265 (also in addition a VEGFR inhibitor), sorafenib (also in addition a VEGFR inhibitor), and XL 281.

Examples for KSP inhibitors are sib, ARRY—520, AZD-4877, CK-1122697, GSK 246053A, GSK-923295, MK-O731, and SB-743921.

Examples for a sro and/or bcr-abl inhibitors are dasatinib, AZD-O530, bosutinib, XL 228 (also an lGF-1 R tor), nib (also a PDGFR and CKit inhibitor), imatinib (also a CKit inhibitor), and NS-187.

An example fora PDK1 inhibitor is BX—517.

An example fora Rho tor is BA-210.

Examples for Pl3 kinase inhibitors are PX-866, BEZ-235 (also an mTor inhibitor), XL 418 (also an Akt inhibitor), XL-147, and XL 765 (also an mTor tor).

Examples for inhibitors of CMet or HGF are XL-184 (also an tor of VEGFR, CKit, Flt3), PF-2341066, MK-2461, XL-880 (also an inhibitor of VEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie2), SU-11274, PHA-665752, AMG-102, and AV-299.

An example for a C-Myc inhibitor is CX—3543.

Examples for Flt3 inhibitors are AC-220 (also an inhibitor of cKit and PDGFR), KW 2449, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC), TG-101348 (also an inhibitor of JAK2), XL-999 (also an inhibitor of cKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR, VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR, and cKit).

Examples for HSP90 inhibitors are tanespimycin, alvespimycin, lPl-504 and CNF 2024.

Examples for JAK/STAT inhibitors are CYT-997 (also interacting with tubulin), TG 101348 (also an tor of Flt3), and XL-019. es for MEK inhibitors are ARRY-142886, PD-325901, AZD-8330, and XL 518. es for mTor inhibitors are temsirolimus, AP-23573 (which also acts as a VEGF tor), everolimus (a VEGF inhibitor in addition). XL-765 (also a Pl3 kinase tor), and BEZ-235 (also a Pl3 kinase tor).

Examples for Akt inhibitors are perifosine, GSK-690693, RX—0201, and triciribine.

Examples for cKit tors are AB-1010, OSl-930 (also acts as a VEGFR inhibitor), AC-220 (also an inhibitor of F|t3 and PDGFR), tandutinib (also an inhibitor of Flt3 and PDGFR), axitinib (also an inhibitor of VEGFR and , XL-999 (also an tor of Flt3, PDGFR, VEGFR, FGFR), sunitinib (also an inhibitor of Flt3, PDGFR, , and XL-820 (also acts as a VEGFR- and PDGFR inhibitor), imatinib (also a bcr—abl inhibitor), nilotinib (also an inhibitor of bcr-abl and PDGFR).

Examples for hedgehog antagonists are lPl-609 and CUR-61414.

Examples for CDK inhibitors are seliciclib, AT-7519, P-276, ZK-CDK (also inhibiting VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509, and AG 024322.

Examples for proteasome inhibitors are bortezomib, carfilzomib, and NPl—0052 (also an inhibitor of NFkappaB).

An example for an NFkappaB pathway inhibitor is NPl-0052.

An example for an ubiquitination pathway inhibitor is HBX-41108.

In preferred embodiments, the additional therapeutic agent is an anti-angiogenic agent.

Examples for anti—angiogenic agents are inhibitors of the FGFR, PDGFR and VEGFR or the respective ligands (e.g VEGF inhibitors like pegaptanib or the EGF dy bevacizumab), EGFL7 inhibitors, such as anti-EGFL7 MAb inhibitors , angiopoietin1/2 such as AMG386, and thalidomides, such agents being selected from, without tion, zumab, nib, GDP-791, SU-14813, telatinib, KRN—951, ZK-CDK (also an inhibitor of CDK), ABT-869, BMS-690514, RAF-265, lMC-KDR, lMC-18F1, lMiDs (immunomodulatory drugs), thalidomide derivative CC-4047, lenalidomide, ENMD 0995, lMC—D11, Ki 23057, brivanib, cediranib, XL-999 (also an inhibitor of cKit and Flt3), 183, GP 868596, IMC 3G3, R-1530 (also an inhibitor of Flt3), nib (also an inhibitor of cKit and Flt3), axitinib (also an inhibitor of cKit), lestaurtinib (also an inhibitor of Flt3 and PKC), nib, tandutinib (also an inhibitor of Flt3 and cKit), pazopanib, GW 786034, PF—337210, lMC-1121B, AVE-0005, AG-13736, E-7080, CHIR 258, sorafenib tosylate (also an inhibitor of Raf), RAF-265 (also an inhibitor of Raf), vandetanib, CP-547632, 0, AEE—788 (also an inhibitor of EGFR and Her2), BAY—57-9352 (also an inhibitor of Raf), BAY4506 (also an inhibitor of Raf), XL 880 (also an inhibitor of cMet), XL-647 (also an inhibitor of EGFR and EphB4), XL 820 (also an inhibitor of cKit), and nilotinib (also an inhibitor of cKit and brc-abl).

The additional therapeutic agent may also be selected from EGFR inhibitors, it may be a small molecule EGFR inhibitor or an anti-EGFR dy. Examples for anti-EGFR antibodies, without limitation, are cetuximab, panitumumab, matuzumab; an example for a small molecule EGFR inhibitor is gefitinib. Another example for an EGFR modulator is the EGF fusion toxin.

Among the EGFR and Her2 inhibitors useful for combination with the ific binding molecule of the invention are lapatinib, gefitinib, nib, cetuximab, trastuzumab, nimotuzumab, zalutumumab, vandetanib (also an inhibitor of , pertuzumab, XL-647, HKl-272, BMS-599626 ARRY-334543, AV 412, mAB-806, BMS-690514, JNJ—26483327, AEE-788 (also an inhibitor of , ARRY-333786, IMO—1 1 F8, Zemab.

Other agents that may be advantageously ed in a therapy with the ific binding molecule of the invention are tositumumab and momab tiuxetan (two radiolabelled anti-CD20 antibodies), alemtuzumab (an anti-CD52 antibody), denosumab, (an osteoclast differentiation factor ligand inhibitor), galiximab (a CD80 antagonist), ofatumumab (a CD20 inhibitor), zanolimumab (a CD4 antagonist), SGN4O (a CD40 ligand receptor modulator), rituximab (a CD20 inhibitor), mapatumumab (a TRAIL-1 receptor agonist), REGN421(SAR153192) or OMP-21 M18 (Dll4 inhibitors).

Other chemotherapeutic drugs that may be used in combination with the ific binding molecule of the present invention are selected from, but not limited to es, hormonal analogues and antihormonals (e.g. tamoxifen, toremifene, fene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, cyproterone acetate, finasteride, buserelin acetate, fludrocortisone, fluoxymesterone, medroxyprogesterone, octreotide, fene, pasireotide, vapreotide), aromatase inhibitors (e.g. anastrozole, letrozole, liarozole, exemestane, atamestane, formestane), LHRH agonists and antagonists (e.g. goserelin acetate, leuprolide, abarelix, elix, deslorelin, histrelin, triptorelin), antimetabolites (e.g. antifolates like methotrexate, pemetrexed, pyrimidine analogues like 5 fluorouracil, capecitabine, decitabine, nelarabine, and gemcitabine, purine and adenosine ues such as mercaptopurine thioguanine, cladribine and pentostatin, cytarabine, fludarabine); antitumor antibiotics (e.g. anthracyclines like doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C, bleomycin dactinomycin, plicamycin, mitoxantrone, pixantrone, streptozocin); platinum derivatives (e.g. cisplatin, oxaliplatin, latin, lobaplatin, satraplatin); ting agents (e.g. estramustine, meclorethamine, melphalan, chlorambucil, busulphan, azine, cyclophosphamide, ifosfamide, hydroxyurea, temozolomide, nitrosoureas such as tine and lomustine, thiotepa); antimitotic agents (e.g. vinca alkaloids like vinblastine, vindesine, vinorelbine, vinflunine and vincristine; and taxanes like paclitaxel, xel and their formulations, larotaxel; simotaxel, and epothilones like ilone, patupilone, ZK-EPO); omerase inhibitors (e.g. epipodophyllotoxins like etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan) and miscellaneous herapeutics such as amifostine, anagrelide, interferone alpha, procarbazine, ne, and porfimer, bexarotene, celecoxib.

The efficacy of bispecific binding molecule of the ion or polypeptides, 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 ation f, depending on the ic disease or disorder of interest. Suitable assays and animal models will be clear to the skilled person, and for example include the assays described herein and used in the Examples below, e.g. a proliferation assay.

The data obtained in the experiments of the invention confirm that bispecific binding molecules of the invention have properties that are superior to those of binding molecules of the prior art. Among such properties are complete inhibition of the VEGF165-VEGFR2 interaction and a low |C50, as can e.g. be taken from the ELISA data of Figure 1 and Table 5 as well as the |C5o (nM) values for VHHs in the AlphaScreen assay as shown in Figures 3, 17, 18 and Table 7; and the affinity KD (nM) of purified VHHs on recombinant human VEGF and mouse VEGF in Table 9, 10 and Figure 5. Also, as shown in Table 13, VEGF binders of the ion have high potency, i.e. in the subnanomolar range, in the HUVEC proliferation assay. This indicates that bispecific binding molecules of the invention are promising ates to have therapeutic efficacy in diseases and disorders associated with VEGF-mediated effects on angiogenesis, such as cancer.

According to another embodiment of the invention, there is ed a method of diagnosing a disease by a) contacting a sample with a binding le of the invention as defined above, and b) ing binding of said binding molecule to said sample, and c) comparing the binding detected in step (b) with a rd, wherein a difference in binding relative to said sample is diagnostic of a disease or disorder associated with VEGF- and/or Ang2—mediated effects on angiogenesis.

Forthis and other uses, it may be useful to further modify a bispecific binding molecule of the invention, such as by uction 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 binding molecule of the invention to another protein, polypeptide or chemical nd that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a bispecific g le 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 bispecific binding molecule of the invention may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or avidin.

Brief description of the Figures: Figure 1: Purified monovalent VHHs block the hVEGF165/hVEGFR2—Fc interaction (ELISA) Figure 2: Purified lent VHHs block the hVEGF165/hVEGFR1-Fc interaction (ELISA) Figure 3: Purified monovalent VHHs block the hVEGF165/hVEGFR2—Fc interaction (AlphaScreen) Figure 4: Purified monovalent VHHs block the hVEGF165/hVEGFR1-Fc ction (AlphaScreen) Figure 5: Binding of monovalent VHHs to recombinant human and mouse VEGF ) Figure 6: Binding of monovalent VHHs to human VEGF121 Figure 7: Purified VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF Figure 8: Formatted VHHs block hVEGF165/hVEGFR2—Fc interaction (ELISA) Figure 9: ted VHHs block hVEGF165/hVEGFR1-Fc interaction (ELISA) Figure 10: Formatted VHHs block hVEGF165/hVEGFR2-Fc interaction (AlphaScreen) PCT/'EP2012/055901 Figure 11:Formatted VHHs block hVEGF165/hVEGFR1-Fc ction (AlphaScreen) Figure 12: Formatted VHHs block mVEGF164/mVEGFR2-Fc ction (AlphaScreen) Figure 13: Formatted VHHs bind to mouse and human VEGF Figure 14: Formatted VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF Figure 15: Formatted VHHs bind to VEGF121 Figure 16: Sequence alignment of VHH VEGFBII23804 with human VH3/JH germline consensus sequence Figure 17: VHH variants of VEGFBII23804 bIOCk hVEGF165/hVEGFR2-FC interaction (AlphaScreen) Figure 18: Sequence-optimized clones of VEGFBII23804 block the hVEGF165/hVEGFR2-Fc interaction (AlphaScreen) Figure 19: Sequence alignment of VHH VEGFBII5BO5 with human VH3/JH germline consensus sequence Figure 20: Description bivalent AngZ VHHs Figure 21: Purified bivalent AngZ VHHs blocking hAngZ-hTie2 (25-1), mAngZ-mTie2 (25-2) and cAngZ-cTie2 (25-3) interaction (ELISA) Figure 22: ed bivalent AngZ VHHs blocking hAng1-hTie2 ction (ELISA) Figure 23: ption trivalent VEGFxAng2 bispecific VHHs Figure 24: Purified trivalent VEGFxAngZ Nanobodies blocking hVEGF-hVEGFR2 interaction (AlphaScreen) Figure 25: Purified trivalent VEGFxAngZ VHHs blocking hAngZ-hTie2 ction (ELISA) Figure 26: Description trivalent and tetravalent VEGFxAngZ bispecific VHHs Figure 27: Purified trivalent and tetravalent VEGFxAngZ VHHs blocking hVEGF- hVEGFR2 (31—1) and hVEGF-hVEGFR1 (31-2) interaction (AlphaScreen) Figure 28: Purified trivalent and tetravalent VEGFxAngZ VHHs blocking hAngZ-hTie2 (32—1), mAngZ—mTie2 (32—2) and cAngZ-cTie2 (32—3) interaction (ELISA) Figure 29: ed trivalent and tetravalent VEGFxAngZ VHHs blocking hAngZ mediated HUVEC survival Figure 30: Description sequence optimized and ty VEGFxAngZ bispecific VHHs Figure 31: Purified VEGFANGBll0002228 VEGFxAngZ VHHs blocking hVEGF- hVEGFR2 (35-1) and hVEGF-hVEGFR1 (35-2) interaction (AlphaScreen) Figure 32: ed VEGFANGBll00022—25-28 VEGFxAng2 VHHs binding to human VEGF165 (36—1) and 21 (36—2) (ELISA) Figure 33: Purified VEGFANGBll0002228 VEGFxAng2 VHHs binding to (A) mouse and (B) rat VEGF164 (ELISA) Figure 34: Purified VEGFANGBll0002228 VEGFxAngZ VHHs binding to (A) human , (B) human VEGF-C, (C) human VEGF-D and (D) human PIGF (ELISA) Figure 35: Purified VEGFANGBll0002228 VEGFxAng2 VHHs blocking hAng2- hTie2 (39-1), mAng2-mTieZ (39-2) and cAngZ-cTieZ (39-3) ction ) Figure 36: Purified GBll0002228 VEGFxAngZ VHHs blocking hAng1- hTie2 interaction (ELISA) Figure 37: Purified VEGFANGBll0002228 VEGFxAng2 VHHs blocking hAng2 mediated HUVEC survival Materials and methods: a) Production and functionality testing of 9 A cDNA encoding the or binding domain of human vascular endothelial growth factor isoform VEGF165 (GenBank: AAM03108.1; AA residues 27 - 135) is cloned into pET28a vector (Novagen, Madison, WI) and overexpressed in E.coli (BL21 Star DE3) as a His-tagged insoluble n. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37°C. Cells are harvested by centrifugation and lysed by sonication of the cell pellet. Inclusion bodies are isolated by centrifugation. After a washing step with 1% Triton X 100 (Sigma-Aldrich), proteins are solubilized using 7.5M guanidine hloride and ed by consecutive rounds of overnight dialysis using buffers with decreasing urea concentrations from 6M till OM. The refolded protein is purified by ion exchange chromatography using a MonoQ5/5OGL (Amersham BioSciences) column followed by gel filtration with a Superdex75 10/300 GL column (Amersheim BioSciences). The purity and homogeneity of the protein is confirmed by SDS-PAGE and Westen blot. In addition, binding activity to VEGFR1, VEGFR2 and zumab is monitored by ELISA. To this end, 1 pg/mL of inant human VEGF109 is immobilized overnight at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). Serial ons of VEGFR1, VEGFR2 or Bevacizumab are added to the 9 coated plate and binding is detected using alkaline phosphatase (AP) conjugated goat anti-human IgG, Fc specific (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and a uent enzymatic reaction in the presence of the substrate PNPP (p- nitrophenylphosphate) (Sigma-Aldrich). VEGF109 could bind to VEGFR1, VEGFR2 and Bevacizumab, indicating that the produced VEGF109 is active. b) KLH conjugation of VEGF165 and functionality testing of KLH-conjugated VEGF165 Recombinant human VEGF165 (R&D s, Minneapolis, MN, USA) is conjugated to mariculture keyhole limpet anin (mcKLH) using the lmject Immunogen EDC kit with mcKLH (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.

Efficient ation of the polypeptide to mcKLH is confirmed by SDS-PAGE.

Functionality of the ated protein is checked by ELISA: 2 ug/mL of KLH conjugated VEGF165 is immobilized overnight at 4°C in a 96—well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). Serial dilutions of VEGFR1 or VEGFR2 are added and binding is detected using a horseradish dase (HRP)—conjugated goat anti-human IgG, Fc specific (Jackson Immuno Research tories Inc, West Grove, PA, USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3’,5,5’-tetramentylbenzidine) (Pierce, Rockford, IL, USA). The KLH conjugated protein could still interact with VEGFR1, VEGFR2 and Bevacizumab, confirming that the relevant epitopes onVEGF165 are still accessible.

Example 1 Immunization with ent VEGF s induces a humoral immune response in llama 1. 1 Immunizations After approval of the Ethical Committee of the faculty of Veterinary ne (University Ghent, Belgium), 4 llamas (designated No. 264, 265, 266, 267) are immunized according to rd protocols with 6 intramuscular injections (100 or 50 e at weekly intervals) of recombinant human VEGF109. The first injection at day 0 is formulated in Complete Freund’s Adjuvant (Difco, Detroit, MI, USA), while the subsequent injections are formulated in Incomplete Freund’s nt (Difco, Detroit, MI, USA). In addition, four llamas (designated No. 234, 235, 280 and 281) are immunized according to the following protocol: 5 intramuscular injections with KLH- conjugated human VEGH165 (100 or 50 ug/dose at biweekly als) followed by 4 intramuscular injections of human VEGF109 (first dose of 100 pg followed 2 weeks later with three 50 ug/dose at weekly interval). 1.2 tion of nduced immune responses in llama To monitor VEGF specific serum titers, an ELISA assay is set up in which 2 ug/mL of recombinant human VEGF165 or VEGF109 is immobilized overnight at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). After on of serum dilutions, bound total IgG is detected using horseradish peroxidase conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc., Montgomery, TX, USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3’,5,5’-tetramentylbenzidine) (Pierce, Rockford, IL, USA). For llamas 264, 265, 266 and 267, an additional ELISA is performed in which the isotype-specific responses against VEGF165 and VEGF109 are evaluated. Isotype specific responses are detected using mouse mAbs ically recognizing conventional llama IgG1 and the heavy-chain only llama IgG2 and IgG3 [Daley et al. (2005). Clin. Diagn. Lab. Imm. -386] followed by a rabbit anti-mouse-HRP conjugate (DAKO). ELISAs are developed using TMB as chromogenic substrate and absorbance is measured at 450nm. The serum titers for each llama are depicted in Table 1.

Table 1: dy-mediated specific serum response against VEGF165 and VEGF109 ELISA (recombinant protein solid phase coated) Recombinant human EGF165 Recombinant human VEGF109 L'ama Total Total lgG VEGF165-KLH 234 ++ n/d n/d n/d ++ n/d n/d n/d VEGF165-KLH 235 ++ n/d n/d n/d ++ n/d n/d n/d + VEGF109 VEGF165-KLH 280 + n/d n/d n/d + n/d n/d n/d + VEGF109 VEGF165-KLH 281 + n/d n/d n/d + n/d n/d n/d + VEGF109 267 VEGF109 nid +/- -- +/- +/- .- n/d, not determined g of the heavy-chain only antibody nt repertoires and preparation of phage Following the final immunogen injection, immune tissues as the source of B-cells that produce the heavy-chain antibodies are collected from the immunized llamas. Typically, two 150-ml blood samples, collected 4 and 8 days after the last antigen injection, and one lymph node biopsy, collected 4 days afterthe last antigen injection are collected per animal. From the blood samples, peripheral blood mononuclear cells (PBMCs) are prepared using FicoII-Hypaque according to the cturer’s instructions ham ences, Piscataway, NJ, USA). From the PBMCs and the lymph node biopsy, total RNA is extracted, which is used as starting material for RT-PCR to y the VHH encoding DNA segments, as described in W02005/044858. For each immunized llama, a library is constructed by pooling the total RNA isolated from all collected immune tissues of that animal. In short, the PCR—amplified VHH repertoire is cloned via specific restriction sites into a vector designed to facilitate phage display of the VHH library. The vector is derived from pUC119 and contains the LacZ promoter, a M13 phage glll protein coding sequence, a resistance gene for ampicillin or icillin, a multiple cloning site and a hybrid glll-pelB leader sequence 0). In frame with the VHH coding sequence, the vector encodes a C-terminal c-myc tag and a His6 tag. Phage are prepared according to standard protocols and stored after filter sterilization at 4°C for further use.

Example 3 Selection of VEGF-specific VHHs via phage display VHH phage libraries are used in different ion strategies applying a multiplicity of selection conditions. Variables include i) the VEGF protein format (rhVEGF165, rhVEGF109 or rmVEGF164), ii) the n presentation method (solid phase: ly coated or via a biotin-tag onto Neutravidin-coated plates; solution phase: incubation in solution followed by capturing on Neutravidin-coated plates), iii) the antigen concentration and iv) the n method (trypsin or competitive elution using VEGFR2).

All selections are carried out in Maxisorp l plates (Nunc, Wiesbaden, y).

Selections are performed as follows: Phage libraries are incubated at RT with variable concentrations of VEGF antigen, either in solution or immobilized on a solid support.

After 2hrs of incubation and extensive washing, bound phage are eluted. In case trypsin is used for phage elution, the protease ty is immediately neutralized by addition of 0.8 mM protease inhibitor AEBSF. Phage outputs that show enrichment over background are used to infect E. coli. Infected E. coli cells are either used to prepare phage for the next selection round (phage rescue) or plated on agar plates (LB+amp+glucose2°/°) for analysis of individual VHH clones. In order to screen a selection output for specific s, single colonies are picked from the agar plates and grown in 1 mL 96—deep-well plates. The IacZ-controlled VHH expression is induced by adding IPTG (0.1-1mM final). Periplasmic extracts (in a volume of ~ 80 uL) are prepared ing to rd methods.

Example 4 Identification of VEGF-binding and VEGF receptor-blocking VHHs Periplasmic extracts are tested for binding to human VEGF165 by ELISA. In brief, 2 pg/mL of recombinant human VEGF165 is lized overnight at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). After addition of typically a 10-fold dilution of the periplasmic extracts, VHH binding is detected using a mouse yc (Roche) and an anti-mouse—HRP conjugate (DAKO).

Clones showing ELISA s of >3-fold above background are considered as VEGF binding VHHs.

In on, periplasmic extracts are screened in a human VEGF165/human VEGFR2 AlphaScreen assay (Amplified Luminescent Proximity Homogeneous Assay) to assess the ng capacity of the VHHs. Human VEGF165 is biotinylated using Sulfo-NHS- LC—Biotin (Pierce, Rockford, IL, USA). Human VEGFR2/F0 chimera (R&D s, Minneapolis, MN, USA) is captured using an anti-humanFc VHH which is coupled to acceptor beads according to the manufacturer’s instructions n Elmer, m, MA, US). To evaluate the neutralizing capacity of the VHHs, periplasmic extracts are diluted 1/25 in PBS buffer containing 0.03 % Tween 20 (Sigma-Aldrich) and preincubated with 0.4 nM biotinylated human VEGF165 for 15 minutes at room temperature (RT). To this mixture the acceptor beads (10ug/ml) and 0.4 nM VEGFR2— hch are added and further incubated for 1 hour at RT in the dark. Subsequently donor beads (10pg/ml) are added followed by incubation of 1 hour at RT in the dark.

Fluorescence is ed by reading plates on the Envision Multi label Plate reader (Perkin Elmer, Waltham, MA, USA) using an excitation ngth of 680 nm and an emission wavelength between 520 nm and 620nm. Periplasmic extract containing irrelevant VHH is used as ve control. Periplasmic extracts containing anti- VEGF165 VHHs which are able to decrease the fluorescence signal with more than 60 % relative to the signal of the ve control are identified as a hit. All hits identified in the AlphaScreen are confirmed in a competition ELISA. To this end, 1 ug/mL of human VEGFR2 chimera (R&D Systems, Minneapolis, MN, USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Fivefold dilutions of the periplasmic extracts are ted in the presence of a fixed concentration (4nM) of biotinylated human 5 in PBS buffer containing 0.1 % casein and 0.05 % Tween 20 (Sigma- Aldrich). Binding of these VHH/bio-VEGF165 complexes to the human VEGFR2 chimera coated plate is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, MO, USA). VHH sequence IDs and the corresponding AA sequences of VEGF-binding (non-receptor—blocking) VHHs and inhibitory (receptor-blocking) VHHs are listed in Table 2 and Table 3, respectively.

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Hw\firmom Thwm> Nv\mo<ow M¢MW WQMBQ< U%¢Mm WQMHQ< #MQMmmwwflmm WQWHQQ QMQmeQW<Mm Mflweflfl HZMHzemmHHmm Q?mm;mm90;w> <<oww><e BZMHZQMmHHmm medgw> <<oww><e BZMHZQMmHHmm camm;mzko;w> <<oww><9 HQMHZQMWHHMM QmmMQmMHOQN> <<QWW><H mewwmmHfl 0mgm><fl wawmmmHfl mm4m>mflw mwzwmmmH< UHHm>mQ% MHWUUmflHfl wmgm><Q Mm3 >>hflm HMUO¢OMm3 >>mmm mmwOflOMME >>mmm MMQO<OMME >>hflm 00mm>QO>m Qmww¢0>qw mwmm<omfim mme wwmm>QO>m QmQUMO>QQ mwm<<omgm mmB mum?>;0>x qmcw¢d>gw mwmdflomqm mmH wwmm>go>m qmawm0>qw mamm<omqm mme Thwm> mv\fio<fi© Thwm> vv\mo<N® TLUE> mv\oHQN® HHmmwm> mv\mommo Dissociation rates of inhibitory VHHs are analyzed on Biacore (Biacore T100 instrument, GE Healthcare). HBS-EP+ buffer is used as running buffer and experiments are performed at 25°C. Recombinant human VEGF165 is irreversibly captured on a CM5 sensor chip via amine coupling (using EDC and NHS) up to a target level of +/- 15OORU.

After immobilization, surfaces are deactivated with 10 min injection of 1M ethanolamine pH8.5. A reference surface is activated and deactivated with respectively S and ethanolamine. Periplasmic extracts of VHHs are injected at a d dilution in running bufferfor 2 min at 45ul/min and allowed to dissociate for 10 or 15 min. n different samples, the surfaces are regenerated with ration buffer. Data are double referenced by subtraction of the cun/es on the reference channel and of a blank running buffer injection. The of the processed curves is evaluated by fitting a two phase decay model in the Biacore T100 Evaluation software v2.0.1. Values for kd-fast, w and % fast are listed in Table 4.

Table 4: Off-rate determination of anti—VEGF receptor-blocking VHHs with Biacore Unique B-cell Representative. .. 0 g level 8:23.22? W, m M (Rm -———— 471 -———- 487 “————— —————— WO 31078 n/d n/d ma 1 19 VEGFBII23E05 1.5OE-02 6.90E-O5 18 275 n/d, not determined Example 5 Characterization of purified anti-VEGF VHHs Three inhibitory EGF VHHs are selected for further characterization as purified protein: VEGFBII23804, VEGFBII24C4 and VEGFBII23A6. These VHHs are expressed in E. coli TGi as c—myc, HisB-tagged proteins. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37°C. After spinning the cell cultures, periplasmic extracts are prepared by freeze-thawing the s. These extracts are used as starting material for VHH cation via IMAC and size exclusion chromatography (SEC). Final VHH preparations show 95% purity as assessed via SDS-PAGE. .1 Evaluation of human VEGF165/VEGFR2 blocking VHHs in human VEGF165/human VEGFR2-Po blocking ELISA The blocking capacity of the VHHs is evaluated in a human VEGF165/human VEGFR2- Fc blocking ELISA. In brief, 1 uglmL of VEGFR2-F0 chimera (R&D Systems, Minneapolis, MN, USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, y). on series (concentration range 1 mM - 64pM) of the purified VHHs in PBS buffer ning 0.1% casein and 0.05% Tween 20 (Sigma) are incubated in the presence of 4 nM biotinlyated VEGF165. Residual binding of bio-VEGF165 to VEGFR2 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, MO, USA) and TMB as substrate. As controls Bevacizumab (Avastin®) and zumab (Lucentis®) are taken along. Dose inhibition curves are shown in Figure 1; the corresponding I050 values and % inhibition are summarized in Table 5.

Table 5: |C50 (nM) values and % inhibition for monovalent VHHs in 65/hVEGFR2-Fc ition ELISA ic50 % VHH '0 inhibition VEGFB||23BO4 VEGFB“23A06 VEGFB||24CO4 Ranibizumab Bevacizumab .2 Evaluation of human VEGF165/VEGFR2 blocking VHHs in human VEGF165/human VEGFRi-Fc blocking ELISA VHHs are also evaluated in a human VEGF165/human VEGFR1-Fc ng ELISA. In brief, 2 uglmL of VEGFR1-Fc chimera (R&D Systems, Minneapolis, MN, USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Dilution series (concentration range 1 mM - 64pM) of the purified VHHs in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) are incubated in the presence of 0.5nM biotinlyated VEGF165. Residual binding of bio-VEGF165 to VEGFR1 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, MO, USA) and TMB as substrate. As controls Bevacizumab, Ranibizumab and an irrelevant VHH (2E6) are taken along. Dose inhibition curves are shown in Figure 2; the corresponding leo values and % inhibition are summarized in Table 6.

Table 6: |C5o (nM) values and % inhibition of monovalent VHHs in hVEGF165/hVEGFR1-Fc competition ELISA inhibition VEGFB||23BO4 VEGFB||23A06 IE.— VEGFB||24CO4 “— Ranibizumab Bevacizumab 1. 5.3 Evaluation of the anti-VEGF 165 VHHs in the human VEGF165/human VEGFR2—Fc blocking creen The blocking capacity of the VHHs is also evaluated in a human VEGF165/human VEGFR2—Fc ng AlphaScreen. Briefly, serial dilutions of purified VHHs (concentration range: 200 nM — 0.7 pM) in PBS buffer containing 0.03 % Tween 20 (Sigma) are added to 4pM bio-VEGF165 and ted for 15 min. Subsequently VEGFR2—Fc (0.4 nM) and anti-Fc VHH-coated acceptor beads (20 ug/ml) are added and this mixture is incubated for 1 hour in the dark. Finally, streptavidin donor beads (20 pg/ml) are added and after 1 hour of incubation in the dark, fluorescence is ed on the Envision microplate reader. Dose-response curves are shown in the Figure 3. The |C50 values for VHHs blocking the human 5 — human VEGFR2—Fc ction are summarized in Table 7.

Table 7: iCso (pM) values and % inhibition for VHHs in hVEGF165/hVEGFR2—Fc competition AlphaScreen tion°/ VHH "3 'C5° (pM) VEGFB||23E304 VEGFB||23A06 VEGFB||24CO4 Ranibizumab .4 Evaluation of the anti-VEGF 165 VHHs in the human VEGF165/human -Fc blocking creen The blocking capacity of the VHHs is also evaluated in a human VEGF165/human VEGFR1-Fc blocking AlphaScreen. Briefly, serial dilutions of ed VHHs (concentration range: 500 nM — 1.8 pM) ) in PBS buffer containing 0.03 % Tween 20 (Sigma) are added to 0.4 nM GF165 and incubated for 15 min. Subsequently VEGFR1-Fc (1 nM) and anti-Fc VHH-coated acceptor beads (20 pg/ml) are added and this mixture is ted for 1 hour in the dark. Finally, streptavidin donor beads (20 pg/ml) are added and after 1 hour of incubation in the dark, fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in the Figure 4. The |C50 values and % inhibition for VHHs blocking the human VEGF165 — human VEGFR1-Fc interaction are summarized in Table 8.

Table 8: |C5o (nM) values for VHHs in hVEGF165/hVEGFR1-Fc competition AlphaScreen VHH ID |C5o (nM) % inhibition VEGFBH2sBO4 “— VEGFBH2sAoe VEGFBII24CO4 —53 Ranibizumab —79 .5 Determination of the affinity of the human VEGF165 —VHH interaction Binding kinetics of VHH VEGFBII23804 with hVEGF165 is analyzed by SPR on a Biacore T100 instrument. Recombinant human VEGF165 is lized ly on a CM5 chip via amine coupling (using EDC and NHS). VHHs are analyzed at different concentrations between 10 and 360nM. Samples are injected for 2 min and allowed to dissociate up to 20 min at a flow rate of 45 pl/min. In between sample injections, the chip surface is regenerated with 100 mM HCI. HBS-EP+ (Hepes buffer pH7.4 + EDTA) is used as running buffer. Binding curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1. The calculated affinities of the anti-VEGF VHHs are listed in Table 9.

Table 9: ty KD (nM) of purified VHHs for recombinant human VEGF165 VEGF165 VEGFBII23804‘3) .2.1E+05 1.4E—02 - 8:33 2.4E—04 VEGFBII23A06‘a) .4.2E+05 2.0E—02 - 532'} 1.0E-04 VEGFBII24C04‘a) .3.2E+05 1.8E—02 - 25323 5 0.4 (a) Heterogeneous binding curve ing in no 1:1 fit, curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1 .6 Binding to mouse VEGF164 Cross-reactivity to mouse VEGF164 is determined using a binding ELISA. In brief, inant mouse VEGF164 (R&D Systems, Minneapolis, MS, USA) is coated overnight at 4°C at 1 pg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, y).

Wells are blocked with a casein on (1% in PBS). VHHs are applied as dilution series (concentration range: 500nM — 32pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP ate (DAKO) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3’,5,5’—tetramenty|benzidine) (Pierce, Rockford, IL, USA) e 5). A mouse VEGF164 reactive mAb is included as positive control. As reference, binding to human VEGF165 is also measured. EC50 values are summarized in Table 10.

Table 10: EC5o (pM) values for VHHs in a recombinant human 5 and mouse VEGF164 binding ELISA —rhVEGF165 rmVEGF164 VHH ID EC5o (pM) EC5o (pM) VEGFBII23B04 NB VEGFBII24C04 NB VEGFBII23A06 NB NB, no binding . 7 Binding to VEGF121 Binding to recombinant human VEGF121 is assessed via a solid phase binding ELISA.

Briefly, recombinant human VEGF121 (R&D s, polis, MS, USA) is coated overnight at 4°C at 1 ug/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany).

Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500nM — 32pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3’,5,5’-tetramenty|benzidine) (Pierce, Rockford, IL, USA) e 6). As positive control serial ons of the VEGFR2 is taken along. E050 values are summarized in Table 11.

Table 11: EC5o (pM) values for monovalent VHHs in a recombinant human VEGF121 binding ELISA VHH ID EC50 (pM) VEGFBII23BO4 VEGFBII24CO4 VEGFBII23A06 5.8 g to VEGF family members VEGFB, VEGFC, VEGFD and PIGF g to VEGFB, VEGFC, VEGFD and PIGF is assessed via a solid phase binding ELISA. In brief, VEGFB, VEGFC, VEGFD and PIGF (R&D Systems, Minneapolis, MS, USA) are coated overnight at 4°C at 1 pg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, y). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500nM — 32pM) and g is detected using a mouse yc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis, MO, USA). As positive controls serial dilutions of the appropriate receptors are taken along and detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgG, Fc specific antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and a uent enzymatic reaction in the presence of the substrate TMB (3,3’,5,5’-tetramenty|benzidine) (Pierce, Rockford, IL, USA). Dose-response curves of VHHs and controls are shown in Figure 7. The results show that there was no detectable binding of the selected VHHs to VEGFB, VEGFC, VEGFD or PIGF. 2012/055901 .9 Epitope binning Biacore-based epitope binning experiments are performed to investigate which VEGF binders bind to a similar or overlapping epitope as VEGFBII23B04. To this end, VEGFBII23B04 is immobilized on a CM5 sensor chip. For each sample, human VEGF165 is passed over the chip surface and reversibly captured by I23B4.

Purified VHHs (100 nM) or periplasmic extracts (1/10 diluted) are then injected with a surface contact time of 240 seconds and a flow rate of 10 uL/minute. Between different samples, the surface is regenerated with regeneration buffer (100 mM HCI). Processed curves are evaluated with Biacore T100 Evaluation software. VHHs could be divided within two groups: group one which gave additional binding to VEGFBII23B04 captured VEGF165 and a second group which is not able to simultaneously bind to VEGFBII23B04 captured VEGF165. Table 12—A summarizes the g es of the tested VHHs.

The same assay set-up is used to assess whether VEGFR1, VEGFR2, Ranibizumab and Bevacizumab are able to bind to human 65 simultaneously with VEGFBII23B04. Table 12—B ts the additional binding responses to VEGFBII23B04—captured VEGF165. Only VEGFR2 is not able to bind to VEGFBII23B04-captured VEGF165, underscoring the ng capacity of VEGFBII23B04 for the VEGF-VEGFR2 interaction. In addition, these data show that the VEGFBII23B04 epitope is different from the Bevacizumab and Ranibizumab epitope.

Table 12-A: Epitope binning of anti-VEGF VHHs — simultaneous binding with VEGFBII23B04 NoorIOW 1C02 1E07 4808 8E0? 8F07 12A07 12801 86C11 86F11 86G08 Edition? 86G10 86G11 87B07 88A01 88A02 88B02 88E02 88G03 88G05 88G11 Inlng 0 23804- 31%;?ng 88H01 89804 89D04 89F09 89G09 89H08 24C04 23A6 27G07 23804 Additional 3D12 5B02 5803 5805 6G02 7D08 8D09 8F06 10C07 10E07 binding to 23B04- 10G04 10G05 11C08 11D09 11E04 11E05 11F12 86H09 41C05 captured VEGF165 * ting same or overlapping epitopes Table 12-3: Epitope binning of VEGFBII23804 — binding of benchmark inhibitors or cognate receptors on VEGFBII23804 captured VEGF165 Injection _m. . Binding (Ru, 100 nM 1727 VEGFBII23804 100 nM — Ranibizumab 100 nM 763 100 “M 1349 VEGFR1 100 nM 1011 VEGFR2 100 nM .10 Characterization of the anti-VEGF VHHs in the HUVEC eration assay The potency of the selected VHHs is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96-well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33ng/mL VEGF. The proliferation rates are measured by [3H] Thymidine oration on day 4. The results of the HUVEC proliferation assay are shown in Table. 2012/055901 Table 13: |C50 (nM) values and % inhibition of monovalent VEGFBII2BBO4, VEGFBII23A06 and VEGFBII24CO4 in VEGF HUVEC proliferation assay VHH ID |C50(nM) % inhibition VEGFBII23804 0.36 VHH ID |C50(nM) % inhibition VEGFBII23A06 4.29 .11 Characten‘zaz‘ion of the EGF VHHs in the HUVEC Erk phosphorylation assay The potency of the selected VHHs is assessed in the HUVEC Erk phosphorylation assay. In brief, y HUVE cells are serum-starved over night and then stimulated in the absence or presence of VHHs with L VEGF for 5 min. Cells are fixed with 4% Formaldehyde in PBS and ERK phosphorylation levels are measured by ELISA using phosphoERK—specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159, Sigma) and polyclonal Rabbit Anti-Mouse-Immunoglobulin-HRP conjugate (P0161, Dako). As shown in Table 14, VEGFBII23804 and Bevacizumab inhibit the VEGF induced Erk phosphoryaltion by at least 90%, with IC5os <1nM.

Table 14: leo (nM) values and % tion of monovalent VEGFBII23804 in VEGF HUVEC Erk orylation assay VHH ID |C50(nM) % inhibition VEGFB||23BO4 __ Bevacizumab __ Example 6 Generation of multivalent anti-VEGF blocking VHHs VHH VEGFBII23804 is cally fused to either VEGFBII23BO4 resulting in a homodimeric VHH (AA sequence see Table 15) or different VEGF binding VHHs resulting in heterodimeric VHHs. To generate the heterodimeric VHHs, a panel of 10 unique VEGF binding VHHs are linked via a 9 or 40 Gly-Serflexible linker in two ent orientations to VEGFBII23804 (AA sequences see Table 15). Homodimeric VEGFBII2BBO4 (VEGFBIIO‘IO) and the 40 heterodimeric bivalent’ VHHs are expressed in E. coli TGi as c-myc, His6—tagged proteins. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37°C. After spinning the cell cultures, periplasmic extracts are prepared by freeze-thawing the pellets. 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Based on potency and maximum level of inhibition, the 5 best bivalent VHHs (VEGFBll021, VEGFBll022, VEGFBl023, 024 and VEGFBll025) are chosen forfurther characterization. An overview of the screening results for the 5 selected bivalent VHHs in the competitive VEGFR2 and VEGFR1 AlphaScreen is shown in Table 16.

Table 16: Potency and cy of 5 best bivalent VHHs in the VEGFNEGFR1 and VEGFNEGFR2 competition AlphaScreen assay VHH ID VEGFR2 VEGFR1 |C5o (PM) % 'C5°(pM) - inhibition VEGFB||021 “_ 100 VEGFB|l022 —-_ 100 l023 91 VEGFBll024 100 I025 82 Example 7 Characterization of formatted EGF VHHs VHHS VEGFBIIO10, |021, VEGFB||022, VEGFBIIO23, VEGFB||024 and VEGFBll025 are compared side-by-side in the VEGFR2 and VEGFR1 blocking ELISA (Figure 8 and 9, Table 17 and Table 18 respectively) and AlphaScreen assay (Figure 10 and 11, Table 19 and 20) as described in Examples 5.1, 5.2, 5.3 and 5.4, respectively.

Table 17: |C50 (pM) values and % inhibition forformatted VHHs in hVEGF165/hVEGFR2-FC competition ELISA VHH ID :33) % inhibition VEGFB||010 VEGFB||021 VEGFBI|022 164 100 VEGFBI|023 213 100 Table 18: |C50 (pM) values and % inhibition of formatted VHHs in VEGF165/hVEGFR1- FC competition ELISA VHH ID ('35,?) % tion VEGFBI|O1O —67 VEGFB||021 7 VEGFB||022 -%- VEGFB||023 1 VEGFB||024 2 |025 1 Bevacizumab 484 9 1 Ranibizumab 594 96 WO 31078 Table 19: |C50 (pM) values and % inhibition forformatted VHHs in hVEGF165/hVEGFR2—Fc competition AlphaScreen VHH ID 38‘5"") % inhibition VEGFBII010 VEGFBII021 VEGFB||022 VEGFB||023 VEGFB||024 VEGFB||025 Ranibizumab m— Table 20: |C50 (pM) values and % inhibition of ted VHHs in VEGF165/hVEGFR1— Fc competition AlphaScreen VHH ID ('pcnjf) % inhibition VEGFBIIOiO 7o VEGFB||021 100 VEGFB||022 n 98 VEGFB||023 87 VEGFB||024 a 98 |025 82 Ranibizumab 87 In addition, formatted VHHs are also tested for their capacity to block the mVEGF164/mVEGFR2—hch interaction. In brief, serial dilutions of purified VHHs (concentration range: 4uM — 14.5 pM) in PBS buffer containing 0.03 % Tween 20 (Sigma) are added to 0.1 nM biotinylated mVEGF164 and incubated for 15 min.

Subsequently mouse VEGFR2-hch (0.1 nM) and anti-hch VHH-coated acceptor beads (20 ug/ml) are added and this mixture is ted for 1 hour. y, streptavidin donor beads (20 pg/ml) are added and after 1 hour of incubation fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in Figure 12. The leo values for VHHs blocking the mouse VEGF164NEGFR2-hFC interaction are summarized in Table 21.

Table 21: leo (pM) values and % inhibition forformatted VHHs in mVEGF164/mVEGFR2-hFc competition creen VHH ID (fa) % inhibition VEGFBIIO22 108 100 VEGFBIIO24 - - mVEGF164 Ranibizumab —— The formatted VHHs are also tested in ELISA for their y to bind mVEGF164 and human VEGF165 (Example 5.6; Figure 13; Table 22); VEGF121 (Example 5.7; Figure 15; Table 23) and the VEGF family members VEGFB, VEGFC, VEGFD and PIGF (Example 5.8; Figure 14). Binding kinetics for human VEGF165 are analyzed as described in Example 5.5. The KD values are listed in Table 24.

Table 22 EC50 (pM) values forformatted VHHs in a inant human 5 and mouse VEGF164 binding ELISA —rhVEGF165 rmVEGF164 VHH ID EC5o (pM) EC5o (pM) VEGFBII010 VEGFB||O21 502 VEGFBII022 464 VEGFB||023 I024 —— VEGFB||025 —— Table 23: ECso (pM) values for formatted VHHs in a recombinant human VEGF121 g ELISA rhVEGF121 VHH ID EC50 (PM) VEGFBII010 VEGFBII022 VEGFBII024 VEGFBII025 Table 24: Affinity KD (nM) of purified formatted VHHs for recombinant human VEGF165 VHH ID (1)68) kd1 (1/8) ka2 (1/8) de (1/8) W VEGFBII010 4.5E+05 1.7E-02 2.9E-02 1.3E-04 VEGFBII021'1.2E+06 1.1E-02 2.3E-02 1.9E-O4 0.07 VEGFBII022® 1.2E+06 9.1E-03 1.4E-02 2.6E-O4 0.14 VEGFBII023 3.0E+05 2 2 2.7E-04 m VEGFBII024 3.0E+05 1.3E-02 2 2.8E-04 VEGFBII025 3.3E+05 2 1.8E-02 3.7E-04 (a) KB: kd1/ka1*(kd2/(kd2+ ka2» 6” Curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1 VHHs VEGFBIIO10, VEGFBII022, VEGFBII024 and VEGFBII025 are also tested in the VEGF-mediated HUVEC proliferation and Erk orylation assay.

The potency of the selected formatted VHHs is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96—well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33ng/mL VEGF. The proliferation rates are measured by [3H] Thymidine incorporation on day 4. The results shown in Table 25 trate that the formatted VHHs and Bevacizumab inhibit the VEGF- induced HUVEC proliferation by more than 90%, with |C5os <1nM.

Table 25: |C50 (nM) values and % inhibition of formatted VHHs in VEGF HUVEC proliferation assay VHH ID leo (nM) % inhibition VEGFB||010 VEGFB||021_E_ VEGFB||022 VEGFB||023 __ VEGFB||024 __ VEGFB||025 Bevacizumab The y of the selected formatted VHHs is assessed in the HUVEC Erk phosphorylation assay. In brief, primary HUVE cells are serum-starved over night and then stimulated in the absence or ce of VHHs with 10ng/mL VEGF for 5 min.

Cells are fixed with 4% Formaldehyde in PBS and ERK phosphorylation levels are measured by ELISA using phosphoERK-specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159, Sigma) and polyclonal Rabbit Anti-Mouse—lmmunoglobulin-HRP conjugate (P0161, Dako). As shown in Table 26, the ted VHHs and Bevacizumab t the VEGF induced Erk phosphoryaltion by more than 90%, with |C5os <1nM.

Table 26: leo (nM) values and % inhibition of formatted VHHs in VEGF HUVEC Erk phosphorylation assay VHH ID |C50(nM) % inhibition VEGFBIIO1O . 92 VEGFBll021-103VEGFBll022 0.18 94 VEGFBIIO23 oo VEGFBll024 4 VEGFBll025 Bevacizumab Example 8 Sequence optimization 8.1 ce optimization of VEGFB/l23BO4 The amino acid sequence of VEGFBII23BO4 is aligned to the human germline sequence VH3-23/JH5, see Figure 16 (SEQ ID NO: 179) The alignment shows that VEGFBII23804 contains 19 framework mutations relative to the reference germline ce. Non—human residues at ons 14, 16, 23, 24, 41, 71, 82, 83 and 108 are selected for substitution with their human germline counterparts.

A set of 8 I23804 variants is generated carrying different combinations of human residues at these positions (AA sequences are listed in Table 27). One additional t is constructed in which the potential ization site at position D5986O (CDR2 region, see Figure 16, indicated as bold italic residues) is removed by introduction of a S6OA mutation.

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Table 28: |C5o (pM) , % inhibition and melting ature (@pH 7) of sequence- zed variants of VEGFBIIZSBO4 mm mm ammo VEGFBII23804 (wt) VEGFBI|111D05 _——=1- VEGFBII111G06 VEGFBIH 12011 VEGFB||113A08 _——‘_ VEGFB||113E03 ”- VEGFBII114CO9 VEGFB||114D02 VEGFBII114D03 VEGFB|I118E10 In a second cycle, tolerated mutations from the humanization effort II111GO6) and mutations to avoid potential posttranslational modification at selected sites (the D16G, the S6OA substitution and an E1 D mutation) are combined resulting in a sequence-optimized clone d from VEGFBII2BBO4: VEGFBIIOOS7. One extra sequence-optimized variant (VEGFB||038) is anticipated which contains the same substitutions as VEGFBIIOOB7, with the exception of the I82M mutation, as this on may be associated with a minor drop in potency. The sequences from both sequence- optimized clones are listed in Table 29. VEGFBI|0037 and VEGFB||0038 are characterized in the VEGF165NEGFR2 blocking AlphaScreen (Example 5.3, Figure 18), the melting temperature is determined in the thermal shift assay as described above and the affinity for binding on VEGF165 is determined in Biacore PCT/'EP2012/055901 (Example 5.5). An ew of the characteristics of the 2 sequence-optimized VHHs is presented in Table 30.

Table 29: AA sequences of sequence-optimized variants of VHH VEGFBII23804 VHH IDI SEQ ID CDR 2 CDR 3 DVQLVES 8YSM WFRQA AISKGG RFTISRDNAKNT SRAYGSSR VEGFB”° GGGLVQP G PGKERE YKYDA VYLQMNSLRPE YEY GGSLRLS FVV VSLEG DTAVYYCAS DVQLVES SYSM WFRQA AISKGG RFTISRDNAKNT SRAYGSSR VEGFB”° GGGLVQP G PGKERE YKYDA VYLQINSLRPED LRLADTYEY GGSLRLS FVV VSLEG TAVYYCAS CAASGRT Table 30: |C5o (pM) values, % inhibition, melting temperature (@pH 7) and affinity (pM) of sequence-optimized clones VEGFB||037 and VEGFBII038 % Tm (oc) VHH .D 'Csoli- @pm VEGFB/l23504 5 8.2 Sequence zation of VEGFBl/5BO5 The amino acid sequence of VEGFBIISBO5 is aligned to the human germline sequence VH3-23/JH5, see Figure 19 (SEQ lDzNO: 179 The alignment shows that VEGFBII5BO5 contains 15 ork ons relative to the reference germline sequence. Non— human residues at positions 23, 60, 83, 105, 108 are selected for substitution with their WO 31078 human germline counterparts while the histidine at position 44 is selected for substitution by glutamine. One humanization variant is constructed carrying the 6 described mutations (AA sequence is listed in Table 31).

Table 31: AA sequences of sequence-optimized ts of VHH I5BO5 (FR, framework; CDR, complementary determining region) VHH IDI FR2 CDR2 CDR3 SEQ ID NO: _.--_-- RISSG RFTISRDNSK VEGFBI|119G GGGLVQP WYRQAP NTVYLQMNS WGQGT 11/ GGSLRLS SMA GKQRELV LRAEDTAVY LVTVSS 125 CAASGIR A YCNT EVQLVES RISSG RFTISRDNSK VEGFBI|120E GGGLVQP WYRQAP NTVYLQMNS / GGSLRLS SMA GKHRELV LKAEDTAVY 126 CVASGIR A YCNT One additional variant is constructed in which the potential oxidation site at position M30 (CDR1 region, see Figure 19 indicated as bold italic residue) is removed by introduction of a M30| mutation. Both variants are tested for their ability to bind hVEGF165 using the ProteOn. In brief, a GLC n Sensor chip is coated with human VEGF165.

Periplasmic extracts of the variants are diluted 1/10 and injected across the chip coated with human VEGF165. Off—rates are calculated and compared to the off-rates of the al VEGFBII5BO5. Off-rates from the 2 variants are in the same range as the off- rates from the parental VEGFBII5BOS indicating that all mutations are tolerated (Table 32).

Table 32: Off-rates sequence-optimized ts VEGFBII5BO5 VHH ID binding level (RU) kd (1/s) VEGFBII5805 242 6. 15E-02 |119G11 234 7.75E-O2 VEGFBII120E1O 257 4.68E-02 In a second cycle, mutations from the humanization effort and the M30| substitution are combined resulting in a sequence-optimized clone of VEGFBII5BO5, designated VEGFBIIO32. The sequence is listed in Table 33. Affinity of IO32 is determined by Biacore (see Example 5.5) and the melting temperature is determined in the thermal shift assay as described above. An overview of the characteristics of the sequence- optimized VHH VEGFBIIO32 is presented in Table 34.

Table 33: AA sequence of sequence—optimized clone VEGFBIIO32 (FR, ork; CDR, complementary determining region) VHHIDI SEQ ID CDR2 FR3 CDR3 FR4 EVQLVES RISSG RFTISRDNSK VEGFBII03 P NTVYLQMNS FSSR WGQGTL 2/ GGSLRLS SMA GKQRELV LRAEDTAVY PNP VTVSS 127 CAASGIR YCNT Table 34: g temperature (@pH 7) and affinity (nM) of sequence-optimized clone VEGFB||032 VHH ID I5BO5(wt) VEGFBI|0032 The potency of the sequence-optimized clones VEGFBII037 and VEGFBIIO38 is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96—well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33ng/mL VEGF. The proliferation rates are ed by [3H] Thymidine incorporation on day 4. The s shown in Table 35, demonstrate that the activity PCT/'EP2012/055901 (potency and degree of inhibition) of the parental VHH I23804 is conserved in the sequence optimized clone VEGFBII038.

Table 35: |C5o (nM) values and % inhibition of the sequence optimized clones I037 and VEGFBII038 in VEGF HUVEC proliferation assay VHH ID |C5o(nM) % inhibition EGFB||23BO4 EGFB||037 38 IE.— Bevacizumab 0.29 Construction, production and characterization of bivalent VHHs targeting AngZ VHHs 1D01 (SEQ ID ), 11807, 00908 and 00027 (SEQ ID No:216) are genetically fused to 1D01 (SEQ ID No: 214), 11807, 00908 and 00027 (SEQ ID No:216), respectively, ing in homodimeric VHHs. The bivalent VHHs are linked via a 9-GlySer or 40—GlySer flexible linker. The encoding DNA sequences of the formatted VHHs are cloned in the expression vector . VHHs are expressed in Pichia pastoris as c—terminally myc - His6 tagged proteins. In brief, BGCM cultures are started from a single colony streak ted over weekend at 30°C (250 rpm). After medium switch to BMCM, cultures are incubated until evening at 30°C (250 rpm) and followed by an induction with 100% methanol. The next day the cultures are induced an additional 3 times (morning, afternoon, evening). The next day cultures are centrifuged for 20 min at 4°C (1 ,500xg). The His6—tagged VHHs present in the supernatant are purified through immobilized metal affinity chromatography (IMAC) followed by desalting (DS) and finally gel filtration (GF) to remove any endotoxins/impurities. An overview of the format and sequence of all bivalent VHHs is depicted in Figure 20 and Table 36-A (linker sequences underlined), SEQ ID Nos 180—185. Expression levels are indicated in Table 36-8.

To e the anti-Ang2 blocking properties in comparison with the monovalent building blocks, bivalent VHHs are analyzed in a human Ang2/hTie2 (Figure 21-1), mouse Ang2/mTie2 e 21-2), cyno Ang2/CTie2 (Figure 21-3) and human Ang1/hTie2 (Figure 22) competition ELISA. A summary of IC50 values is shown in Table 37.

Table 36-A Sequences of bivalent VHH ing Ang2 ANGEIIOOOOl EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEQEGVSCIRCSDGSTYYADSVKG? FTISSDNAK TVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGG-VQAGGSLRLSCAASGFTFDDYALGWtRQAAG<<R4GVSCIRCSDGSTYYADSVKGRF TISSDNAK r1VYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLV‘I‘VSS (SEQ ID NO: 180) ANGBIIOOOOZ EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR F"ISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGGS GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWF QAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYEQMNSLKPED"AVYYCAASIVPRS EPYEYDAWGQGTLVTVSS (SEQ ID NO: 181) ANGBIIOOOO3 SGGGLVQVGDSLQLSCAASGRTFSTYLMVGWFRQAPG{EREFAAGIWSSGDTAYADSVRGR FTISRDNAKNTVYLQMN LKTEDTAVYYCAGSYDGNYYIPGFYKDWGQGTLVTVSSGGGGSGGGSEV QEVESGGGAVQVGDSLRLSCAASGRTFSTYLMVGWFRQAPGKEREFAAGIWSSGD"AYADSVRGRFT ISRDNAKN"VYLQMNSLKTEDTAVYYCAGSYDGNYYIPGFYKDWGQGTLVTVSS SEQ ID NO: 182) ANGBIIOOOO4 EVQ-VESGGG-VQAGGSLRESCAASGFTLDDYAIGWtRQA?GK~R4GVSSIRD DGSTYYADSVKGR FTISSDNDKN"VYVQMNSVKPEDTAVYYCAAVPAGRLRFGEQWYPEYEYDAWGQG"LVTVSSGGGGS GGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSD DKVTVYLQMNSEKPEDTAVYYCAAVPAGRERFGEQWYPLYEYJAWGQGTLVTVSS (SEQ ID NO: 183) ANGBIIOOOO5 EVQLVESGGG-VQAGGSLRLSCAASGFTLDDYAIGWtRQA?GK~R4GVSSIRD DGSTYYADSVKGR NDKN"VY-QMNSLKPED"AVYYCAAVPAGR-RFGFQWYP-YEYDAWGQGTLVDVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGEVQAGGSLQLSCAASGFTLDDY A:GWFRQAPGKEREGVSSIRDVDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAV PAGRLRFGEQWYPEYEYDAWGQG"LVTVSS (SEQ ID NO: 184) ANGBIIOOOO6 GGG-VQPGGSLRLSCAASGITLDDYAIGWtRQAPGK~R~GVSSIRD GGSTYYADSVKGR F"ISSDNSK r1VY-QMNST.RPED"AVYYCAAVPAGRRYGF‘QWYPIYEYDAWGQGTLV‘EVSSGGGGS GGGSFVQLLESGGGLVQPGGS-R-SCAASGITLDDYAIGWERQAPGKEREGVSSIRDNGGSTYYADS VKGRFTISSDNSKNTVYLQMNSERPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS (SEQ ID NO: 185) Table 36-3 Expression VHH ID Format (mg/L) ANGBII00001 1 ANGBII00002 7 ANGB||00003 8 ANGBII00004 51 ANGBII00005 03 ANGBII00006 VT 3 Table 37: |C5o values (pM) in human Ang2/human Tie2, mouse Ang2/mouse Tie2, cyno AngZ/cyno Tie2 and hAng1/hTie2 competition ELISA. hAng1/hAngZ hAngZ |C5o |C5o (PM) (PM) 6,973 10,455 9,484 0 1 1 18 ,205 _\ 00 3 52 8 >192,014 _\ O 2 25 6,052,631 4 5,600 n.d., not determined WO 31078 Example 10 Construction, production and terization of trivalent bispecific VHHs targeting VEGF and Ang2 using anti-serum abumin as half-life ion The anti-VEGF VHH VEGFBII00038 (US 2011/0172398 A1) and the anti-AngZ VHH 00027 (SEQ ID No:216) are used as building blocks to generate bispecific VHHs VEGFANGBII00001-00004. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a triple Ala or 9 Gly- Ser flexible linker. VHHs are produced and purified as described in Example 9. An overview of the format and ce of all four bispecific VHHs is depicted in Figure 23 and Table 37-A (linker sequences underlined), SEQ ID Nos 186-189. Expression levels are indicated in Table 38-B.

Table 38-A Sequences of bispecific VHH targeting VEGF and Ang2 EGFANGBIIOOOOI DVQ-VESGGG-VQPGGSLR_LSCAASGRTFSSYSWGWtRQAPGKLR tVVAISKGGYKYJAVSLEGRF I'1ISQDNAKNHVYLQINSLRE’EDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS 1VQLVESGGGVVQPGNSLRVSCAASGFTFSSFGWSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGQ FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTLJDYAIGWtRQAPGKLRLGVSSIRD DGSTYYADSVKGRFTISSDNDK I'1VY QMNSL(P1D"AVYYCAAVPAGRLRFGLQWYP-Y*YDAWGQG"LVTVSS (SEQ ID NO: __86) VEGFANGBIIOOOOZ 1VQ VESGGGLVQAGGSLRL.SCAASGFTLDDYAIGWERQAPGKLRLGVSSIRDNDGSTYYADSV<G FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGG GGGSEVQLVESGGGLVQPGVSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYAD ISRDNAK"TLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG1.4VTVSSGGGGSGGGSDVQ-VV SGGGLVQPGGS-R-SCAASGRTFSSYSMGWFRQAPGKEREFVVAIS<GGYKYDAVSL.EGRFTISRD AKNTVYLQINS-RP1DTAVYYCASSRAYGSSRLRLADTYEYWGQG"LV'1VSS (SEQ ID NO: 187) VEGFANGBIIOOOO3 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPED"AVYYCAAVPAGRLRFGEQWYPLYEYDAWGQG1.4VTVSSGGGGS GGGSDVQLV1SGGG-VQPGGS-R SCAASGR1FSSYSMGWFRQAPGKEREFVVAIS(GGYKYDAVSL EGRFTISRD AK I'1VYLQINSRP1DTAVYYCASSRAYGSSRLRLADTYE'.YWGQG1LV'11VSSGGGGS GGGSFVQT.V1SGGG-VQPGNS- q SCAASGF1FSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNA<””LYLQM TAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 188) GBIIOOOO4 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWtRQAPGKLRLtVVAIS<GGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPED’1AVYYCASSRAYGSSRLRLADTY1YWGQGT AAEVQ-V1 SGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSD1LYADSVKGRF1ISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG1LVTVSSAAA1VQT.VESGGG VQAGGSVR-SC AASGFTLDDYAIGWFRQAPGKLR1GVSSIRDNDGS"YYADSVKGRFTISSDNDKN1VYLQMNSLKPE DTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 189) Table 38-3 To explore the anti-VEGF ng properties in comparison with the monovalent building block VEGFB||00038, all four bispecific VHHs are analyzed in the VEGFNEGFRZ-Fc (Figure 22) competition AlphaScreen. The assay is slightly adjusted compared to Example 12.3 described in patent US 2011/0172398 A1. Both human VEGF165 and human VEGFR2-Fe are added at 0.05 nM. This competition assay is also performed after preincubation of the VHH with 25 uM human serum n. A summary of IC50 values and % inhibition is shown in Table 39.

Table 39: |C5o values (nM) in human VEGF165/human VEGFR2 competition AlphaScreen GBIIOOOO1 VEGFANGBII00002 VEGFANGBIIOOOO3 ‘ VEGFANGBIIOOOO4 To explore the anti-Ang2 blocking properties in comparison with the monovalent building block 00027 (SEQ ID Noz216) all four bispecific VHHs are analyzed in a human Ang2/hTie2—Fc (Figure 25) ition ELISA. This assay is also performed after incubation of the VHH with 0,5 uM human serum albumin. A y of |C5o values is shown in Table 40.

Table 40: |C5o values (pM) in human Ang2/human Tie2 competition ELISA 00027 VEGFANGBIIOOOO1 VEGFANGBIIOOOOZ VEGFANGBIIOOOO3 VEGFANGBIIOOOO4 AMG386 n.d., not determined 2012/055901 Example 11 Construction, tion and characterization of ent and tetravalent bispecific VHHs targeting VEGF and Ang2 using anti-serum albumin binding as half-life extension Ten bispecific VHHs targeting VEGF and AngZ are constructed (VEGFANGBII00005- 00015). In these constructs monovalent and bivalent 1D01 (SEQ ID NO:214), monovalent and nt 7G08 (SEQ ID NO:215) and bivalent 00027 (SEQ ID NO:216) anti—AngZ ng blocks are included. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly- Ser flexible . VHHs are produced and purified as described in Example 8. An overview of the format and sequence of all ten bispecific VHHs is depicted in Figure 26 and Table 41—A r sequences underlined), SEQ ID Nos 190-199. Expression levels are indicated in Table 41 -B.

Table 41 -A Sequences of bispecific VHH targeting VEGF and Ang2 EGFANGBIIOOOO5 -VESGGG-VQPGGSL?SSCAASGRTFSSYSMGWtRQAPGK7R7tVVAISKGGYKYDAVSLEGRF QDNAKNTVYLQINSL??EDTAVYYCASSRAYGSSRLRLADTYIEYWGQGTLVTVSSGGGGSGGGS 7VQ7VESGGG'VQPGGSTQVSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGSVQPGNSDRSSCAASGFTFSSFGMSWVRQAPG<G.7WVSSISGSGSDTLYAJSVKG RFTISRDNAKm1 -RP7DTAVYYCTIGGSLSRSSQGT.JV'1VSS (SEQ ID ) EGFANGBIIOOOO6 -VESGGG-VQPGGSLQSSCAASGRTFSSYSMGWtRQA?GK7R7 tVVAIS<GGYKYDAVS-7GRF "1ISRDNAK\I"1 SQINSLQ?IEDTAVYYCASSRAYGSSRLR'ADTY7YWGQGT'VTVSSGGGGSGGGS EVQLVESGG RLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR 7TISRDNAZ . DRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG SVQPGGSLRSSCAASGFADJYYAIGWtRQVPGK7R7GVSCISSSDGITYYVDSVKGRFTISRD AKN - YCATDSGGYIDYDCMGDGYDYWGQGTSVTVSS (SEQ ID NO:191) EGFANGBIIOOOO7 DVQ- -VQPGGSL?SSCAASGRTFSSYSWGWtRQAPGK7R7tVVAIS<GGYKYDAVS77GRF "ISRDNAKVTVYLQINSL??I3DTAVYYCASSRAYGSSRLRVAD7Y7YWGQGT'VTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFALDYYA-GWFRQVPGKEREGVSCISSSDGITYYVDSVKGR "ISRDNAKN1VYLQMNSDK3EDTAVYYCA-DSGGYIDYDCMGAGYDYWGQG-LVTVSSGGGGSGGG I SVQPGNSARSSCAASGFTFSSFGWSWVRQAPG<G.7WVSSISGSGSDTLYADSVKG 11-Y QMNS-RP7DTAVYYC7IGGSLSRSSQGTSV’VSSGGGGSGGGS VQLV7 SGG VQPGGS -SCAASGEASDYYAIGWFRQVPGK7R7GVSCISSSDGITYYVDSVKGRFTISRDNAK ’1VYTQMNS-<P7D”AVYYCAT3SGGYIDYDCMGSGYDYWGQG70VTVSS (SEQ ID NO: 192) VEGFANGBIIOOOO8 DVQLVESGG L QPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYSQINSLRPEDTAVYYCASSRAYGSSRLRDAD7YEYWGQGTLVTVSSGGGGSGGGS ESGGG VQAGGSLRSSCAASGFTLDDYAIGWtRQAPGK7R7 GVSSIRDNDGSTYYADSVKGR FTISSDNDK '1VYLQMNSL(PED7AVYYCAAVPAGRLRFG7QWYP-Y7YDAWGQGTLVTVSSGGGGS GGGSFVQLV7SGGG-VQAGGS-R-SCAASGF"LDDYAIGWTRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSD DKNHVYLQWNSL<PEDTAVYYCAAVPAGRVRFG7QWYPLYFYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYSQWNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 193) 2012/055901 EGFANGBIIOOOO9 DVQ-VESGGG-VQPGGSLRJSCAASGRTFSSYSWGWtRQAPGK~R<tVVAISKGGYKYJAVSLEGRF r1ISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQVVESGGG-VQPGNSLRLSCAASGFTFSSFGWSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG JVQAGGSDRLSCAASGFTLJJYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDK r1VY-QMNST.KP_'3""AVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLV SGGGLVQAGGS-R-SCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISS DK TVYVQM S-<PEDTAVYYCAAVPAGR-RFG7QWYPLYEYDAWGQGTLVTVSS (SEQ ID 0: 194) VEGFANGBIIOOOIO DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYJQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQ-VESGGG-VQAGGSLRLSCAASGFTLDDYAIGWtRQAPGK~R4GVSSIRDNDGSTYYADSVKGR FTISSDNDK MNSLKPED"AVYYCAAVPAGRT.RFGF‘QWYPX‘YDAWGQGTLVTVSSGGGGS GGGSEVQVVESGGGLVQPGNS-R-SCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDVAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG"LVTVSSGGGGSGGGSEVQLVE SGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDVDGSTYYADSVKGRFTISSD NDKNTVYDQM TAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 195) EGFANGBIIOOOll DVQLVESGGGJ QPGGSLQLSCAASGRTFSSYSMGWtRQAPG<<R4tVVAISKGGYKYDAVS'7GRF TISRDNAKV"VYJQINSLQPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTVVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR TISSDNAKVTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE GJVQAGGSLRDSCAASGFTFDDYAJGWtRQAAGK R4GVSCIRCSDGS"YYADSVKGRF r1VYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV .VQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKG.7WVSSISGSGSDTJYADSVKGRFT DNAKTTJYLQMNSLR?EDTAVYYCTIGGSJSRSSQGTLVTVSS (SEQ ID NO: 196) VEGFANGBIIOOOIZ SLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF ISRDNAKVTVYLQINSLR?EDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQ GG-VQPGNSLRASCAASGFTFSSFGWSWVRQAPG<G EWVSSISGSGSDTJYADSVKGR 1"1-Y-QMNS._.RPEDFKATVYYCTIGGSLS1"-§SSQGT.J\/"“\/SSGGGGSGGGSEVQLVESGGG .SCAASGFTTDDYAJGWbRQAAG<4R4GVSCIRCSDGSTYYADSVKGRF"ISSDNAKN 7D"AVYYCAASIVPRSKLEPYEYDAWGQGHLVTVSSGGGGSGGGSEVQLVESGGGL 4SCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFT:SSDNAKNT DTAVYYCAASIVPRSKLEPYEYDAWGQGTAVTVSS (SEQ ID NO: 197) VEGFANGBIIOOOI3 -VQPGGSLRASCAASGRTFSSYSWGWtRQAPGK<R4tVVAISKGGY<YDAVSLEGRF NSLRPEDTAVYYCASSRAYGSSRLRAAD"YEYWGQGTLVTVSSGGGGSGGGS .VQAGGSLQJSCAASGFTFDDYAJGWtRQAAGK~R4GVSCIRCSDGS"YYADSVKGR FTISS3NAKN"VYLQMNSVK?7DTAVYYCAASIVPRSKLE?YEYDAWGQGTLVTVSSGGGGSGGGS: VQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDVAKTT-Y-QMNSLRPEDTAVYYCTIGGSJSRSSQGTLVflVSSGGGGSGGGSEVQLVESGGGJ VQAGGSLRJSCAASGFTFDJYALGWJ:RQAAG<<R4GVSCIRCSDGSTYYADSVKGRF"ISSDNAKNr1 VYLQMNSL<PED"AVYYCAASIVPRSKLEPYEYDAWGQGTAVTVSS (SEQ ID NO: 198) VEGFANGBII00014 DVQ-VESGGG-VQPGGSL?JSCAASGRTFSSYSWGWtRQA?GK~R<tVVAISKGGYKYDAVSLEGRF r1ISRDVAKVP‘VYLQINSLQEEDTAVYYCASSQAYGSSRLR'ADTYEYWGQGTTNTVSSGGGGSGGGS EVQLVESGG LVQPGNSL LSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKT"-Y-QMNSDRPED"AVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG DSCAASGFTFDDYAJGWERQAAG<~R4GVSCIRCSDGSTYYADSVKGRFTISSDNAKN .(P7D"AVYYCAASIVPRSKLEPYEYDAWGQG"JVTVSS (SEQ ID NO: 199) Table 41 -B Expression (mg/L) VEGFANGBIIOOOO8 VEGFANGBII00009 GBIIOOO10 VEGFANGBIIOOO11 VEGFANGBIIOOO12 VEGFANGBIIOOO13 VEGFANGBIIOOO14 To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFBIIOOOS8, all ten ific VHHs are ed in the VEGFNEGFRZ-Fc (Example 10; Figure 27-1) and VEGFNEGFR1 (Figure 27-2) competition AlphaScreen. The VEGFR1 assay is slightly adjusted compared to Example 12.4 as described in patent US 2011/0172398 A1. Human VEGF165 and human -F0 are added at 0.05 nM. These competition assays are also performed after preincubation of the VHH with 25 uM human serum albumin. A summary of |C5o values is shown in Table 42.

Table 42: |C5o values (nM) in human VEGF165/human VEGFR2 and human VEGF165/human VEGFR1 competition AlphaScreen VEGFANGBIIOOOO5 VEGFANGBII00006 VEGFANGBIIOOOO7 -m--- VEGFANGBHOOOOg ; —m--- VEGFANGBHOOOOQ _ ----- VEGFANGBHOOMO A " -mm-- VEGFANGBIIOOOO1 VEGFANGBIIOOO11 VEGFANGBIIOOO12 GBIIOOO13 VEGFANGBIIOOO14 Ranibizumab n.d., not ined To explore the anti-Ang2 blocking properties in comparison with their respective monovalent building block 7GO8 (SEQ ID No:215), 1D01 (SEQ ID No:214) and 00027 (SEQ ID ), all ten bispecific VHHs are analyzed in the human Ang2/hTie2-Fc (see Example 5.1; Figure 28-1), mouse Ang2/mTie2-Fc (see Example 5.2; Figure 28-2) and cyno Ang2/cTie2-Fc (see Example 5.2; Figure 28-3) competition ELISA. The human assay is also performed after incubation of the VHH with 0.5 uM human serum albumin. Additionally, a hAng2 ed HUVEC survival assay is performed (see Example 5.5; Figure 29). A summary of |C5o values and % inhibition is shown in Table 43.

Nm_._. oc>o\mmc< :5 mg? oc>o Om>DI m 925 mac. mwsoENoc< .3228 . M,w 3308 85: “a: .Nm_._. BEBE Eaafifiafial m;“_-m-.mnw . “my cmEJEch< was} “gm + Eaafiaafial EEEHEHfiHI cmEsc 8:532 c_ g Gd coEQEE Em £0 829 cam :25 mcto 31% A23 82 829, gm 3,2 <m3m woON =mOZ<LOm> moooo =mOZ<mOm> ooooo =m02<m0m> noooo owm02< =m02<m0m> woooo E .3 8:58:50 llllllfillléEaaaalfial II a Eafilfill w Jr“ W.; W?” Ifiaalalfill Eaaafilfia W\x w‘g lfial Wm -@nfl--flm- EEEEEHEH -@Efl--mm- + III =m02<m0m> moooo =mOZ<LOm> orooo ome§< =mOZ<LOm> rrooo =m02<m0m> Nwooo =mOZ<LOm> wrooo =mOZ<mOm> wrooo omm0§< UmEELBmU Affinities of for human serum albumin have been determined and are shown in Table 44. Briefly, human serum albumin (Sigma, St Louis, MO, USA) is immobilized on a CM5 chip via amine coupling. A multicycle kinetic approach is used: increasing concentrations of VHH (2—8—31500 nM) are injected and allowed to associate for 2 min and to dissociate for 10 min at a flow rate of 100 uL/min. Between VHH injections, the surfaces are regenerated with a 10 sec pulse of 10 mM Glycine-HCI pH 1.5 and 60 sec stabilization period. Association/dissociation data are evaluated by fitting a 1:1 interaction model (Langmuir g) or geneous Ligand model. The affinity nt K9 is calculated from resulting association and dissociation rate constants ka and kd (Table 44).

A<m_>_ Hail aW a WW WWW .WWWW WWWW £8326 No-m_N.m No- WMWWW m5. .WWWW ,WWWW E23 @ mo+m_@.© mo+m_N_\ m WWW 339: WW ,WWWWW Ucm II I WW ,WWWWW A<mov memo mo- mo- um Wm Mm WWWW 098 _\ v v 29.: mo+m_m m.o+m_w mo+m_m W: WW v F _‘ SEE; “I I WW WOW, wII> mo-m_w. mo- moww. m5. 'EE WWWW _\ v v 85qu mo+Mm. mowa mo+wo. WW W6 v N N 3EE< .3» _\_\m_._< 89::ng 03m... Foooo__m02<n_0w> moooo__m_OZ<u_Om_> moooo__m_02<u_0m> woooo=m02<m0m> owooo=m®z<m0w> :ooo__m_oz<u_Om> m02<n_0m_> :oo__m_OZ<u_Om_> Woc Example 12 Construction, production and characterization of sequence optimized and affinity matured bispecific VHHs targeting VEGF and Ang2 using anti-serum albumin g as half-life extension 14 bispecific VHHs targeting VEGF and Ang2 are constructed (VEGFANGBIIOOO15- 00028). In these constructs bivalent 00921 (a ce optimized 1D01 variant) (SEQ ID No:220), monovalent VHHs 00908 — 00932 — 00933 — 00934 — 00935 — 00936 — 00937 — 00938 nce optimized/affinity matured 28D10 ts) (SEQ ID No:222), bivalent 00956 (SEQ ID NO:223) (sequence optimized 28D10 variant) and monovalent 00928 (SEQ ID NO:221) (sequence optimized 37F02 variant) anti-AngZ building blocks are included. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly-Ser flexible linker. An overview of the format and ce of all 14 bispecific VHHs is depicted in Figure 30 and Table 45-A (linker sequences underlined), SEQ ID Nos 200-213. sion levels are indicated in Table 45-B.

Table 45-A ces of bispecific VHH targeting VEGF and Ang2 GBIIOOOlS -VESGGG-VQPGGSLRASCAASGRTFSSYSMGWtRQAPG<<R4tVVAISKGGYKYDAVSLEGRF " RDNAKN"VYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS G-VQPGNSL?SSCAASGFTFSSFGMSWVRQAPG<G.7WVSSISGSGSDTLYADSVKGR ""LYLQMNSVQPTDTAVYYCTIGGSLSRSSQGTSVflVSSGGGGSGGGSEVQLVESGGG JVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKN ”VYLQMNSL.?EDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS (SEQ ID NO: EGFANGBIIOOOl6 -VESGGG-VQPGGSLRSSCAASGRTFSSYSWGWtRQAPG<*R*tVVAISKGGYKYJAVSLEGRF " RDNAKNTVYLQINSLQPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS 7VQ1VESGGG-VQPGNSLQVSCAASGFTFSSFGWSWVRQAPG{GLEWVSSISGSGSDTSYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG SVQPGGSLRLSCAVSGITSJJYAIGWFRQAPGKEREGVSSIRJNGGSTYYADSVKGRFTISSDNSKN " -QMNSLRP7DTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: EGFANGBIIOOOl7 -VESGGG-VQPGGSL?SSCAASGRTFSSYSWGWtRQAPG<~R~tVVAISKGGYKYJAVSLEGRF ' r1VYLQINSL'R’NE'.DTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGG LVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKT"-Y-QMNSSRPEDTAVYYCTIGGSLSRSSQGTAVTVSSGGGGSGGGSEVQLVESGGG SVQPGGSLRSSCAASGITADDYAIGWERQAPGK<R4GVSAIRDNGGSTYYADSV<GRF"ISSDNSKN " -QMNSLRPTD"AVYYCAAVPAGRLRYG*QWYPIY~YDAWGQGTLVTVSS SEQ ID NO: EGFANGBIIOOOIB DVQ-VESGGG-VQPGGSLR_JSCAASGRTFSSYSWGWtRQAPGKEREtVVAISKGGYKYDAVSLEGRF EISQDNAKNTVYLQINSLQPEDTAVYYCASSRAYGSSRLRLADTYIEYWGQGTLVTVSSGGGGSGGGS EVQEVESGGG-VQPGNSL?LSCAASGFTFSSFGWSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTELYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG EVQPGGSLRLSCAASGITEJJYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFEISSDNSKN RPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: VEGFANGBIIOOOI9 -VESGGG-VQPGGSL?ESCAASGRTFSSYSWGWtRQAPGKEREtVVAISKGGYKYlAVSLEGRF 1ISRDNAKNEVYLQINSLQPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS E RESCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR EDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG DDYAIGWtRQAPGKEREGVSAIRSSGGSTYYADSV<GRFEISSDNSKN EDEAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: VEGFANGBIIOOOZO DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF EISRDNAKNEVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQEVESGGGEVQPGNSLRESCAASGFTFSSFGWSWVRQAPGKG EWVSSISGSGSDTEYADSVKGR MNS_.RPEDTAVYYCTIGGSLSRSSQGTLVEVSSGGGGSGGGSEVQLVESGGG .SCAVSGITLDDYAIGWtRQAPGKEREGVSAIRDNGGSTYYADSV<GRFEISSDNSKN EDEAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: VEGFANGBIIOOOZI DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF EISRDNAKNEVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQ-VESGGG-VQPGNSL3ESCAASGFTFSSFGWSWVRQAPG<G EWVSSISGSGSDTEYADSVKGR FTISRDNAKEELYLQMNSVRPEDTAVYYCTIGGSLSRSSQGTEVEVSSGGGGSGGGSEVQLVESGGG .VQPGGSLR-SCAVSGITLDDYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFEISSDNSKN EVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 206) VEGFANGBIIOOOZZ G-VQPGGSLRESCAASGRTFSSYSWGWERQAPG<EREtVVAISKGGYKYJAVSLEGRF RDNAKNEVYLQINSLRPIDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS .VESGGG-VQPGNSL?ESCAASGFTFSSFGWSWVRQAPG<G EWVSSISGSGSDTEYADSVKGR 1E-YQMNS'RPEDTAVYYCTIGGSLSRSSQGTLVEVSSGGGGSGGGSEVQLVESGGG ESCAVSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFEISSDNSKN EDEAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: EGFANGBIIOOOZ3 -VESGGG-VQPGGSLRESCAASGRTFSSYSWGWtRQAPGKEREtVVAISKGGYKYJAVSLEGRF RDNAKNTVYLQINSLQPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS 'VQEVESGGG-VQPGNSLQLSCAASGFTFSSFGWSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTELYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG EVQPGGSERLSCAASGFTEJJYAIGWFRQAPGKEQEGVSAIRDNGGSTYYADSVKGRFEISSDNSK ’ QMNST.RP CAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVI PGGS-R-SCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSV<GRFTISS S-RPFDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: GBIIOOOZ4 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYJQINSLRPEDTAVYYCASSRAYGSSRLRLAJTYEYWGQGTLVTVSSGGGGSGGGS EVQ-VESGGG-VQPGGSLRLSCAASGFTLDDYAIGWtRQAPG<EREGVSAIRDNGGSTYYADSVKGR FTISSDNSK EVYLQMNSVRPEDEAVYYCAAVPAGRLRFGEQWYP-YEYDAWGQGTLVTVSSGGGGS GGGSFVQEVESGGGLVQPGGS-R-SCAASGFELDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADS VKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLREGEQWYPLYEYDAWGQGTLVTVSSE GSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFEISRDNAKTELYJQMNSLRPEDEAVYYCTIGGSESRSSQGTLVEVSS SEQ ID NO: 209) EGFANGBIIOOOZ5 DVQEVESGGGLVQPGGSLQLSCAASGRTFSSYSMGWtRQAPG<EREtVVAISKGGY<YDAVSLEGRF EISRDNAKVEVYLQINSLQPEDTAVYYCASSRAYGSSRLREADTYEYWGQGTVVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGEEWVSSISGSGSDTLYADSVKGR FTISRDN KETLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVEVSSGGGGSGGGSEVQLVESGGG EVQPGGSERESCAASGFTFDDYALGWtRQAPG<EREGVSCIRCSGGSTYYADSVKGRFTISSDNSK EVY-QMNS-RPEDTAVYYCAASIVPRSKLEPYEYDAWGQGELVTVSSGGGGSGGGSEVQLVESGGG.

VQPGGSLQESCAASGFTFDDYALGWtRQAPG EREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNE VYLQMNSEQPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS SEQ ID NO: 210) BII00026 DVQ-VESGGG-VQPGGSLRESCAASGRTFSSYSMGWtRQAPGK~R4tVVAISKGGYKYDAVSLEGRF TISRDNAK INSLQPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQDVESGGG-VQPGGSL?LSCAASGFTFDDYALGWFRQAPGK4R4GVSCIRCSGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAS:VPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGAVQPGGSLRDSCAASGFTFDDYAJGWFRQAPGKEREGVSCIRCSGGS"YYADSVKGRF T K r1VYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV Q-V:SGGG-VQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKG.7WVSSISGSGSDTJYADSVKGRFT ISRDNAKTTJYLQMNSLR?EDTAVYYCTIGGSJSRSSQGTLVTVSS SEQ ID NO: 211) VEGFANGBII00027 3VQVVESGGG-VQPGGSLQLSCAASGRTFSSYSWGWFRQAPGK*R<FVVAISKGGY<YDAVSLEGQF ISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS IVQAVESGGGJVQPGNSLRASCAASGFTFSSFGWSWVRQAPGKG.7WVSSISGSGSDTLYADSVKGR NA<""-Y-QMNS_.RPEDTAVYYCTIGGSLSRSSQGT.J\/"VSSGGGGSGGGSEVQLVESGGG .VQPGGS-R-SCAASGFAJDYYAIGWtRQAPGK<R4GVSCISSSGGITYYADSVKGRFTISRDNSKN 1VY-QMNS'RP':'D"A.VYYCATDSGGYIDYDCSGJGYDYWGQG"LVTVSS SEQ ID 0: 212) VEGFANGBII00028 DVQWVESGGGVVQPGGSL LSCAASGRTFSSYSWGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQAVESGGGJVQPGNSLRASCAASGFTFSSFGWSWVRQAPGKG.7WVSSISGSGSDTLYADSVKGR FTISRDNAK""-Y-QMNSARPEDTAVYYCTIGGSLSRSSQGTJV"VSSGGGGSGGGSEVQLVESGGG .VQPGGSVR-SCAASGFTLDDYAIGWtRQAPGK<R4GVSAIRSSGGSTYYADSVKGRFTISSDNSK r1VY-QMNST.RP?D"A.VYYCAAVPAGRLRFGEQWYELYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVIZ SGGGLVQPGGSJRLSCAASGFTLDDYAIGWFRQAPGKEREGVSA:RSSGGSTYYADSVKGRFTISS3 VSKVTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: 213) Table 45-3 sion VHH ID (mg/L) VEGFANGBII00022 VEGFANGBII00025 VEGFANGBII00028 To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFBIIOOOS8, the bispecific VHHs are analyzed in the VEGFNEGFRZ- Fc (Example 10; Figure 31-1) and VEGFNEGFR1 (Example 11; Figure 31-2) competition AlphaScreen. These competition assays are also performed after preincubation of the VHH with 25 uM human serum albumin. A summary of |C5o values is shown in Table 46-A. 2012/055901 Table 46-A |C50 values (nM) in human 5/human VEGFR2 and human VEGF165/human VEGFR1 competition AlphaScreen |C5o ICSO VHH ID Format HSA % inh % inh (nM) (WI) 3/1”? 9?? gag: ‘ -- El“- - um“- ----- VEGFANGBHOOOZg _"-m- n.d., not determined Binding kinetics of the bispecific VHHs on human VEGF165 is analyzed by SPR on a Biacore T1OO instrument (see Example 12.5 described in patent US 2011/0172398 A1).

Monovalent Nanobody VEGFB||00038 is taken along as reference (Table 46-B).

Table 46-B: Overview of kinetic ters in hVEGF165 Biacore assay. ka1 kd1 ka2 kd2 KD1 (1/Ms) (1/3) (1/3) (1/3) (M) VEGFBIIOOO38 2.6E+05 1.3E-02 1.3E-02 1.9E-O4 7.5E-1O The ability of the VHHs to bind to human isoform VEGF121 is determined in a binding ELISA. Binding of a dilution series of VHH to 1 ug/mL directly coated human VEGF121 (R&D) (human VEGF165 as reference) is detected using ylated anti-VHH 1A4 followed by extravidin-HRP. 1A4 is a anti-VHH VHH (generated in—house by Ablynx NV). The benchmark Avastin serves as positive l and is detected using a HRP ated anti-human Fc antibody. An irrelevant VHH serves as negative control.

Representative binding response curves on VEGF165 and VEGF121 are shown in Figure 46 corresponding EC50 values are summarized in Table 46-C.

Table 46-C: Overview of EC50 values in hVEGF165 and hVEGF121 binding ELISA. 165 21 —(M) (M) VEGFANGBII00022 1.4E-09 2.3E-09 VEGFANGBII00025 9 2.5E-09 VEGFANGBII00028 1.2E-09 2.1E-09 Binding to rat and mouse VEGF164 is assessed in a binding ELISA. VHHs binding to 1 ug/mL directly coated murine or rat VEGF164 (R&D) are detected using biotinylated anti—VHH 1A4 followed by extravidin-HRP. As positive control the mouse/rat cross— reactive monoclonal antibody B20-4.1 tech) is titrated and detected with an HRP conjugated anti-human Fc dy. An irrelevant VHH serves as negative control.

Results are shown in Figure 33. All 3 bispecific VHH are not cross-reactive to mouse and rat VEGF.

Binding to human VEGF-B, VEGF-C, VEGF-D and PIGF is assessed via a binding ELISA. Binding of VHHs to 1 ug/mL directly coated VEGF-B (R&D), VEGF-C (R&D), VEGF-D (R&D) and PIGF (R&D) was detected using biotinylated anti—VHH 1A4 followed by extravidin-HRP. As positive controls a series of ons of the appropriate ors (hVEGFR1-Fc for hVEGF—B and hPIGF, hVEGFR2—Fc for hVEGF-C, anti-hVEGF-D mAb (R&D) for hVEGF-D) are taken along. An irrelevant VHH serves as negative control. Results are shown in Figure 34. All 3 bispecific VHH are not binding to VEGF family members.

To explore the anti-Ang2 ng properties in comparison with their respective monovalent building block 00921 (SEQ ID NO:220) and 00938 (SEQ ID NO:222), all 3 bispecific VHHs are analyzed in the human Ang2/hTie2—Fc (see Example 5.1; Figure 35-1), mouse Ang2/mTie2—Fc (see Example 5.2; Figure 35-2) and cyno Ang2/cTie2—Fc (see Example 5.2; Figure 35-3) competition ELISA. The human assay is also performed after incubation of the VHH with 0.5 uM human serum n.

Additionally, bispecific VHHs are tested in the hAng1/hTie2 competition ELISA (see Example 5.3; Figure 36) and the Ang2 mediated HUVEC survival assay (see Example .5; Figure 37). A y of IC50 values and % inhibition is shown in Table 47-A.

HUVEC survival % . . . inh 100 00 0 0 0 0 1 n .d 0 1 100 0 1 0 1 .d n 1 0 100 100 n .d IC50 (nM) 4.3 1.9 n.d. 1.4 18.8 1.0 2.2 n.d. 1.2 6.8 3.7 n.d. hAng1 IC ratio 50 hAng2 > 60,395 > 39,902 > 44,771 5,656 > 125 5,194 > 160,694 > 133,660 5,632 > 1,979 > 66,222 > 50,816 % inh 100 100 100 100 100 100 100 100 100 100 100 100 ELISA cAng2 IC 50 M) (p 86 101 115 13 43,500 14 38 37 15 1,294 65 78 mAng2 % inh 100 100 100 100 100 100 100 100 100 100 100 100 IC 50 (pM) 58 56 68 2 27,990 1,816 72 17 69 15 2 3 Ang2 % inh 100 100 100 100 100 100 100 100 100 100 100 100 IC 50 (pM) 33 50 45 5 15,940 12 15 5 1,010 30 39 4 HSA - - + - - - + - - - + - 00921 00956 00921 00938 00956 ALB11 ALB11 ALB11 00938 00038 00921 00038 00956 00038 values (nM) in hAng2 mediated HUVEC survival assay Format 50 values (pM) in human Ang2/human Tie2, mouse Ang2/mouse Tie2 and cyno Ang2/cyno Tie2 competition ELISA, IC hAng1 ELISA and IC 50 47- A: VHH ID 00938 VEGFANGBII00022 AMG386 00921 VEGFANGBII00025 AMG386 00956 VEGFANGBII00028 AMG386 n.d., not determined Table Affinities of GBI|00022—25-28 for human, mouse, cyno and rat AngZ (see Example 5.4) have been determined and are shown in Table 47-B.

Table 47-B: Affinity KD of purified VHHs for recombinant human, cyno, mouse and rat AngZ —human LD cyno AngZ-FLD ka kd KD ka kd KD (1/MS) (1/8) (M) (1/MS) (1/8) (M) VEGFANGBIIOOOZZ 9.7E+05 1.5E-05 1.6E-11 1.5E+06 1.3E-05 8.1E-12 -------ka kd KD ka kd KD Affinities of VEGFANGBI|00022—25-28 for human, mouse and cyno serum albumin have been ined (Example 11) and are shown in Table 48. The m affinity constant K9 is calculated from resulting association and dissociation rate constants ka and kd (Table 48).

Table 48: ty KD (nM) of purified VHHs for recombinant human, mouse and cyno serum albumin using (A) 1:1 ction model or (B) heterogeneous ligand model amos kd KD (1/Ms) (1/5) (nM) .9E+05 3.0E-02 51 .2E+05 5.-4E03 150 ———- ka1 kd1 ka2 kd2 KD1 KDZ (1/Ms) (1/5) (1/5) (1/5) (nM) (nM) VEGFANGBII00025 6.2E+05 9.9E-02 4.7E+04 5.7E-04 -- VEGFANGBII00028 5.9E+04 6.9E-04 5.7E+o5 9.4E-02 * describes 70% or more of the interaction mvooo ”$862 a 89 ammuoz omooo 89 ”C852 880 8v 89 xmmmuoz MD<NMBmGQmOmHO 0E>mD>MMEHGQmmmHU wM>mD<NMBmmeDmHU mm>B>onowz mm>e>oewooz mm>B>OBw003 mm>B>QBw #mm mm>B>QBUODE mm>B>QBwODE wm>mo<wwemwwmomHo wwewammmHo wm>mm<wwemwwmmmH< wmemwwmmmH< DE mm>B>QBOOGB mm>B>QBwODS mm>E>QBQOOB 2%.: Q GU UM>mD<MMBmODZQmHm 0M>mQ<NMBmmeomHo wm>mD<MMEHmemmHo N38 0me macucanoo xemnoz 988 Q émmuoz NMh m>wmfimmw<<0mm3 m>wmmmxom>0mm3 m>wmmmmwm<omm3 ¢QMEMmEvamm>Hm MQMOAGZOQMHHWOOWQ <dmqumMBOmwmmamo<m> m>wmmmmwm<dw m3 mwm<dflm3 m>wmmmmwm<dmm3 m>wmmmxwm<dmm3 m>wmmmxwm<dmm3 m0m<dmm3 89 9 (flwmwmfléxmmwOHm mODMDwame m:_uc_n-~m:< HMQU wq<mm oH<Mw wH<wm HMQU GH<MD DQ<MD Mao <dwqumMEOrwuwémw<m>. <Qwflwmflévmmm>Hm m>wmmmmwm<dmmz;wq<wm wH<NM DH<ND 0H<NQ (memeWSOEwwmémw<m>. <dwmwdmwgamwmmamw<m> OH<MM R0 due ”3552 wmooo <<wa Q 89 ”ammuoz Dmhhwm<<omflmu <<UMW><BDEmm 89 0me xim 580 Ow<0>qooo‘m>fl0>m QQ<MOm<<0mnmqm00m0>gwwwmm>do>m QQBmwm<<om1¢flmww<d>dwwwmm> Ddfimwm<<om1QOwme>quwmm> Dd<mwm<<om1¢flmwwm0>quwmm> DhBfiUm<<om1¢fimwwm0>dwwwmm>1 QQBme><Om_mémwwm0>quwmm>F QQBmOm<<omflmqm00m0>qw®©mm>fl fiHDEmm_mZEO1M>BZMmZQmmH <BDEmm_mZEOFM>BZMmZQmmH HMh o>m mfim <<UMM><BDEmMFmZEOQM>BZM<ZQmmHBmm B<OMM><BQmmMJmZZOQW>BZM<ZQmm“Emm <<OMM><EDmmMJmZZOQM>EZMQZQmmuEmm Q>m omEmwm<<0mflQOwme>quwmm>FO>E maflmwm<<omflmqm00m0>quwmm>fl0>m ”6562 Hg mmm <<UMM><BDEmm_mZEO_%>BZMmZDmmHBmm mZEOFM>BZMmZDmmHBmm o>m Q>m o>m 0>m m Bmm Bmm <<UMW><BQEmmFmZEOFM>BZMmZQmmHBmm <<OMM><EDmmmflmZZOHM>EZMmZQmmHEmm 89 9 So: due HoQH womb bNo HoDH moww bmo Nwooo omooo mwooo ammoo mmmoo mmmoo ommoo ammoo mmmoo mmmoo ommoo

Claims (12)

Claims:

1. A bispecific binding le comprising - at least one VEGF-binding component, - at least one serum albumin binding component, and 5 - at least one Ang2-binding component and, wherein said Ang2-binding component binds to Ang2 with a potency at least 5,000 times higher than to Ang1 or to Ang4, and wherein said VEGF-, serum albumin and Ang2-binding components are globulin single variable domains, each immunoglobulin single variable 10 domain consisting of four framework regions and three complementarity determining s (CDRs), and wherein said bispecific binding molecule is selected from the group ting of bispecific binding molecules having (i) the CDR sequences as present in VEGFANGBII00022 (SEQ ID NO: 207), 15 (ii) the CDR sequences as present in VEGFANGBII00025 (SEQ ID NO: 210), (iii) the CDR sequences as present in VEGFANGBII00028 (SEQ ID NO: 213).

2. A ific binding le of claim 1, wherein said immunoglobulin single variable domains are VHHs. 20

3. The bispecific binding le of claim 2, selected from the group consisting of bispecific binding molecules (i) to (iii), the molecules (i) comprising - a VEGF binding domain sing the CDRs 1 to 3 of the VEGFBII00038 VHH domain (SEQ ID NO:57), 25 - the Alb11 VHH domain (SEQ ID NO: 254) as the serum albumin binding component, and - the ANGBII00938 VHH domain (SEQ ID NO: 222) as the Ang2-binding domain, in this order; 30 the molecules (ii) comprising - a VEGF binding domain comprising the CDRs 1 to 3 of the VEGFBII00038 VHH domain (SEQ ID NO:57), - the Alb11 VHH domain (SEQ ID NO: 254) as the serum albumin binding component, and - two ANGBII00921 VHH domains (SEQ ID NO: 220) as the Ang2-binding domains, 5 in this order; the molecules (iii) comprising - a VEGF binding domain comprising the CDRs 1 to 3 of the VEGFBII00038 VHH domain (SEQ ID NO:57), 10 - the Alb11 VHH domain (SEQ ID NO: 254) as the serum albumin g component, and - two ANGBII00956 VHH domains (SEQ ID NO: 223) as the Ang2-binding domains, in this order.

4. The bispecific binding molecule of claim 3, selected from the group ting VEGFANGBII00022 (SEQ ID NO: 207), VEGFANGBII00025 (SEQ ID NO: 210), and 20 VEGFANGBII00028 (SEQ ID NO: 213).

5. A ific g molecule as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples.

6. A nucleic acid molecule encoding a bispecific binding molecule of any one of claims 1 to 5. 25

7. A vector containing a c acid of claim 6.

8. A host cell containing a nucleic acid molecule of claim 6 or a vector of claim 7, wherein said cell is not within a human being.

9. A pharmaceutical composition containing at least one bispecific binding le of any one of claims 1 to 5 as the active ingredient. 30

10. The pharmaceutical composition of claim 9 for the treatment of a disease that is associated with VEGF- and/or Ang2-mediated effects on angiogenesis.

11. The pharmaceutical composition of claim 9 or claim 10 for the treatment of cancer and cancerous diseases.

12. The pharmaceutical composition of claim 9 or claim 10 for the treatment of eye 5 Boehringer Ingelheim International GmbH By the Attorneys for the Applicant SPRUSON & FERGUSON Per: 1 / 125