NZ614243B2

NZ614243B2 - BISPECIFIC BINDING MOLECULES BINDING TO Dll4 AND Ang2

Info

Publication number: NZ614243B2
Application number: NZ614243A
Authority: NZ
Inventors: Joachim Boucneau; Marieange Buyse; Erik Depla; Andreas Gschwind; Rene Georg Ott
Original assignee: Boehringer Ingelheim International Gmbh
Priority date: 2011-04-01
Filing date: 2012-03-30
Publication date: 2015-07-28

Abstract

Discloses a bispecific binding molecule comprising at least one Ang2-binding component, at least one Dll4-binding component, and - at least one serum albumin binding component, wherein said Ang2-, Dll4- and serum albumin 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 DLLANGBII00017 (SEQ ID NO: 476), (ii) the CDR sequences as present in DLLANGBII00018 (SEQ ID NO: 477), and (iii) the CDR sequences as present in DLLANGBII00019 (SEQ ID NO: 478). lin 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 DLLANGBII00017 (SEQ ID NO: 476), (ii) the CDR sequences as present in DLLANGBII00018 (SEQ ID NO: 477), and (iii) the CDR sequences as present in DLLANGBII00019 (SEQ ID NO: 478).

Description

Bispecific binding molecules binding to D||4 and Ang2 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 approximately 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 (Ferrara et al., Nat Rev Drug Discov. 2004 May;3(5):391—400.) by neutralizing VEGF (Avastin) or its ors (Sutent and nib). Recent studies in mice have shown, that Angiopoietin2 (Ang2), a ligand of the Tie2 receptor, controls vascular remodeling by enabling the ons of other angiogenic factors, such as VEGF. Ang2 is primarily expressed 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 tin 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 d angiogenesis (Falcén 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 26;103(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.). In CRC cancer Ang2 serum levels correlate with eutic 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 Mar;10(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 Ang1 mediate ng functions upon Tie2 binding and activation. Ang2- mediated Tie2 activation results in endothelial cell tion, te iation, vessel leakage and induction of vessel sprouting. In contrast to Ang2, Ang1 signaling maintains vessel integrity 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 coiled-coil domain and a C-terminal fibrinogen-like domain, the latter is required for Tie2 interaction. Ang2 is primarily sed by endothelial cells and strongly induced by hypoxia and other angiogenic factors, including VEGF.

Tie2 is found on endothelial cells, haematopoietic stem cells and tumor cells. Ang2- Tie2 has been demonstrated 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 elial cells (HUVEC). Ang2 induces tyrosine phosphorylation of cally 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 nduced endothelial cell responses has been described.

Ang2 —deficiency has been shown to result in a profound lymphatic ning defect in mice. Although the loss of Ang2 is dispensable for nic vascular development, AngZ —deficient mice have persistent ar defects 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 enic factors, such as VEGF.

The Ang2-Tie2 system exerts crucial roles during the angiogenic switch and later stages of tumor angiogenesis. Ang2 expression 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 growth.

Therapeutic ng of Ang2 with Ang2 mAbs has shown broad efficacy in a variety of tumor xenograft models.

As summarized in US 2008/0014196, angiogenesis is implicated in the pathogenesis of a number of ers, including solid tumors and metastasis.

In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to sia, and for providing nourishment for the growth and metastasis of the tumor. Folkman et al., Nature 339 -58 (1989), 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 5):391-400.

The Notch signaling pathway is important for cell-cell communication, which involves gene regulation mechanisms that control multiple cell entiation processes during embryonic development and in adult organisms. Notch signaling is ulated in many cancers, e.g. in T-cell acute lymphoblastic leukemia and in solid tumors a et al. 2007, Cell Cycle 6 (8): 927—30; Shih etal., Cancer Res. 2007 Mar 1;67(5): 1879-82).

Dll4 (or Delta like 4 or delta-like ligand 4) is a member of the Delta family of Notch ligands. The extracellular domain of Dll4 is composed of an N-terminal domain, a Delta/Serrate/Lag—2 (DSL) domain, and a tandem of eight epidermal growth factor (EGF)-like repeats. Generally, the EGF domains are ized as comprising amino acid residues 1 (EGF-1; domain 1), 252-282 (EGF-2; domain 2), 284-322 (EGF-3; domain 3), 324-360 ; domain 4), and 362-400 (EGF-5; domain 5), with the DSL domain at about amino acid residues 173—217 and the inal domain at about amino acid residues 27-172 of thl4 ().

It has been reported that Dll4 exhibits highly selective expression by vascular endothelium, in particular in arterial endothelium (Shutter et al. (2000) Genes Develop. 14: 1313-1318). Recent studies in mice have shown that Dll4 is induced by VEGF and is a negative feedback regulator that restrains vascular sprouting and branching. Consistent with this role, the deletion or inhibition of Dll4 results in excessive angiogenesis (Scehnet eta/., Blood. 2007 Jun 1;109(11):4753-60). This unrestrained angiogenesis paradoxically decreases tumor growth due to the ion of non—productive vasculature, even in tumors resistant to EGF therapies (Thurston et al., Nat Rev Cancer. 2007 May;7(5):327-31; ; Noguera-Troise et al., Nature. 2006 Dec 21; 444(7122)). rmore, the combined inhibition of VEGF and D||4 is shown to provide superior anti-tumor ty compared to anti-VEGF alone in xenograft models of multiple tumor types (Noguera-Troise et al., Nature. 2006 Dec 21; 444(7122):1032-7; Ridgway et al., Nature. 2006 Dec 21 ;444(7122):1083-7).

Due to these results, D||4 is being considered a ing target for cancer therapy, and several ical compounds that target D||4 are in (pre-)c|inica| development have been described: REGN—421 (= SAR153192; Regeneron, Sanofi-Aventis; WO2008076379) and OPM-21M18 (OncoMed) (Hoey et a/., Cell Stem Cell. 2009 Aug 7; 5(2):168—77), both fully human D||4 antibodies; YW152F tech), a humanized D||4 antibody (Ridgway et al., Nature. 2006 Dec 21; 444(7122):1083—7); DII4-Fc (Regeneron, Sanofi-Aventis), a recombinant fusion n composed of the extracellular region of D||4 and the Fc region of human lgG1 (Noguera-Troise et al., Nature. 2006 Dec 21 ;444(7122)).

However, the state-of—the art monoclonal antibodies (MAbs) and fusion proteins have several shortcomings in view of their therapeutic application: To prevent their degradation, they must be stored at near freezing temperatures. Also, since they are quickly digested in the gut, they are not suited for oral administration. Another major restriction of MAbs for cancer therapy is poor transport, which results in low concentrations and a lack of targeting of all cells in a tumor.

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

It has been a r object of the invention to provide methods for the prevention, treatment, alleviation and/or diagnosis of such diseases, ers or conditions, involving the use and/or administration of such binding molecules and compositions comprising them. In particular, it is has been an object of the ion to provide such pharmacologically active binding les, compositions and/or methods that provide advantages compared to the , compositions and/or methods currently used and/or known in the art. These advantages include improved therapeutic and/or pharmacological ties and/or other advantageous properties, e.g. for manufacturing purposes, especially as ed 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 immunoglobulin single variable domains like VHHs and domain antibodies, which comprises at least one DLL4-binding component and at least one Ang2-binding component in a single molecule. These bispecific binding molecules may comprise a further binding component, preferably a binding omponent binding to serum albumin.

More specifically, herein disclosed is a bispecific binding molecule which essentially comprises (i) at least one Dll4-binding component ically binding to at least one epitope of Dll4 and (ii) at least one Ang2-binding component specifically binding to at least an epitope of Ang2, n the components are linked to each other in such a way that they simultaneously bind to Dll4 and Ang2 or that they bind to either Dll4 or Ang2 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 dies, and/or any other sort of immunoglobulin single variable domains, such as VL s, as defined herein, provided that each of these immunoglobulin single le s will bind the antigen, i.e. Dll4 or Ang2, respectively.

According to an embodiment of the invention, there is provided a bispecific binding molecule comprising - at least one Ang2-binding component - at least one Dll4-binding component, and - at least one serum albumin binding component, wherein said Ang2-, Dll4- and serum albumin binding components are immunoglobulin single variable domains, each immunoglobulin single variable domain consisting of four framework s and three mentarity 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 t in DLLANGBII00017 (SEQ ID NO: 476), (ii) the CDR sequences as present in DLLANGBII00018 (SEQ ID NO: 477), and (iii) the CDR sequences as present in DLLANGBII00019 (SEQ ID NO: 478).

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 antibodies.

According to a particularly preferred embodiment, all globulin single variable domains are VHHs, preferably humanized (or “sequence-optimized”, as defined herein) VHHs. Accordingly, the ion relates to bispecific binding les comprising an (optionally humanized or ce-optimized) anti-Dll4 VHH and an (optionally humanized or sequence-optimized) anti-Ang2 VHH.

However, it will be clear to the skilled person that the ng herein may be applied analogously to bispecific binding molecules ing other anti-Dll4 or anti-Ang2 immunoglobulin single variable domains, such as domain antibodies. - 5a - In another aspect, the invention relates to nucleic acids encoding the bispecific binding molecules of the ion as well as host cells containing same.

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

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

The invention further relates to applications and uses of the ific binding molecules, nucleic acids, host cells, products and compositions described herein, as well as to s for the prevention and/or treatment for es and disorders that can be modulated by inhibition of Dll4.

It has been found that the Ang2-binding component of the bispecific binding molecules according to the present invention binds to 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 Angl—mediated signalling, which would counter the intended ngiogenetic effect.

It has further been found that the DLL4-binding ent of the bi—specific binding molecules according to the present invention binds to DLL4-A with an affinity of at least 1,000 times higher than to DII1, Jagged1 and preferably also against Jagged2.

Due to this selectivity unwanted side reactions can be avoided.

In a preferred embodiment the bispecific binding les of the present invention are provided as linked VHH domains. Such molecules are significantly smaller than conventional antibodies and have thus the potential for penetrating into a tumor deeper than such conventional antibodies. This t is further accentuated by the specific ces disclosed herein after being free of glycosylation sites.

Further, due to the bispecific nature (Dll4— and Ang2-binding components in one le) the tumor penetration of both functionalities will be necessarily equal, which will ensure that the beneficial s of the combined antagonism of Dll4 and Ang2 will be provided within the whole depth of penetration of the tumor. This is an advantage over the combination of individual antagonists against these targets, since WO 31076 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 half-like due to a serum albumin g component such as a serum albumin binding molecule as described herein.

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

DEFINITIONS Unless indicated or defined othen/vise, 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, (1990), and Roitt et al,, "Immunology" (2nOl Ed.), Gower l Publishing, London, New York (1989), as well as to the general background art cited herein; Furthermore, unless indicated otherwise, all methods, steps, techniques and manipulations that are not ically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled . Reference is for example again made to the standard handbooks, to the general background art referred to above and to the further references cited therein.

The term “bispecific binding molecule” refers to a le comprising at least one inding molecule (or “Ang2-binding component”) and at least one Dll4-binding molecule (or “Dll4—binding component”). A bispecific binding le may contain more than one Ang2-binding molecule and/or more than one Dll4-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 Dll4-binding molecule, in the part of the molecule that binds to Ang2 or to Dll4, i.e. in its “Ang2-binding component” (or ng2 component) or “Dll4—binding component” (or anti-Dll4 component), respectively. The word cific” in this context is however not to be construed as to exclude further binding components with binding specificity to molecules other than D||4 and Ang2 from the bispecific g molecule. miting examples of such further binding components are binding components binding to serum albumin.

Unless ted otherwise, the terms "immunoglobulin" and "immunoglobulin sequence" - whether used herein to refer to a heavy chain antibody or to a conventional 4-chain dy — are used as l terms to include both the full- size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or 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 "protein sequence"), should generally be understood to e 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 retation.

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 independently of the rest of the protein. Generally, domains are responsible for te functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.

The term "immunoglobulin domain" as used herein refers to a globular region of an dy chain (such as e.g. a chain of a tional 4—chain antibody or of a heavy chain antibody), or to a polypeptide that essentially ts 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 arranged 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 domains.

The term "immunoglobulin variable domain" as used herein means an immunoglobulin domain essentially consisting of four "framework s" which are ed to in the art and hereinbelow as "framework region 1" or "FR1"; as "framework region 2" or"FR2”; as "framework region 3” or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are upted by three "complementarity determining regions" or "CDRs", which are referred to in the art and hereinbelow as "complementarity 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 le domains like VHHs and domain antibodies are preferred.

The term "immunoglobulin single le " as used herein means an immunoglobulin variable domain which is capable of ically binding to an epitope of the antigen without pairing with an additional variable immunoglobulin domain.

One example of immunoglobulin single variable domains in the g of the present invention are "domain antibodies", such as the immunoglobulin single variable s VH and VL (VH s and VL domains). r example of immunoglobulin single variable domains are "VHH domains" (or simply "VHHs") from camelids, as defined hereinafter.

In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (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 disulphide linked Fv or a scFv nt, or a diabody (all known in the art) derived from such tional 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 e of the tive antigen.

"VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been bed 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 S, 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 s that are present in conventional 4-chain antibodies (which are referred 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 referred to herein as "VL domains" or "VL domains"). VHH domains can specifically bind to an epitope 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 , VHH antibody fragment, VHH antibody, as well as "Nanobody®" and "Nanobody® domain ("Nanobody" being a trademark of the y Ablynx N.V.; Ghent; m) are used interchangeably and are representatives of immunoglobulin single variable domains (having the structure R1-FR2—CDR2—FR3—CDR3-FR4 and ically 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. W02009/109635, 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. ence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from ds, as shown e.g. in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, -38 (1999). 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 positions 36-49, - CDR2 comprises the amino acid residues at positions 50-65, - FR3 ses the amino acid residues at positions 66-94, - CDR3 comprises the amino acid residues at positions , 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 s 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 es indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual SEQUENCE.

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 sequences 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 therapy as functional n—binding molecules. In ular, and without being limited o, VHH domains (which have been "designed" by nature to functionally bind to an antigen without pairing with a light chain variable domain) can function as single, relatively small, functional n— binding ural units.

Due to their unique properties, immunoglobulin single variable domains, as defined , like VHHs or VHs (or VLs) - either alone or as part of a larger ptide, e.g. a biparatopic molecule - offer a number of icant advantages: 0 only a single domain is required to bind an antigen with high affinity and with high selectivity, so that there is no need to have two separate domains present, nor to assure that these two domains are present in the right spacial conformation and configuration (i.e. through the use of especially designed s, as with scFv's); -1]- immunoglobulin 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 multivalent 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 prepared, 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 ed using microbial fermentation (e.g. as further described below) and do not require the use of mammalian expression systems, as with for example conventional antibodies; globulin single variable domains are relatively small (approximately kDa, or 10 times r than a conventional lgG) compared to conventional 4-chain antibodies and antigen-binding fragments f, and therefore show r) penetration into tissues (including but not limited to solid tumors and other dense tissues) and can be administered in higher doses than such conventional 4-chain dies and n—binding fragments f; VHHs have specific so—called “cavity—binding properties” (inter alia due to their extended CDR3 loop, compared to VH domains from n antibodies) and can therefore also access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding fragments f; VHHs have the particular advantage that they are highly soluble and very stable and do not have a tendency to aggregate (as with the mouse-derived antigen-binding domains described by Ward et al., Nature 341: 544-546 (1989)).

The globulin single variable domains of the invention are not limited with respect to a ic ical source from which they have been obtained or to a specific method of preparation. For example, obtaining VHHs may e 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) expressing a nucleic 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 humanized VHH; (4) "camelizing" (as described below) a globulin single variable heavy domain from a naturally ing dy 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) "camelizing" a VH, or expressing a nucleic acid molecule encoding such a zed VH; (6) using techniques for ing synthetically or semi—synthetically proteins, polypeptides or other amino acid sequences; (7) preparing a nucleic 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 selections 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 /O40153 and W02006/122786.

WO 31076 According to specific embodiments, the immunoglobulin single variable domains of the invention or present in the polypeptides of the invention are VHH s with an amino acid sequence that ially corresponds to the amino acid sequence of a naturally occurring VHH , but that has been "humanized" or “sequence- optimized” (optionally after affinity-maturation), i.e. by ing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a le heavy domain of a conventional 4-chain antibody 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 ic 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 thereto, 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 \ﬁ genes such as DP 47, DP 29 and DP 51) either alone or in combination. le framework regions (FR) of the immunoglobulin single variable domains of the invention can be selected from those as set out e.g. in and specifically, include the so-called "KERE" and "GLEW" classes. Examples are immunoglobulin single variable domains having the amino acid sequence G-L-E-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 zing tution for VHHs belonging to the 103 P,R,S- group and/or the GLEW—group (as defined below) is 1080 to 108L. Methods for humanizing immunoglobulin single variable domains are known in the art.

Binding immunoglobulin single variable domains with improved properties in view of therapeutic ation, e.g. enhanced affinity or sed immunogenicity, may be obtained from individual binding molecules by techniques known in the art, such as 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 ty may be obtained by affinity- tion of r g molecule, the latter representing, with respect to the ty-matured molecule, the “parent” binding molecule.

Methods of ing 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 ds can be "humanized" (also termed “sequence-optimized” herein, “sequence-optimizing” may, in addition to humanization, ass an additional modification of the ce by one or more mutations that furnish the VHH with improved properties, such as the removal of potential post translational modification sites) by replacing one or more amino acid es in the amino acid sequence of the original 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 4-chain 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 f, 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 arks by the GlaxoSmithKline group of companies) have been described in e.g. Ward, E.S., et a/.: "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli"; Nature 341: 6 (1989); Holt, L.J. eta/.: "Domain antibodies: proteins for therapy"; TRENDS in Biotechnology 21(11): 484-490 (2003); and W02003/002609.

Domain antibodies essentially correspond to the VH or VL domains of antibodies from non-camelid mammals, in particular human 4-chain antibodies. In order to bind an epitope as a single antigen binding domain, i.e. without being paired with a VL or VH domain, respectively, specific selection for such antigen binding properties is required, e.g. by using ies of human single VH or VL domain sequences.

Domain antibodies have, like VHHs, a lar weight of approximately 13 to approximately 16 kDa and, if d from fully human sequences, do not require 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 d person that it is possible to "graft" one or more of the CDR's mentioned above onto other "scaffolds", ing 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 conventional antibodies or the polypeptides of the invention, and more particularly by the antigen-binding site of said molecules.

Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an globulin.

A polypeptide (such as an globulin, 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 y fof' and/or that "has city fof' a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be st" or "directed against" said epitope, antigen or protein or is a "binding" molecule with respect to such epitope, n or protein. In this context, a Dll4-binding component may also be referred to as “Dll4-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 n (such as an globulin single variable domain of the invention) molecule can bind. The specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a e for the binding strength between an epitope and an antigen-binding site on the antigen— binding protein: the lesser the value of the KB, the stronger the binding strength between an epitope and the antigen-binding molecule (alternatively, the affinity can also be sed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person (for example on the basis of the r sure 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 polypeptides containing 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 otherwise, the term “Dll4—binding molecule” or “Ang2—binding molecule” includes anti-Dll4 or anti-Ang2 antibodies, anti-Dll4 antibody or anti-Ang2 antibody fragments, “anti-Dll4 antibody-like molecules” or Ang2 antibody-like molecules”, as defined herein, and conjugates with any of these. Antibodies e, but are not limited to, onal and chimerized monoclonal antibodies. The term ,,antibody“ encompasses complete immunoglobulins, like onal antibodies produced by recombinant expression in host cells, as well as antibody fragments or “antibody-like molecules”, including single—chain antibodies and linear antibodies, so- called “SMlPs” (“Small r |mmunopharmaceuticals”), as e.g described in WC 02/056910; Antibody-like molecules include immunoglobulin single variable domains, as defined herein. Other examples for antibody-like molecules are immunoglobulin super family antibodies (lgSF), or CDR—grafted molecules.

“Ang2—binding molecule” or “DIM-binding molecule” respective/y, refers to both lent target—binding molecules (i.e. molecules that bind to one epitope of the respective target) as well as to bi- or alent binding molecules (i.e. binding molecules that bind to more than one epitope, e.g. atopic” molecules as defined hereinbelow). Ang2(or binding molecules containing more than one Ang2(or DIl4)—binding immunoglobulin single variable domain are also termed “formatted” g molecules, they may, within the target-binding component, in addition to the immunoglobulin single variable domains, comprise linkers and/or moieties with or functions, e.g. half-life—extending es like albumin-binding immunoglobulin single variable s, and/or a fusion partner like serum albumin and/or an attached polymer like PEG.

The term "biparatopic Ang2(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 erlapping epitopes of the respective n. The biparatopic binding molecules are composed of immunoglobulin single variable domains which have different specificities with respect to the epitope. The part of an n-binding molecule (such as an antibody or an immunoglobulin single variable domain of the ion) that recognizes the epitope is called a paratope.

A ted 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 antigen. 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 ion will bind with a dissociation constant (KB) of 10E-5 to 10E-14 moles/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 10E-13, as measured e.g. in a Biacore or in a Kinexa assay), and/or with an association constant (KA) of at least 10E7 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 ecific binding. Preferably, a polypeptide of the invention will bind to the desired n, i.e.

VEGF or Dll4, 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 competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition , 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 comparing two amino acid sequences, the term "amino acid difference" refers to insertions, deletions or substitutions 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 substitution(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 influence on the function, activity or other ical properties of the polypeptide.

Such conservative amino acid substitutions are well known in the art, for example from WO 98/49185, wherein conservative amino acid substitutions preferably are tutions in which one amino acid within the following groups (i) — (v) is substituted 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 tic, nonpolar residues: Met, Leu, lle, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. ularly 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 le is ered 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 ed - 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, ty or minor component. In particular, a polypeptide or nucleic acid molecule is considered "essentially isolated" when it has been ed at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to lOOO-fold or more. A polypeptide or nucleic acid molecule that is "in essentially isolated form" is preferably ially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as rylamide gel electrophoresis.

"Sequence identity' between two Dll4-binding molecule sequences or between two Ang2-binding molecule sequences indicates the percentage of amino acids that are identical between the sequences. It may be calculated or ined as described in paragraph f) on pages 49 and 50 of . "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent vative amino acid tutions.

Alternative methods for numbering the amino acid es 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 domains as described above will be followed, unless indicated otherwise.

An "affinity-matured” inding molecule or Ang2-binding molecule, in particular a VHH or a domain antibody, has one or more tions in one or more CDRs which result in an improved affinity for Dll4 or Ang2, as compared to the respective parent Dll4-binding molecule or Ang2—binding molecule. Afffinity-matured DIl4-binding les or Ang2-binding molecules of the invention may be prepared by methods known in the art, for example, as bed by Marks et a/., 1992, Biotechnology 10:779-783, or Barbas, eta/., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier eta/., 1995, Gene 169:147-155; Yelton eta/., 1995, Immunol. 155: 1994-2004; Jackson eta/., 1995, J. Immunol. 154(7):3310-9; and Hawkins eta/., 1992, J. Mol.

Biol. 226(3): 889 896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

For the present invention, an “amino acid sequences of SEQ ID NO: x”: includes, if not otherwise stated, an amino acid ce that is 100% identical with the sequence 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 ce shown in the respective SEQ ID NO: x.

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

Examples of cancer to be treated with a bispecific binding molecule of the invention, include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers, as suggested for ent with Dll4 antagonists in US 2008/0014196, include squamous cell cancer, small-cell lung cancer, all cell lung cancer, adenocarcinoma of the lung, squamous oma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, trial or uterine oma, salivary gland carcinoma, kidney cancer, liver , prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and various types of head and neck cancer.

Dysregulation of angiogenesis can lead to many disorders that can be treated by compositions and methods of the invention. These disorders include both nonneoplastic and neoplastic conditions. Neoplasties include but are not limited those described above.

Non-neoplastic disorders include, but are not limited to, as suggested for treatment with Dll4 antagonists in US 2008/0014196, red or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), sis, psoriatic plaques, dosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age—related macular degeneration, diabetic r edema, l neovascularization, corneal graft neovascularization, corneal graft ion, retinal/choroidal neovascularization, neovascularization of the angle sis), ocular neovascular disease, vascular restenosis, arteriovenous mations (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias ding Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, acute lung injury/ ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusions, cerebral edema (e.g., associated with acute stroke/ closed head injury/ trauma), synovial inflammation, pannus formation in RA, myositis ossificans, hypertropic bone ion, osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, 3ml spacing of fluid es (pancreatitis, compartment syndrome, burns, bowel e), uterine fibroids, premature labor, -2]- 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, derma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial on (such as that associated with pericarditis), and l effusion.

DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention relates to a bispecific binding molecule comprising at least one Dll4-binding component and at least one inding component.

In a preferred embodiment, the present ion relates to a bispecific binding molecule comprising at least one Dll4-binding ent and at least one Ang2- binding component which further comprises at least a further g component, ably a serum albumin binding ent (serum albumin binding molecule).

In a preferred embodiment, the serum albumin binding component of the binding molecule of the present invention is an isolated immunoglobulin single variable domain or a polypeptide ning one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework 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 N03: 522, 525, 528, 531, 534, 537, or 540.

More preferably, said one or more immunoglobulin single variable domain of the serum albumin binding component contain a. a CDR3 with an amino acid ce selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs N08: 522, 525, 528, 531, 534, 537, or 540; b. a CDR1 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID N03: 520, 523, 526; 529, 532, 535, or 538; c. a CDR2 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID N03: 521, 524, 527, 530, 533, 536, or 539.

In a more preferred embodiment, said one or more immunoglobulin single variable domains of the serum albumin binding component are VHHs, preferably having an amino acid sequence shown in SEQ ID NOs: 98 or 519.

According to preferred embodiments, said Dll4-binding component and said inding component comprise at least one Dll4-binding immunoglobulin single variable domain and at least one inding immunoglobulin single variable domain, respectively.

In a preferred aspect, said Dll4—binding component and said Ang2—binding component each comprise at least one Ang2—binding globulin single variable domain and at least one inding 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 anti-Dll4 and/or the anti-Ang2 component contained in the bispecific binding molecules of the invention may include two (or more) anti-Dll4 (or anti-Ang2, respectively) immunoglobulin single variable domains, wherein the immunoglobulin single variable domains are ed t different es within the Dll4 (or Ang2) target. Thus, the two immunoglobulin single variable domains in a bispecific g molecule will have different antigen specificity and therefore different CDR sequences.

Such bivalent binding molecules are also named "biparatopic single domain dy constructs" (if the immunoglobulin single variable domains t or essentially consist of single domain antibodies), or "biparatopic VHH ucts" (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 molecule of the invention, one or both of the binding les may be bivalent; e.g. the Ang2—binding component may be biparatopic and the DII4-binding component may be one immunoglobulin single variable domain, or WO 31076 the Ang2—binding component may be one immunoglobulin single variable domain and the Dll4-binding component may be biparatopic.

In bispecific binding molecules of the invention, it is preferably the Ang2—binding component that contains a bivalent Ang2—binding immunoglobulin single variable domain, e.g. a biparatopic VHH.

The Dll4-binding component comprises at least a variable domain with four framework regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, n said CDR3 has an amino acid sequence selected from amino acid sequences shown in a) SEQ ID NOs: 1 to 166 and 458, b) SEQ ID NOs: 333 to 353, or C) SEQ ID N08: 375 to 395.

An amino acid sequence a), selected from a first group of SEQ ID NOs: 1 to166 and 458, is ned as partial sequence in a corresponding amino acid ce selected from a second group of sequences shown in Table 5 and in SEQ ID NO: 167 to 332 and 459.

An amino acid sequence b), selected from a first group of SEQ ID N05: 333 to 353, is contained as partial sequence in a corresponding sequence selected from a second group of sequences shown in Table 16-A and in SEQ ID NOs: 354 to 374.

An amino acid sequence 0) selected from a first group of SEQ ID NOs: 375 to 395 is contained as partial sequence in a corresponding sequence selected from a second group of sequences shown in Table 16-B and in SEQ ID NOs: 396 to 416.

In a second , said Dll4—binding component is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single le domain ts of four framework s 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 a) SEQ ID NOs: 1 to 166 and 458, b) SEQ ID NOs: 333 to 353, or c) SEQ ID NOs: 375 to 395.

In a further aspect, said immunoglobulin single variable domain of the Dll4-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: 1 to 166 and 458; b) a CDR1 and a CDR2 with an amino acid sequence that is contained, as ted in Table 5, as partial sequence in a sequence selected from a second group of amino acid sequences shown in SEQ ID NOs: 167 to 332 and 459; n a SEQ ID NO: x of said first group, for SEQ ID Nos 1- 166: corresponds to SEQ ID NO: y of said second group in that y = x +166.

In a further aspect said immunoglobulin single variable domain contains a) a CDR3 with an amino acid sequence selected a said first group of amino acid sequences shown in SEQ ID NOs: 333 to 353; b) a CDR1 and a CDR2 with an amino acid sequence that is contained, as indicated in Table 16—A, as a partial sequence in a sequence selected from a second group of ces shown in SEQ ID NOs: 354 to 374; wherein a SEQ ID NO: x of said first group corresponds to SEQ ID NO: y of said second group in that y = x +21.

In a further aspect said immunoglobulin single le domain has a) a CDR3 with an amino acid sequence selected a said first group of amino acid sequences shown in SEQ ID N03: 375 to 395; b) a CDR1 and a CDR2 with an amino acid ce that is contained, as indicated in Table 16—8, as a partial sequence in a sequence selected from a second group of sequences shown in SEQ ID NOs: 396 to 416; wherein a SEQ ID NO: x of said first group corresponds with SEQ ID NO: y of said second group in that y = x +21.

In a red embodiment, the immunoglobulin single variable domain is a VHH.

In a further aspect, the VHH has an amino acid ce selected from amino acid sequences shown in Table 5 and in SEQ ID NOs: 167 to 332 and 459.

The Ang2-binding component ses at least a variable domain with four framework regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, n said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID N05: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518.

In a second , said Ang2—binding component is an isolated globulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid ces shown in SEQ ID NOs: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518 In a further aspect, said immunoglobulin single variable domain of the Ang2-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 N08: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518 (see also Table 36); b. a CDR1 with an amino acid sequences that is contained, as indicated in Table 22-A or 28, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 489, 492, 495, 498, 501, 504, 507, 510, 513, or 516 (see also Table 36); c. a CDR2 with an amino acid sequences that is contained, as ted in Table 22-A or 28, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID N05: 490, 493, 496, 499, 502, 505, 508, 511, 514, or 517 (see also Table 36).

Preferably, the immunoglobulin single variable domain of the Ang2-binding component is a VHH, preferably having amino acid sequence selected from amino acid ces shown in SEQ ID N03: 479, 480, 481, 482, 483, 484, 485, 486, 487, or 488.

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 AngZ—binding component as described herein.

The present invention thus also relates to an Ang2—binding VHH with an amino acid sequence selected from acid sequences shown in SEQ ID N03: 479, 480, 481, 482, 483, 484, 485, 486, 487, or 488.

Dll4—and/or Ang2—binding components with improved properties in view of therapeutic application, e.g. enhanced ty or decreased immunogenicity, may be obtained from individual Dll4- or Ang2—binding components of the invention by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, humanizing, ing fragments derived from different globulin sequences, PCR assembly using overlapping primers, and r techniques for ering globulin sequences well known to the skilled ; or any suitable combination of any of the foregoing. Reference is, for example, made to standard handbooks, as well as to the further description and Examples.

Preferably, a Dll4—binding component of the invention with sed affinity is obtained by affinity-maturation of another Dll4-binding component, the latter representing, with t to the affinity-matured molecule, the “parent” Dll4-binding component. The same holds true for the inding component.

Thus, in yet another preferred embodiment, a Dll4-or Ang2—binding molecule of the invention is an immunoglobulin single variable domain that has been obtained by affinity maturation of a parent immunoglobulin single variable domain defined above.

In yet another preferred embodiment, the invention relates to an globulin single variable domain obtained by affinity—maturation of a VHH.

Suitable parent Dll4-binding ents for affinity maturation are, by way of example, the above-described VHHs with amino acid sequences shown in SEQ ID NOs: 167 to 332 and 459.

Suitable parent Ang2—binding components for affinity maturation are, by way of example, the described VHHs with amino acid sequences shown in SEQ ID NOsz479, 480, 481, 482, 483, or 484. ingly, the ion also relates to Ang2—binding molecules that have been obtained by affinity maturation and/or sequence optimization of an defined VHH, e.g. to a VHH that has been obtained by sequence optimization of a VHH having an amino acid sequence shown as SEQ ID N03: 482, 483, 484, 485, 486, 487, 488. The “source” amino acid ces that were used to generate the latter VHHs are shown in SEQ ID NOs: 479, 480, or 481. Also these amino acid sequences are suitable Ang2—binding components that can be applied in the binding molecules of the present invention.

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 Dll4-binding component, at least one Ang2- binding 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 Figure 16 or 23, e.g., the Dll4-, Ang2—or serum albumin binding component can be N—terminal or C-terminal. y, “00042”, “00045” or “00050” as referred to in the legend of Figure 16 stand for Ang2—binding components, while “00018” stands for a Dll4—binding component and “ALB1 1” stands for a serum albumin binding component. None of them is to be ued to a specific sequence, but stands for a Ang2—, Dll4- and serum albumin binding component in general when used in the context of possible set-ups of binding molecules of the present invention.

However, it is preferred that the serum albumin binding component is in between the Dll4- and Ang2-binding component (or vice , while it is particularly preferred that at least one Ang2—binding component is inal, ed by at least one serum albumin binding component, ed by at least one Dll4-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 les comprising at least one Dll4-binding component, at least one Ang2-binding component and at least one serum albumin binding ent having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 460—478.

“At least one” binding component (Ang2, Dll4 or serum albumin) when used herein includes that a binding molecule of the present invention may contain one, two, three, four or five Ang2—, Dll4, and/or serum albumin binding components (i.e., entities/units) which are preferably represented by an immunoglobulin singly variable domain as described herein.

In yet another preferred embodiment, the invention relates to a Dll4 immunoglobulin single variable domain that has been obtained by affinity maturation of a VHH with an amino acid sequence shown in SEQ ID NO: 197.

In yet another embodiment, said immunoglobulin single variable domain that is derived from a VHH with the amino acid sequence shown in SEQ ID NO: 197 is selected from immunoglobulin single le s with amino acid ces shown in SEQ ID N08: 354 to 374.

In a preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO: 358.

In an even more preferred ment, the immunoglobulin single variable domain has been obtained by humanization of a VHH with an amino acid ce shown in SEQ ID NO: 358.

In another preferred embodiment, the immunoglobulin single le domain is a VHH with an amino acid sequence shown in SEQ ID NO: 356.

In an even more red embodiment, the invention relates to an immunoglobulin single variable domain that has been obtained by humanization of a VHH with an amino acid sequence shown in SEQ ID NO: 356.

In yet another preferred embodiment, the invention relates to an immunoglobulin single variable domain that has been obtained by affinity maturation of a VHH with an amino acid sequence shown in SEQ ID NO: 224.

In yet r embodiment, said immunoglobulin single variable domain derived from a VHH with the amino acid sequence shown in SEQ ID NO: 224 is selected from globulin single variable domains with amino acid sequences shown in SEQ ID N05: 396 to 416.

In another preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO: 402.

In an even more preferred embodiment, the globulin single variable domain has been obtained by humanization of the VHH with the amino acid sequence shown in SEQ ID NO: 402.

In another preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO: 416.

In an even more preferred embodiment, the immunoglobulin single variable domain has been obtained by humanization of the globulin single variable domain with the amino acid sequence shown in SEQ ID NO: 416 In r preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO:407.

In an even more preferred embodiment, the immunoglobulin single variable domain has been obtained by humanization of the immunoglobulin single variable domain with the amino acid sequence shown in SEQ ID NO: 413.

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

In yet another embodiment, the representatives of the class of Dll4—and/or Ang2— binding immunoglobulin single variable domains of the invention or present in the polypeptides of the invention have amino acid ces 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 naturally occurring variable heavy chain from a tional 4-chain 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 med in a manner known per se, which will be clear to the skilled person, and reference is additionally be made to . Such zation 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 ). A detailled description of such ization" and "camelization" techniques and preferred framework region sequences consistent ith can additionally be taken from e.g. pp. 46 and pp. 98 of and pp. 107 of .

The Dll4-or Ang2-binding components of the invention, e.g. immunoglobulin single variable domains and or polypeptides containing them, have icity for Dll4 or Ang2, respectively, in that they comprise one or more immunoglobulin single variable domains specifically binding to one or more epitopes within the Dll4 or Ang2 le, respectively.

Specific binding of an Dll4-and/or Ang2 binding component to its antigen Dll4 or Ang2, respectively, can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or competitive binding assays, such as mmunoassays (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 Dll4, a Dll4-binding component of the invention, e.g. an immunoglobulin single variable domain, is not limited with regard to the species.

Thus, the globulin single variable domains of the invention or polypeptides containing them ably bind to human Dll4, if ed for therapeutic purposes in humans. However, immunoglobulin single variable domains that bind to Dll4 from another mammalian species, or polypeptides containing them, are also within the scope of the invention. An immunoglobulin single variable domain of the invention binding to one species form of Dll4 may react with Dll4 from one or more other species. For e, immunoglobulin single variable domains of the invention binding to human Dll4 may exhibit cross reactivity with Dll4 from one or more other species of primates and/or with Dll4 from one or more species of animals that are used in animal models for diseases, for example monkey (in particular lgus or Rhesus), mouse, rat, rabbit, pig, dog or) and in particular in animal models for diseases and disorders associated with Dll4-mediated effects on angiogenesis (such as the species and animal models mentioned herein). Immunoglobulin single le domains of the invention that show such cross-reactivity are advantageous in a -3]- research and/or drug pment, since it allows the immunoglobulin single le domains of the invention to be tested in acknowledged disease models such as monkeys, in particular Cynomolgus or Rhesus, or mice and rats. The same is true for Ang2.

Also, the inding components of the invention are not limited to or defined by a ic domain or an antigenic determinant of Dll4 against which they are directed.

Preferably, in view of cross-reactivity with one or more Dll4 molecules from species other than human that is/are intended for use as an animal model during development of a therapeutic Dll4 nist, a Dll4-binding component recognizes an epitope in a region of the Dll4 of interest that has a high degree of ty with human Dll4. By way of example, in view of using a mouse model, an immunoglobulin single variable domain of the invention recognizes an epitope which is, totally or in part, located within the EGF-2 domain, which shows a high identity n human and mouse. The same is true for Ang2.

Therefore, according to a preferred embodiment, the invention relates to a Dll4- g component, in particular an immunoglobulin single variable domain or a polypeptide containing same, wherein said immunoglobulin single variable domain is selected from the group that binds to an epitope that is totally or partially contained within the EGF-2 domain that corresponds to amino acid residues 252-282 of SEQ ID NO: 417.

If a polypeptide of the ion is a biparatopic molecule as defined herein, which contains more than one immunoglobulin single variable domain of the invention, at least one of the immunoglobulin single le domain components binds to the epitope within the EGF-2 domain, as defined above.

Preferably, an immunoglobulin single le domain of the invention binds to Dll4 and/or Ang2 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM (as determined by Surface Plasmon Resonance analysis, as described in Example 5.7).

Preferably, the immunoglobulin single variable domains of the invention have |C5o values, as measured in a competition ELISA assay as described in Example 5.1. in the range of 10'6 to 104° moles/litre or less, more preferably in the range of 10'8 to '1O 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 preferred ment of the invention, Dll4-binding immunoglobulin single variable domains of the invention or polypeptides containing them bind to Dll4 with an dissociation constant (KB) of 10'5 to 10'12 moles/liter (M) or less, and preferably 10'7 to 10'12 moles/liter (M) or less and more preferably 10'8 to '12 moles/liter (M), and/or with an association constant (KA) of at least 107 M1, preferably at least 108 M1, 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 Dll4 can be determined. The same is true for Ang2.

In a further embodiment, the invention relates to Dll4-binding components comprising two or more immunoglobulin single variable domains that bind to the antigen Dll4 or Ang2, respectively, at different non-overlapping es. More specifically, such polypeptide of the invention essentially consists of or comprises (i) a first immunoglobulin single variable domain specifically binding to a first epitope of Dll4 or Ang2, tively, and (ii) a second immunoglobulin single variable domain specifically binding to a second epitope of Dll4 or Ang2, respectively, wherein the first epitope of Dll4/Ang2 and the second epitope of Dll4/Ang2 are not identical es.

In other words, such ptide of the invention comprises or essentially consists of two or more immunoglobulin single variable s that are directed against at least two different epitopes present in ng2, wherein said immunoglobulin single variable domains are linked to each other in such a way that they are capable of simultaneously binding Dll4/Ang2. 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 contains at least two g sites for Dll4/Ang2.

Such Dll4-binding component of the invention includes (at least) two anti-Dll4 globulin single variable s, wherein (the) two immunoglobulin single variable domains are directed against ent epitopes within the Dll4 molecule.

Thus, these two immunoglobulin single variable domains will have a ent antigen specificity and therefore ent CDR sequences. For this reason, such polypeptides of the invention will herein also be named "biparatopic polypeptides", or atopic single domain antibody constructs" (if the immunoglobulin single variable domains t 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. The same is true for Ang2, mutatis mutandis.

According to a specific embodiment of the invention, in case that the polypeptide of the invention includes more than two anti—Dll4 immunoglobulin single variable domains, i.e. three, four or even more anti-Dll4 immunoglobulin single variable domains, at least two of the anti-Dll4 immunoglobulin single variable domains are directed against different epitopes within the Dll4 molecule, wherein any further immunoglobulin single variable domain may bind to any of these two different epitopes and/or a further epitope present in the Dll4 molecule. The same is true for Ang2, mutatis mutandis.

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

Dll4 or Ang2, respectively.

The detailed ption of the binding components is primarily provided for the Dll4- binding component. However, all features and options ed herein for the DM- binding component also apply lently for the Ang2—binding ent, mutatis mutandis.

According to preferred embodiments, the binding molecules present in the bispecific g molecules (the Ang2-binding molecules within the Ang2-binding ent or the Dll4-binding molecules within the Dll4-binding component or the two adjacent Ang2— and Dll4-binding components) may be connected with each other directly (i.e. without use of a linker) or via a . The linker is preferably a linker peptide and will be selected so as to allow binding of the two different binding molecules to each of non-overlapping epitopes of the targets, either within one and the same target molecule, or within two different molecules.

In the case of biparatopic binding molecules, ion of linkers within the Ang2— or the Dll4-binding ent will inter a/ia depend on the epitopes and, specifically, the ce 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 herein, optionally after some limited degree of routine experimentation.

Two binding 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 immunoglobulin single variable domain or via suitable linkers). Such an additional VHH or domain dy 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. atively, the two or more immunoglobulin single variable domains that bind to the respective target may be linked in series (either ly 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 ic polypeptides 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 sequence may be a naturally occurring sequence or a non-naturally occurring ce. If used for therapeutic purposes, the linker is preferably non- immunogenic in the subject to which the bispecific binding molecule of the invention is administered.

One useful group of linker sequences are linkers d from the hinge region of heavy chain dies as described in WC 1996/34103 and .

Other examples are poly-alanine linker sequences such as Ala- Ala— Ala.

Further red examples of linker sequences are Gly/Ser linkers of different length such as (glyxsery)Z linkers, including (gly4ser)3 , (gly4ser)4, (gly4ser), (gly3ser), gly3, and (glygser2)3.

Some non-limiting examples of linkers are shown in Figures 40 and 48, e.g. the linkers GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS; SEQ ID NO: 90); GGGGSGGGS (9GS; SEQ ID NO: 91); GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (4OGS; SEQ ID NO: 92).

If a bispecific binding molecule is modified by the ment of a polymer, for example of a polyethylene glycol PEG (polyethylene glycol) moiety, the linker sequence ably includes an amino acid e, such as a cysteine or a lysine, allowing such modification, e.g. PEGylation, in the linker region.

Examples of linkers useful for PEGylation are: GGS (“GSQ,C5”, SEQ ID N093); GGGGCGGGGSGGGGSGGGGSGGGGS (“GSZ5,CS, SEQ ID N0294) GGGSGGGGSGGGGCGGGGSGGGGSGGG ("GSZ7,C14", SEQ ID , GGGGSGGGGSGGGGCGGGGSGGGGSGGGGSGGGGS ("GSB5,CI5", SEQ ID NO:96), and GGGGCGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (“GS35,C5”, SEQ ID N029?) Furthermore, the linker may also be a thylene glycol) moiety, as shown in e.g.

. In another embodiment, the immunoglobulin single variable domains are linked to each other via another moiety (optionally via one or two linkers), such as another polypeptide which, in a preferred but non-limiting ment, 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 desired properties to the polypeptide. For example, and without tion, the moiety may improve the half-life of the protein or polypeptide, and/or may reduce its immunogenicity or improve any other desired property.

According to a red embodiment, a bispecific binding molecule of the invention includes, ally 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 "half-life" is defined as the time it takes for the serum tration 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 moiety, a fragment of an albumin moiety, an albumin binding moiety, such as an anti-albumin immunoglobulin single variable domain, a transferrin binding , 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) tive.

In another embodiment, the bispecific binding molecule of the ion comprises a moiety which binds to an n found in blood, such as serum albumin, serum immunoglobulins, thyroxine-binding protein, ogen or transferrin, thereby conferring an increased half-life in vivo to the resulting polypeptide of the ion.

According to a specifically preferred embodiment, such moiety is an albumin-binding globulin and, especially 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 domain. 2012/055897 Immunoglobulin single variable domains binding to human serum n are known in the art and are bed in further detail in e.g. . Specifically, useful albumin binding VHHs are ALB 1 and its humanized counterpart, ALB 8 (). Other albumin binding VHH domains 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 contains the amino acid sequence shown in SEQ ID NO: 98 or 519.

According to a further embodiment of the invention, the two immunoglobulin single variable domains, in preferably VHHs, may be fused to a serum albumin le, such as described e.g. in WOO1/79271 and 9934. As e.g. described in , the fusion protein may be obtained by conventional recombinant technology: a DNA molecule coding for serum albumin, or a fragment thereof, is joined to the DNA coding for the bispecific binding molecule, the obtained uct is inserted into a plasmid suitable for expression in the selected host cell, e.g. a yeast cell like Pichia pastoris or a bacterial cell, and the host cell is then transfected with the fused nucleotide sequence and grown under le conditions. The sequence of a useful HSA is shown in SEQ ID NO: 99.

According to another embodiment, a ife extending modification of a polypeptide of the invention (such modification also reducing immunogenicity of the polypeptide) comprises attachment of a suitable cologically acceptable polymer, such as ht or branched chain poly(ethylene glycol) (PEG) or derivatives thereof (such as methoxypoly(ethylene ) or mPEG). Generally, any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to 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 (2003); Harris and Chess, Nat. Rev.

Drug. Discov. 2 (2003); and .

Various reagents for PEGylation of polypeptides are also commercially available, for e 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-100MA, Sunbright® ME-200MA, and Sunbright® ME—400MA.

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

Preferably, for the ptides of the invention, 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 PEGylation, 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, preferably in such a way that said PEGylation either (1) increases the half-life in vivo; (2) reduces immunogenicity; (3) provides one or more r beneficial 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 affinity by more than 50 %, and more preferably not by more than 10%, as determined by a le assay described in the art); and/or (4) does not affect any of the other desired properties of the bispecific binding les of the invention. Suitable PEG- groups and s for attaching them, either specifically or non-specifically, will be clear to the skilled person. Various reagents for PEGylation of polypeptides are also commercially available, for e from Nektar Therapeutics, USA, or NOF Corporation, Japan, such as the ght® EA Series, SH Series, MA Series, CA Series, and ME Series, such as Sunbright® ME-100MA, Sunbright® ME-ZOOMA, 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, n the PEG moiety is attached to the polypeptide in a linker region and, specifially, at a Cys residue at position 5 of a G89- linker peptide as shown in SEQ ID NO:93, at position 14 of a GS27-Iinker peptide as shown in SEQ ID NO:95, or at position 15 of a GSBS—linker peptide as shown in SEQ ID N096, or at position 5 of a 35GS-linker peptide as shown in SEQ ID NO:97. 2012/055897 A bispecific binding molecule of the ion may be PEGylated with one of the PEG reagents as mentioned above, such as "Sunbright® MA", as shown in the following chemical formula: GHSQ—{GHECHEDjn—CHECHEGHENHCS-(GHQ;g-Ng Bispecific binding molecules that contain linkers and/or half-life extending functional groups are shown in SEQ ID NO: 81 and in Figure 48.

According to another embodiment, the globulin single variable s are domain antibodies, as defined herein. lmmunoglobulin 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 replacing one or more amino acid residues in the amino acid sequence of a naturally occurring variable heavy chain from a conventional 4-chain 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 med in a manner known per se, which will be clear to the skilled person, and reference is additionally be made to WO 94/04678.

Such camelization may preferentially occur at amino acid positions which are present at the VH-VL interface and at the so-called Camelidae rk residues (see for example also WO 94/04678). A detailled ption 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 and pp. 107 of .

The binding components have specificity for Ang2 or Dll4, 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 AngZ molecule or within the Dll4 molecule, tively.

Specific binding of a binding component to its antigen Ang2 or Dll4 can be ined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or competitive g assays, such as 2012/055897 radioimmunoassays (RIA), enzyme assays (EIA and ELISA) and sandwich ition assays, and the different variants thereof known per se in the art.

With regard to the antigen Ang2 or Dll4, respectively, an immunoglobulin single variable domain is not limited with regard to the species. Thus, the immunoglobulin single variable domains preferably bind to human Ang2 or to human Dll4, respectively, if intended for therapeutic purposes in humans. However, immunoglobulin single variable domains that bind to Ang2 or Dll4, respectively, from another mammalian species, or polypeptides containing them, are also within the scope of the invention. An globulin single variable domain binding to one species form of Ang2 or Dll4 may cross-react with the respective antigen from one or more other s. For example, immunoglobulin single variable domains binding to the human antigen may t cross reactivity with the respective antigen from one or more other species of primates and/or with the antigen from one or more species of animals that are used in animal models for diseases, for e 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). Immunoglobulin single variable s 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 s, in particular lgus or Rhesus, or mice and rats.

Also, the binding components are not d to or defined by a specific domain or an antigenic determinant of the antigen against which they are ed. 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 Ang2/Dll4 antagonist, a binding ent recognizes an epitope in a region of the the respective antigen 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 bispecific binding molecules of the invention recognizes an epitope which is, totally or in part, located within the EGF—2 domain of Ang2, which shows a high identity between human and mouse.

Therefore, according to a preferred embodiment, the bispecific binding molecule of the ion comprises a Dll4-binding molecule which is an immunoglobulin single variable domain that is selected from the group that binds to an epitope that is totally or partially ned within the EGF-2 domain that corresponds to amino acid residues 2 of SEQ ID NO:101.

In another aspect, the invention relates to nucleic acid molecules that encode ific binding molecules of the ion. 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 construct, as defined herein. A nucleic acid of the invention may be c DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been ically adapted for expression in the intended host cell or host organism).

According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated form, as defined hereabove.

The c 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 expression of the bispecific binding molecule in vitro and/or in vivo (i.e. in a le 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 promoter(s), enhancer(s), terminator(s), and the like. Such elements and their selection in view of expression of a specific sequence in a specific host are common dge of the skilled person. Specific examples of regulatory elements and other elements useful or necessary for expressing ific 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 The nucleic acids of the ion 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 information 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 invention; and/or that contain a nucleic acid of the invention. According to a particularly preferred embodiment, 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 bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains such as s of Bacillus, Streptomyces, Staphylococcus, and Lactococcus.

Suitable fungal cell include cells from s of Trichoderma, Neurospora, and Aspergi/lus. Suitable yeast cells e cells from species of Saccharomyces (for example Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizosaccharomyces pombe), Pichia (for e Pichia is and Pichia methano/ica), and Hansenu/a.

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 invention 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 molecule under ions that allow expression of the bispecific g molecule of the invention; and - recovering or isolating the ptide expressed by the host cells from the culture; and - optionally further purifying and/or modifying and/or formulating the bispecific g molecule of the invention.

For tion on an industrial scale, preferred host sms include strains of E. coli, Pichia is, and S. cerevisiae that are suitable for large scale expression, production and fermentation, and in particular for large scale pharmaceutical expression, production and fermentation.

WO 31076 The choice of the specific expression system depends in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a bispecific g molecule of the invention for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to ylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression.

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 ) and then isolated from the host cells and optionally further purified; or they can be produced ellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and ally further purified.

Methods and reagents used for the recombinant production of ptides, such as specific suitable expression vectors, transformation or transfection methods, ion markers, methods of induction of protein expression, culture conditions, and the like, are known in the art. rly, n isolation and purification techniques useful in a method of manufacture of a polypeptide of the invention are well known to the skilled person.

In a further aspect, the invention relates to a peptide having an amino acid sequence of a CDR3 contained in an anti-DII4-VHH having an amino acid sequence selected from sequences shown in SEQ ID NOs: 1 to 166 and 458, SEQ ID N05: 333 to 353, or SEQ ID N03: 375 to 395, 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 globulin chain, or for insertion into a non— immunoglobulin scaffold, e.g. a protease inhibitor, DNA-binding protein, cytochrome b562, a helix-bundle protein, a disulfide-bridged peptide, a |ipoca|in or an anticalin, thus conferring target-binding properties 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 usually comprises ng 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 ucleotide annealing or by means of overlapping PCR fragments, as e.g. described by Daugherty et a/., 1991, Nucleic Acids Research, Vol. 19, 9, 2471-2476. A method for inserting a VHH CDR3 into a non-immunoglobulin scaffold has been bed by Nicaise eta/., 2004, Protein Science, 13, 1882-1891.

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

For pharmaceutical use, a bispecific binding molecule of the invention or a polypeptide containing same may be formulated as a pharmaceutical preparation or ition sing at least one bispecific binding le of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non—limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral stration (such as by intravenous, uscular 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 le administration forms - which may be solid, semi—solid or liquid, depending on the manner of administration - as well as methods and rs for use in the preparation thereof, will be clear to the skilled person, and are further described herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one bispecific binding molecule, in particular one immunoglobulin single variable domain of the invention or a ptide containing same and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and ally one or more further active substances. 2012/055897 The bispecific binding molecules of the invention may be ated and administered in any suitable manner known per se: Reference, in particular for the immunoglobulin single variable domains, is for example made to , , , and , as well as to the standard oks, 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 ns. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, uminal, arterial or intrathecal administration) or for l (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection.

Suitable carriers or diluents for such preparations for e include, without limitation, sterile water and pharmaceutically acceptable aqueous buffers and solutions such as logical phosphate-buffered saline, Ringer's ons, dextrose solution, and Hank's solution; water oils; glycerol; 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 mixtures thereof. Usually, aqueous ons or suspensions will be preferred.

Thus, the bispecific binding molecule of the ion may be systemically administered, e.g., , in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the bispecific g molecule of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, , and the like. Such compositions and preparations should contain at least 0.1% of the Dll4-binding -46— 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 g molecule of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, pills, capsules, and the like may also contain binders, excipients, disintegrating agents, ants and sweetening or ring agents, for example those mentioned on pages 143-144 of WO 08/020079. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid r, such as a vegetable oil or a hylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the bispecific g 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 . 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 binding molecules of the invention may be incorporated into sustained— e preparations and s.

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

The bispecific binding molecules of the invention may also be administered intravenously or intraperitoneally by infusion or injection, as further described on pages 144 and 145 of .

For topical administration of the bispecific binding molecules of the invention, it will generally be desirable to administer them to the skin as compositions or ations, in combination with a dermatologically acceptable r, which may be a solid or a liquid, as further described on page 145 of . 2012/055897 Generally, the concentration of the bispecific binding 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 olid 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 bispecific binding molecule ed, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the ant physician or clinician. Also, the dosage of the bispecific binding molecules of the invention varies depending on the target cell, tumor, tissue, graft, or organ. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub- doses per day. The se itself may be further d, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

An administration regimen may include long-term, daily treatment. By term” is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

See Remington’s Pharmaceutical Sciences n, E.W., ed. 4), Mack Publishing 00., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any cation.

According to a further embodiment, the invention relates to the use of ific binding molecules of the invention, e.g. immunoglobulin single variable s or polypeptides containing them, for therapeutic purposes, such as - for the prevention, treatment and/or alleviation of a disorder, disease or condition, especially in a human being, that is associated with Dll4-mediated and/or AngZ- related effects on angiogenesis or that can be prevented, treated or alleviated by modulating the Notch signaling pathway and/or the Tie2 signalling pathway with a bispecific binding molecule according to the invention, - in a method of treatment of a patient in need of such therapy, such method comprising administering, to a t in need thereof, a pharmaceutically active -48— 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 ions associated with Dll4-mediated and/or Ang2— ed effects on enesis; - as an active ingredient in a pharmaceutical composition or medicament used for the above purposes.

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

According to r aspect, the disease is an eye disease associated with associated with Dll4-mediated and/or Ang2-mediated effects on enesis or which can be treated or alleviated by modulating the Notch signaling pathway and/or the Tie2 signalling pathway with a bispecific binding molecule.

Depending on the cancerous disease to be treated, a bispecific binding molecule of the invention may be used on its own or in ation with one or more additional eutic agents, in particular selected from chemotherapeutic agents like DNA damaging agents or therapeutically active compounds that inhibit angiogenesis, signal transduction pathways or mitotic checkpoints in cancer cells.

The additional eutic agent may be administered simultaneously with, optionally as a component of the same pharmaceutical preparation, or before or after administration of the bispecific binding molecule.

In certain embodiments, the additional therapeutic agent may be, without tion, one or more inhibitors selected from the group of inhibitors of EGFR, VEGFR, HERZ- neu, Her3, AuroraA, AuroraB, PLK and Pl3 kinase, FGFR, PDGFR, Raf, Ras, KSP, PDK1, PTK2, lGF—R or IR.

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, NFkappaB, the proteasome, Rho, an tor of wnt signaling 2012/055897 or an inhibitor of the tination pathway or another tor of the Notch signaling pathway.

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

An example for a PLK inhibitor is GSK-461364.

Examples for raf inhibitors are BAY4506 (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 ispinesib, 20, AZD-4877, CK—1122697, GSK 246053A, GSK-923295, MK—O731, and SB-743921.

Examples for a src and/or bcr-abl tors are dasatinib, AZD-O530, bosutinib, XL 228 (also an IGF-1 R inhibitor), nilotinib (also a PDGFR and oKit inhibitor), imatinib (also a oKit inhibitor), and NS—187.

An example for a PDK1 inhibitor is BX-517.

An example for a Rho inhibitor is BA—210.

Examples for Pl3 kinase tors are PX—866, 5 (also an mTor inhibitor), XL 418 (also an Akt inhibitor), XL-147, and XL 765 (also an mTor inhibitor).

Examples for tors of (Net or HGF are XL-184 (also an inhibitor of VEGFR, cKit, Flt3), PF-2341066, MK—2461, XL-88O (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-22O (also an inhibitor of oKit and PDGFR), KW 2449, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC), 348 (also an inhibitor of JAK2), XL-999 (also an inhibitor of oKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR, VEGFR and oKit), and inib (also an inhibitor of PDGFR, and oKit).

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

Examples for AT inhibitors are 7 (also interacting with tubulin), TG 101348 (also an tor of Flt3), and XL-O19.

Examples for Mek inhibitors are ARRY-142886, PD-325901, AZD-8330, and XL 518.

Examples for mTor tors are temsirolimus, AP-23573 (which also acts as a VEGF inhibitor), everolimus (a VEGF tor in addition). XL-765 (also a Pl3 kinase inhibitor), and 5 (also a Pl3 kinase inhibitor).

Examples for Akt tors are perifosine, GSK—690693, RX-O201, and triciribine. es for cKit inhibitors are AB-1010, OSl-930 (also acts as a VEGFR inhibitor), AC-22O (also an inhibitor of Flt3 and PDGFR), tandutinib (also an tor of Flt3 and PDGFR), axitinib (also an tor of VEGFR and PDGFR), XL-999 (also an inhibitor of Flt3, PDGFR, VEGFR, FGFR), sunitinib (also an inhibitor of Flt3, PDGFR, VEGFR), and XL—82O (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, FHA-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—OO52.

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

In preferred ments, 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 anti-VEGF antibody bevacizumab), and thalidomides, such agents being selected from, without limitation, bevacizumab, motesanib, 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 -5]- tor of cKit and Flt3), 1B3, CP 868596, IMC 3G3, R—1530 (also an inhibitor of Flt3), sunitinib (also an inhibitor of cKit and Flt3), axitinib (also an inhibitor of cKit), lestaurtinib (also an inhibitor of Flt3 and PKC), vatalanib, tandutinib (also an inhibitor of Flt3 and cKit), pazopanib, GW 786034, PF-337210, IMO-1121B, AVE-0005, 36, E-7080, CHIR 258, sorafenib tosylate (also an inhibitor of Raf), 5 (also an inhibitor of Raf), vandetanib, 632, OSl—930, AEE-788 (also an inhibitor of EGFR and Her2), BAY9352 (also an inhibitor of Raf), BAY4506 (also an tor of Raf), XL 880 (also an inhibitor of cMet), XL-647 (also an inhibitor of EGFR and , 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 antibody. 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 bispecific binding le of the invention are lapatinib, gefitinib, erlotinib, cetuximab, zumab, nimotuzumab, zalutumumab, vandetanib (also an tor of VEGFR), pertuzumab, , HKl-272, BMS-599626 ARRY-334543, AV 412, mAB-806, BMS—690514, JNJ—26483327, AEE-788 (also an inhibitor of VEGFR), ARRY-333786, lMC-1 1 F8, Zemab.

Other agents that may be advantageously combined in a therapy with the bispecific binding molecule of the invention are tositumumab and ibritumomab tiuxetan (two radiolabelled anti—CD20 antibodies), zumab (an anti-CD52 antibody), denosumab, (an osteoclast differentiation factor ligand inhibitor), galiximab (a CD80 antagonist), ofatumumab (a CD20 inhibitor), mumab (a CD4 antagonist), SGN4O (a CD40 ligand receptor modulator), rituximab (a CD20 inhibitor) or mapatumumab (a TRAIL-1 receptor agonist).

Other chemotherapeutic drugs that may be used in combination with the bispecific binding le 5 of the present invention are selected from, but not limited to hormones, hormonal analogues and antihormonals (e.g. tamoxifen, fene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, cyproterone e, finasteride, buserelin acetate, fludrocortisone, mesterone, medroxyprogesterone, octreotide, arzoxifene, pasireotide, vapreotide), aromatase tors (e.g. anastrozole, letrozole, liarozole, exemestane, atamestane, formestane), LHRH agonists and antagonists (e.g. goserelin acetate, leuprolide, abarelix, cetrorelix, deslorelin, lin, relin), antimetabolites (e.g. antifolates like rexate, pemetrexed, pyrimidine analogues like 5 fluorouracil, capecitabine, decitabine, nelarabine, and gemcitabine, purine and ine ues such as mercaptopurine thioguanine, cladribine and pentostatin, cytarabine, fludarabine); antitumor antibiotics (e.g. cyclines like doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C, bleomycin dactinomycin, plicamycin, mitoxantrone, pixantrone, streptozocin); platinum derivatives (e.g. cisplatin, oxaliplatin, carboplatin, lobaplatin, satraplatin); alkylating agents (e.g. estramustine, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazine, cyclophosphamide, ifosfamide, hydroxyurea, temozolomide, nitrosoureas such as carmustine and lomustine, thiotepa); antimitotic agents (e.g. vinca alkaloids like vinblastine, vindesine, vinorelbine, vinflunine and vincristine; and taxanes like paclitaxel, docetaxel and their formulations, larotaxel; simotaxel, and lones like ixabepilone, patupilone, ZK—EPO); topoisomerase inhibitors (e.g. ophyllotoxins like etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan) and laneous chemotherapeutics such as amifostine, anagrelide, interferone alpha, procarbazine, mitotane, and porfimer, bexarotene, celecoxib. ularly preferred combination partners of the bispecific binding molecules of the present invention are VEGF antagonists, like bevacizumab in®), Vargatef®, Sorafenib and Sunitinib.

The efficacy of bispecific binding molecules of the invention or polypeptides containing them, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell- based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the ic disease or er of interest. Suitable assays and animal models will be clear to the d 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 Dll4-binding components of the invention have properties that are superior to those of Dll4-binding molecules of the prior art, as can e.g. be taken from the ELISA data of Figure 10, showing that affinity-matured VHHs block hDLL4/hNotch1-Fc ction in a complete manner, as well as the |C5o (nM) values for affinity d VHHs in hDLL4/hNotch1-Fc competition ELISA; and the affinity KD (nM) of purified affinity matured VHHs on recombinant human DLL4 and mouse DLL4. This indicates that Dll4-binding components of the invention are promising candidates to have eutic efficacy in diseases and disorders associated with Dll4—mediated effects on angiogenesis, such as cancer.

According to r embodiment of the ion, there is provided a method of diagnosing a disease by a) contacting a sample with a Dll4-and/or Ang2 binding component of the invention as defined above, and b) detecting binding of said Dll4-and/or Ang2-binding component to said sample, and c) comparing the binding detected in step (b) with a standard, wherein a ence in g ve to said sample is diagnostic of a disease or disorder associated with Dll4—mediated effects on angiogenesis.

For this and other uses, it may be useful to further modify a bispecific binding component of the invention, such as by introduction of a functional group that is one part of a specific binding pair, such as the -(strept)avidin binding pair. Such a onal group may be used to link the bispecific binding molecule of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a bispecific binding molecule of the invention may be conjugated to biotin, and linked to r protein, ptide, 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 streptavidin.

Brief description of the s: Figure 1: Amino acid sequence alignment of human, rhesus and cynomolgus DLL4.

Figure 2: Human and mouse DLL4 deletion mutants (amino acid domain boundaries in superscript).

Figure 3: Purified VHHs blocking hDLL4/hNotch1-Fc interaction (ELISA).

Figure 4: Purified VHHs blocking hDLL4/hNotch1-Fc interaction (AlphaScreen).

Figure 5: Purified VHHs blocking CHO-hDLL4/hNotch1-Fc and CHO—mDLL4/hNotch1-Fc ction .

Figure 6: Purified VHHs blocking DLL4 mediated Notch1 cleavage (reporter).

Figure 7: Binding of purified VHHs to recombinant human and mouse DLL4 (ELISA).

Figure 8: Binding of purified VHHs to recombinant human DLL1 and human Jagged-1 ).

Figure 9: Binding of purified VHHs to human/mouse/cynomolgus DLL4 (FACS).

Figure 10: Affinity matured VHHs blocking hDLL4/hNotch1-Fc interaction (ELISA).

Figure 11: Purified affinity matured VHHs blocking CHO-hDLL4/hNotch1-Fc and CHO-mDLL4/hNotch1-Fc interaction (FMAT).

Figure 12: Binding of purified VHHs to human/mouse DLL4 (ELISA).

Figure 13: Binding of purified affinity matured VHHs to recombinant human DLL1 and human Jagged-1 (ELISA).

Figure 14: Binding of purified VHHs to human/mouse/cynomolgus DLL4 (FACS).

Figure 15: tion of VHH effects on Dll4-mediated inhibition of HUVEC proliferation.

Figure 16: Description cycle 1 DLL4xAngZ VHHs Figure 17: Purified cycle 1 DLL4xAngZ VHHs blocking hDLL4-hNotch1 interaction (ELISA) Figure 18: Purified cycle 1 DLL4xAngZ VHHs blocking CHO-hDLL4/Notch1 (44-1) and LL4/Notch1 (44—2) interaction (FMAT) Figure 19: Purified cycle 1 DLL4xAngZ VHHs binding to human, mouse and cynomolgus DLL4 overexpressing CHO cells (FACS) Figure 20: Purified cycle 1 DLL4xAngZ VHHs binding to human, mouse and rat DLL4 (ELISA) Figure 21: Purified cycle 1 DLL4xAngZ VHHs binding to human DLL1 and Jagged-1 (ELISA) Figure 22: Purified cycle 1 DLL4xAngZ VHHs blocking hAngZ-hTie2 , mAng2- mTie2 (48-2) and cTie2 (48-3) interaction ) Figure 23: Description cycle 2 DLL4xAngZ bispecific VHHs Figure 24: Purified cycle 2 DLL4xAngZ VHHs ng hDLL4-hNotch1 ction (ELISA) Figure 25: Purified cycle 2 DLL4xAngZ VHHs blocking CHO-hDLL4/Notch1 (51-1) and CHO-mDLL4/Notch1 (51—2) interaction (FMAT) Figure 26: Purified cycle 2 DLL4xAngZ VHHs blocking hDLL4 mediated Notch1 activation (reporter gene assay) Figure 27: Purified cycle 2 DLL4xAngZ VHHs binding to human, mouse and cynomolgus DLL4 overexpressing CHO cells (FACS) Figure 28: Purified cycle 2 DLL4xAngZ VHHs binding to human, mouse and rat DLL4 (ELISA) Figure 29: Purified cycle 2 DLL4xAngZ VHHs binding to human DLL1 and Jagged—1 (ELISA) Figure 30: Purified cycle 2 DLL4xAngZ VHHs blocking hAngZ-hTie2 (56-1), mAng2- mTie2 (56-2) and cTie2 (56-3) interaction (ELISA) Figure 31: Purified cycle 2 DLL4xAng2 VHHs blocking hAng1-hTie2 ction (ELISA) Figure 32 : Purified cycle 2 DLL4xAng2 VHHs blocking hAng2 mediated HUVEC survival. als and methods a) Generation CH0 and HEK293 cell lines overexpressing human, mouse and cynomolgus Dll4 The cDNAs encoding human (SEQ ID NO: 417; NM_O19074.2) and mouse D|I4 (NM_O19454.3) are amplified from a Human Adult Normal Tissue Heart cDNA library (BioChain, Hayward, CA, USA) and a Mouse Heart Tissue cDNA library (isolated from C57/Bl6 strain), respectively, using oligonucleotides designed in the 5’ and 3’ UTR of the corresponding ce (see Table 1; SEQ ID NO:421 to 426).

Amplicons are cloned into the mammalian expression vector pCDNA3.1(+)—neo (Invitrogen, Carlsbad, CA, USA).

Table 1: Oligonucleotide sequences used for ication of DLL4 gene full length ogues.

Human DLL4 Mouse DLL4 Cynomolgus DLL4 >FWd_hDLL4 >FWd_mDLL4 >FWd_CDLL4 GCGAACAGAGCCAG GAGCGACATCCCTA GCGAACAGAGCCAG ATTGAGG (SEQ ID ACAAGC (SEQ ID ATTCAGG (SEQ ID NO:421) NO:423) NO:425) >Rev_hDLL4 >Rev_mDLL4 >Rev_cDLL4 GGATGTCCAGGTAGG CTCTGTTCC CCAGACAGACACCC CTCCTG (SEQ ID CTTGG (SEQ ID AAAGGT (SEQ ID NO:422) NO:424) NO:426) Cynomolgus Dll4 cDNA is amplified from a Cynomolgus Normal Tissue Heart cDNA library (BioChain, d, CA, USA), using primers designed on the 5’ and 3’ UTR of the Dll4 encoding sequence of the closely d s rhesus (Macaca mulatta Dll4, SEQ ID NO:418; XM_OO1099250.1) (see Table 1). The final amplicon is cloned in the mammalian sion vector pCDNA3.1(+)-neo (lnvitrogen, Carlsbad, CA, USA). The amino acid sequence of cynomolgus Dll4 was shown to be 100% identical to , and 99% cal to human (see Figure 1; differences from the human sequence are indicated as bold-underlined).

To establish Chinese Hamster Ovary (CHO) cells overexpressing human Dll4, mouse Dll4 or cynomolgus Dll4, parental CHO cells are electroporated with pCDNA3.1(+)- l4, pcDNA3.1(+)-neo—lel4 or pcDNA3.1(+)—neo-chl4, respectively. Human Embyonic Kidney (HEK293) cells overexpressing human Dll4 and mouse Dll4 are generated by lipid—mediated transfection with Fugene (Roche) of pCDNA3.1(+)-neo- thl4 or lel4 plasmids, respectively, in the HEK293 parental cell line. For all conditions, transfectants are selected by adding 1 mg/mL geneticin (lnvitrogen, Carlsbad, CA, USA). b) Generation of monoclonal anti-D/l4 IgG and Fab fragment In US 2008/0014196 (Genentech) a human/mouse cross-reactive Dll4 mAb is described that was used by y et al. (2006) to show additive effects of VEGF mAb and Dll4 mAb on tumor growth in a number of xenograft models. This anti—Dll4 mAb and its corresponding Fab are purified to assess the properties of this antibody (fragment) in biochemical/cellular assays and xenograft models and for specific elutions during phage selections. The published variable heavy and light chain sequences of Dll4 mAb are cloned into a hlgG2aK framework, transiently expressed in HEK293 cells and purified from atants using protein A chromatography.

Purified Dll4 mAb shows binding to human Dll4 and mouse Dll4 in ELISA and FACS (using CHO-lel4 and CHO-thl4 , sub-nanomolar ties to both growth factor orthologues in Biacore.

The corresponding Dll4 Fab fragment is constructed via gene assembly based on back-translation and codon optimization for expression in E. coli using Leto’s Gene Optimization software (www.entechelon.com). Oligonucleotide primers for the assembly of the variable light chain (VL), variable heavy chain (VH), constant light chain (CL) and constant domain 1 of the heavy chain (CH1) are designed and an ly PCR is performed. The cDNA seqments encoding VL+CL and VH+CH1 are cloned into a pUC119—derived vector, which contains the LacZ promotor, a resistance gene for kanamycin, a multiple cloning site and a hybrid gIII-pelB leader sequence, using the restriction sites Sﬁl and Asc/ and the restriction sites Kpni and Not], respectively. In frame with the Fab coding sequence, the expression vector encodes a C-terminal HA and His6—tag. The Fab fragment is expressed in E. coli as HisS- tagged n and subsequently purified from the culture medium by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC).

Relevant amino acid sequences of the variable heavy and variable light chain are ed (SEQ ID NO: 1 and SEQ ID NO: 2; respectively, of US 2008/0014196); the amino acid sequences of the te heavy and light chain are shown in SEQ ID N05: 419 and 420, respectively. 0) Generation of DII4 mutants for epitope g To identify the region in the extracellular domain (ECD) of DII4 that comprises the e recognized by the anti—DII4 VHHs, ssive deletion mutants of the DII4 ECD are generated. The mammalian expression vector pSecTag2/Hygro (Invitrogen, Carlsbad, CA, USA) comprising a CMV promotor upstream of polynucleotides encoding a nested series of deletion fragments of the DII4 ECD fused to a polyHis- tag are generated using standard recombinant DNA technology (see Figure 2; amino acid domain boundaries in superscript).). These recombinant ns are expressed in transiently transfected HEK293 cells using the Freestyle 293 Expression System (Invitrogen, Carlsbad, CA, USA) from which conditioned medium is collected and purified via IMAC. Only DII4 mutants lacking the EGF2-Iike domain showed ed binding to the humanized human/mouse cross-reactive II4 mAb described above (immobilized via a capturing anti-human IgG coated Biacore sensor chip). This IgG is known to have a specific binding epitope in this DII4 domain (patent application Genentech, US 2008/0014196A1). d) Generation of BM reporter assay ds A reporter assay is developed based on the y-secretase mediated cleavage of Notch1 and nuclear translocation of the ellular domain of Notch1 (NICD) upon stimulation with DII4, essentially as described (Struhl and Adachi, Cell. 1998 May 15; 2012/055897 649—60). Gal4NP16 coding sequences are inserted into the NICD-coding ce. The potent hybrid transcriptional activator GAL4-VP16, which consists of a DNA binding fragment of yeast GAL4 fused to a Herpes simplex viral transcriptional activator domain VP16, is inserted carboxy—terminal to the transmembrane domain of Notch1. Cleavage of this construct by y-secretase results in the release of the Gal4NP16 NICD fusion protein which will translocate to the nucleus where it will bind to and transcriptionally activate a nsfected luciferase reporter plasmid, containing a strong GAL4-UAS promoter sequence (Struhl, G. and Adachi, A., Cell, vol. 93, 649-660, 1998). The human Notch1-Gal4NP16 sion cassette is cloned in pcDNA3.1(+)-neo rogen, Carlsbad, CA, USA). The pGL4.31[LchP/Gal4UAS/Hygro] vector (Promega, Madison, WI, USA) is used as luciferase reporter plasmid.

Example 1 Immunization with D||4 from different species induces a humoral immune response in llama 1. 1. Immunizations After approval of the l Committee of the faculty of Veterinary Medicine (University Ghent, Belgium), 4 llamas (designated No. 208, 209, 230, 231) are immunized with 6 intramuscular ions (100 or 50 ug/dose at weekly intervals) of recombinant human Dll4 (R&D Systems, Minneapolis, MN, US). The Dll4 antigen is formulated in Stimune (Cedi Diagnostics BV, Le|ystad, The Netherlands). Three additional llamas (designated No. 127b, 260, 261) are immunized according to standard protocols with 4 subcutaneous injections of alternating human Dll4 and mouse Dll4 overexpressing CHO cells which are established as described above.

Cells are re-suspended in D-PBS and kept on ice prior to injection. rmore, three additional llamas (designated No. 282, 283, 284) are immunized according to standard protocols with 4 intramuscular injections (100 or 50 ug/dose at biweekly intervals) of alternating recombinant human Dll4 and mouse Dll4 (R&D Systems, polis, MN, US). The first injection at day 0 with human Dll4 is ated in Complete Freund’s Adjuvant (Difco, Detroit, MI, USA), while the subsequent injections with human and mouse Dll4 are formulated in Incomplete Freund’s Adjuvant , Detroit, MI, USA). 1.2. Evaluation of d immune responses in llama To evaluate the induction of an immune responses in the animals against human Dll4 by ELISA, sera are collected from llamas 208, 209, 230 and 231 at day 0 (pre- ), day 21 and day 43 (time of peripheral blood lymphocyte [PBL] collection), from llamas 127b, 260 and 261 at day 0 and day 51, and from llamas 282, 283 and 284 at day 0, day 28 and day 50. In short, 2 ug/mL of recombinant human Dll4 or mouse Dll4 (R&D Systems, Minneapolis, MN, USA) are 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, specifically bound immunoglobulins are detected using a horseradish peroxidase (HRP)—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), showing that a significant antibody-dependend immune response against Dll4 is induced. The antibody response is mounted both by conventional and heavy-chain only antibody expressing B-cell oires since specifically bound globulins can be detected with antibodies specifically recognizing the conventional llama lgG1 antibodies or the heavy chain only llama lgG2 or lgG3 dies (Table 2-A). In all llamas injected with mouse Dll4, an antibody se is mounted by conventional and heavy chain only antibody expressing B-cells specifically against mouse Dll4. Additionally, serum titers of cell zed animals are confirmed by FACS analysis on human and mouse Dll4 overexpressing HEK293 cells (Table 2-B). The Dll4 serum titer responses for each llama are depicted in Table 2. -6]- Table 2: Antibody mediated specific serum response against DLL4.

A) ELISA (recombinant n solid phase coated) Recombinant human Recombinant mouse llllillllllllllllllllllllllllllllllllllllllll llllllillllllllll|I||||llllllIlllllllllllll||| ND: not determined 2012/055897 B) FACS (natively expressed protein on HEK293 cells) -64— reClillll‘lilillllilillllilillll-l||||lililllllilillllillll ND: not determined Example 2 Cloning of the heavy-chain only anti-Dll4 antibody fragment repertoires and preparation of phage Following the final immunogen injection, immune tissues as the source of B-cells that produce the chain antibodies are ted from the immunized llamas.

Typically, two 150—ml blood s, collected 4 and 8 days after the last antigen injection, and one lymph node biopsy, collected 4 days after the last antigen injection are collected per animal. From the blood samples, peripheral blood mononuclear cells (PBMCs) are prepared using Ficoll-Hypaque according to the manufacturer’s instructions (Amersham 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 amplify the VHH encoding DNA segments, as described in WC 05/044858. 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 tate phage display of the VHH library. The vector is derived from pU0119 and contains the LacZ promoter, a M13 phage glll protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a hybrid glll-pelB leader sequence 0). ln 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. e 3 Selection of BM specific VHHs via phage display VHH repertoires obtained from all llamas and cloned as phage library are used in different selection strategies, applying a licity of selection conditions. Variables include i) the Dll4 protein format (C-terminally His-tagged recombinantly expressed extracellular domain of human Dll4 Pro524) and mouse Dll4 (Met1-Pro525) (R&D Systems, Minneapolis, MN, USA), or full length human Dll4 and mouse Dll4 present on Dll4-overexpressing CHO or HEK293 cells, ii) the antigen presentation method (plates directly coated with Dll4 or Neutravidin plates coated with Dll4 via a biotin-tag; solution phase: incubation in solution followed by capturing on vidin- coated plates), iii) the antigen concentration and iv) different elution methods (non— specific via trypsin or specfic via cognate receptor Notch1/Fc chimera or ll4 b). All selections are done in Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).

Selections are performed as follows: Dll4 antigen ations for solid and solution phase selection formats are ted as described above at multiple concentrations. After 2h tion with the phage ies followed by extensive washing, bound phage are eluted with trypsin (1 mg/mL) for 30 minutes. In case trypsin is used for phage elution, the protease ty is immediately neutralized ng 0.8 mM protease inhibitor ABSF. As control, selections w/o antigen are performed in parallel. Phage outputs that show ment over background (non- antigen control) are used to infect E. coli. Infected E. coli cells are either used to e 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 binders, single colonies are picked from the agar plates and grown in 1 mL 96-deep-well plates. LacZ-controlled VHH expression is induced by adding IPTG (0.1-1mM final) in the absence of glucose. Periplasmic extracts (in a volume of ~ 80 uL) are ed according to rd protocols Example 4 Screening of periplasmic extracts in Dll4-Notch1 AlphaScreen and FMAT competition assay Periplasmic extracts are screened in a human DII4/human Notch1 creen assay to assess the blocking capacity of the expressed VHHs. Human Dll4 is biotinylated using biotin (Sigma, St Louis, MO, USA) and biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sigma, St Louis, MO, USA).

Notch1/Fc chimera (R&D Systems, Minneapolis, MN, USA) is captured using an anti- Fc VHH which is coupled to acceptor beads according to the manufacturer’s instructions n Elmer, Waltham, MA, US). To evaluate the neutralizing capacity of the VHHs, dilution series of the periplasmic extracts are pre-incubated with biotinylated human DII4. To this mixture, the acceptor beads and the streptavidin donor beads are added and r incubated for 1 hour at room temperature.

Fluorescence is measured by reading plates on the Envision Multilabel Plate reader (Perkin Elmer, Waltham, MA, USA) using an excitation wavelength of 680 nm and an emission wavelength of 520 nm. Decrease in fluorescence signal indicates that the binding of biotinylated human Dll4 to the human Notch1/Fc receptor is blocked by the VHH expressed in the periplasmic extract.

Alternatively, CHO-thl4 and CHO-lel4 cells are used in a human /Fc FMAT (Fluorometric Microvolume Assay logy) competition assay. Recombinant human /Fc chimera (R&D Systems, Minneapolis, MN, USA) is randomly d with Alexa—647 (lnvitrogen, Carlsbad, CA, USA). In brief, 5 uL periplasmic material is added to 100 pM or 175 pM d human /Fc together with 7,500 CHO-thl4 or CHO-lel4 overexpressing cells, tively, and readout is performed after 2 hours of tion. To set the no-competition baseline, at least replicates of cells with human Notch1/Fc~Alexa647 are included and the percentage of inhibition is calculated from this baseline. All calculations are based on the FL1_total signal which comprises the average of the fluorescence per well times the number of counts per well.

From this ing, inhibiting VHHs are selected and sequenced. ce analysis revealed 167 unique VHHs belonging to 40 different B-cell lineages. The total number of variants found for each B-cell lineage is depicted in Table 3. An overview of periplasmic screening data is given in Table 4. The amino acid sequences of all obtained unique VHHs are shown in the Sequence Listing (SEQ ID NO:167 — 332 and 459) and in Table 5 (CDRs and framework regions are indicated).

Table 3: Selection parameters used for the identification of DLL4 specific VHH B—cell lineages. selection librar VHH ID variant Y format e 5 DLLBII8A ( 231 trypsm DLLBI|5B ( 231 trypsm RI: biot- thLL4 DLLB||7B (3 nM) 3 21 231 trypsin Rll: biot- thLL4 (0.03 DLLBII6B biot—thLL4 4 13 31 11 (3 M) W0 2012/131076 Rkbbb thLL4 DLLB||8C (SnND RH:Mob thLL4 (3nND DLLB||19 Mot¢hDLL4 D10 l (3nND DLLBII33 lilllll LL4 C5 (2E6hnL) DLLBH28 nnDLL4 B6 (0.5 ug/mL) DLLBH17 Mot¢hDLL4 G10 CBnM) DLLBH17 Mot¢hDLL4 C 1 (3nND DLLBH19 Mot¢hDLL4 F4 Illllll (3nND DLLBH17 Mot¢hDLL4 F10 Illllll (3nND DLLBII17 Mot¢hDLL4 BS lilllll (3nND DLLB||19 lilllll Mot¢hDLL4 F12 CBnM) RI: biot- thLL4 2 (3 nM) RII: biot- thLL4 (3 nM) RI: biot- thLL4 DLLBH47 (3 nM) 1 230 RH: biot- thLL4 (3 nM) RkCHO- mDLL4 DLLBH56 (2E6/mL) 230 RH: CHO- mDLL4 (2E6/mL) RI: CHO- mDLL4 DLLBH95 (2E6/mL) RII: CHO- mDLL4 (2E6/mL) RI: CHO- mDLL4 DLLBII96 (2E6/mL) 19 trypsin RII: CHO- mDLL4 (2E6/mL) RI: CHO- mDLL4 (2E6/mL) RII: CHO- DLLBI|1O mDLL4 (2E6/mL) _ trypsm RIII: biot- (RIII) thLL4 (+thLL4) RI: CHO- mDLL4 (2E6/mL) RII: CHODLLBII 21 mDLL4 trypsm_ RIII: biot- (RIII) thLL4 (0.01 | RI: CHO- mDLL4 DLLBI|11 (2E6/mL) 22 trypsin RII: CHO- mDLL4 (2E6/mL) RI: CHO- mDLL4 (2E6/mL) RII: CH0- DLLBI|1O 23 2 230 mDLL4 (2E6/mL) _ trypsm RIII: biot- (RIII) thLL4 (0.01 RI: CHO- mDLL4 (2E6/mL) RII: CHODLLBII 24 mDLL4 (2 E6/m L) trypsm_ RIII: biot- (RIII) thLL4 (0.1 WO 31076 RI: CHO- mDLL4 DLLBII11 (2E5/mL) trypsin RII: CHO- mDLL4 (2E6/mL) RI: CHO- mDLL4 (2E6/mL) RII: CH0- DLLBI|1O 1 mDLL4 (2E6/mL) trypsin RIII: biot- (RIII) thLL4 (0.1 RI: CHO- mDLL4 (2E6/mL) RII: CH0- DLLBII1O 1 mDLL4 (2E6/mL) trypsin RIII: biot- (RIII) thLL4 (1 nM) RI: CHO- mDLL4 DLLBI|11 (2E6/mL) 28 |!%H|||||| trypsin RII: CHO- mDLL4 (2E6/mL) RI: CHO- mDLL4 RII: CHO- DLLBI|10 29 mDLL4 (2E6/mL) trypsin RIII: biot- (RIII) thLL4 (1 nM) RI: CHO- mDLL4 (2E6/mL) RII: CHO- DLLBII’IO mDLL4 4A3 (2E6/mL) trypsin RIII: biot- (RIII) thLL4 (1 nM) + thLL4 RI: CHO- mDLL4 (2E6/mL) rhNotCh RH:CHO- 1/Fc DLLBH1O InDLL4 31 (RLRH) 404 (2E6hnL) trypsin RHkbmb (RIII) thLL4 m nM)+ thLL4 RI: CHO- mDLL4 (2E6/mL) rhNotCh RH:CHO- 1/Fc DLLBH1O rnDLL4 32 (RLRH) 4B5 L) trypsin RHkbbb (RIII) thLL4 m nM)+ thLL4 RI: CHO- mDLL4 DLLB||1O (2E6/mL) RH:CHO- rnDLL4 (2E6HnL) RI: biot- thLL4 DLLBII58 (3 nM) rhNotch 4 260 A“ “F0 RII: biot- rmDLL4 (3 RI: HEK293H- hDLL4 (2E6/mL) DLLBII61 1 260 t _ RII.. rVPSln HEK293H- hDLL4 (2E6/mL) RI: HEK293H- hDLL4 (2E6/mL) DLLBII61 1 260 t _ RII.. rVPSln HEK293H— hDLL4 (2E6/mL) RI: H- hDLL4 (2E6/mL) DLLB||62 1 260 t _ RII.. rVPSln HEK293H- mDLL4 (2E6/mL) ||||||| RI: CHO- mDLL4 (2E6/mL) RII: CHO- mDLL4 DLLBII11 (2E6/mL) 5A5 trypsin RIII: biot- (RIII) thLL4 (1 nM) trypsin RIV:CHO mDLL4 (2E6/mL) I RI: CHO- mDLL4 DLLBII83 (2E6/mL) RI: CHO- hDLL4 (2E6/mL) liillll RI: CHO- hDLL4 DLLB|I8O (2E6/mL) RI: CHO- hDLL4 (2E6/mL) Table 4: Screening of periplasmic extracts containing expressed anti—DLL4 VHH Represen- _ umque Alpha FM Biacore tative FMAT Screen (a) VHH ID sequen DLLB||8A09 M DLLBIISB’H (JO DLLBII7BO5 .1 DLLBII6B11 :84 DLLBII8011 .57 19D1 I CI) (TI DLLBII33CO \1 (TI DLLB||28BO DLLBII17G1 -78— Iiilll DLLBH17CO a)NJ CDA Illlll DLLBH19FO 1 (0 (N Iiilll DLLBH17F1 1.1503 / O slamm) IHHIII DLLBH17BO 3 / 0) ‘4 3 225mm) IIHIII DLLBH19F12 IIHIII DLLBH4ZBO7 DLLBH47DO 16 \I Ililll DLLBH56AO A IHHIII DLLBH95FO CD _\ _\ Iiilll DLLBH96CO N(N CD (0 IHHIII DLLBH104G GDJ; IHIIII DLLBH102F \I Ch COCh 08 Illiilll|||||ii|||||IllliilllIlllillll|||||i|||||||||il|||Illiilll||||il|||||||i||||Illillll|||||||||||||i|||||| DLLBH112A 22 1 ”\lM CO \I DLLBH102G 23 2 DLLBH101G 24 1 CO A toM M DLLBH112A 1 \l (TI DLLBH101H 26 1 00\l \1 (TI DLLBH101H 27 1 (1)01 CI) (.0 DLLBH112E 28 1 00 Ch |%H||| 01Fo1 Illllll CD0'1 \l P3 c: |'|'|bN lgilll DLLBH104A Illllll CD 00 IHIIII DLLBH104C Illllll 00\l co 00 _\ |%!||| DLLBH104B05 l \l CI) |%H||| DLLBH107C '\I O1 03 Illllll IHHIII DLLBH58A1 l|||||| ' (.0 0‘1 \l 00 -80— WO 31076 DLLBII61FO DLLBII61FO DLLBII62C1 DLLBII115A 38 1 DLLBII83GO DLLBII8OEO (a) if multiple unique variants within a B-cell lineage are identified, the range (max-min) in off-rate or the off-rate of a e member is given between brackets in italics). (b) heterogeneous fit: fast and slow off-rate determined.

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Selected VHHs are expressed in E. coli TG1 as c-myc, agged proteins. sion 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. These extracts are used as starting material and VHHs are purified via IMAC and size exclusion chromatography (SEC) ing in 95% purity as assessed via SDS-PAGE. .1. Evaluation of DII4 blocking VHHs in ELISA The blocking capacity of the VHHs is evaluated in a human DII4 — human Notch1/Fc blocking ELISA. In brief, 1 pg/mL of human Notch1/Fc chimera (R&D Systems, Minneapolis, MN, USA) is coated in a 96—well MaxiSorp plate (Nunc, Wiesbaden, Germany). A fixed concentration of 15 nM ylated human DII4 is preincubated with a dilution series of the VHH for 1 hour, after which the mixture is incubated on the coated Notch1 receptor for an additional hour. Residual g of biotinylated human DII4 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St. Louis, MO, USA) (Figure 3). Human DII4 is biotinylated as described above. The IC50 values for VHHs blocking the human DII4 — human Notch1/Fc interaction are depicted in Table 6.

Table 6: |C50 (nM) values forVHHs in hDLL4/hNotch1-Fc competition ELISA VHH ID 6811 _\ O‘l 55D12 56A09 LO 56CO4 56H08 57C11 62C11 96C03 101GO8 O‘I 104G01 1 15A05 LO _\ antiDLL4 Fab 0.7 .2. Evaluation of BM biocking VHHs in AlphaScreen In brief, 1 nM biotinylated human D||4 is captured on streptavidin-coated donor beads (20 ug/mL), while 0.4 nM of the receptor human Notch1 (as a Fc fusion protein) is captured on anti-human Fc VHH-coated acceptor beads (20 ug/mL).

Both loaded beads are incubated er with a dilution range of the competing VHH e 4). The |C50 values for VHHs blocking the human Dll4 — human Notch1/Fc interaction are depicted in Table 7.

Table 7: |C5o (nM) values for VHHs in hNotCh1 competition AlphaScreen -_8A09 antiDLL4 (a) partial inhibitor .3. Inhibition by anti-D/l4 VHHs of human Notch 1/Fc binding to human or mouse D/l4 expressed on the CHO cells The blocking capacity of the VHHs is evaluated in a human and mouse D|l4 — human Notch1/Fc competitive FMAT assay (Figure 5) as outlined in Example 4.

The |C5o values for VHHs blocking the interaction of human Notch1/Fc to human or mouse D||4 expressed on CHO cells are depicted in Table 8.

Table 8: (Mean) |C5o values (nM) of purified VHHs blocking the ction of human Notch1/Fc to human or mouse DLL4 expressed on CHO cells (FMAT) VHH ID |C50(nM) 19F04 33.0 55D12 39.1 41.0 56A09 10.6 15.0 56C04 28.7 49.6 56H08 22.0 33.7 57C11 53.9 49.5 62C11 172.2 106.3 96C03 160.8 28.8 101G08 24.6 92.1 115A05 22.0 43.0 anmmab 5.4 WO 31076 .4. Evaluation of D/l4 blocking VHHs in reporter assay To evaluate the potency of the ed VHHs, a reporter assay is set up which is based on the v-secretase mediated cleavage of Notch1 and release of the intracellular domain of Notch1 (NICD) upon ation with DII4. The Notch1- GAL4NP16 construct is cotransfected with the pGL4.31[Luc2P/Gal4UAS/Hygro] reporter plasmid in HEK cells resulting in a transient expression of the fusion protein. These transiently transfected cells are stimulated for 24 hours by co- culture with a HEK293-thl4 stable cell line. Forty—eight hours post-transfection, the readout is performed. The VHHs are preincubated with the HEK293—hDIl4 cells 1 hour before the start of the co-culture and are included during the co-culture (Figure 6). The |C5o values of the VHHs for blocking the Dll4-mediated cleavage of Notch1 and subsequent translocation of its NICD to the s of the receptor cell are depicted in Table 9.

Table 9: (Mean) ICso values (nM) of purified anti-Dll4 VHHs in a DLL4/Notch1 reporter assay .5. Epitope binning In order to determine whether VHHs can bind simultaneously to D||4 when e.g. a benchmark antibody is bound, e binning experiments are carried out (via Surface Plasmon Resonance (SPR) on a Biacore T100 ment). Anti-DII4 Fab fragment is irreversibly immobilized on the reference and on the active flow cell of a CM5 sensor chip. For each sample (cycle), human D||4 is injected on the active and reference flow cell and reversibly captured by anti-DII4 Fab. onal binding of VHHs is evaluated by ion over the immobilized surface. All VHHs and anti- D||4 Fab are injected at 100 nM with a surface contact time of 120 seconds and a flow rate of 10 uL/minute. Surface is regenerated using 10 mM glycine ).

Processed curves are evaluated with Biacore T100 tion software.

Table 10—A represents the sequential injection/regeneration path of analysed VHHs and controls. VHHs DLLBII56A09 (SEQ ID NO: 300), DLLBII96C03 (SEQ ID NO: 326), DLLBII101GO8 (SEQ ID NO: 197) and DLLBII115AO5 (SEQ ID NO: 224) are shown not to additionally bind to human D||4 captured by D||4 Fab.

Injection of D||4 Fab also failed to additionally bind human D||4 ting that all epitopes are saturated. Therefore, it can be concluded that these VHHs recognize an epitope overlapping with D||4 Fab for binding human Dll4. Human-only VHHs DLLBIISB11 (SEQ ID NO: 174) and DLLBII104GO1 (SEQ ID NO: 215) show additional binding on D||4 Fab captured human DII4, indicating that these VHHs that are specific for human D||4 recognize a different epitope than the mouse cross-reactive VHHs.

Table 10-A: e binning of anti-DLL4 VHHs — simultaneous binding with DLL4 Fab Injection Binding] Binding level step Regeneration (RU) .6. Epitope mapping using D/l4 deletion s Binding of the VHHs to these Dl|4 mutants is assessed in Biacore. In brief, VHHs DLLBIIIOfGO8 (SEQ ID N019?) and DLLBII115A5 (SEQ ID NO: 224) are coated on a CM4 Sensorchip and 200 nM of each deletion mutant is injected across the chip. Binding is qualitatively assessed. No binding of DLLBII56A09 (SEQ ID NO: 300), DLLBII101GO8 (SEQ ID NO: 197) and DLLBII115AO5 (SEQ ID NO: 224) is observed to human and mouse Dll4 mutants thl4.1 and lel4.8, respectively, lacking EGF—Iike 2 domain (Table 10—B). Indirect evidence using a thl4/DII4 IgG competitive ELISA already pointed to this observation. In brief, 1 pg/mL of Dll4 IgG is coated in a l MaxiSorp plate (Nunc, Wiesbaden, y). A fixed concentration of 6 nM biotinylated human Dll4 is ubated with a dilution series of the VHH for 1 hour, after which the mixture is incubated on the coated IgG for an additional hour. Residual binding of biotinylated human Dll4 is detected using horseradish peroxidase ated extravidin (Sigma, St. Louis, MO, USA) (data not shown). Human Dll4 is biotinylated as described above. It is known from patent literature that the monoclonal anti-Dll4 IgG (Genentech, US 2008/OO14196A1) binds to an epitope within the EGF-like 2 domain of Dll4.

Table 10-3: Epitope mapping of anti—DLL4 VHHs — binding to DLL4 deletion mutants DLLBII56A9 DLLBII101G8 DLLBII115A5 Binding Binding Binding kd (1/s) kd (1/s) kd (1/s) (RU) (RU) (RU) hDLL4 81 4 373 2.0E-O3 24 3.5E O3- 3 3 nO nO hDLL4.1 no binding binding g hDLL4.3 25 7.4E-O4 O3 37 3.5E-O3 2012/055897 hDLL4.5 1.2E-03 2.19E-03 4.2E-03 hDLL4.6 1.1E-03 2.20E-O3 4.1E-O3 no no mDLL4.8 no binding binding_ _ binding_ _ mDLL4.1O 1.1E-03 5.14E-03 3.8E-03 mDLL4.11 3 6.16E-03 6.6E—03 mDLL4.12 1.3E-03 4.52E-03 3.1E-03 . 7. ining the afﬁnity of the hD/i4 — VHH interaction Kinetic analysis to determine the affinity of the Dll4 — VHH interaction is performed by Surface Plasmon Resonance (SPR) on a Biacore T100 instrument.

Recombinant human Dll4 is immobilized onto a CM5 chip via amine ng using EDC and NHS) or biotinylated human Dll4 is captured on a SA chip (streptavidin surface). Purified VHHs or Fab fragment are injected for 2 minutes at different concentrations en 10 and 300 nM) and allowed to dissociate for 20 min at a flow rate of 45 ul/min. Between sample injections, the surfaces are regenerated with 10 mM e pH1.5 and 100 mM HCl. HBS—N (Hepes buffer pH7.4) is used as g buffer. If possible, data are evaluated by fitting a 1:1 interaction model (Langmuir g) onto the binding curves. The affinity constant KD is calculated from resulting association and dissociation rate constants (ka) and (kd). The affinities of the anti-Dll4 VHHs are depicted in Table 11. —124— Table 11: Affinity KD(anl) of purified VHHs for recombinant human DLL4 _mom VHH .D mum (a) heterogeneous binding curve resulting in no 1:1 fit .8. Binding to ortho/ogues (mD/l4, cD/l4) and family s (hJagged—1,hDLL 1) In order to ine cross-reactivity to mouse DII4 a binding ELISA is performed.

In brief, recombinant mouse DII4 (R&D s, Minneapolis, MS, USA) is coated overnight at 4°C at 1 ug/mL in a 96-well MaxiSorp plate (Nunc, den, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis, MO, USA) (Figure 7). As reference, binding to human DII4 is measured. EC50 values are summarized in Table 12.

WO 31076 Table 12: E050 (nM) values for VHHs in a recombinant human DLL4 and mouse DLL4 binding ELISA _mm mm VHH ID EC5o (nM) EC5o (nM) 7A02 7805 8A09 8C11 17F1O 19FO4 55D12 56A09 56CO4 56HO8 . 8.7 57C11 83.4 62C11 13.1 96C03 86.5 101G08 _ 53.9 WO 31076 In order to ine the logus cross-reactivity of the VHHs, a FACS binding experiment is performed. Cynomolgus Dll4 expressing HEK293 cells (transient or stable ection) are used for a titration binding experiment of the VHHs. After a 30 minutes incubation on ice, all samples are washed and detection is performed by applying anti-c—myc~Alexa647 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Human and mouse Dll4 overexpressing HEK293 cells are taken as reference. The mean MCF value is determined on the FACS Array and used for calculation of the EC50 value (see Figure 9).

Absence of binding to homologous ligands human DLL1 and human Jagged-1 is assessed via solid phase binding assay (ELISA). In brief, human DLL1 (Alexis, San Diego, CA, USA) and human Jagged-1 (Alexis, San Diego, CA, USA) are 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 and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St. Louis, MO, USA). All anti-Dll4 VHHs are considered as being non-cross reactive to these homologous ligands (Figure 8). .9. tion of VHHs in blocking Dll4- mediated HUVEC proliferation The potency of the selected VHHs is evaluated in a proliferation assay, as described by Ridgway et al., Nature. 2006 Dec 21 122):1083-7), in ed form. In brief, 96-well tissue culture plates are coated with purified Dll4-His (RnD s; C—terminal His—tagged human Dll4, amino acid 27-524, 0.75mllwell, ng/ml) in coating buffer (PBS, 0.1% BSA). Wells are washed in PBS before 4000 HUVE cells/well are seeded in quadruplicate. Cell proliferation is measured by [3H]-Thymidine incorporation on day 4. The results, shown in Figure 15, trate that the DLL4 VHHs DLLB||101G08, DLLB||104GO1, DLLB||115AO5, DLLB||56A09 and the DLL4 Fab inhibit the DLL4-dependent effect on HUVEC eration in a dose-dependent manner, the |C5o values are summarized in Table 13. The tested VHHs achieve a complete inhibition of the DLL4-dependent effect at 10uM.

Table 13 |C50 values obtained in the DLL4 proliferation assay (nee eeeemee e Example 6 Affinity tion of selected anti-Dll4 VHHs VHHs DLLBII101GO8 and DLLB||115AO5 are subjected to two cycles of affinity maturation.

In a first cycle, amino acid substitutions are introduced randomly in both framework (FW) and complementary ining regions (CDR) using the error- prone PCR method. Mutagenesis is performed in a two-round PCR-based ch (Genemorph ll Random Mutagenesis kit obtained from Stratagene, La Jolla, CA, USA) using 1 ng of the DLLB||101GO8 or DLLB||115AO5 cDNA template, followed by a second error—prone PCR using 0.1 ng of product of round 1. After a polish step, PCR products are inserted via unique restriction sites into a vector designed to facilitate phage display of the VHH library. Consecutive rounds of in—solution selections are performed using decreasing concentrations of biotinylated recombinant human DLL4 (biot-thLL4) and trypsin elutions. Affinity- driven selections in a third round using cold thLL4 (at least 100x excess over biot-thLL4) are also med. No selections on murine DLL4 are included as (conservation of) cross-reactivity is assessed at the screening level. Individual mutants are produced as recombinant protein using an expression vector derived from , which contains the LacZ promoter, a resistance gene for ampicillin, a multiple g site and an ompA leader sequence (pAX50). E. coli TG1 cells are transformed with the expression vector library and plated on agar plates (LB + Amp + 2% glucose). Single colonies are picked from the agar plates and grown in 1 mL p-well plates. VHH expression is d by adding IPTG (1 mM). Periplasmic extracts (in a volume of ~ 80 uL) are ed according to standard methods and ed for binding to recombinant human and mouse Dll4 in a ProteOn (BioRad, Hercules, CA, USA) off-rate assay. In brief, a GLC n Sensor chip is coated with recombinant human Dll4 on the “ligand channels” L2 and L4 (with L1/L3 as reference channel), while “ligand channels” L3 and L6 is coated with mouse Dll4. Periplasmic extract of affinity matured clones is diluted 1/10 and injected across the “analyte channels” A1 -A6. An average off-rate is calculated of the wild type clones present in the plate and served as a reference to calculate te improvements.

In a second cycle, a atorial library is created by simultaneously randomising the susceptible positions identified in cycle one. For this, the full length DLLBII101 G8 or DLLBII115AO5 cDNA is synthesized by overlap PCR using oligonucleotides degenerated (NNS) at the randomisation positions and a rescue PCR is med. A list of the primers used for generating the combinatorial library can be found in Table 14 and SEQ ID NOs: 427 to 457. The randomised VHH genes are inserted into a phage display vector (pAX50) using ic restriction sites as described above (Example 2). Preparation of periplasmic extracts of individual VHH clones is performed as described before.

Table 14: Oligonucleotides affinity maturation libraries 101G08 combinatorial library 115A5 combinatorial library oligonucleotides oligonucleotides >101 GO8CL_de1-bis >115AO5CL_de_1 gaggtgoaattggtggagtotgggGGTGG gaggtgoaattggtggagtotgggGGTGGTCT TCTGGTTCAGGCTGGT (SEQ ID GGTTCAGCCAGGT (SEQ ID ) NO:427 ) >115A5CL_rev1-bis >1 o1 GO8CL_fwd_2 TGAGGAGACGGTGACCTGGGTCCC TCCTGCGCAGCTTCTGGTCGTA CTGACCCC (SEQ ID NO:444) CCTTCTCCAGCTACGCGATGG >1 15AO5CL_fwd_2 CT (SEQ ID NO:428) CTTCCGGCTTTACGWTCGG >101 GO8CL_fwd_3 CTCCTACGACATGTCTTGGG (SEQ CCAGGCAAAGAACGCGAGTWC ID ) GTAGCCGCAATCCGTTGGAGC >1 15A05CL1_rev_2 GGT (SEQ ID ) ACGCACCCCAGTATTCACCCTGACG >101 G080L_fwd_4 CGCCCAAATGTAGCGATCTGCAGC CTGATTCCGTTCAGGGTCGTTT (SEQ ID NO:446) CACCATCTCTCGTGACAACGC >1 15AO5CL_fwd_3 G (SEQ ID NO:430) AGGTCCGGAATGGGTGTCCKCTATC >101 fwd_5 AACTCTGGTGGTGGTAGCAC (SEQ CTGCAGATGAACTCTCTGAAAC lD NO:447) CGGAAGATACGGCAGTCTACT >1 15AO5CL_rev_3 AC (SEQ ID NO:431) TCTTCCGGTTTCAGGCTGTTCATCTG >101 GO8CL_fwd_6-4 CAGGTACAGCGTGTTTTTG (SEQ ID GACACTCGTCTGcgtCCGTACctg NOI448) TACGACYATTGGGGTCAGGGT >1 15AO5CL_fwd_4 A (SEQ ID NO:432) AAAGGTCGTTTCACCATCTCTCGTGA >101 GO8CL_fwd_6-3 CAACGCCAAAAACACGCTG (SEQ ID GACACTCGTCTGGVACCGTACct NO:449) gTACGACYATTGGGGTCAGGGT >1 15AO5CL_rev_4 A (SEQ ID NO:433) TGAAACGACCTTTTWCGWAGTCGGY >101 GO8CL_fwd_6-2 GGTGCTACCACCAC (SEQ GACACTCGTCTGcgtCCGTACG ID NO:450) AGTACGACYATTGGGGTCAGG >1 15AO5CL_fwd_5 GTA (SEQ ID NO:434) TGAAACCGGAAGATACCGCGGTATA >101 GO8CL_fwd_6-1 CGCTGCAGATCGCT (SEQ GACACTCGTCTGGVACCGTAC ID NO:451 ) GACYATTGGGGTCAG >1 15AO5CL_rev_5 GGTA (SEQ ID NO:435) CCATTCCGGACCTTTACCCGGAGAA >101 G080L_rev_2-2 CGACGAACCCAAGACATGTC (SEQ CAGACGAGTGTchgCGCACGG ID NO:452) TTTGCACAGTAGTAGACTGCCG >1 15AOSCL_fwd_6—1 T (SEQ ID NO:436) TACTGGGGTGCGTACGHATACGACT >101 GO8CL_rev_2—1 GTCAGGGTAC (SEQ ID CAGACGAGTGTCTRCCGCACG NO:453) GTTTGCACAGTAGTAGACTGCC >1 15AO5CL_fwd_6-2 GT (SEQ ID NO:437) TACTGGGGTGCGTACcagTACGACTA >101 GO8CL_rev_3 CTGGGGTCAGGGTAC (SEQ ID AGAGTTCATCTGCAGATAGACG ) GTGTTTTTCGCGTTGTCACGAG A(SEQ ID NO:438) >115AO5CL rev 6 >101 GO8CL_rev_4 CCGGAAGCTGCACAGCTCAGACGCA CTGAACGGAATCAGSGTAATAC GAGAACCACCTGGCTGAACC (SEQ GCAGTTYCACCGCTCCAACGG ID NO:455) AT (SEQ ID NO:439) >1 15AO5CL2_rev_2—2 >101 GO8CL_rev_5 CCCAGTAGTAACCCTGACG GCGTTCTTTGCCTGGAGCCTG CGCCCRAATGTAGCGATCTGCAGC CCAAGCCATCGCGTA (SEQ ID NO:456) GCT (SEQ ID NO:440) >115AO5CL2 rev 21 >101 GO8CL_rev_6 ACGCACCCCAGTAKTCACCCTGACG AATGTAGCGATCTGCAGC AGAAGCTGCGCAGGACAGACG (SEQ ID NO:457) GAGAGAGCCACCAGCCTGAAC CAG (SEQ ID NO:441) >101 GO8CL_rev1-bis GACGGTGACCTGGGT CCCCTGACCCCAAT (SEQ ID NO:442) Screening for binding to recombinant human D||4 in a ProteOn off-rate assay identifies Clones with up to 38-fold (DLLBII101GO8) and 11-fold (DLLBII115A05) improved off—rates (Table 15).

W0 2012/131076 Table 15: Off-rate screening of DLLBII101GO8 and DLLBII115AO5 affinity-matured Clones.

DLLBH101GO8 DLLBH129DO8 DLLBH129HO4 DLLBH129G1O 29HO7 DLLBH129802 29E11 DLLBH130FO6 DLLBH13OBOS DLLBH129DO1 DLLBH130DO6 DLLBH129G09 DLLBH129805 DLLBH130E03 DLLBH129HO5 DLLBH130AO5 DLLBH1SOBOZ 2012/055897 DLLB||129H02 DLLBII1SOBO4 DLLBII129EO7 DLLBII129EO3 DLLBII129A03 The best top DLLBII101GO8 variants and DLLBII115AO5 variants are cloned into sion vector pAX100 in frame with a C-terminal c-myc tag and a (His)6 tag.

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Characterization data are summarized in Table 21. l, the affinity matured VHHs show clear improvements in ty and potency, while their binding to lel4 and cyno D||4 is maintained and no binding to hDLL1 or hJAG1 is observed Table 17: |C50 (nM) values for affinity matured VHHs in hDLL4/hNotch1-Fc competition ELISA 2012/055897 2-0_ _1.4 Table 18: |C5o values (nM) of purified affinity matured VHHs blocking the interaction of human Notch1/Fc to human or mouse DLL4 expressed on CHO cells (FMAT) 015 21.2 Table 19: Affinity KD (nM) of purified ty matured VHHs on recombinant human DLL4 and mouse DLL4 -thLL4 rmDLL4 701608 5'65 4. 8E+04 2. 35—03 48.0 9.4E+04 (wt) 03 129Aos 2.1805 1.2304 ---- 129805 2.3E+05 7.9E—05 .2.7E+05 S41E- 129DO8 1.8E+O5 6.4E—O5 2.7E+O5 3403 u 2.9E- 129E11 1.9E+05 9.0E—05 0.5 2.5E+05 12 129W wees rse—os ---- 130303 2.2E+os asE-os ---- 13OFO6 2.0E+O5 _OE—05 anti- DLL4 5 3. 4E-04 _-hDLL4 115405 4. 0E— 2. 5E+05 1. 7E+05 9. OE- 133A09 4. 4E+05 :--013.5E+O5 4. 7E— 133GO5 5. 9E+O5 ::-I 4. 7E+05 3. 9E- 136CO7 6. 2E+05 5. OE+O5 4. 7E— anti- 3. 4E— DLL4 2. 3E+05 2012/055897 Table 20: E050 (nM) values of affinity matured VHHs for binding on CHO-hDLL4, CHO—mDLL4 and CHO-CDLL4 (FACS) —--- —--- WO 31076 Table 21: Characteristics of affinity-matured VHHs derived from DLLBII101GO8 and DLLBII115AO5 FM FMA ELI AT T FA SA hD mDL CS LL4 L4 (nM EC EC EC (nM) |C5o |C5o ) IC (nM (nM 50 50 5o 50 hD (nM) (nM (nM (nM LL1 mD ) L4 ) ) ) "I III-- ...-...“.- ..“...“.

........... ........... ........... DLL4 1.5 1.5 5.5 3.0 5.6 2.1 2.5 WO 31076 $1“ng rim: FA FA FA ELI CS CS CS SA 4 -Cl) 4 3 0' 3 O— nb: no binding 2012/055897 Example 8 Construction, production and characterization of ific VHHs targeting DLL4 and Ang2 using anti-serum albumin binding as half-life extension In a first cycle, the anti-DLL4 VHH DLLBII00018 (US 172398 A1) and the cycle 1 sequence optimized anti-AngZ VHHs 00042 (SEQ ID NO: 482), 00045 (SEQ ID NO: 484) and 00050 (SEQ ID NO:483) are used as building blocks to generate bispecific VHHs DLLANGBI|00001-00016. 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 5. An overview of the format and sequence of all bispecific VHHs is ed in Figure 16 and Table 22-A (linker sequences underlined), SEQ ID Nos 460-475. Expression levels are indicated in Table 22—B.

Table 22-A Sequences of ific VHH targeting DLL4 and Ang2 DLLANGBIIOOOOI DVQLVESGGGLVQPGGSSRLSCAASGR"FSSYAMAWYRQAPGKEREYVAAIRWSGG"AYYADSVKGR FTISRDVAKNTVYLQMNSLRPEDTAVYYCA.RAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYC"IGGSASRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG QPGGS-R-SCAASGFTFDDYALGWFRQAPG(EREGVSCIRCSDGSTYYADSVKGRF"ISSDNSK r1V YLQMNSSRPEDTAVYYCAASIVPRSK-*PY<YDAWGQG"SVTVSSGGGGSGGGSEVQLVESGGG-VQ PGGSLRSSCAASGFTFDDYALGWFRQAPG<<R“GVSCIRCSDGSTYYADSVKGRFTISSDNSKN"VY JRPEDTAVYYCAASIVPRSKL4PY4YDAWGQGTSVTVSS (SEQ ID NO: 460) DLLANGBIIOOOOZ DVQLVESGGGLVQPGGSLRLSCAASGETFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGR FTISS3VSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVISGGGLVQPGGSSRLSCAASGFTFDDYALGWFRQAPG<EREGVSCIRCSDGSTYYADSVKGRF D SKNTVYLQMNS-RP7DTAVYYCAASIVPRSKL*FY”YDAWGQGTLVTVSSGGGGSGGGSEV . SGGGLVQPG S-R-SCAASGF"FSSFGMSWVRQAPGKG.7WVSSISGSGSDTSYADSVKGRFT ISR AK"TLYLQWNS-RPEDTAVYYC"IGGSLSRSSQGTLV"VSSGGGGSGGGSEVQLVFSGGG-V Q?GGS-RVSCAASGR"FSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGRF”ISRDNAKN"V YLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSS (SEQ ID NO: 461) DLLANGBIIOOOO3 SGGGLVQPGGSERASCAASGR"FSSYAMAWYRQAPG<EREYVAAIRWSGGTAYYADSVKGR F"ISRDNAKNTVY-QMNS-QPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV Q-VESGGGLVQPG S-R-SCAASGF"FSSFGMSWVRQAPG(G.7WVSSISGSGSDTSYADSVKGRFT ISRDNAK"TLYLQWNS-RPEDTAVYYC"IGGSLSRSSQGTSV"VSSGGGGSGGGSEVQLVFSGGG-V Q?GGS-RWSCAASGF"SDDYAIGWFRQAPGKEREGVSSIRD ADSVKGRF”ISSDVSKN"V LQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 462) DLLANGBIIOOOO4 DVQSVESGGGLVQPGGSERASCAASGF"LDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYAJSVKGR F"ISSDNSKNTVY - -RPEDTAVYYCAAVPAGRLRFGEQWYPLYEYJAWGQGTSVTVSSGGGGS GGGSEVQLVESGGG- SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAK"’- -QWNSLRPED"AVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGS*VQLV~ SGGG-VQPGGSLRVSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGG"AYYADSVKGR?TISR3 NAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSS (SEQ ID NO: 463) DLLANGBIIOOOO5 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR F"ISRDNAKNTVYAQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTSVTVSSGGGGSGGGSEV Q-VESGGGLVQPG S-R-SCAASGFTFSSFGMSWVRQAPG(GLEWVSSISGSGSDTLYADSVKGRFT ISRDNAK"TLYLQMNS-RP7DTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGIV R-SCAASGFTIDDYAIGWFRQAPGKEREGVSSIRD DGSTYYADSVKGRFTISSDNSK r1V YLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESG GGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNS KNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 464) DLLANGBIIOOOO6 DVQ-V7SGGGLVQPGGSIRLSCAASGR"FSSYAMAWYRQAPG(EREYVAAIRWSGGTAYYADSV<GR F"ISRDNAKNTVYIQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGS Q-VESGGGLVQPG SLR-SCAASGFTFSSFGMSWVRQAPGKG.7WVSSISGSGSDTIYADSVKG ISRDNAKTTLYLQWNSLRPEDTAVYYC"IGGSLSRSSQGTLV"VSSGGGGSGGGSEVQLVFSGGG QPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSDGITYYADSVKGRFTISRDNSKNTV LQMNSLRPEDTAVYYC TDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID V0: 465) DLLANGBIIOOOO7 VQPGGSJQLSCAASGFAJDYYAIGWFRQAPGKEREGVSCISSSDGITYYADSVKGR D SKNTVY-QMNS-RPEDTAVYYCA"DSGGYIDYDCMGIGYDYWGQGTLVTVSSGGGGSGGG 4SGGGLVQPGNSIRLSCAASGF"FSSFGMSWVRQAPG<GLEWVSSISGSGSDTLYADSVKG RDNAKTTLYIQMNSLRPEDTAVYYC"IGGSLSRSSQGTIVTVSSGGGGSGGGSEVQLVESGG LVQPGGSLRLSCAASGRHFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGRFTISRDNAK QMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTL _VSS (SEQ ID NO: 466) DLLANGBIIOOOOB ESGGGLVQPGGSIRLSCAASGF"LDDYAIGWFRQAPGKEREGVSSIRD DGSTYYADSVKGR F"1 I S S 3N S KNTVYLQMN S IR}? EDTAVYYCAAVPAGRLRFGIIQWYPLYEYJAWGQGTLVTVS S GGGGS GGGSEVQLVESGGG-VQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSDNSKN"VYIQMNSLRPED"AVYYCAAVPAGR-RtG4QWYPVY4YDAWGQGTLVTVSS§ GGGSGGGSFVQLVFSGGGVVQPGNSVR-SCAASGFTFSSFGWSWVRQAPGKGLEWVSSISGSGSDTJ YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQIE’GGSLRJSCAASGR'TFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGRF"1 KNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYD{WGQGTLVﬂVSS (SEQ ID NO: 467) DLLANGBIIOOOO9 DVQ-VESGGGLVQPGGS-R-SCAASGRTFSSYAMAWYRQAPG(EREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQWNSLRPEDTAVYYCA.RAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGRFT ISSDNSKVTVYLQMVSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQ LVESGGGAVQPGGS-R-SCAASGFTFDDYA4GWFRQAPG<4R4GVSCIRCSDGS"YYADSVKGRFTI SS3 SKN"VYLQMNS-RPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVEVSSGGGGSGGGSEVQL ESGGGLVQPGNSLR-SCAASGFTFSSFGMSWVRQAPG<G.7WVSSISGSGSDTIYADSVKGRFTIS (TTVYLQMNS-RP7DTAVYYCTIGGSISRSSQGTIVTVSS (SEQ ID NO: 468) BIIOOOIO DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNSKNTVYLQMN LRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE ESGGGLVQPGGS-Q-SCAASGFTFDDYALGWFRQAPG(EREGVSCIRCSDGSTYYADSVKGRF DNSKNTVYLQMNSIRPEDTAVYYCAASIVPRSKL~PY<YDAWGQGTIVTVSSGGGGSGGGSEV ESGGGTN’QPGGSR-SCAASGR""FSSYAMAWYRQAPGPGR4YVAAIRWSGG""AYYADSVKGRF"1 RD AKNTVYLQMNS-RPEDTAVYYCANRAPDTRLAPYEYD{WGQGTLV“VSSGGGGSGGGSEVQ.

SGGGLVQPGNSVR-SCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTIYADSVKGRETIS NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 469) DLLANGBIIOOOll IVESGGGLVQPGGSIRASCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR NAKNTVYJQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTIVTVSSGGGGSGGGSEV Q-V7SGGGLVQPGGSLR-SCAASGF"LDDYAIGWFRQAPGK~R4GVSSIRDNDGSTYYADSVKGRFT ISSDNSKNTVYLQWNSLRPEDTAVYYCAAVPAGRLRFGEQWYP.YFYDAWGQGTIVEVSSGGGGSGG ESEVQ-VFSGGGLVQPG SVRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 470) DLLANGBIIOOOIZ DVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISS3NSKNTVYAQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYJAWGQGTAVTVSSGGGGS GGGSEVQLVESGGG-VQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADS VKGRF"ISRDNAK "VYIQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTIVEVSSGGGGSGG ESEVQ-VFSGGGLVQPG S-RLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRETISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 471) DLLANGBII00013 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYAQM SLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV Q-VESGGGLVQPGGS-R-SCAASGFTLDDYAIGWFRQAPGK~R4GVSSIRDNDGSTYYADSVKGRFT K TVYLQMNS-RP7DTAVYYCAAVPAGRLRFG*QWYP-Y*YDAWGQGTLVEVSSGGGGSGG VFSGGGLVQPGGS-RLSCAASGF"LDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVK GQETISSUNSKNTVYIQWNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSEEE GSGGGSEVQLVESGG SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYA DSVKGRFTISRDNAK""LY4QMNSLRPED"AVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 472) DLLANGBIIOOOl4 DVQLVESGGGLVQPGGSERLSCAASGF"LDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISS3NSKNTVYLQMNSERPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS QLVESGGG-VQPGGSLRLSCAASGF"LDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSDNSKN"VYEQMNSLRPED"AVYYCAAVPAGR-RFGFQWYPLYEYDAWGQGTLVTVSSE GGGSGGGSEVQLVESGG LVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAY YADSVKGRFTISRDNAKVTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYJHWGQGTLVTVSSEEE GSGGGSEVQLVESGGGEVQPGNSLRLSCAASGFTFSSFGMSWVQQAPGKG-EWVSSISGSGSDTLYA DSV<GRFTISRDNAKT"-Y-QMNSLRPEDTAVYYCTIGGSLSRSSQGTLVﬂVSS (SEQ ID NO: 473) DLLANGBIIOOOl5 DVQ-VESGGGLVQPGGS-¥-SCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FT:SRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDiWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSDGITYYADSVKGRFT IS?) SKNTVYLQMNSERPEDTAVYYCA"DSGGYIDYDCMGEGYJYWGQG"EVTVSSGGGGSGGGSE VQ-VESGGGLVQPGNS-R-SCAASGFTFSSFGMSWVRQAPG<G EWVSSISGSGSDTLYADSVKGRF TISRD AK"TLYLQMNS-RPEDTAVYYC"IGGSLSRSSQGTEVﬂVSS (SEQ ID NO: 474) DLLANGBIIOOOI6 DVQ-VESGGGLVQPGGSERLSCAASGFALDYYAIGWFRQAPG(EREGVSCISSSDGITYYADSVKGR " 3NSKNTVYLQMNSLRPEDTAVYYCATDSGGYIDYDCW LGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKG RF"ISQDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYJHWGQGTLVTVSSGGGGSGGGSE VQ-VESGGGLVQPGNSLRESCAASGFTFSSFGMSWVRQAPG<G SGSGSDTLYADSVKGRF TISRD AKTTLYLQMNSLRPEDTAVYYC"IGGSLSRSSQGTEVﬂVSS (SEQ ID NO: 475) Table 22-3 Expression (mg/L) DLLAI\GBIIOOOOZ DLLANGBIIOOOO3 DLLAI\GB1100004 U] \] DLLANGBIIOOOOS 00 b.) DLLA\GB1100006 DLLANGBIIOOOO7 DLLAVGBHOOOOS H 00 DLLAI\GB1100009 D.) O DLLA\IGBIIOOO 10 )—t W DLLAI\GBIIOOOI 1 DLLAVGBIIOOO 12 GB1100013 DLLANGBIIOOO 14 DLLA\GBIIOOO 15 DLLANGBIIOOO 16 n.e.: no expression performed To explore the anti-DLL4 blocking properties in comparison with the monovalent building block DLLBIIOOO18, all purified bispecific VHHs are analyzed in the hDLL4/hNotch1 competition ELISA (see Example 5.1 as described in patent US 2011/0172398 A1) e 17) and the CHO-hDLL4 / CHO-mDLL4 competition FMAT (see Example 5.3 as described in patent US 2011/0172398 A1) (Figure 18). Here, the ELISA ition assay is performed with a fixed concentration of 8 nM biotinylated hDLL4. Both ELISA and the FMAT competition assay are also performed after preincubation of the VHH with 12.5 uM and 25 uM human serum albumin, tively.

A summary of |C5o values and % inhibition is shown in Table 23.

Table 23: |C5o values (nM) and % tion in hDLL4/hNotch1 competition ELISA and CHO—hDLL4 and CHO-mDLL4 competition FMAT.

W0 2012/131076 BIIOOOO2 . E“? $1: -.4; \o 92 10.5 \J-"M ' BIIOOOO9 .U' u: 14.0 -.U‘ N 95 14.4 .\‘ 4:- DLLANGBIIOOO 10 -10.0 100 8iv P“ \o 97 9° \0 85 DLL4 Fab - -9°9N9°9°9°9°9°9°9°9°9°9°9°9°9°9°¥9°f° 4;43O\U)NU1WNU1r—-\lO43Nﬁ—*0\,_.3:4Ch3““ PFL 9° N 85 - .w N 65 DLLB1100018 3“» 9° u: 70 - 87 .0 N 70 P‘P’P’r‘!‘ moow\)oo DLLAI\GBIIOOOO3 92 11.4 - 92 9° >— .U‘ — ,_1 OO DLLANGBIIOOOO4 97 12.1 H O ._. - 87 >1 w O\ . 4:. \0 .p DLLAI\GBIIOOOO5 83 13.1 - 93 9° u] DLLANGBIIOOOO6 94 15.4 00 . 00 - 87 1—9 DLLAI\GBIIOOOll 9° >— 7 - 9° 9.) 2 DLLANGB1100012 ° 9—! o D—l - 00 ; f1 T‘P'i‘ﬂoo— DLLANGBIIOOO 13 - \ON DLLANGBIIOOO 14 DLL4 Fab - 4.2 DLLB1100018 DLLANGBIIOOOO7 DLLANGBIIOOOO8 DLLANGBIIOOO 15 DLLANGBIIOOO 16 -—--Emn n.d., not determined Additionally, in order to determine cross-reactivity of the bispecific VHHs to murine and cynomolgus DLL4, a FACS binding ment is performed. Briefly, CHO cells overexpressing mouse and cynomolgus DLL4 are used for a titration binding ment of the VHHs. After a 30 min incubation on ice, all samples are washed and a 2—step detection using anti-c-myc followed by goat-anti mouse IgG-PE labeled is performed. CHO cells overexpressing human DLL4 are taken as reference. The mean MCF value is determined using a FACS Array and used for calculation of the E050 value (Table 24; Figure 19).

Table 24: E050 values of bispecific VHHs binding to human, mouse and cyno DLL4 overexpressed on CHO cells (FACS) DLLBIIOOOIS BIIOOOO 1 DLLAI\GB1100003 DLLA\GBIIOOOOS DLLANGBIIOOOO7 In order to determine cross-reactivity to mouse DLL4 and rat DLL4, a binding ELISA is med. In brief, recombinant mouse DLL4 (R&D Systems, Minneapolis, MI, USA) and rat DLL4 is coated overnight at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, y). Wells are blocked with a 1% casein solution. VHHs are applied as dilution series and binding is detected biotinylated anti-VHH 1A4 followed by extravidin-HRP. 1A4 is an anti-VHH VHH (generetad in-house by Ablynx NV). As nce binding to human DLL4 is measured. EC50 values are summarized in Table 25 and Figure 20.

Table 25: EC50 values of bispecific VHHs g to human, mouse and rat DLL4 (ELISA) ---hDLL4 EC50 mDLL4 rDLL4 DLLBIIOOOIS 2.6 DLLANGBIIOOOOI 3 .4 Absence of binding to the gous human ligands DLL1 and Jagged-1 is assessed via a solid phase binding assay (ELISA). In brief, 1 ug/mL of recombinant human DLL1 (Alexis, San Diego, CA, USA) or recombinant human Jagged-1 (Alexis, San Diego, CA, USA) is coated ght at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a 1% casein solution. VHHs are applied as dilution series and binding is detected biotinylated anti-VHH 1A4 ed by idin—HRP. All bispecific VHH are considered as being non-cross reactive to these homologous ligands. Results are shown in Figure 21.

To explore the anti-Ang2 blocking properties in comparison with the monovalent anti- Ang2 building blocks 00042, 00045 and 00050, all purified bispecific VHHs are analyzed in a human Ang2/hTie2-Fc (Figure 22-1), mouse Ang2/mTie2 (Figure 22-2) and cyno Ang2/cTie2 (Figure 22-3) competition ELISA. This assay is also performed after tion of the VHH with 0.5 uM human serum albumin. A summary of |C5o and % inhibition values is shown in Table 26.

W0 2012/131076 Table 26: |C5o values (pM) and % inhibition in human, mouse and cyno AngZ/Tie2 competition ELISA A"d:5 ,_..g -O\\] "oox U.) 100 U] C\ \O - ._. O DLLANGBIIOOOO 1 DLLANGB1100002 DLLANGBIIOOOO9 NM \O-P -U.) 00 DLLANGBIIOOO 10 AMG386 LAUI 4300 \00 DLLAI\GBIIOOOO3 - DLLANGBIIOOOO4 UIUI coc7\,_.o r—I OO -D.) i— OO DLLANGBIIOOOOS U] i— OO -U.) 4; )—‘ OO DLLANGBIIOOOO6 .P r—I OO DLLANGBIIOOO 1 1 NU.) N-Ik \IN p—d OO OO - >—I OO DLLANGBIIOOO 12 U.) C\ r—a OO -U.) i—i DLLANGB1100013 H OO GB1100014 LAD-)9.) mmq DLLANGBIIOOO 15 DLLANGB1100016 i n.d., not determined ties of certain DLL4-Ang2 ific VHHs for human serum albumin have been determined (see Example 5) and are shown in Table 27. The affinity constant KD is calculated from resulting association and dissociation rate constants ka and kd.

Table 27: Affinity KD of purified VHHs for human serum albumin (HSA) ka kd I(D (l/Ms) (l/s) (nM) ,2E+05 1.8E-03 4 7.8E+04 4.9E-03 63 1.2E+05 4.7E-03 39 7.5E+04 4.6E-03 61 DLLANGBIIOOOO7 i 5 4.6E-03 42 DLLANGBIIOOO 12 i 8 . 4E+04 5 . 5E-03 DLLANGBIIOOO 14 - i 5.7E+04 5.8E-03 102 In a second cycle, the anti-DLL4 VHH DLLBII00018 (US 2011/0172398 A1) and the final sequence optimized anti—Ang2 VHHs 00921 (SEQ ID NO: 485), 00938 (SEQ ID ) and 00956 (SEQ ID NO:488) are used as building blocks to generate bispecific VHHs DLLANGBII00017-00019. A c fusion to a serum albumin binding VHH is used as ife extension methodology. ng blocks are linked via a 9 Gly-Ser flexible linker. An overview of the format and sequence of all bispecific VHHs is depicted in Figure 23 and Table 28 (linker sequences underlined), SEQ ID Nos 476-478.

Table 28 Sequences of bispecific VHH targeting DLL4 and Ang2 DLLANGBIIOOOl7 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR r1ISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV .VTSGGG-VQPGNS-R-SCAASGFTFSSFGWSWVRQAPGKG.7WVSSISGSGSDTSYADSVKGRFT SRDNAK""LYLQMNSLRPEDTAVYYCTIGGSLSRSSQG"LV"VSSGGGGSGGGSEVQLVESGGGSV PGGS-R-SCAASGF"FDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRF":SSDNSKN"V LQWNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTSVTVSSGGGGSGGGS? Q7VESGGGLVQ PGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFT_SSDNSKNTVY LQMNSSRPEDTAVYYCAASIVPRSKLEPYEYJAWGQGTLVTVSS (SEQ ID NO: 476) DLLANGBIIOOOlB SGGGLVQPGGS-R-SCAVSGI"LDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGR FTISSDNSKNTVYSQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTSVTVSSGGGGS GGGSTVQ-VESGGG-VQPGGSLRLSCAASGR"FSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADS VKGRF"ISRDNAKV"VYSQMNSLRPED"AVYYCANRAPDTRLAPYEYDHWGQGTLVHVSSGGGGSGG ESEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRF"ISRJNAKTTSYLQWVSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 477) BIIOOOl9 DVQ-VTSGGGLVQPGGS-R-SCAASGF"LDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGR FTISSDNSKNTVY-QMNS-RPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTSVTVSSGGGGS GGGSTVQ-VESGGG-VQPGGSLRLSCAASGF"LDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADS VKGRFTISSDNSK r1VYSQVINSLRPED"AVYYCAAVPAGRRFGF‘QWYPVYTYDAWGQGTLVTVSSE GGGSGGGSFVQLVFSGGGDVQPGGSLRVSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAY YADSVKGRFTISRDNAKVTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSEEE GSGGGSEVQLVESGGGSVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYA DSVKGRFTISRDNAKT".YLQMNSLRPTDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 478) To explore the anti-DLL4 blocking properties in ison with the monovalent building block DLLBII00018, all purified bispecific VHHs are analyzed in the hDLL4/hNotch1 competition ELISA (see Example 5.1 as described in patent US 2011/0172398 A1) (Figure 24), the CHO-hDLL4 / CHO-mDLL4 ition FMAT (see Example 5.3 as described in patent US 2011/0172398 A1) (Figure 25) and the hDLL4 mediated Notch1 activation (reporter gene) assay (see Example 5.4 as described in patent US 2011/0172398 A1) (Figure 26). Here, the ELISA competition assay is performed with a fixed concentration of 8 nM biotinylated hDLL4. The ELISA competition assay, the FMAT competition assays and the reporter gene assay are also performed after preincubation of the VHH with 12.5 uM, 25 uM and 162 uM human serum n, respectively. A summary of |C5o values and % inhibition is shown in Table 29. co_EmQEoo 3.58-010 Suwbcwocoo ucm Ego: stone .659; 3.55-010 “w wocmgmtmug .<m_._m_ 549:6:0 >wmmm cozzoquo 823w >mwwm 340:-030 coE>> EBoZEvjoc 8ch 3:30. :03: 54%. <m5m .659: m cozmbcmocoo c_ vac II> coEQEE om wme 8 “$59; “we vmm: E vcm Eoﬁoz 99> 020%: :25 8:2va ” II> 5c mo:_m> .98 *0 v.30: S w a 82 H H mco_&m> coEQEE vcm w 5N H ﬂaw... _n_ ooodmxim oooimﬁﬂéﬁiﬂ oooim—OZANAQQ oooimﬁuZ<AAQ gm 88$ 25::me ism 93 3V Binding to human DLL4, mouse DLL4 and rat DLL4 is assessed in Biacore. Briefly, kinetic analysis of the bispecific VHHs is performed by SPR on a Biacore T1OO ment. Recombinant human DLL4 (R&D Systems, Minneapolis, MI, USA) and mouse DLL4 (R&D Systems, Minneapolis, MI, USA) are immobilized on a CM5 chip via amine coupling. VHHs are injected over these surfaces at ent concentrations n 2.5 and 1,800 nM. Samples are injected for 2 min and d to dissociate for min at a flow rate of 45 ul/min. Between sample injections, the es were regenerated with a 100s pulse of 10mM glycine pH 1.5. Association/dissociation data are evaluated by fitting a 1:1 interaction model (Langmuir binding). The affinity constant KD is calculated from resulting association and dissociation rate nts ka and kd (Table 30).

Table 30: Binding kinetcs of bispecific VHHs for binding to human and mouse DLL4 (Biacore) — hDLL4 mDLL4 ka ka KD ka k8 KD (1/MS) (1/5) (nM) (1/MS) (1/S) (nM) DLLANGBII00017 1.6E+05 9.55-05 “9.1905 2.6E-04 Additionally, in order to determine cross-reactivity of the bispecific VHHs to murine and cynomolgus DLL4, a FACS g experiment is performed. Briefly, CHO cells overexpressing mouse and cynomolgus DLL4 are used for a titration g experiment of the VHHs. After a 30 min incubation on ice, all samples are washed and a 2—step detection using biotinylated anti-VHH 1A4 followed by PE labeled streptavidin is performed. CHO cells overexpressing human DLL4 are taken as reference. The mean MCF value is determined using a FACS Array and used for calculation of the E050 value (Table 31; Figure 27).

Table 31: E050 values of bispecific VHHs binding to human, mouse and cyno DLL4 overexpressed on CHO cells (FACS) CHO-hDLL4 CHO—mDLL4 CHO-CDLL4 EC50 (11M) EC50 (11M) EC50 (11M) In order to determine cross-reactivity to mouse DLL4 and rat DLL4, a binding ELISA is performed. In brief, recombinant mouse DLL4 (R&D Systems, polis, MI, USA) and rat DLL4 is coated overnight at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a 1% casein solution. VHHs are applied as dilution series and binding is detected biotinylated anti—VHH 1A4 followed by extravidin—HRP. As reference binding to human DLL4 is measured. E050 values are summarized in Table 32 and Figure 28.

Table 32: E050 values of bispecific VHHs binding to human, mouse and rat DLL4 hDLL4 mDLL4 cDLL4 EC50 (11M) EC50 (11M) EC50 (11M) Absence of g to the homologous human ligands DLL1 and Jagged-1 is assessed via a solid phase binding assay (ELISA). In brief, 1 ug/mL of recombinant human DLL1 (Alexis, San Diego, CA, USA) or recombinant human Jagged-1 (Alexis, San Diego, CA, USA) is coated overnight at 4°C in a l MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a 1% casein solution. VHHs are d as dilution series and binding is detected biotinylated anti-VHH 1A4 ed by extravidin-HRP. All bispecific VHH are considered as being non-cross reactive to these homologous s. Results are shown in Figure 29.

To explore the anti-Ang2 blocking properties in comparison with the final sequence zed monovalent anti-AngZ building blocks 00921, 00938 and 00956, all purified bispecific VHHs are analyzed in a human AngZ/hTie2 (Figure 30-1), mouse AngZ/mTie2 (Figure 30-2), cyno Ang2/cTie2 (Figure 30-3), a hAng1/hTie2 (Figure 31) competition ELISA and the hAng2 mediated HUVEC survival assay (Figure 32). A summary of |C5o and % inhibition values is shown in Table 33. cozzquoo 0B5: 3:33 BEE 3 h<m_._m_ Nwiﬁmia 05$ cozzoquo 39 moEch< oc>o 0cm I'I'l .WJ.£ 3308 02 I 59:35 IEIEIEI IEIIIIIII c_ >88 IIIIIIIIIIIEIIIIIIII IEIIEIEIEIIIIEIIIIIIII 828%: .93st o\o om>oz umEELBmU 0cm mg 53 9 336 BEBE ﬁg 3,2 NOSE Sooozm—Ozamwiﬂ ﬁmEELBmU ”mm Em 0.9m... <m3m 6.: Affinities of DLLANGBIIOOOl719 for human, mouse, cyno and rat Ang2 (see Example 5) have been determined and are shown in Table 34.

Table 34: Binding kinetics of purified VHHs for recombinant human, cyno, mouse and rat Ang2 —human AngZ-FLD cyno AngZ-FLD ka kd KD ka kd KD (l/Ms) (l/s) (M) (l/Ms) (l/s) (M) 1.90E+06 1.30E-02 6.60E-09 2.50E+06 1.20E-02 09 —mouse AngZ-FLD rat AngZ-FLD k3l kd KD ka kd K1) (l/MS) (1/S) (M) (l/MS) (1/S) (M) 9.10E+05 1.50E-02 1.70E-08 6.70E+05 3.30E-02 4.90E-08 Affinities of DLLANGBIIOOOl719 for human, mouse and cyno serum albumin have been determined (Example 5) and are shown in Table 35. The ty nt KD is calculated from resulting ation and dissociation rate constants ka and kd.

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Claims

What we claim is:

1. A bispecific binding molecule comprising - at least one Ang2-binding component, - at least one Dll4-binding component, and - at least one serum albumin binding component, wherein said Ang2-, Dll4- and serum albumin binding components are immunoglobulin single le domains, each globulin 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 ific g les having (i) the CDR sequences as present in DLLANGBII00017 (SEQ ID NO: 476), (ii) the CDR ces as present in DLLANGBII00018 (SEQ ID NO: 477), (iii) the CDR sequences as present in DLLANGBII00019 (SEQ ID NO: 478).

2. The bispecific binding molecule of claim 1, wherein said immunoglobulin single variable s are VHHs.

3. The bispecific binding molecule of claim 2, selected from the group consisting of bispecific binding molecules (i) to (iii), the molecules (i) comprising - the DLLBII00018 VHH domain as the Dll4-binding component, - the Alb11 domain (SEQ ID NO: 519) as the serum albumin binding component, and - two ANGBII000921 VHH domains (SEQ ID NO: 485) as the inding domains, in this order; the molecules (ii) comprising - one Ang2-binding domain, having a CDR1 having the sequence DYAIG (= SEQ ID NOs: 495, 498, 513, 516), a CDR2 having the sequence AIRSSGGSTYYADSVKG (= SEQ ID NOs: 514, 517), and a CDR3 having the sequence VPAGRLRYGEQWYPIYEYDA (= SEQ ID NO: 515), - the DLLBII00018 domain as the Dll4-binding component, and - the Alb11 domain (SEQ ID NO: 519) as the serum albumin binding component, in this order; the molecules (iii) comprising - two Ang2-binding domains, each having a CDR1 having the sequence DYAIG (= SEQ ID NOs: 495, 498, 513, 516), a CDR2 having the sequence AIRSSGGSTYYADSVKG (= SEQ ID NOs: 514, 517), and a CDR3 having the sequence VPAGRLRFGEQWYPLYEYDA (= SEQ ID NO: 518), - the DLL00018 domain as the Dll4-binding component, and - the Alb11 domain (SEQ ID NO: 519) as the serum albumin g component, in this order.

4. The bispecific binding molecule of claim 3, selected from the group consisting of (i) DLLANGBII00017 (SEQ ID NO: 476), (ii) DLLANGBII00018 (SEQ ID NO: 477), and (iii) DLLANGBII00019 (SEQ ID NO: 478).

5. A ific binding molecule as d in claim 1, substantially as before described with reference to any one of the examples.

6. A nucleic acid molecule ng a bispecific binding molecule of any one of claims 1 to 4.

7. A vector containing a nucleic acid molecule 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 molecule of any one of claims 1 to 5 as the active ingredient.

10. The pharmaceutical ition of claim 9 for the treatment of a disease that is associated with Dll4-mediated 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 diseases. Boehringer Ingelheim International GmbH By the Attorneys for the Applicant N & FERGUSON Per: