WO2016059057A1 - Vegfr-2 binding polypeptides - Google Patents

Vegfr-2 binding polypeptides Download PDF

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Publication number
WO2016059057A1
WO2016059057A1 PCT/EP2015/073687 EP2015073687W WO2016059057A1 WO 2016059057 A1 WO2016059057 A1 WO 2016059057A1 EP 2015073687 W EP2015073687 W EP 2015073687W WO 2016059057 A1 WO2016059057 A1 WO 2016059057A1
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seq
vegfr
binding
sequence
binding polypeptide
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PCT/EP2015/073687
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French (fr)
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Fredrik Frejd
Susanne KLINT
Stefan STÅHL
John LÖFBLOM
Filippa FLEETWOOD
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Affibody Ab
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds

Definitions

  • the present disclosure relates to a class of engineered polypeptides having a binding affinity for vascular endothelial growth factor receptor 2 (in the following referred to as VEGFR-2).
  • VEGFR-2 vascular endothelial growth factor receptor 2
  • the present disclosure also relates to the use of such a VEGFR-2 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.
  • angiogenesis plays a central role in the process of solid tumor growth and metastasis as well as in healing and tissue remodeling.
  • Tumor angiogenesis is a complex process dependent on a multi-faceted program of endothelial cell activation, stromal cell and endothelial-progenitor recruitment,
  • Angiogenesis is regulated in normal and malignant tissues by the balance of angiogenic stimuli and angiogenic inhibitors that are produced in the target tissue and at distant sites (Fidler IJ et al., (1998) Cancer J. Sci. Am vol.4, no.1; 58-66).
  • a key angiogenesis signaling system that regulates proliferation and migration of endothelial cells, comprises proteins of the vascular endothelial growth factor (VEGF) family and their receptors (vascular endothelial growth factor receptor 1, 2 and 3, abbreviated VEGFR-1, VEGFR- 2 and VEGFR-3, respectively).
  • VEGF-A also known as vascular permeability factor, VPF
  • VPF vascular permeability factor
  • VEGF-A is a multifunctional cytokine that is induced by hypoxia and oncogenic mutations and can be produced by a wide variety of tissues (Kerbel RS et al., (1998) Mol. Med., 4(5):286-295; Mazure NM et al., (1996) Cancer Res., 56:3436-3440).
  • the recognition of VEGF-A as a primary stimulus of angiogenesis in pathological conditions has led to various attempts to block VEGF-A activity.
  • the VEGF family of soluble agonists includes VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF). These factors bind to the three cognate receptors VEGFR-1 (also known as fms-like tyrosine kinase 1/Flt-1), VEGFR-2 (also known as Flk-1/KDR) and VEGFR-3 (also known as Flt-4) (Clarke JM et al., (2013) Expert Opin. Biol. Ther.13(8):1187-1196). VEGF-A binds to VEGFR-1 and VEGFR-2.
  • VEGFR-1 also known as fms-like tyrosine kinase 1/Flt-1
  • VEGFR-2 also known as Flk-1/KDR
  • VEGFR-3 also known as Flt-4
  • VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF-A and is widely considered to be the main receptor driving angiogenesis.
  • the VEGFRs belong to the immunoglobulin subclass of the receptor tyrosine kinase (RTK) super family and have seven Ig-like extracellular domains with a single transmembrane helix with an intracellular kinase region (Olsson AK et al., (2006) Nat Rev Mol Cell Biol 7(5):359-71; Ferrara N et al., (2004)
  • VEGFR-2 Upon binding of VEGF-A, VEGFR-2 can form either a homodimer or a heterodimer complex with VEGFR-1, resulting in intracellular tyrosine phosphorylation (Olsson AK et al., (2006) supra; Ferrara N et al., (2004) supra).
  • VEGFR stimulation leads to downstream activation of the mitogen-activated protein kinase (MAPK) pathway and promotion of endothelial cell proliferation as well as phosphatidylinositol 3’ kinase (PI3K) activity, ultimately leading to increased cell survival through AKT/PKB, cell migration and vascular permeability via expression of endothelial nitric oxide synthase. Additionally, activation of VEGFR promotes vascular permeability, actin remodeling, and cell migration by way of the Src/TSAd and P38/MAPK pathways (Ferrara N et al., (2004) supra).
  • VEGFRs are expressed at high levels in many types of human solid tumors, including lung, breast, renal, ovarian, glioma and gastrointestinal tract carcinomas (Clarke JM et al., (2013) Expert Opin. Biol. Ther.13(8):1187- 1196), and the inhibition of VEGFR signaling has recently emerged as a potential therapy method for cancers.
  • VEGFR-2 has been shown to play an important role in the direct regulation of angiogenesis and mitogenic signaling and therapies targeting VEGFR-2 signaling have been clinically validated with the FDA approvals of the VEGFR-2 inhibitor bevacizumab, a fully humanized mAb (Presta LG et al., (1997) Cancer Res.57:4593-4599), and the small- molecule VEGFR signaling inhibitors sorafenib (Wilhelm SM et al., (2004) Cancer Res.64:7099-101) and sunitinib (Mendel DB et al., (2003) Clin Cancer Res.9:327-337, O’Farrel AM et al., (2003) Blood.101:3597-3605) for the treatment of multiple malignancies including colorectal, non-small cell lung (NSCLC), renal cell and hepatocellular carcinomas (Hurwitz H et al., (2004) N Engl J Med.350(23):2335-42; Sandler A e
  • VEGFR antagonists for example lenvatinib (Matsui J et al., (2008) Clin Cancer Res.14:5459-5465) and motesanib (Polverino A et al., (2006) Cancer Res.66:8715-8721)
  • pazopanib and regorafenib were recently approved for treatment of renal cell carcinoma and colorectal cancer, respectively.
  • VEGFR-2 signaling which culminate in angiogenesis include a potent increase in vascular permeability and promotion of endothelial cell migration, proliferation and survival (Olsson AK et al., (2006) supra).
  • anti-angiogenic therapy with the potential for both high affinity and high specificity blockade of VEGFR-2 is an attractive goal for the treatment of malignancies and various diseases associated with excessive angiogenesis and cell migration, such as cancers manifesting solid tumors.
  • monoclonal antibodies are not always optimal for targeting solid tumors (neither for diagnostic nor for therapeutic pay-load purposes).
  • Therapeutic effect is dependent on an efficient distribution of the drug throughout the tumor and molecular imaging depends on a high ratio between tumor uptake and surrounding normal tissue. Since tumor penetration rate (including extravasation) is negatively associated with the size of the molecule, the relatively large antibody molecule inherently has poor tissue distribution and penetration capacity.
  • tumor penetration rate including extravasation
  • the extraordinarily long in vivo half-life of antibodies results in relatively high blood signals and thereby relatively poor tumor-to-blood contrasts.
  • agents with a high affinity for VEGFR-2 remains a matter of substantial interest within the field. Of high importance is also the provision of agents of dual or even multiple affinities, such as agents with a high affinity for VEGFR-2 and for one or more additional factor(s) associated with a disease state or disorder. Additionally, the provision of uses of such molecules in the treatment and diagnosis of disease is of great interest. Summary of the invention
  • VEGFR-2 binding agents which could for example be used for therapeutic, prognostic and diagnostic applications.
  • VEGFR-2 binding polypeptide comprising a VEGFR-2 binding motif BM, which motif consists of an amino acid sequence selected from: i) ENX 3 X 4 ASX 7 EIA X 11 LPNLX 16 DX 18 QY IAFIYX 26 LLX 29 wherein, independently from each other, X 3 is selected from L and Y;
  • X 4 is selected from E, F, I, K, M, V, W and Y;
  • X 7 is selected from K, N and R;
  • X 11 is selected from F, H, L and N;
  • X 16 is selected from N and T;
  • X 18 is selected from A, D, E, G, H, K, Q, S and T;
  • X 26 is selected from K and S;
  • X 29 is selected from D and R;
  • VEGFR-2 binding polypeptides a class of sequence related, VEGFR-2 binding polypeptides is based on a statistical analysis of a number of random polypeptide variants of a parent scaffold, that were selected for their interaction with VEGFR-2 in selection experiments.
  • the identified VEGFR-2 binding motif, or“BM” corresponds to the target binding region of the parent scaffold, which region constitutes two alpha helices within a three-helical bundle protein domain.
  • the varied amino acid residues of the two BM helices constitute a binding surface for interaction with the constant Fc part of antibodies.
  • the random variation of binding surface residues and subsequent selection of variants have replaced the Fc interaction capacity with a capacity for interaction with
  • VEGFR-2 VEGFR-2.
  • the function of any polypeptide is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof.
  • the disclosure encompasses modified variants of the VEGFR-2 binding polypeptide, which have retained VEGFR-2 binding characteristics.
  • VEGFR-2 binding polypeptide comprising an amino acid sequence with 82 % or greater identity to a polypeptide as defined in i).
  • said polypeptide may comprise a sequence which is at least 86 %, such as at least 89 %, such as at least 93 %, such as at least 96 % identical to a polypeptide as defined in i).
  • an amino acid residue belonging to a certain functional grouping of amino acid residues e.g.
  • hydrophobic, hydrophilic, polar etc) could be exchanged for another amino acid residue from the same functional group.
  • such changes may be made in any position of the sequence of the VEGFR-2 binding polypeptide as disclosed herein.
  • such changes may be made only in the non-variable positions, also denoted scaffold amino acid residues.
  • changes are not allowed in the variable positions, i.e. positions denoted with an“X” in sequence i).
  • the term "% identity", as used throughout the specification, may for example be calculated as follows.
  • the query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al., (1994) Nucleic Acids Research, 22: 4673-4680). A comparison is made over the window corresponding to the shortest of the aligned sequences.
  • the shortest of the aligned sequences may in some instances be the target sequence.
  • the query sequence may constitute the shortest of the aligned sequences.
  • the amino acid residues at each position are compared and the percentage of positions in the query sequence that have identical
  • X n and“X m ” are used to indicate amino acids in positions n and m in the sequence i) as defined above, wherein n and m are integers which indicate the position of an amino acid within said sequence as counted from the N-terminal end of said sequence.
  • X 3 and X 7 indicate the amino acid in position three and seven, respectively, from the N- terminal end of sequence i).
  • polypeptides wherein X n in sequence i) is independently selected from a group of possible residues according to Table 1.
  • X n may be selected from any one of the listed groups of possible residues and that this selection is independent from the selection of amino acids in X m , wherein n ⁇ m.
  • any of the listed possible residues in position X n in Table 1 may be independently combined with any of the listed possible residues any other variable position in Table 1.
  • a polypeptide wherein X 4 in sequence i) is selected from E, F, I, M, V, W and Y
  • a polypeptide wherein X 4 in sequence i) is selected from F, I, K, M, V, W and Y
  • the listed embodiments may be freely combined in yet other embodiments.
  • one such combined embodiment is a polypeptide in which X 4 is selected from F, I, K, M, V, W and Y, while X 7 is selected from K and R, and X 18 is selected from A, E, K and T.
  • X 3 is selected from L and Y;
  • X 4 is selected from E, F, I, M, V and Y;
  • X 7 is selected from K, N and R;
  • X 11 is selected from F, H, L and N;
  • X 16 is T
  • X 18 is selected from A, D, E, K, S and T;
  • X 26 is K
  • X 29 is D.
  • X 3 X 7 X 11 is selected from LNH and LKN.
  • X 3 X 4 X 7 is selected from LKN, LMN, YIK, YMK, LFK and LVK.
  • X 3 X 4 X 11 is selected from LKH, LMN, YIN, YMN, LFN and LVN.
  • BM VEGFR-2 binding motif
  • sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:5-25.
  • sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
  • sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:23.
  • the BM as defined above“forms part of” a three-helix bundle protein domain This is understood to mean that the sequence of the BM is“inserted” into or“grafted” onto the sequence of the original three-helix bundle domain, such that the BM replaces a similar structural motif in the original domain.
  • the BM is thought to constitute two of the three helices of a three-helix bundle, and can therefore replace such a two-helix motif within any three-helix bundle.
  • the replacement of two helices of the three-helix bundle domain by the two BM helices has to be performed so as not to affect the basic structure of the polypeptide.
  • the overall folding of the C ⁇ backbone of the polypeptide according to this embodiment of the invention is substantially the same as that of the three-helix bundle protein domain of which it forms a part, e.g. having the same elements of secondary structure in the same order etc.
  • a BM according to the present disclosure “forms part” of a three-helix bundle domain if the polypeptide according to this embodiment has the same fold as the original domain, implying that the basic structural properties are shared, those properties e.g. resulting in similar CD spectra.
  • the skilled person is aware of other parameters that are relevant.
  • the VEGFR-2 binding motif thus forms part of a three-helix bundle protein domain.
  • the BM may essentially constitute two alpha helices with an interconnecting loop, within said three-helix bundle protein domain.
  • said three- helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different three- helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof.
  • the three-helical bundle protein domain is a variant of protein Z, which is derived from domain B of staphylococcal Protein A.
  • the VEGFR-2 binding polypeptide may comprise a binding module (BMod), the amino acid sequence of which is selected from: iii) K-[BM]-DPSQSX a X b LLX c EAKKLX d X e X f Q; wherein
  • [BM] is a VEGFR-2 binding motif as defined herein
  • X 29 is D
  • X a is selected from A and S;
  • X b is selected from N and E;
  • X c is selected from A, S and C;
  • X d is selected from E, N and S;
  • X e is selected from D, E and S;
  • X f is selected from A and S; and iv) an amino acid sequence which has at least 83 % identity to a
  • the VEGFR-2 binding polypeptide may beneficially exhibit high structural stability, such as resistance to chemical modifications, to changes in physical conditions and to proteolysis, during production and storage, as well as in vivo.
  • the VEGFR-2 binding polypeptide may comprise a binding module (BMod), the amino acid sequence of which is selected from: v) K-[BM]-QPEQSX a X b LLX c EAKKLX d X e X f Q, wherein
  • [BM] is a VEGFR-2 binding motif as defined herein
  • X 29 is R
  • X a is selected from A and S;
  • X b is selected from N and E;
  • X c is selected from A, S and C;
  • X d is selected from E, N and S;
  • X e is selected from D, E and S;
  • X f is selected from A and S; and vi) an amino acid sequence which has at least 83 % identity to a
  • sequence iv) and vi) have at least 85 %, such as at least 87 %, such as at least 89 %, such as at least 91 %, such as at least 93 %, such as at least 95 %, such as at least 97 % identity to a sequence defined by iii) and v), respectively.
  • X a in sequence iii) or v) is A.
  • X a in sequence iii) or v) is S.
  • X b in sequence iii) or v) is N.
  • X b in sequence iii) or v) is E.
  • X c in sequence iii) or v) is A.
  • X c in sequence iii) or v) is S.
  • X c in sequence iii) or v) is C.
  • X d in sequence iii) or v) is E.
  • X d in sequence iii) or v) is N.
  • X d in sequence iii) or v) is S.
  • X e in sequence iii) or v) is D.
  • X e in sequence iii) or v) is E.
  • X e in sequence iii) or v) is S.
  • X d X e in sequence iii) or v) is selected from EE, ES, SD, SE and SS.
  • X d X e in sequence iii) or v) is ES.
  • X d X e in sequence iii) or v) is SE.
  • X d X e in sequence iii) or v) is SD.
  • X f in sequence iii) or v) is A.
  • X f in sequence iii) or v) is S.
  • X a is S; X b is E; X c is A and X f is A. In one embodiment, in sequence iii) or v), X a is A; X b is N; X c is C and X f is A.
  • X a is A; X b is N; X c is A; X d X e is ND and X f is A.
  • X a is S; X b is E; X c is A; X d X e is ND and X f is A.
  • X a is A; X b is N; X c is C; X d X e is ND and X f is A.
  • X a is S; X b is E; X c is S; X d X e is ND and X f is S.
  • X a is S; X b is E; X c is C; X d X e is ND and X f is S.
  • X a is A; X b is N; X c is A; X d X e is SE and X f is A.
  • X a is S; X b is E; X c is A; X d X e is SE and X f is A.
  • X a is A; X b is N; X c is C; X d X e is SE and X f is A.
  • X a is S; X b is E; X c is S; X d X e is SE and X f is S.
  • X a is S; X b is E; X c is C; X d X e is SE and X f is S.
  • X a is A; X b is N; X c is A; X d X e is ES and X f is A.
  • X a is S; X b is E; X c is A; X d X e is ES and X f is A.
  • X a is A; X b is N; X c is C; X d X e is ES and X f is A.
  • X a is S; X b is E; X c is S; X d X e is ES and X f is S.
  • X a is S; X b is E; X c is C; X d X e is ES and X f is S.
  • X a is A; X b is N; X c is A; X d X e is SD and X f is A.
  • X a is S; X b is E; X c is A; X d X e is SD and X f is A.
  • X a is A; X b is N; X c is C; X d X e is SD and X f is A.
  • X a is S; X b is E; X c is S; X d X e is SD and X f is S.
  • X a is S; X b is E; X c is A; X d X e is SD and X f is S.
  • sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25 presented in Figure 1.
  • sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5-25.
  • sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:5-25. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
  • sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:23.
  • a VEGFR-2 binding polypeptide which comprises an amino acid sequence selected from: vii) YA-[BMod]-AP;
  • [BMod] is a VEGFR-2 binding module as defined herein; and viii) an amino acid sequence which has at least 83 % identity to a
  • VEGFR-2 binding polypeptide which comprises an amino acid sequence selected from: ix) FN-[BMod]-AP;
  • [BMod] is a VEGFR-2 binding module as defined herein; and x) an amino acid sequence which has at least 83 % identity to a
  • sequence viii) and x) may for example be at least 84 %, such as at least 86 %, such as at least 88 %, such as at least 90 %, such as at least 92 %, such as at least 94 %, such as at least 96 %, such as at least 98 % identical to a sequence defined by vii) and ix), respectively.
  • the VEGFR-2 binding motif may form part of a polypeptide comprising an amino acid sequence selected from
  • VEGFR-2 binding polypeptide comprises an amino acid sequence selected from:
  • [BM] is a VEGFR-2 binding motif as defined herein; and xii) an amino acid sequence which has at least 82 % identity to the
  • the VEGFR-2 binding polypeptide comprises an amino acid sequence selected from:
  • [BM] is a VEGFR-2 binding motif as defined herein; and xiv) an amino acid sequence which has at least 82 % identity to the
  • the VEGFR-2 binding polypeptide comprises an amino acid sequence selected from:
  • [BM] is a VEGFR-2 binding motif as defined herein; and xvi) an amino acid sequence which has at least 82 % identity to the
  • sequence xii), xiv) and xvi) may for example be at least 84 %, such as at least 86 %, such as at least 87 %, such as at least 89 %, such as at least 91 %, such as at least 93 %, such as at least 94 %, such as at least 96 %, such as at least 98 % identical to a sequence defined by xi), xiii) and xv), respectively.
  • Sequence xi) or xiii) in such a polypeptide may be selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25 presented in Figure 1.
  • said sequence is selected from the group consisting of SEQ SEQ ID NO:3 and SEQ ID NO:5-25.
  • said sequence is selected from the group consisting of SEQ ID NO:5-25.
  • said sequence is selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
  • said sequence is SEQ ID NO:23.
  • the VEGFR-2 binding polypeptide as disclosed herein it consists of any one of the sequences disclosed herein, having from 0 to 15, such as from 0 to 7, additional N-terminal residues. In one
  • the VEGFR-2 binding polypeptide consists of any one of the sequences disclosed herein, having from 0 to 15, such as from 0 to 4, additional C-terminal residues. In a combination of these embodiments, the VEGFR-2 binding polypeptide consists of any one of the sequences disclosed herein, having from 0 to 15, such as from 0 to 7, additional N-terminal residues, and from 0 to 15, such as from 0 to 4, additional C-terminal residues.
  • the terms“VEGFR-2 binding” and”binding affinity for VEGFR-2” as used in this specification refer to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance technology.
  • VEGFR-2 binding affinity may be tested in an experiment in which VEGFR-2, or a fragment thereof, is immobilized on a sensor chip of the instrument, and the sample containing the polypeptide to be tested is passed over the chip.
  • the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing VEGFR-2, or a fragment thereof, is passed over the chip.
  • the skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for VEGFR-2. If a quantitative measure is desired, for example to determine a K D value for the interaction, surface plasmon resonance methods may also be used.
  • Binding values may for example be defined in a Biacore (GE Healthcare) or ProteOn XPR36 (Bio-Rad) instrument.
  • VEGFR-2 is suitably immobilized on a sensor chip of the instrument, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected in random order. K D values may then be calculated from the results using for example the 1:1 Langmuir binding model of the BIAevaluation 4.1 software, or other suitable software, provided by the instrument manufacturer.
  • the terms“albumin binding” and“binding affinity for albumin” as used in this disclosure refer to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance technology, such as in a Biacore instrument or ProteOn XPR36 instrument, in an analogous way to the example described above for VEGFR-2.
  • the VEGFR-2 binding polypeptide is capable of binding to VEGFR-2 such that the K D value of the interaction with VEGFR-2 is at most 1 x 10-6 M, such as at most 1 x 10-7 M, such as at most 1 x 10-8 M, such as at most 1 x 10-9 M, such as at most 1 x 10-10 M.
  • Binding of a polypeptide as defined herein to VEGFR-2 may interfere either with signaling via VEGFR-2 in vivo or in vitro.
  • VEGFR-2 binding polypeptide as defined herein which is capable of blocking VEGFR-2 dependent signaling.
  • Blocking activity may for example be quantified by the half maximal inhibitory concentration (IC50), which is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. This quantitative measure indicates how much of a particular substance is needed to inhibit a given biological process by half, and is commonly used in the art.
  • IC50 half maximal inhibitory concentration
  • said VEGFR-2 is human VEGFR-2. In another embodiment, said VEGFR-2 is murine VEGFR-2.
  • the VEGFR-2 binding polypeptide disclosure herein retains its ⁇ -helical conformation at temperatures higher than the body temperature of a fever free human subject.
  • Fever also known as pyrexia or febrile response
  • Elevated body temperature can be classified into fever (>37.5– 38.3 °C), hyperthermia (>37.5– 38.3 °C) and hyperpyrexia (>40.0– 41.5 °C).
  • said VEGFR-2 binding polypeptide retains its conformation at temperatures above the hyperthermia range, such as above the hyperpyrexia range.
  • VEGFR-2 binding polypeptide which has a melting temperature of at least 40 °C, such as at least 45 °C.
  • a melting temperature of at least 40 °C, such as at least 45 °C.
  • a VEGFR-2 binding polypeptide as described herein, which polypeptide has been extended by and/or comprises additional amino acids at the C terminus and/or N terminus.
  • a polypeptide should be understood as a polypeptide having one or more additional amino acid residues at the very first and/or the very last position in the polypeptide chain.
  • a VEGFR-2 binding polypeptide may comprise any suitable number of additional amino acid residues, for example at least one additional amino acid residue. Each additional amino acid residue may individually or collectively be added in order to, for example, improve and/or simplify production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
  • Such additional amino acid residues may comprise one or more amino acid residues added for the purpose of chemical coupling.
  • One example of this is the addition of a cysteine residue.
  • Additional amino acid residues may also provide a”tag” for purification or detection of the polypeptide, such as a His 6 tag, a (HisGlu) 3 tag (“HEHEHE” tag) or a ”myc” (c-myc) tag or a”FLAG” tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of a His 6 -tag.
  • a His 6 tag such as a His 6 tag, a (HisGlu) 3 tag (“HEHEHE” tag) or a ”myc” (c-myc) tag or a”FLAG” tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of a His 6 -tag.
  • IMAC immobilized metal affinity chromatography
  • the further amino acids as discussed above may be coupled to the VEGFR-2 binding polypeptide by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as
  • VEGFR-2 binding polypeptide as a fusion protein or joined in any other fashion, either directly or via a linker, for example an amino acid linker.
  • the further amino acids as discussed above may for example comprise one or more polypeptide domain(s).
  • a further polypeptide domain may provide the VEGFR-2 binding polypeptide with another function, for example another binding function, an enzymatic function, a toxic function, a fluorescent signaling function or combinations thereof.
  • a further polypeptide domain may moreover provide another VEGFR-2 binding moiety.
  • a VEGFR-2 binding polypeptide in a multimeric form is understood to comprise at least two VEGFR-2 binding polypeptides as disclosed herein as monomer units, the amino acid sequences of which may be the same or different.
  • Multimeric forms of the polypeptides may comprise a suitable number of domains, each having a VEGFR-2 binding motif, and each forming a monomer within the multimer. These domains may have the same amino acid sequence, but alternatively, they may have different amino acid sequences.
  • the VEGFR-2 binding polypeptide of the invention may form homo- or heteromultimers, for example homo- or heterodimers.
  • VEGFR-2 binding polypeptide wherein said monomer units are covalently coupled together.
  • said VEGFR-2 binding polypeptide monomer units are expressed as a fusion protein.
  • VEGFR-2 binding polypeptide in dimeric form there is provided.
  • said dimeric form is a homodimeric form.
  • said dimeric form is a
  • VEGFR-2 binding polypeptide comprising a first monomer unit as defined herein and a second monomer unit consisting of a different VEGFR-2 binding polypeptide.
  • the VEGFR-2 binding polypeptide comprises
  • a second monomer unit consisting of a second VEGFR-2 binding polypeptide, comprising a second VEGFR-2 binding motif BM2, which motif consists of an amino acid sequence selected from: xvii) EFX 3 X 4 ADX 7 EIR X 11 LPNLX 16 HGQX 20 X 21 AFIX 25 X 26 LYX 29 wherein, independently from each other, X 3 is selected from Q and R;
  • X 4 is selected from A, D, H, K, L, M, R, S and V;
  • X 7 is selected from A, I and R;
  • X 11 is selected from A, D and G;
  • X 16 is selected from N and T;
  • X 20 is selected from F and W;
  • X 21 is selected from K and Y;
  • X 25 is selected from K and V
  • X 26 is selected from K, N and S;
  • X 29 is selected from D and R;
  • said second monomer unit consisting of a second VEGFR-2 binding polypeptide, has affinity for a different epitope of VEGFR-2 than said first monomer unit.
  • said first and second monomer units bind to different epitopes on VEGFR-2.
  • the above definition of a second, different class of sequence related, VEGFR-2 binding polypeptides is also based on a statistical analysis of a number of random polypeptide variants of a parent scaffold.
  • This group of second VEGFR-2 binding polypeptides were selected for their interaction with VEGFR-2 in several different selection experiments as described in the Examples section of the present disclosure.
  • the identified second VEGFR-2 binding motif, or“BM2” corresponds to the target binding region of the parent scaffold, which region constitutes two alpha helices within a three-helical bundle protein domain.
  • the varied amino acid residues of the two BM2 helices constitute a binding surface for interaction with the constant Fc part of antibodies.
  • the random variation of binding surface residues and subsequent selection of variants replaced the Fc interaction capacity of the parent scaffold with a capacity for interaction with VEGFR-2.
  • the disclosure encompasses VEGFR-2 binding polypeptides in heterodimeric form, wherein the second monomer unit comprises second VEGFR-2 binding polypeptides which have retained VEGFR-2 binding characteristics.
  • VEGFR-2 binding polypeptide wherein said second monomer unit comprises a second VEGFR-2 binding polypeptide comprising an amino acid sequence with 82 % or greater identity to a polypeptide as defined in xvii).
  • the second polypeptide may comprise a sequence which is at least 86 %, such as at least, 89 %, such as at least 93 %, such as at least 96 % identical to a polypeptide as defined in xvii).
  • VEGFR-2 binding polypeptides in heterodimeric form comprising any VEGFR-2 binding polypeptide defined by sequence i) or having at least 82 % identity to a sequence defined in i) as a first monomer unit, and a second VEGFR-2 binding polypeptide having a sequence defined in xvii) or having at least 82 % identity to a sequence defined in xvii) as a second monomer unit.
  • sequence identity of encompassed modified variants of VEGFR-2 binding polypeptides above is equally relevant for the second VEGFR-2 binding polypeptide and will not be repeated here for the sake of brevity.
  • VEGFR-2 binding polypeptides in heterodimeric form comprising second monomer units wherein X n in sequence xvii) is independently selected from a group of possible residues according to Table 2 are encompassed by the present disclosure.
  • X n may be selected from any one of the listed groups of possible residues and that this selection is independent from the selection of amino acids in X m , wherein n ⁇ m.
  • any of the listed possible residues in position X n in Table 2 may be
  • VEGFR-2 binding polypeptide comprising a second monomeric unit, wherein, in sequence xvii), X 3 is selected from Q and R;
  • X 4 is selected from A, K, R, S and V;
  • X 7 is R
  • X 11 is selected from A and G;
  • X 16 is T
  • X 20 is selected from F and W;
  • X 21 is Y
  • X 25 is V
  • X 26 is selected from K and N;
  • sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42. In one embodiment, sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:26-42.
  • sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as from the group consising of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:42.
  • sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as the group consisting of SEQ ID NO:34 and SEQ ID NO:41.
  • sequence xvii) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:41.
  • a binding motif as defined above “forms part of” a three-helix bundle protein domain. This is equally valid for BM2 as for BM described above.
  • the second VEGFR-2 binding motif (BM2) forms part of a three-helix bundle protein domain.
  • the BM2 may essentially constitute two alpha helices with an interconnecting loop, within said three- helix bundle protein domain.
  • said three-helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different three-helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof.
  • the three-helical bundle protein domain is a variant of protein Z, which is derived from domain B of
  • BM2 may replace BM in any one of the 49-, 53- or 58-mer contexts disclosed herein.
  • the VEGFR-2 binding polypeptide comprises
  • a second monomer unit consisting of a second VEGFR-2 binding polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:26-42.
  • any VEGFR-2 binding polypeptide defined by sequence i) or having at least 82 % identity to a sequence define in i) may for example be combined with any one of the second VEGFR-2 binding polypeptides listed in Figure 1, i.e. any polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:26-42.
  • said first monomer unit is selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23. In one particular embodiment, said first monomer unit is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23.
  • said second monomer unit is selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:42.
  • said second monomer unit is selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39; SEQ ID NO:41 and SEQ ID NO:42.
  • a VEGFR-2 binding polypeptide as defined herein may comprise any one of the listed first monomer units and any one of the listed second monomer units.
  • VEGFR-2 binding polypeptide is in one embodiment, in
  • heterodimeric form and comprises SEQ ID NO:12 and SEQ ID NO:34; SEQ ID NO:12 and SEQ ID NO:39; SEQ ID NO:12 and SEQ ID NO:41; SEQ ID NO:12 and SEQ ID NO:42; SEQ ID NO:13 and SEQ ID NO:34; SEQ ID NO:13 and SEQ ID NO:39; SEQ ID NO:13 and SEQ ID NO:41; SEQ ID NO:13 and SEQ ID NO:42; SEQ ID NO:20 and SEQ ID NO:34; SEQ ID NO:20 and SEQ ID NO:39; SEQ ID NO:20 and SEQ ID NO:41; SEQ ID NO:20 and SEQ ID NO:42; SEQ ID NO:23 and SEQ ID NO:34; SEQ ID NO:23 and SEQ ID NO:39; SEQ ID NO:23 and SEQ ID NO:41; or SEQ ID NO:23 and SEQ ID NO:42.
  • VEGFR-2 binding polypeptide in one embodiment, in
  • heterodimeric form comprises SEQ ID NO:23 and SEQ ID NO:41.
  • the first and second monomer units of said VEGFR-2 binding polypeptide bind to different epitopes of VEGFR-2.
  • the term“different epitopes of VEGFR- 2” refers to non-overlapping epitopes, such that binding of the first monomer unit does not sterically interfere with the binding of the second monomer unit to VEGFR-2.
  • binding to two different epitopes may result in stronger binding, as measured by association rate k a , dissociation rate k d and/or affinity K D , than binding to one epitope or to two partially overlapping epitopes.
  • a VEGFR-2 binding polypeptide in heterodimeric form which is capable of binding to VEGFR-2 such that the dissociation rate constant k d of the interaction with VEGFR-2 is at least 2 times, such as at least 5 times, such as at least 10 times, such as at least 15 times, such as at least 20 times lower, such as at least 30 times lower than the dissociation rate constant k d of a comparable polypeptide in homodimeric form.
  • VEGFR-2 binding polypeptide in heterodimeric form, which is capable of binding to VEGFR-2 such that the dissociation rate constant k d of the interaction with VEGFR-2 is at most 10-2 s-1 , such as at most 10-3 s-1 , such as at most 10-4 s-1.
  • VEGFR-2 binding polypeptide is used to encompass VEGFR-2 binding polypeptides in all forms, i.e. monomeric and multimeric forms, for example dimeric forms, and in particular the heterodimeric form discussed immediately above.
  • polypeptide as defined herein is part of a fusion protein or a conjugate comprising a second or further moieties. Second and further moiety/moieties of the fusion polypeptide or conjugate in such a protein may suitably have a desired biological activity.
  • a fusion protein or a conjugate comprising a first moiety consisting of a
  • VEGFR-2 binding polypeptide according to the first aspect, and a second moiety consisting of a polypeptide having a desired biological activity.
  • said fusion protein or conjugate may additionally comprise further moieties, comprising desired biological activities that can be either the same as or different from the biological activity of the second moiety.
  • Non-limiting examples of a desired biological activity comprise a therapeutic activity, a binding activity and an enzymatic activity.
  • the second moiety having a desired biological activity is a therapeutically active polypeptide.
  • Non-limiting examples of therapeutically active polypeptides are biomolecules, such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.
  • binding activities are binding activities which increase the in vivo half-life of the fusion protein or conjugate, and binding activities which act to block a biological activity.
  • the binding activity is an albumin binding activity which increases the in vivo half-life of the fusion protein or conjugate.
  • said albumin binding activity is provided by the albumin binding domain of streptococcal protein G or a derivative thereof.
  • said fusion protein may for example comprise a VEGFR-2 binding polypeptide in monomeric or multimeric form (such as a homodimeric or heterodimeric form) as defined herein and an albumin binding domain of streptococcal protein G or a derivative thereof.
  • said binding activity is binding to a cancer associated factor.
  • cancer associated factors include platelet-derived growth factor receptor ⁇ (PDGFR- ⁇ ), platelet-derived growth factor receptor ⁇ (PDGFR- ⁇ ), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B), platelet-derived growth factor C (PDGF-C), platelet-derived growth factor D (PDGF-D), epidermal growth factor receptor 1 (EGFR), epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor 3 (HER3), epidermal growth factor receptor 4 (HER4), epidermal growth factor, tumor growth factor ⁇ , epigen, epiregulin, neuregulins 1-4, c-kit, Raf kinases such as B-Raf and C-raf, rearranged during transfection (RET) receptor, colony stimulating factor 1 receptor (CSF- 1R) and fms-like tyrosine kinase 3 (Flt-3).
  • RET colony stimulating
  • said binding activity is binding to an angiogenesis associated factor.
  • angiogenesis associated factors include fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF-1), basic FGF, angiogenin 1 (Ang-1), angiogenin 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase with immunoglobulin-like domains 1 (TIE-1), tyrosine kinase with immunoglobulin-like domains 2 (TIE-2), vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor 3 (VEGFR-3), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor
  • said binding activity is binding to an immune response associated factor.
  • immune response associated factors include T-cell recruitment factors such as CD3, CD28, T- cell receptor ⁇ (TCR ⁇ ), T-cell receptor ⁇ (TCR ⁇ ), cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), and programmed cell death protein 1 (PD-1); NK-cell recruitment factors such as CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1) and the natural cytotoxicity receptors NKp30 and NKp40, programmed death- ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2) -, B7 homolog 3 (B7- H3), B7 homolog 4 (B7-H4), herpes virus entry mediator (HVEM)/B- and T- lymphocyte attenuator (BTLA), killer inhibitory receptor (KIR), lymphocyte- activation gene 3 (LAG3), galectin-9 (Gal) T-cell recruitment factors
  • the polypeptide comprises an immunostimulatory cytokine.
  • said immunostimulatory cytokine is selected from the group consisting of IL-2, IL-7, IL-9, IL-12, IL-15, IL-21 and interferons and G-CSF.
  • a VEGFR-2 binding polypeptide, fusion protein or conjugate which comprises an anti-cancer agent.
  • Non-limiting examples of anti-cancer agents include agents selected from the group consisting of auristatin, anthracycline, calicheamycin, combretastatin, doxorubicin, duocarmycin, the CC-1065 anti-tumor-antibiotic, ecteinsascidin, geldanamycin, maytansinoid, methotrexate, mycotoxin, taxol, ricin, bouganin, gelonin, pseudomonas exotoxin 38 (PE38), diphtheria toxin (DT), and their analogues, and derivates thereof and combinations thereof.
  • auristatin anthracycline
  • calicheamycin combretastatin
  • doxorubicin duocarmycin
  • CC-1065 anti-tumor-antibiotic ecteinsascidin
  • geldanamycin maytansinoid
  • methotrexate mycotoxin
  • taxol
  • VEGFR-2 binding polypeptide, fusion protein or conjugate which comprises an anti-angiogenic agent.
  • Non-limiting examples of anti-angiogenic agents include agents selected from the group consisting of angiostatin, endostatin, tumstatin, angiopoietin, thrombospondin, IFN- ⁇ , IL-12, cartilage-derived angiogenesis inhibitors, matrix metalloproteinase inhibitors, and derivatives thereof and combinations thereof.
  • anti-angiogenic agents include bevacizumab, ranibizumab, etaracizumab, itraconazole, suramin, 2-methoxyestradiol, tasquinimod, linomide, axitinib, cediranib, motesanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, vandetanib and vatalanib.
  • Non-limiting examples of immune response modifying agents include agents selected from the group consisting of disease-modifying antirheumatic drugs (DMARDs), such as gold salts, azathioprine, methotrexate and leflunomide; calcineurin inhibitors, such as cyclosporin A or FK 506;
  • DMARDs disease-modifying antirheumatic drugs
  • gold salts such as gold salts, azathioprine, methotrexate and leflunomide
  • calcineurin inhibitors such as cyclosporin A or FK 506
  • mTOR inhibitors such as rapamycin
  • ascomycin having immuno-suppressive properties glucocorticoids
  • corticosteroids corticosteroids; cyclophosphamide; immunosuppressive monoclonal antibodies; adhesion molecule inhibitors, such as LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; anti-TNF agents, such as etanercept; blockers of proinflammatory cytokines; IL-1 blockers such as anakinra or IL-1 trap; IL-17 blockers; chemokine blockers; non steroidal anti-inflammatory drugs (NSAIDs) such as aspirin; and anti- infectious agents and other immune response modulating agents.
  • adhesion molecule inhibitors such as LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists
  • anti-TNF agents such as etanercept
  • blockers of proinflammatory cytokines such as anakinra or IL-1 trap
  • IL-17 blockers chemokine blockers
  • anti-cancer, anti-angiogenic and immune response modifying agents include all possible variants of said agents, for example the agent auristatin is intended to include for example auristatin E, auristatin F, auristatin PE, and derivatives thereof.
  • agent auristatin is intended to include for example auristatin E, auristatin F, auristatin PE, and derivatives thereof.
  • multispecific agents such as antibodies with the ability to bind to more than one antigen, for example through engineering of the complementarity determining regions (CDRs) to address two antigens in a single antibody combining site (Bostrom et al, 2009, Science 323(5921):1610-1614;
  • a polypeptide with affinity for VEGFR-2 as disclosed herein may also exhibit affinity for another factor, such as a factor associated with cancer or an angiogenesis related disorder, or an immune response associated factor.
  • another factor such as a factor associated with cancer or an angiogenesis related disorder, or an immune response associated factor.
  • the term“complex” is intended to refer to two or more associated polypeptide chains, one having an affinity for VEGFR-2 by virtue of its VEGFR-2 binding motif as defined above, and the other being an antibody or an antigen binding fragment thereof. These polypeptide chains may each contain different protein domains, as described amply above for the VEGFR-2 binding polypeptide of the first aspect, and the resulting multiprotein complex can have multiple functions.“Complex” intends to refer to two or more polypeptides as defined herein, connected by covalent bonds, for example two or more polypeptide chains connected by covalent bonds through expression thereof as a recombinant fusion protein, or associated by chemical conjugation.
  • the third aspect provides a complex comprising an antibody or an antigen binding fragment thereof.
  • antibodies are immunoglobulin molecules capable of specific binding to a target (an antigen), such as a carbohydrate, polynucleotide, lipid, polypeptide or other, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • antibody or an antigen binding fragment thereof encompasses not only full-length or intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof, such as Fab, Fab′, F(ab′) 2 , Fab 3 , Fv and variants thereof, fusion proteins comprising one or more antibody portions, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies and covalently modified antibodies.
  • antigen-binding fragments thereof such as Fab, Fab′, F(ab′) 2 , Fab 3 , Fv and variants thereof, fusion proteins comprising one or more antibody portions, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, te
  • modified antibodies and antigen binding fragments thereof include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular
  • a full-length antibody comprises two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variable region (V H ) and first, second and third constant regions (C H 1, C H 2 and C H 3).
  • Each light chain contains a light chain variable region (V L ) and a light chain constant region (C L ).
  • V H heavy chain variable region
  • C H 1 first, second and third constant regions
  • Each light chain contains a light chain variable region (V L ) and a light chain constant region (C L ).
  • IgA, IgD, IgE, IgG, IgM and IgY and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • full-length antibody refers to an antibody of any class, such as IgD, IgE, IgG, IgA, IgM or IgY (or any sub-class thereof).
  • the subunit structures and three-dimensional configurations of different classes of antibodies are well known.
  • An“antigen binding fragment” is a portion or region of an antibody molecule, or a derivative thereof, that retains all or a significant part of the antigen binding of the corresponding full-length antibody.
  • An antigen binding fragment may comprise the heavy chain variable region (V H ), the light chain variable region (V L ), or both.
  • V H and V L typically contains three complementarity determining regions CDR1, CDR2 and CDR3.
  • the three CDRs in V H or V L are flanked by framework regions (FR1, FR2, FR3 and FR4).
  • examples of antigen binding fragments include, but are not limited to: (1) a Fab fragment, which is a monovalent fragment having a V L -C L chain and a V H -C H 1 chain; (2) a Fab’ fragment, which is a Fab fragment with the heavy chain hinge region, (3) a F(ab′) 2 fragment, which is a dimer of Fab’ fragments joined by the heavy chain hinge region, for example linked by a disulfide bridge at the hinge region; (4) an Fc fragment; (5) an Fv fragment, which is the minimum antibody fragment having the V L and V H domains of a single arm of an antibody; (6) a single chain Fv (scFv) fragment, which is a single polypeptide chain in which the V H and V L domains of an scFv are linked by a peptide linker; (7) an (scFv) 2 , which comprises two V H domains and two V L domains, which are associated through the two V H domains via dis
  • Antigen binding fragments can be prepared via routine methods.
  • F(ab′) 2 fragments can be produced by pepsin digestion of a full- length antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of F(ab′) 2 fragments.
  • fragments can be prepared via recombinant technology by expressing the heavy and light chain fragments in suitable host cells (e.g., E. coli, yeast, mammalian, plant or insect cells) and having them assembled to form the desired antigen- binding fragments either in vivo or in vitro.
  • a single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region.
  • a flexible linker may be incorporated between the two variable regions. The skilled person is aware of methods for the preparation of both full-length antibodies and antigen binding fragments thereof.
  • this aspect of the disclosure provides a complex as defined herein, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments, Fab’ fragments, F(ab’) 2 fragments, Fc
  • said at least one antibody or antigen binding fragment thereof is selected from full-length antibodies, Fab fragments and scFv fragments. In one particular embodiment, said at least one antibody or antigen binding fragment thereof is a full-length antibody.
  • the antibody or antigen binding fragment thereof is selected from the group consisting of monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and antigen-binding fragments thereof.
  • the term“monoclonal antibodies” as used herein refers to antibodies having monovalent affinity, meaning that each antibody molecule in a sample of the monoclonal antibody binds to the same epitope on the antigen, whereas the term“polyclonal antibodies” as used herein refers to a collection of antibodies that react against a specific antigen, but in which collection there may be different antibody molecules for example identifying different epitopes on the antigen.
  • Polyclonal antibodies are typically produced by inoculation of a suitable mammal and are purified from the mammal’s serum.
  • Monoclonal antibodies are made by identical immune cells that are clones of a unique parent cell (for example a hybridoma cell line).
  • human antibody refers to antibodies having variable and constant regions corresponding substantially to, or derived from, antibodies obtained from human subjects.
  • chimeric antibodies refers to recombinant or genetically engineered antibodies, such as for example mouse monoclonal antibodies, which contain polypeptides or domains from a different species, for example human, introduced to reduce the antibodies’ immunogenicity.
  • humanized antibodies refers to antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans, in order to reduce immunogenicity.
  • a complex as defined herein may be beneficial for a complex as defined herein to, in addition to being capable of binding VEGFR-2, target at least one additional antigen, such as an antigen selected from the group consisting of an antigen associated with cancer, an antigen associated with an angiogenesis related disorder and an antigen associated with the immune response.
  • said additional antigen is associated with cancer.
  • said additional antigen is associated with angiogenesis.
  • said additional antigen is associated with the immune response.
  • the antigen is selected from the group consisting of platelet-derived growth factor receptor ⁇
  • PDGFR- ⁇ platelet-derived growth factor receptor ⁇
  • PDGFR- ⁇ platelet-derived growth factor A
  • PDGF-B platelet-derived growth factor B
  • PDGF-C platelet-derived growth factor C
  • PDGF-D platelet-derived growth factor D
  • epidermal growth factor receptor 1 EGFR
  • epidermal growth factor receptor 2 HER2
  • epidermal growth factor receptor 3 HER3
  • epidermal growth factor receptor 4 HER4
  • epidermal growth factor epidermal growth factor receptor 1
  • HER2 epidermal growth factor receptor 2
  • HER3 epidermal growth factor receptor 3
  • HER4 epidermal growth factor receptor 4
  • epidermal growth factor epidermal growth factor
  • tumor growth factor ⁇ epigen, epiregulin, neuregulins 1-4, c-kit, Raf kinases such as B-Raf and C-raf, rearranged during transfection (RET) receptor, colony stimulating factor 1 receptor (CSF-1R) and f
  • said antibody or fragment thereof is selected from the group consisting of olaratumab, tovetumab, cetuximab, nimotuzumab, panitumumab, zalutumumab, trastuzumab, pertuzumab, MM-111,
  • the antigen is associated with angiogenesis and is selected from the group consisting of fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF-1), basic FGF, angiogenin 1 (Ang-1), angiogenin 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase with immunoglobulin-like domains 1 (TIE-1), tyrosine kinase with immunoglobulin-like domains 2 (TIE- 2), vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor 3 (VEGFR-3), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-
  • said antibody or fragment thereof is selected from the group consisting of AMG 780, AMG 386, MEDI-3617, nesvacumab, CVX-241, bevacizumab, ranibizumab, VGX100, CVX-241, ABP 215, PF-06439535, fresolimumab, metelimumab, onartuzumab, emibetuzumab and tarextumab.
  • the antigen is associated with the immune response and is selected from the group consisting of T-cell recruitment factors such as CD3, CD28, T-cell receptor ⁇ (TCR ⁇ ), T-cell receptor ⁇ (TCR ⁇ ), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and programmed cell death protein 1 (PD-1); NK-cell recruitment factors such as CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1) and the natural cytotoxicity receptors NKp30 and NKp40; programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), herpes virus entry mediator (HVEM)/B- and T-lymphocyte attenuator (BTLA), killer inhibitory receptor (KIR), lymphocyte-activation gene 3 (LAG3), galectin-9 (Gal9)/T cell immunoglobin, T
  • said antibody or fragment thereof is selected from the group consisting of visilizumab, otelixizumab, ipilimumab, tremelimumab, pembrolizumab, nivolumab, pidilizumab,
  • the complex as described herein may for example be present in the form of a fusion protein or a conjugate.
  • said at least one VEGFR-2 binding polypeptide and said at least one antibody, or antigen binding fragment thereof may be coupled by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the complex as a fusion protein or joined in any other fashion, either directly or via a linker, for example an amino acid linker.
  • a complex as defined herein wherein said complex is a fusion protein or a conjugate.
  • said complex is a fusion protein.
  • said complex is a conjugate.
  • said VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of said antibody or antigen binding fragment thereof.
  • said VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the light chain of said antibody or antigen binding fragment thereof.
  • said VEGFR-2 binding polypeptide is attached to the N-terminus and/or C-terminus of the light chain and heavy chain of said antibody or antigen binding fragment thereof.
  • the VEGFR-2 binding polypeptide may be attached to only the N-terminus of the heavy chain(s), only the N-terminus of the light chain(s), only the C-terminus of the heavy chain(s), only the C-terminus of the light chain(s), both the N-terminus and the C-terminus of the heavy chain(s), both the N-terminus and the C- terminus of the light chain(s), only the C-terminus of the light chain(s) and the N-terminus of the heavy chain(s), only the C-terminus of the heavy chain(s) and the N-terminus of the light chain(s), of said antibody or antigen binding fragment thereof.
  • the construction of a fusion protein often involves use of linkers between functional moieties to be fused.
  • linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
  • Linkers have been used to for example increase stability or improve folding of fusion proteins, to increase expression, improve biological activity, enable targeting and alter pharmacokinetics of fusion proteins.
  • the VEGFR-2 binding polypeptide, fusion protein, conjugate or complex as defined herein further comprises at least one linker, such as selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
  • said linker is arranged between a first moiety consisting of a VEGFR-2 binding polypeptide as defined herein and a second moiety consisting of a polypeptide having a desired biological activity.
  • said linker is arranged within said first moiety.
  • one or more linker(s) may be arranged between monomeric units of the polypeptide as defined herein, such as between the monomer units in a homodimer or heterodimer as described above in connection with an embodiment of the first aspect.
  • said linker may be arranged between said first monomer unit and said second monomer unit.
  • said linker is arranged between said VEGFR-2 binding polypeptide and said antibody or antigen binding fragment thereof.
  • linker arranged in any of above mentioned contexts does not exclude the presence of additional linkers in the same or any other context.
  • Flexible linkers are often used when the joined domains require a certain degree of movement or interaction, and may be particularly useful in some embodiments of the VEGFR-2 binding polypeptide, fusion protein, conjugate or complex as defined herein.
  • Such linkers are generally composed of small, non-polar (for example G) or polar (for example S or T) amino acids.
  • Some flexible linkers primarily consist of stretches of G and S residues, for example (GGGGS) p and (SSSSG) p . Adjusting the copy number“p” allows for optimization of the linker in order to achieve appropriate separation between the functional moieties or to maintain necessary inter-moiety interaction.
  • G and S linkers other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues, such as T, A, K and E, to maintain flexibility, as well as polar amino acid residues to improve solubility.
  • said linker is a flexible linker comprising glycine (G), serine (S) and/or threonine (T) residues.
  • n 1-5.
  • m 0-5.
  • p 1-5.
  • said linker is selected from the group consisting of S 4 G, (S 4 G) 3 , (S 4 G) 4 and (S 4 G) 8 . In one embodiment, said linker is selected from the group consisting of S 4 G, (S 4 G) 3 and (S 4 G) 4 . In one embodiment, said linker is selected from the group consisting of S 4 G, (S 4 G) 3 and (S 4 G) 8 . In one embodiment, said linker is S 4 G. In one embodiment, said linker is (S 4 G) 3 . In one embodiment, said linker is (S 4 G) 8 . In another embodiment, said linker is (S 4 G) 4 . In one embodiment, said linker is (G 4 S) 3 .
  • said linker is VDGS.
  • first, second and further moieties is made for clarity reasons to distinguish between VEGFR-2 binding polypeptide or polypeptides according to the invention on the one hand, and moieties exhibiting other functions on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of the fusion protein, conjugate or complex. Similarly, the designations first and second monomer units are made for clarity reasons to distinguish between said units.
  • said first moiety may without restriction appear at the N-terminal end, in the middle, or at the C-terminal end of the fusion protein, conjugate or complex.
  • the above aspects furthermore encompass polypeptides in which the VEGFR-2 binding polypeptide according to the first aspect, the VEGFR-2 binding polypeptide as comprised in a fusion protein or conjugate according to the second aspect or in a complex according to the third aspect, further comprises a label, such as a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides and radioactive particles. Such labels may for example be used for detection of the
  • such labeled polypeptide may for example be used for labeling tumors with high expression of VEGFR- 2.
  • the labeled VEGFR-2 binding polypeptide is present as a moiety in a fusion protein, conjugate or complex also comprising a second moiety having a desired biological activity.
  • the label may in some instances be coupled only to the VEGFR-2 binding polypeptide, and in some instances both to the VEGFR-2 binding polypeptide and to the second moiety of the fusion protein or conjugate and/or the antibody or antigen binding fragment thereof the complex.
  • the label may be coupled to a second moiety, or antibody or antigen binding fragment thereof only and not to the VEGFR-2 binding moiety.
  • VEGFR-2 binding polypeptide comprising a second moiety, wherein said label is coupled to the second moiety only.
  • a labeled polypeptide may contain only the VEGFR-2 binding polypeptide and e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide, or contain the VEGFR-2 binding polypeptide, a therapeutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy.
  • a therapeutic radionuclide which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide, or contain the VEGFR-2 binding polypeptide, a therapeutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy.
  • a labeled polypeptide may contain a VEGFR-2 binding polypeptide in heterodimeric form and e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide, or contain the VEGFR-2 binding polypeptide in heterodimeric form, a therapeutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy.
  • a complex which contains a VEGFR-2 binding polypeptide as defined herein, an antibody or antigen binding fragment thereof and a e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide or to the antibody or antigen binding fragment thereof.
  • a therapeutic radionuclide which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide or to the antibody or antigen binding fragment thereof.
  • such a radiolabeled polypeptide may comprise a radionuclide.
  • a majority of radionuclides have a metallic nature and metals are typically incapable of forming stable covalent bonds with elements presented in proteins and peptides. For this reason, labeling of proteins and peptides with radioactive metals is performed with the use of chelators, i.e. multidentate ligands, which form non-covalent compounds, called chelates, with the metal ions.
  • the incorporation of a radionuclide is enabled through the provision of a chelating environment, through which the radionuclide may be coordinated, chelated or complexed to the polypeptide.
  • a chelator is the polyaminopolycarboxylate type of chelator.
  • Two classes of such polyaminopolycarboxylate chelators can be distinguished: macrocyclic and acyclic chelators.
  • the VEGFR-2 binding polypeptide, fusion protein, conjugate or complex comprises a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the VEGFR-2 binding polypeptide via a thiol group of a cysteine residue or an epsilon amine group of a lysine residue.
  • the most commonly used macrocyclic chelators for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides and radiolanthanides are different derivatives of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid).
  • a chelating environment of the VEGFR- 2 binding polypeptide, VEGFR-2 binding polypeptide in heterodimeric form, fusion protein, conjugate or complex is provided by DOTA or a derivative thereof.
  • a chelating polypeptide encompassed by the present disclosure is obtained by reacting the DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10- maleimidoethylacetamide (maleimidomonoamide-DOTA) with said DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10- maleimidoethylacetamide (maleimidomonoamide-DOTA) with said
  • a chelating polypeptide encompassed by the present disclosure is obtained by reacting the DOTA derivative DOTAGA (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid) with said polypeptide.
  • 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and derivatives thereof may be used as chelators.
  • NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid
  • a chelating environment of the VEGFR-2 binding polypeptide, VEGFR-2 binding polypeptide in heterodimeric form, fusion protein, conjugate or complex is provided by NOTA or a derivative thereof.
  • a chelating polypeptide encompassed by the present disclosure is obtained by reacting the NOTA derivative NODAGA (2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1- yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid) with said NOTA derivative NODAGA (2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1- yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid) with said NOTA derivative NODAGA (2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1- yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)d
  • polypeptide The most commonly used acyclic polyaminopolycarboxylate chelators are different derivatives of DTPA (diethylenetriamine-pentaacetic acid).
  • polypeptides having a chelating environment provided by
  • diethylenetriaminepentaacetic acid or derivatives thereof are also provided.
  • a polynucleotide encoding a VEGFR-2 binding polypeptide, fusion protein or complex as described herein; an expression vector comprising said
  • polynucleotide and a host cell comprising said expression vector.
  • VEGFR-2 binding polypeptide, fusion protein or complex as described above comprising culturing said host cell under conditions permissive of expression of said polypeptide from its expression vector, and isolating the polypeptide.
  • VEGFR-2 binding polypeptide, fusion protein or complex of the present disclosure may alternatively be produced by non-biological peptide synthesis using amino acids and/or amino acid derivatives having protected reactive side-chains, the non-biological peptide synthesis comprising
  • VEGFR-2 binding polypeptide or fusion protein as described herein to at least one antibody or antigen binding fragment thereof.
  • conjugation methods such as conventional chemical conjugation methods, for example using charged succinimidyl esters or carbodiimides.
  • the VEGFR-2 binding polypeptide according to the present disclosure may be useful as a therapeutic, diagnostic and/or prognostic agent in its own right or as a means for targeting other therapeutic or diagnostic agents, with e.g. direct or indirect effects on
  • VEGFR-2 A direct therapeutic effect may for example be accomplished by inhibiting VEGFR-2 signaling.
  • a composition comprising a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex as described herein and at least one pharmaceutically acceptable excipient or carrier.
  • said composition further comprises at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents.
  • additional active agents that may prove useful in such combination are anti-cancer agents, anti-angiogenic agents and immune response modifying agents as described herein.
  • VEGFR-2 binding polypeptides of the present disclosure confer several advantages over conventional monoclonal antibody based therapies. Such advantages include advantages in formulation, modes of administration, such as alternative routes of administration, administration at higher doses than antibodies and absence of Fc-mediated side effects. Also, many diseases and disorders, such as cancers and angiogenesis related disorders, are associated with more than one factor and thus a complex as defined herein confers the advantage of targeting an additional antigen together with VEGFR-2. In another aspect of the present disclosure, there is provided a
  • a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the treatment, diagnosis or prognosis of a VEGFR-2 related disorder.
  • said VEGFR-2 binding polypeptide, VEGFR-2 binding polypeptide in heterodimeric form, fusion protein, conjugate or composition is provided for use as a medicament.
  • a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein for use as a medicament to modulate VEGFR-2 function in vivo.
  • the term “modulate” refers to changing the activity, such as rendering VEGFR-2 function hypomorph, partially inhibiting or fully inhibiting VEGFR-2 function.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the treatment of a VEGFR-2 related disorder. In one embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the diagnosis of a VEGFR-2 related disorder.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the prognosis of a VEGFR-2 related disorder.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in diagnosis or prognosis to distinguish subjects who respond to anti angiogenic therapy from subjects who do not respond to said therapy. It is to be understood that said VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition may be used as the sole diagnostic or prognostic agent or as a companion diagnostic or prognostic agent.
  • VEGFR-2 related disorder comprising administering to a subject in need thereof an effective amount of a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein.
  • the VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein modulates VEGFR-2 function in vivo.
  • the term“VEGFR-2 related disorder” refers to any disorder, disease or condition in which VEGFR-2 signalling plays a regulatory role.
  • said VEGFR-2 related disorder is selected from cancer and angiogenesis related disorders.
  • Non-limiting examples of VEGFR-2 related disorders are cancers selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, melanomas, cancer of the oral cavity and any cancer manifested by solid tumors with VEGFR-2 expression; as well as other VEGFR-2 related disorders including angiogenic related disorders including inflammatory diseases, retinopathies, age related macular degeneration, diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinopathy of prematurity and macular edema secondary to retinal vein occlusions.
  • VEGFR-2 has been linked to the prognosis and poor survival in patients suffering from squamous cell carcinoma of the lung and bladder cancer (Holtzer TR et al., (2013) PLoS One, Nov 14;8(11); Xia G et al., (2006) J Urol. Apr;175(4):1245-52).
  • said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma,
  • said angiogenic related disorder is selected from the group consisting of inflammatory diseases, retinopathies, age related macular degeneration, diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinopathy of prematurity and macular edema secondary to retinal vein occlusions.
  • a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein together with at least one second drug substance, such as an anti-cancer agent, anti-angiogenic agent or an immune response modifying agent.
  • a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in prognosis and/or diagnosis together with at least one cell proliferation marker.
  • contemplated cell proliferation markers are those selected from the group consisting of Ki-67, AgNOR, choline, claspin, cyclin A, CYR61, Cdk1, histone H3, HsMCM2, IL-2, Ki-S1, Ki-S2, LigI, MCM2, MCM6, MCM7, mitosin, p120, PCNA, PDPK, PLK, STK1, TK-1, topoisomerase II alpha and TPS.
  • a method of detecting VEGFR-2 comprising providing a sample suspected to contain VEGFR-2, contacting said sample with a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein, and detecting the binding of the VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition to indicate the presence of VEGFR-2 in the sample.
  • said method further comprises an
  • intermediate washing step for removing non-bound polypeptide, fusion protein, conjugate, complex or composition, after contacting the sample.
  • said method is a diagnostic or prognostic method for determining the presence of VEGFR-2 in a subject, the method comprising the steps:
  • VEGFR-2 binding polypeptide a fusion protein, conjugate, complex or composition as described herein, and
  • said method further comprises an intermediate washing step for removing non-bound polypeptide, fusion protein, conjugate or composition, after contacting the subject or sample and before obtaining a value.
  • said method further comprises a step of comparing said value to a reference.
  • Said reference may be by a numerical value, a threshold or a visual indicator, for example based on a color reaction.
  • the skilled person will appreciate that different ways of comparison to a reference are known in the art and may be suitable for use.
  • said subject is a mammalian subject, such as a human subject.
  • said method is performed in vivo. In another embodiment, said method is performed in vitro. While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be
  • Figure 1 is a listing of the amino acid sequences of examples of VEGFR-2 binding polypeptides of the present disclosure (SEQ ID NO:1, 3 and 5-25), second VEGFR-2 binding polypeptides (SEQ ID NO:2, 4 and 26- 42), albumin binding polypeptide ABD053 (SEQ ID NO:47); control polypeptides (SEQ ID NO:48, 49 and 50); as well as the amino acid sequences of human VEGFR-2 (SEQ ID NO:43), murine VEGFR-2 (SEQ ID NO:44), human VEGF-A (SEQ ID NO:45) and murine VEGF-A (SEQ ID NO:46) used for selection, screening and/or characterization of the invention.
  • VEGFR-2 binding motifs (BMs and BM2s) of the VEGFR-2 binding polypeptides disclosed herein extend from residue 8 to residue 36 in the sequences with SEQ ID NO:1-42.
  • the amino acid sequences of the 49 amino acid residues long polypeptides (BMod) predicted to constitute the complete three-helix bundle within each of these Z variants extend from residue 7 to residue 55.
  • Figure 2 shows results from evaluation of (A) Z05752 (SEQ ID NO:1) and (B) Z05993 (SEQ ID NO:2) in terms of binding specificity and epitope on VEGFR-2 using ELISA.
  • the absorbance is represented on the Y axis and the different VEGFRs tested and their respective concentrations are indicated by numbers 1-10 on the X axis (see Example 1).
  • Figure 3 shows the result from flow-cytometric analysis of Z17681 (SEQ IN NO:3) and Z17682 (SEQ ID NO:4) displayed on the surface of S. carnosus.
  • Z variants are represented on the X axis, and a ratio of FL-1 fluorescence intensity, corresponding to binding of (A) human VEGFR-2 or (B) murine VEGFR-2, to FL-6 fluorescence intensity, corresponding to surface expression level, is represented on the Y axis.
  • Figure 3C shows a ratio of FL-1 fluorescence intensity, corresponding to binding of (A) human VEGFR-2 or (B) murine VEGFR-2, to FL-6 fluorescence intensity, corresponding to surface expression level.
  • Figure 3C shows a
  • VEGFR-2 binding signal FL-1 is represented on the Y axis and the surface expression level (FL-6) is represented on the X axis.
  • Figure 4 shows circular dichroism (CD) spectra of the first generation VEGFR-2-binding polypeptides.
  • Figure 4A shows CD spectra of Z17681 and Z17682 at wavelengths ranging from 250 to 195 nm at 20 °C before and after variable temperature measurement (VTM). The spectra recorded before and after VTM overlap well for both variants.
  • Figure 4B shows graphs of variable temperature measurement (VTM) spectra obtained at 221 nm while heating Z17681 and Z17682 from 20 to 90 °C.
  • Figure 5 shows sensorgrams obtained from affinity measurements of VEGFR-2 binding polypeptides (A) Z17681 and (B) Z17682 for human and murine VEGFR-2 using surface plasmon resonance (SPR) analysis.
  • SPR surface plasmon resonance
  • Figure 6 shows the results from SPR-based competition assays of Z17681 and Z17682 binding to human and murine VEGFR-2.
  • Figure 6A shows sensorgrams obtained from a double injection, where 1 ⁇ M of Z17681 was injected (1), immediately followed by a second injection (2) of either a combination of 1 ⁇ M of Z17681 and 1 ⁇ M of Z17682, or 2 ⁇ M of Z17681, over immobilized human or murine VEGFR-2-Fc.
  • Figure 6B shows sensorgrams obtained from a double injection, where 1 ⁇ M of Z17682 was injected (1), immediately followed by a second injection (2) of either a combination of 1 ⁇ M of Z17682 and 1 ⁇ M of Z17681, or 2 ⁇ M of Z17682 over immobilized human or murine VEGFR-2-Fc.
  • An increase in response signal upon injection of a second Z variant indicates that Z17681and Z17682 can bind simultaneously to VEGFR-2.
  • Figure 7 shows the results obtained from SPR-based competition assays of Z17681 and Z17682 with human and murine VEGF-A.40 nM of human or murine VEGFR-2/Fc, which had been pre-incubated for 40 min with a 25 x molar excess of Z17681 or Z17682, was injected over a surface of immobilized human or murine VEGF-A. As shown in the SPR sensorgrams, pre-incubation with either of the VEGFR-2 specific Z variants blocked the interaction of VEGFR-2 with VEGF-A.
  • Figure 8 shows the result from flow-cytometric analysis of the alanine scan described in Example 5.
  • the 13 residues in the VEGFR-2-binding Z variants that were substituted with alanine are represented on the X axis, and the fold change in normalized binding signal (a ratio of FL-1 fluorescence intensity, corresponding to VEGFR-2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level) compared to the corresponding non-mutated binder (Z17681 or Z17682) is represented on the Y axis.
  • Binding to human (black bar) or murine (grey bar) VEGFR-2 is shown.
  • Figure 9A and 9B show scatter plots from fluorescence activated cell sorting of the affinity maturation library Z17681 matlib and Z17682 matlib , respectively, displayed on S. carnosus.
  • the VEGFR-2 binding signal (FL-1) is represented on the Y axis and the surface expression level (FL-6) is represented on the X axis.
  • the scatter plots show cells from the original unsorted library as well as cells isolated in the 1st, 2nd, 3rd and 4th selection round, respectively. For the 4th selection round, scatter plots are shown for selections including an off-rate step of 0 min, 30 min or 4.5 h. Scatter plots obtained for Z17681 and Z17682 are shown for comparison.
  • Figure 10A and 10B show the result from flow-cytometric analysis of the on-cell ranking experiment for maturated Z variants obtained from
  • the normalized binding signal to human VEGFR-2 after an off-rate incubation step of 30 min (black bar) or 4.5 h (grey bar) is shown.
  • the Z variants are represented on the X axis and a ratio of FL-1 fluorescence intensity, corresponding to VEGFR-2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level, is represented on the Y axis.
  • Figure 11A and 11B show the result from flow-cytometric analysis of binding of the top ten ranked Z variants obtained from Z17681 matlib and Z17682 matlib , respectively, to murine VEGFR-2.
  • the Z variants are
  • the fold change in normalized binding signal (a ratio of FL-1 fluorescence intensity, corresponding to VEGFR-2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level) compared to the corresponding non-mutated binder (Z17681 and Z17682, respectively) is represented on the Y axis.
  • Figure 12A and 12B show CD spectra obtained from VEGFR-2-binding Z variants.
  • Figure 13A and 13B show CD spectra from heat stability
  • VTM spectra obtained at 221 nm while heating the Z variants Z17701, Z17698, Z17691, Z17690, Z17720, Z17717, Z17712 and Z17719 from 20 to 90 °C are shown.
  • Figure 14 shows sensorgrams obtained for human VEGFR-2 of affinity maturated VEGFR-2 binding polypeptides. Injections of 5 nM, 10 nM and 20 nM Z17701 (SEQ ID NO:23), Z17698 (SEQ ID NO:20), Z17690 (SEQ ID NO:12) and Z17719 (SEQ ID NO:41) as indicated over immobilized human VEGFR-2-Fc were analyzed.
  • Figure 15A and 15B show the results obtained for murine VEGFR-2 of affinity matured VEGFR-2 binding polypeptides evaluated by SPR.
  • Figure 16 shows the results from an SPR-based competition assay of VEGFR-2 binding polypeptides Z17701 (SEQ ID NO:23) and Z17719 (SEQ ID NO:41).
  • Figure 17 shows the results from an SPR-based competition assay of VEGFR-2 binding polypeptides Z17701 and Z17719 with human and murine VEGF-A.40 nM of human or murine VEGFR-2/Fc, which had been pre- incubated for 40 min with a 25 x molar excess of Z17701 or Z17719, was injected over a surface of immobilized human or murine VEGF-A (as indicated). The SPR sensorgrams show that pre-incubation with either of the Z variant molecules blocked VEGFR-2 interaction with VEGF-A.
  • Figure 18 shows sensorgrams obtained from SPR analysis of dimeric VEGFR-2 binding Z variants as indicated.200 nM of Z17681-ABD053- Z17682, Z17682-ABD053-Z17681, Z17681-ABD053-Z17681 or Z17682- ABD053-Z17682 was injected over a surface of immobilized HSA followed by injections of (A) human or (B) murine VEGFR-2 at the indicated
  • Figure 19 shows the results of flow-cytometric analysis of binding of VEGFR-2-specifc dimeric Z variants (A) to VEGFR-2 expressing 293/KDR cells or (B) to non-VEGFR-2 expressing HEK293 cells in vitro. Binding of the Z variants (FL6) or the positive control antibody (FL1) is shown on the X axis and cell count is shown on the Y axis. Binding of heterodimers, homodimers or negative controls is indicated. A higher shift in fluorescence intensity was observed for the heterodimeric constructs than for the homodimeric constructs upon binding to VEGFR-2-expressing cells.
  • Figures 20A and 20B show the results from flow cytometric analysis of the binding of dimeric Z variant constructs to VEGFR-2-expressing HUVEC and 293/KDR cells, respectively.
  • the different constructs tested are shown on the X axis and the normalized binding signal (FL-6 fluorescence intensity of each sample divided by the FL-6 fluorescence intensity of the negative control construct Z03638-ABD053-Z03638) is shown on the Y axis.
  • Figure 21 shows the results from flow cytometric analysis of the IC50 evaluation of dimeric Z variant constructs blocking VEGF-A binding to
  • 293/KDR cells The concentrations of Z variant construct are shown on the X axis and the percentage of retained binding (FL-2 fluorescence intensity of each sample divided by the FL-2 fluorescence intensity of cells incubated with only VEGF-A) is shown on the Y axis.
  • Figure 22 shows the results from the VEGFR-2 phosphorylation blocking activity of dimeric Z variant constructs on 293/KDR cell.
  • the different combinations of Z variant construct and stimulation with VEGF-A or PBS are shown on the X axis, and the percentage of phosphorylation (OD450 for each sample, compared to OD450 for cells stimulated with VEGF-A after treatment with no Z variant construct) is shown on the Y axis.
  • Figure 23 shows inhibition of VEGF-A induced downstream
  • Figure 24 shows in vitro inhibition of sprout formation on HUVECs 3-4 days after the indicated treatments, in A) confocal microscopy images, B) a diagram showing the number of sprouts per bead and C) a diagram showing the average sprout length.
  • Figure 25 shows inhibition of VEGF-A induced proliferation by the dimeric Z variant Link2.
  • Figure 26 shows retained binding by a NODAGA-conjugated
  • (HE) 3 -Z17701- (S 4 G)-Z17719-Cys-NODAGA (grey) and Link2 (black) were injected at concentrations of 50 nM, 100 nM and 500 nM.
  • VEGFR-2 binding polypeptides described herein were sequenced, and the corresponding amino acid sequences are listed in Figure 1 and denoted by the identifiers SEQ ID NO:1- 42.
  • the Examples also describe the characterization of VEGFR-2 binding polypeptides as well as their in vitro functionality.
  • VEGFR-2/Fc (hVEGFR-2/Fc, R&D Systems) essentially as described in Grönwall et al., 2007(Grönwall et al., (2007) J Biotechnol, 128:162-183).
  • the library variants were fused to a gene encoding a Taq polymerase binding Z variant, denoted Ztaq (Gunneriusson E et al., (1999). Protein Eng. 12(10):873-8).
  • the selection buffer was phosphate buffered saline (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4, PBS) supplemented with 0.1 % Tween 20 (PBST 0.1) and 3 % BSA (Sigma). PBST 0.1 was used as wash buffer.
  • Pre-selections were performed in cycles 1-3 by incubation of phage stock with biotin-SP conjugated human IgG, Fc (Jackson Immuno Research Laboratories) immobilized on streptavidin coated paramagnetic beads (SA-beads, Invitrogen) and with biotin conjugated anti-IgG Z variant (Affibody AB) immobilized on SA-beads (anti IgG-Z conjugated SA-beads).
  • SA-beads streptavidin coated paramagnetic beads
  • Affibody AB biotin conjugated anti-IgG Z variant
  • the selection was performed in solution in four cycles, with one track in the first cycle and subsequently divided into six tracks in the last cycle.
  • An overview of the selection strategy describing an increased stringency in subsequent cycles obtained using a lowered target concentration and an increased number of washes, is shown in Table 3.
  • Phage particles bound to Fc fused target were captured on anti-IgG-Z conjugated SA-beads before washing and phage particles were finally eluted at pH 2.2 (50 mM glycine-HCl).
  • a constant high amount of phage particles was used for each round in half of the tracks in cycles 2-4 (see Table 3).
  • the number of phage particles used in the selection was about 2000 times the number of eluted phage particles in the previous cycle.
  • Table 3 Overview of the phage display selection using human VEGFR-2/Fc as tar et
  • Z variants for ELISA: The Z variants were produced by inoculating single colonies of Escherichia coli (E. coli) containing phagemids from the selections into culture medium supplemented with 1 mM isopropyl- ⁇ -D-1-thiogalactopyranoside (IPTG). Bacteria containing Z variants expressed as fusion proteins with Ztaq were subjected to repeated freeze- thaw cycles. Cells were pelleted by centrifugation to extract the proteins in the periplasmic fractions.
  • E. coli Escherichia coli
  • IPTG isopropyl- ⁇ -D-1-thiogalactopyranoside
  • ELISA-based screening for VEGFR-2 binding Z variants Binding of Z05752 and Z05993 to hVEGFR-2/Fc and murine VEGFR-2/Fc (mVEGFR- 2/Fc) was investigated using a standard sandwich ELISA.
  • half-area 96 well plates were coated with a polyclonal goat anti-Z IgG (Affibody AB) at a concentration of 4 ⁇ g/ml in PBSC (PBS supplemented with 0.5 % casein). After washing the plates in water, the periplasmic samples containing the soluble Z-Ztaq variants (VEGFR-2-binding Z variants fused to Ztaq) were added to the wells.
  • PBS supplemented with 0.05 % Tween 20 0.05
  • Plates were washed 4 times in PBST 0.05 followed by addition of hVEGFR- 2/Fc or mVEGFR-2/Fc diluted in PBSC.
  • the target concentrations used for screening were 3.6 nM or 7.2 nM hVEGFR-2/Fc and 0.9 nM or 9 nM mVEGFR-2/Fc.
  • the plates were developed by addition of a horseradish peroxidase-conjugated goat anti- human IgG (anti-human IgG-HRP, Southern Biotechnology) diluted 1:10000 in PBSC followed by addition of TMB substrate (Thermo Scientific) according to the supplier’s recommendations. Plates were measured at 450 nm using a microplate reader (Victor3, Perkin Elmer).
  • ELISA-based specificity and epitope analysis The same ELISA set up as for said screening was used to investigate the specificity of Z05752 and Z05993 as well as the ability of these variants to interfere with the binding of VEGF-A to hVEGFR-2.
  • Selectivity to human and murine VEGFR- 2/Fc, human and murine VEGFR-1/Fc (R&D Systems) and human and murine VEGFR-3/Fc (R&D Systems) were assayed at target concentrations of 1.8 nM hVEGFR-2/Fc, 1.8 nM hVEGFR-2/Fc, 20 nM hVEGFR-1 or 20 nM hVEGFR-3/Fc according to the setup below.
  • hVEGFR-2/Fc at a concentration of 1.8 nM was premixed with a 15-fold molar excess of human VEGF-A (R&D Systems) before addition to the ELISA plates.
  • the ELISA assays were developed as described above and the plates were measured at 450 nm using a microplate reader (Victor3, Perkin Elmer) For each Z variant the experimental set up was a follows:
  • Phage display selection and screening of Z variants with affinity for VEGFR-2 A naive Z variant library of 1.4 ⁇ 1010 complexity displayed on M13 filamentous phage (monovalent phagemid system) was used to select binders to hVEGFR-2/Fc. After four selection rounds, a total of 279
  • This Example describes the subcloning of the amino acid residues corresponding to helix 1 and 2 of Z05752 and Z05993 into a staphylococcal display vector to obtain Z variants Z17681 (SEQ ID NO:3) and Z17682 (SEQ ID NO:4). Flow cytometric analysis of Z17681 and Z17682 displayed on S. carnosus as well as purification of said variants is described. Materials and methods
  • the genes encoding said Z variants were amplified by PCR, cleaved by the restriction enzymes XhoI and SacI (New England Biolabs) and ligated into the staphylococcal display vector pSCZ1 (Kronqvist, N et al., (2008) Protein Eng Des Sel 21(4): 247-255) which had been cleaved with the same enzymes. Ligation was performed using T4 DNA ligase (New England Biolabs) followed by transformation to E. coli RR1 ⁇ M15 cells. Plasmids encoding Z17681 (SEQ ID NO:3) and Z17682 (SEQ ID NO:4) were thus obtained.
  • the plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen) and transformed into S. carnosus by electroporation as previously described in Löfblom et al., 2007 (Löfblom J et al., (2007) Journal of Applied Microbiology 102(3): 736-747).
  • TSB medium (30 g/l; Merck) supplemented with 20 ⁇ g/ml chloramphenicol, and cultivated for 16 h at 37 °C and 150 rpm.
  • Approximately 106 cells were washed with 1 ml PBS supplemented with 0.1 % Pluronic F108 NF Surfactant (PBSP; pH 7.4; BASF Corporation), re-suspended in 200 nM of hVEGFR-2/Fc or 150 nM mVEGFR-2/Fc, and incubated at room temperature for 1 h under conditions of gentle mixing.
  • PBSP Pluronic F108 NF Surfactant
  • Z17682 The genes encoding Z17681 and Z17682 were amplified by PCR using Phusion DNA polymerase (Finnzymes) and cleaved by restriction enzymes NdeI and XhoI (New England Biolabs). The genes were ligated into a pET-26b(+) vector (Merck), cleaved with the same enzymes, using T4 DNA ligase (New England Biolabs) and transformed into E. coli RR1 ⁇ M15 cells (Rüther U (1982), supra). Plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen) and transformed into E. coli Rosetta (DE3) cells (Merck) using heat shock.
  • Colonies were inoculated to TSB medium (30 g/l; Merck), supplemented with 5 g/l yeast extract (Merck) and 50 ⁇ g/ml kanamycin (Sigma-Aldrich Company Ltd, Dorset, UK), and cultivated over night at 37 °C and 150 rpm. After 16 h, the cultures were diluted to an OD value of 0.05-0.1 in TSB medium (30 g/l; Merck). When an OD value of 0.5-1 was reached, protein expression was induced with isopropyl-beta-D- thiogalactopyranoside (IPTG) (Apollo Scientific Ltd.) and cultures were incubated for 20 h at 25 °C and 150 rpm. Cells were harvested by
  • Recombinant staphylococcal cells were incubated with either 150 nM mVEGFR-2/Fc or 200 nM hVEGFR-2/Fc, together with fluorescently labeled goat anti-human IgG and human serum albumin (HSA) labeled with a different fluorophore.
  • Both Z17681 and Z17682 were functionally expressed on the cell surface and showed binding to human as well as murine VEGFR-2 ( Figure 5).
  • the measured binding signal for both human and murine VEGFR- 2 was slightly higher for Z17681 than for Z17682.
  • a VEGFR-2 binding Z variant displayed on S. carnosus was used as a negative control.
  • Z17681 and Z17682 Protein production and purification of Z17681 and Z17682: The genes encoding Z17681 and Z17682 were sub-cloned into the expression vector pET-26b(+) for production of soluble protein. Proteins were expressed in the cytoplasm and recovered by sonication of the cells followed by IMAC purification. The purity of the soluble proteins was analyzed using SDS- PAGE, and single bands corresponding to the correct molecular weight ( ⁇ 6.5 kDa) were observed for both variants.
  • Z17681 and Z17682 were further characterized using circular dischroism (CD) spectroscopy and surface plasmon resonance analysis. Materials and methods
  • Circular dichroism analysis of Z17681 and Z17682 Purified Z variants Z17681 and Z17682 were analyzed by CD spectroscopy using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB). CD spectra were obtained at 250-195 nm and 20 °C. Thermal unfolding was analyzed at 221 nm and a temperature range of 20-90 °C.
  • Binding kinetics analysis of Z17681 and Z17682 Surface plasmon resonance experiments were performed using a ProteOn XPR36 instrument (Biorad Laboratories), employing PBS buffer with 0.1 % Tween (PBST) as a running buffer and 10 mM HCl or PBST for regeneration. Human and murine VEGFR-2/Fc (R&D Systems) were immobilized at 4200 and 4800 RU, respectively, by amine coupling on two surfaces of a GLM sensor chip (Biorad Laboratories).
  • PBST PBS buffer with 0.1 % Tween
  • Binding of Z17681 and Z17682 to both human and murine VEGFR-2 was analyzed by injections of five different concentrations (50 nM, 100 nM, 200 nM, 350 nM and 500 nM) at 100 ⁇ l/min over the immobilized VEGFR-2/Fc for 60 s followed by a dissociation phase of 600 s.
  • PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration.
  • Binding kinetics analysis of Z17681 and Z17682 The kinetics of the binding of Z17681 and Z17682 to human and murine VEGFR-2 was analyzed by surface plasmon resonance. Binding of Z17681 and Z17682 to both human and murine VEGFR-2 was detected by injecting the Z variants over human and murine VEGFR-2/Fc immobilized by amine coupling. Equilibrium dissociation constants (K D ) for human VEGFR-2 were determined to be around 160 nM for Z17681 and 80 nM for Z17682. The K D value for murine VEGFR-2 was determined to be around 260 nM for Z17681 and 90 nM for Z17682 ( Figure 5).
  • Z17681 and Z17682 were analyzed using SPR to determine if they could bind simultaneously to VEGFR-2, and whether they competed with VEGF-A for binding to VEGFR-2. Materials and methods
  • Z17682 or an injection of 2 ⁇ M Z17681 as a control.
  • Z17682 was injected first, followed a mixture of Z17681 and Z17682, or an injection of 2 ⁇ M Z17682 as a control.
  • PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration.
  • VEGF-A (R&D Systems) was immobilized on a sensor chip surface.40 nM of human or murine VEGFR- 2/Fc (R&D Systems), which had been pre-incubated with a 25-fold molar excess of Z17681, Z17682 or PBS (as a control) for 40 min, was injected over the surface. PBS buffer with 0.1% Tween (PBST) was used as a running buffer and 10 mM NaOH was used for regeneration. Results
  • Z17681 and Z17682 were analyzed using SPR to determine if they could simultaneously bind to VEGFR-2.
  • a saturating concentration (1 ⁇ M) of Z17681 was injected, directly followed by an injection of a mix of 1 ⁇ M Z17681 and 1 ⁇ M Z17682, or an injection of 2 ⁇ M Z17681 (control).
  • a large increase in response signal was observed for the mix of Z17681 and Z17682, but not for Z17681 alone, indicating that Z17681 and Z17682 were able to bind simultaneously to VEGFR-2 (Figure 6A).
  • the results indicate that Z17681 and Z17682 bind to distinct and non- overlapping epitopes on VEGFR-2.
  • Single-point mutations of Z17681 and Z17682 for alanine scan Single-point mutations of Z17681 and Z17682 with codon substitution to alanine at residues 9, 10, 11, 13, 14, 17 (not in Z17681), 18 (not in Z17682), 24, 25, 27, 32 or 35 were constructed by PCR amplification using oligonucleotides encoding each mutation. Residues Ala17 in Z17681 and Ala18 in Z17682 were mutated to valine. The mutated genes were cloned into the staphylococcal display vector pSCZ1, using restriction enzymes XhoI and SacI (New England Biolabs) and T4 DNA ligase (New England Biolabs) according to the supplier’s recommendations.
  • Plasmids were prepared from E. coli RR1 ⁇ M15 cells using a QIAprep Spin Miniprep kit (Qiagen) and sequences were confirmed by BigDye Thermo Cycle Sequencing using an ABI Prism 3700 instrument (Applied Biosystems). Plasmids were transformed into S. carnosus by electroporation as described in Example 2.
  • PBSP Surfactant
  • H+L Alexa Fluor® 488 goat anti- human IgG (H+L) antibody
  • Invitrogen 150 nM Alexa Fluor® 647-HSA conjugate.
  • Cells were incubated on ice for 45 min in the dark.
  • the cells were washed with 200 ⁇ l ice-cold PBSP and re-suspended in 200 ⁇ l ice-cold PBSP for flow-cytometric analysis.
  • Alanine scan mutagenesis of Z17681 and Z17682 Alanine and valine scan mutagenesis was used to analyze the individual contribution from residues in Z variants Z17681 and Z17682 to the interaction with VEGFR-2. Thirteen mutants of each binder were created and all were mutated to alanine except position Ala17 in Z17681 and Ala18 in Z17682, which were mutated to valine. The 26 mutants were expressed and displayed on the surface of staphylococci and binding to VEGFR-2 was analyzed by flow cytometry (Figure 8). The results obtained were used for the design of two affinity maturation libraries as described in Example 6.
  • Z17681 matlib contained approximately 4.1 x 107 individual clones
  • Each position was randomized with 18 codons corresponding to amino acids: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, Y, V, W (excluding C and P in all positions) with the amino acid residues based on the sequences of the VEGFR-2 binding Z variants Z17681 and Z17682 spiked at a higher proportion to generate an average mutation frequency of approximately three mutations per molecule.
  • the randomization frequency in each position was also normalized with the results from the alanine scan described above, resulting in less mutations in positions important for VEGFR-2 binding and more mutations in positions of less importance (Table 4).
  • Tables 4A and B show the library design of Z17681 matlib and
  • Oligonucleotides encoding helix 1 and 2 of Z variants containing the randomizations described above were purchased from Isogenica.
  • the libraries were amplified by 15 cycles of PCR using Phusion DNA polymerase (Finnzymes) and the PCR products were purified using QIAquick PCR purification kit (Qiagen GmbH).
  • the PCR products were digested with restriction enzymes XhoI and SacI (New England Biolabs) followed by purification using preparative gel electrophoresis (2 % agarose gel) and a QIAquick gel extraction kit (Qiagen).
  • the staphylococcal display vector pSCZ1 was digested with the same restriction enzymes and purified by preparative gel electrophoresis as described above.
  • the libraries were ligated into pSCZ1 using T4 DNA ligase (New England Biolabs) according to the supplier’s recommendations. Plasmids were purified and concentrated using phenol chloroform extraction and ethanol precipitation, and subsequently transformed into E. coli SS320 (MC1061 F’) electrocompetent cells (Lucigen). Plasmids were prepared using a Jetstar Maxi Kit (Genomed) and transformed into S. carnosus by electroporation as previously described (Löfblom J et al., (2007), supra).192 colonies from each library were sequenced by BigDye Thermo Cycle Sequencing using an ABI Prism 3700 instrument (Applied Biosystems). Results
  • Flow-cytometric cell sorting of Z variant libraries Aliquots of the libraries containing 1.2 x 109 cells (30 times the estimated library size) were inoculated to 500 ml TSB+YE (Merck) with 10 ⁇ g/ml chloramphenicol, and incubated at 150 rpm and 37 °C for 24 h. Approximately 109 cells were harvested by centrifugation (3500 x g, 6 min at 4 °C) and washed twice with PBSP, followed by re-suspension in 1.5 ml of 20 nM hVEGFR-2/Fc (R&D Systems) in PBSP and incubation for 2 h at room temperature under conditions of gentle mixing.
  • sorting round 2-4 approximately 10 x the library size was labeled for sorting. Those approximately 0.5 % of the population that exhibited the highest VEGFR-2-binding signal were gated and sorted to TSB+YE (Merck). The cells were grown overnight in TSB+YE with 10 ⁇ g/ml chloramphenicol. In the first round, the libraries were incubated with 50 nM hVEGFR-2/Fc and in the second and third round with 5 nM hVEGFR-2/Fc. In the fourth round, an off- rate selection strategy was applied. First, cells were incubated with 20 nM hVEGFR-2/Fc (R&D Systems) as described above.
  • Flow-cytometric cell sorting was used for isolation of staphylococcal cells displaying Z variants with increased affinity for VEGFR-2.
  • Four rounds of sorting were performed, using a starting concentration of 50 nM hVEGFR-2/Fc fusion and decreasing down to 20 nM in the third round ( Figure 9).
  • Target binding was detected using a
  • On-cell ranking of maturated Z variants Individual clones from the fourth sorting round occurring more than twice among the sequenced colonies from Z17681 matlib and all unique variants from Z17862 matlib were inoculated in 3 ml TSB+YE (Merck) and incubated overnight at 150 rpm and 37 °C. Cells were washed with 800 ⁇ l PBSP and incubated with 20 nM hVEGFR-2/Fc (R&D Systems) for 1.5 h. After washing with ice-cold PBSP, cells were incubated at room temperature with 50 nM unlabeled VEGFR-2 (Sino Biological) for 30 min or 4 h under conditions of gentle mixing.
  • the cells were incubated in PBSP with Alexa Fluor® 488 goat anti-human IgG (H+L) antibody (Invitrogen) (1:1000 dilution) and 150 nM Alexa Fluor® 647- HSA conjugate for 45 min on ice in the dark.
  • the cells were washed once with ice-cold PBSP and then analyzed using a Gallios flow cytometer (Beckman Coulter).
  • Cells displaying binders Z17681 and Z17682 were included for comparison and cells displaying the TNF ⁇ -binding Z variant Z00185 (SEQ ID NO:48) were included as a negative control.
  • On-cell affinity ranking of maturated VEGFR-2-binding Z variants The affinities of individual clones were compared by flow-cytometric analysis of recombinant staphylococci.21 Z variants (denoted SEQ ID NO:5-25) from Z17681 matlib which occurred more than twice among the sequences, and the 17 unique Z variants (denoted SEQ ID NO:26-42) from Z17682 matlib were included in the assay ( Figures 10A and 10B).
  • the samples were subjected to 30 min or 4.5 h incubations with unlabeled VEGFR-2 after incubation with hVEGFR-2/Fc.
  • the target binding signal was normalized against the expression level as described above. All analyzed clones showed a slower dissociation compared to the primary binders Z17681 and Z17682.
  • Z17681 matlib Z17687 (SEQ ID NO:9), Z17688 (SEQ ID NO:10), Z17689 (SEQ ID NO:11), Z17690 (SEQ ID NO:12), Z17691 (SEQ ID NO:13), Z17694 (SEQ ID NO:16), Z17698 (SEQ ID NO:20), Z17699 (SEQ ID NO:21) and Z17701 (SEQ ID NO:23)) as well as the top eight ranked clones from Z17682 matlib (Z17705 (SEQ ID NO:27), Z17706 (SEQ ID NO:28), Z17712 (SEQ ID NO:34), Z17713 (SEQ ID NO:35), Z17714 (SEQ ID NO:36), Z17716 (SEQ ID NO:38), Z17717 (SEQ ID NO:39) and Z17720 (SEQ ID NO:42)) were also analyzed using mVEGFR-2. These binders exhibited retained cross reactivity to mVEGFR-2 ( Figures 11A and 11B).
  • Z17690, Z17691, Z17698 and Z17701 corresponding to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, respectively
  • Z017681 matlib and Z17712, Z17717, Z17719 and Z17720 corresponding to SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, respectively
  • Z17682 matlib were chosen for further characterization.
  • the four Z variants from Z017681 matlib were chosen because they showed the highest signals after 30 min and 4.5 h of off-rate incubation.
  • Example 8 the eight selected maturated Z variants from Example 8 were further characterized using CD analysis and surface plasmon resonance analysis. Material and methods
  • Z017681 matlib and Z17712, Z17717, Z17719 and Z17720 (corresponding to SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, respectively) from Z017682 matlib were sub-cloned to pET26-b(+) and transformed into BL21(DE3) cells (Merck). Soluble proteins were produced and purified as described in Example 2 for binders Z17681 and Z17682.
  • Circular dichroism analysis of maturated Z variants Circular dichroism analysis was performed at 195-205 nm and 20 °C using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) in a cell with an optical path- length of 1 mm. Variable temperature experiments were performed by measuring the ellipticity while heating the Z variants from 20 °C to 90 °C. A concentration of 0.2 mg/ml Z variant was used.
  • the Z variants were immobilized on a GLC sensor chip (Biorad Laboratories) using a ProteOn XPR36 instrument (Biorad Laboratories).5 nM, 10 nM or 20 nM mVEGFR-2 (Sino Biological) was passed over the surfaces. PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration. Results
  • Circular dichroism Secondary structure content of the four selected candidates from each library was analyzed using CD spectroscopy and the melting temperature was determined by heating the samples from 20 °C to 90 °C ( Figure 12). Refolding after heating to 90 °C was evaluated after allowing the samples to cool to 20 °C. All clones showed an alpha-helical content, although Z17691 (SEQ ID NO:13) had a lower degree of alpha- helical content than the other clones. All clones demonstrated similar secondary structure content after heating, indicating complete refolding ( Figure 12). The melting temperatures of the clones obtained from
  • Z17682 matlib containing the K33N mutation (Z17720, Z17717 and Z17712) had a lower melting temperature than both the primary binder Z17682 and the second generation binder Z17719, which both lack the K33N mutation ( Figure 13 and Table 6).
  • the melting temperatures for these clones were around 37 °C, while the melting temperature for Z17719 was 45 °C.
  • Table 6 Melting temperatures of maturated VEGFR-2-binding Z variants.
  • Binding kinetics analysis of second-generation binders Binding kinetics of Z17701, Z17698, Z17690 and Z17719 were analyzed using surface plasmon resonance ( Figure 14 and 15). Different concentrations (5 nM, 10 nM and 20 nM) of each Z variant were injected over immobilized human VEGFR-2/Fc. The equilibrium dissociation constant for Z17719 for mVEGFR-2 was determined by injection of Z17719 over immobilized mVEGFR-2/Fc. Z17701, Z17698 and Z17690 were immobilized on a sensor chip and 5 nM, 10 nM or 20 nM mVEGFR-2 (not fused to Fc in order to avoid avidity effects) was passed over the surfaces.
  • the equilibrium dissociation constant (K D ) was determined from the association and dissociation rates calculated from sensorgrams using a monovalent binding equation and non- linear regression.
  • Equilibrium dissociation constants for human VEGFR-2 were determined to be 5.0 nM-10.9 nM for the four candidates (Table 7A), representing about a 30-fold and an 8-fold increase in affinity compared to the original binders Z17681 and Z17682, respectively.
  • the affinities for mVEGFR- 2 were determined to be 7.8 nM-11.9 nM, representing around a 15-fold increase in affinity compared to primary binders (Table 7B).
  • Table 7A Affinit constants of maturated Z variants for human VEGFR-2.
  • the Z variants were injected in the opposite order, i.e.1 ⁇ M Z17719 was injected first, followed by a second injection of either a combination of 1 ⁇ M Z17701 and 1 ⁇ M Z17719, or of 2 ⁇ M Z17719.
  • PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration.
  • VEGF-A (R&D Systems) was immobilized on a sensor chip surface.40 nM of human or murine VEGFR- 2/Fc (R&D Systems), which had been pre-incubated for 40 min with a 25-fold molar excess of Z17701 or Z17719 (or PBS as a control), was injected over the surface. PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM NaOH was used for regeneration. Results
  • VEGFR-2/Fc was immobilized on a sensor chip and detection of simultaneous binding was performed using a double injection.1 ⁇ M of the first Z variant was injected, immediately followed by a second injection of either a combination of 1 ⁇ M of the first Z variant and 1 ⁇ M of the second Z variant, or 2 ⁇ M of the first Z variant (as control). The second injection of 2 ⁇ M of the first Z variant did not give rise to any substantial increase in binding signal, whereas the addition of 1 ⁇ M of the second Z variant resulted in a substantial increase in signal (Figure 16).
  • VEGFR-2 simultaneously to VEGFR-2 and that both of them compete with human as well as murine VEGF-A for binding to VEGFR-2.
  • dimeric maturated Z variants were produced and characterized using SPR analysis.
  • the dimeric Z variants were also shown to be able to bind to VEGFR-2 expressed on the surface of mammalian cells.
  • pET26b-Z02891-ABD053- Z03638 encoded a dimeric Z variant construct comprising the HER2-binding Z variant Z02891 (SEQ ID NO:49) and the Taq polymerase-binding Z variant Z03638 (SEQ ID NO:50), separated by the albumin binding domain ABD053 (SEQ ID NO:47).
  • Z variant molecules were replaced by VEGFR-2- binding Z variants to generate the following four constructs: Z17701-(S 4 G) 4 -ABD053-(S 4 G) 4 -Z17719,
  • the genes encoding the first Z variants were amplified using Phusion DNA polymerase (Finnzymes). PCR products and vector were digested with NdeI and AscI (New England Biolabs), ligated using T4 DNA ligase (New England Biolabs) and transformed into E. coli RR1 ⁇ M15 cells (Rüther U (1982), supra). Plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen). Z03638 was replaced by the second respective VEGFR-2-binding Z variant using restriction enzymes SacII and XhoI essentially as described above.
  • plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen) and transformed into E. coli BL21 (DE3) cells (Merck) using heat shock. Colonies were inoculated to TSB medium (30 g/l; Merck),
  • the dissociation constants of the homodimeric and heterodimeric constructs were compared by injections of 15 nM, 30 nM and 60 nM of human or murine VEGFR-2 following an injection of 200 nM of each of the homodimeric constructs; or by injections of 5 nM, 15 nM and 45 nM human or murine VEGFR-2 following an injection of 200 nM of the heterodimeric constructs.
  • the dissociation constants were calculated from sensorgrams using a monovalent binding equation and non-linear regression.
  • Table 8B Dissociation rate constants for dimeric constructs against murine VEGFR-2.
  • HEK293 cells with no expression of VEGFR-2 were incubated with the same dimeric Z variants.
  • dimeric maturated Z variants with different linker lengths were produced and characterized in terms of binding to VEGFR-2, blocking of VEGF-A binding to VEGFR-2 expressed on the surface of mammalian cells, as well as inhibition of VEGFR-2 mediated phosphorylation and angiogenesis.
  • Link4 Z17701-(S 4 G) 8 -Z17719-(S 4 G) 3 -ABD053.
  • the genes encoding these constructs in the vector pJ411 were ordered from DNA2.0. Colonies were inoculated to TSB medium (30 g/l; Merck), supplemented with 5 g/l yeast extract (Merck) and 50 ⁇ g/ml kanamycin (Sigma-Aldrich) and cultivated over night at 37 °C and 150 rpm. After 16 h, the cultures were re-inoculated to an OD 600 of 0.05-0.1 in TSB medium (30 g/l; Merck).
  • IPTG isopropyl-beta-D-thiogalactopyranoside
  • 293/KDR cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10 % FBS and 2 mM L-glutamine. Cells grown on petri dishes were washed with PBS and harvested by pipetting. HUVECs (Lonza) were cultivated in Endothelial Growth Medium-2 (EGM-2) (Lonza) supplemented with EGM-2 Bullet Kit (Lonza). Cells grown on petri dishes were washed with PBS, harvested by trypsination, centrifuged at 220 rcf for 5 min and pellets were re-suspended in PBS with 1 % BSA (PBSB).
  • DMEM Modified Eagle Medium
  • the cells were incubated for 20 min on ice with 200 nM of each of the four dimer constructs, the negative control construct Z03638-(S 4 G) 4 - ABD053-(S 4 G) 4 -Z03638, or the positive control mouse IgG anti-human VEGFR-2 antibody (R&D Systems), in PBSB.
  • the cells were pelleted by centrifugation at 300 rpm for 4 min, and re-suspended in 150 nM Alexa Fluor® 647-HSA conjugate (or 1 ⁇ g/ml Alexa Fluor® 488 Rabbit Anti-Mouse IgG (H+L) antibody (Invitrogen).
  • IC50 evaluation for VEGF-A blocking 293/KDR cells (Sibtech Inc.) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10 % FBS and 2 mM L-glutamine. Cells grown on petri dishes were washed with PBS and harvested by pipetting.
  • DMEM Modified Eagle Medium
  • the cells were incubated for 15 min on ice with 1 nM, 4 nM, 16 nM, 64 nM, 256 nM or 1024 nM of each of the four dimeric Z constructs, or 4 nM, 64 nM or 1024 nM of the negative control construct Z03638-(S 4 G) 4 - ABD053-(S 4 G) 4 -Z03638, or no Z variant, in PBSB.
  • the cells were pelleted by centrifugation at 300 rpm for 4 min, re-suspended in 50 nM biotinylated VEGF-A (Acro Biosystems), and incubated on ice for 15 min.
  • SPR analysis SPR was used to analyze the binding of Link1-Link4 to VEGFR-2 using a ProteOn XPR36 system.
  • HSA was immobilized on a GLM sensor chip (Bio-Rad Laboratories) at 700 RU and 2300 RU. Each dimeric construct was injected at a concentration of 100 nM over the HSA
  • PKH67 Green Fluorescent Cell linker kit (Sigma-Aldrich) was used to label HUVECs according to the
  • Approximately 25000 WI38 cells in EBM-2 supplemented with 2 % FBS and 50 ng/ml VEGF-A were plated in each well and incubated for 24 h at 37 °C. After incubation, the wells were treated with 50 nM or 10 nM Link2, 50 nM or 10 nM of the VEGFR-2 binding monoclonal antibody ramucirumab (ImClone Systems) or 50 nM of the negative control variant Z03638-(S 4 G) 4 -ABD053- (S 4 G) 4 -Z03638. A positive control with only VEGF-A added and a negative control without any VEGF-A added were also included. The number of sprouts and average sprout length were imaged 3-4 days later using a Zeiss700 confocal microscope and analysis was done using Image J.
  • VEGF-A induced 293/KDR proliferation assay Approximately 1000 293/KDR cells (cultivated in DMEM supplemented with 10 % FBS and 2 mM L-glutamine) per well were seeded in different 96 well plates and incubated for approximately 20 h. After 7 h, 10 ⁇ l of CCK8 agent (from cell counting kit- 8; Sigma Aldrich) was added to one of the plates, which was analyzed to confirm that cells were distributed evenly. After 20 h, medium was removed and 200 ⁇ l fresh medium was added. Cells were then treated with 100 nM Link2 + 10 nM VEGF-A, 10 nM VEGF-A only (positive control), or PBS
  • Example 11 demonstrated that the heterodimeric constructs Z17701- ABD053-Z17719 and Z17719-ABD053-Z17701 could bind to VEGFR-2 with a higher apparent affinity than the homodimeric constructs, as a result of simultaneous binding of Z17701 and Z17719.
  • new heterodimeric constructs based on Z17701 and Z17719 were constructed.
  • the genes encoding Z17701 (SEQ ID NO:23) and Z17719 (SEQ ID NO:41) were fused together with ABD053 in the four constructs denoted Link1, Link2, Link3 and Link4.
  • the four constructs were expressed in E. coli and purified by affinity chromatography using HSA-Sepharose. SDS-PAGE confirmed that the proteins were pure and of the correct size.
  • IC50 evaluation for VEGF-A blocking To verify that dimeric Z variants with different linker lengths could block VEGF-A binding to VEGFR-2 expressed on the surface of mammalian cells, a flow-cytometry based analysis was performed.293/KDR cells (Sibtech Inc.) were incubated with concentrations varying from 1 nM to 1024 nM of the four dimeric constructs Link1-4.
  • biotinylated VEGF-A After incubation with dimeric Z constructs, cells were incubated with biotinylated VEGF-A (Acro Biosystems). Cell binding of the biotinylated VEGF-A was detected using streptavidin-R-phycoerythrin conjugate (SAPE, Invitrogen) (or Alexa Fluor® 488 Rabbit Anti-Mouse IgG Antibody for the positive control).
  • SAPE streptavidin-R-phycoerythrin conjugate
  • Alexa Fluor® 488 Rabbit Anti-Mouse IgG Antibody for the positive control.
  • IC50 values demonstrate that all constructs can block VEGF-A binding to VEGFR-2 equally well, and that a linker length as short as 5 amino acids can be used without the introduction of steric constraint, which may be useful for future in vivo imaging applications, where a small protein size is desirable.
  • VEGFR-2 phosphorylation was stimulated by addition of human VEGF-A to the cells. After VEGF-A incubation, further phosphorylation was prevented by the presence of PhosStop (Roche), a phosphatase inhibitor cocktail, in the cell lysis buffer. De-phosphorylation was prevented by the presence of Na orthovanadate and protein degradation was prevented by the presence of phenylmethylsulfonyl fluoride (PMSF).
  • PhosStop a phosphatase inhibitor cocktail
  • VEGFR-2 downstream activation is an important part of signaling for angiogenesis progression (Gourlaouen et al. (2013), J Biol Chem 288:7467-7480).
  • SPR analysis SPR was used to determine the kinetics of binding between dimeric Z variants with different linker lengths and human or murine VEGFR-2. HSA was immobilized on a chip surface followed by injection of the dimeric Z variants and then of monomeric hVEGFR-2 or mVEGFR-2. This particular setup both allowed determination of dissociation constants for the different constructs (Table 9A and 9B) and confirmed their ability to bind to VEGFR-2 while interacting with HSA. The dimeric Z variant with the shortest linker, Link 2, showed the slowest dissociation rate.
  • the K D values for the interaction of Link2 with hVEGFR-2 and mVEGFR-2 were determined to be 313 ⁇ 30 pM and 292 ⁇ 11 pM, respectively.
  • Table 9A Dissociation rate constants a ainst human VEGFR-2.
  • Table 9B Dissociation rate constants a ainst murine VEGFR-2.
  • HUVEC sprouting assay In vitro HUVEC sprouting assay: In this assay, the anti-angiogenic effect of a dimeric VEGFR-2 binding Z variant was investigated by analysis of inhibition of HUVEC sprout formation. An ample decrease in both length and number of sprouts were shown for cells treated with Link2 or with the VEGFR- 2 binding monoclonal antibody ramucirumab, included for comparison, compared to cells incubated with negative control dimeric Z variant or untreated cells ( Figure 24).
  • VEGF-A induced 293/KDR proliferation assay The anti-angiogenic effect of Link2 was further investigated in a proliferation assay using 293/KDR cells. Cells treated with VEGF-A exhibit a significant increase in growth rate compared to untreated cells. The results from cells treated with both VEGF-A and Link2 show that the dimeric Z variant significantly inhibits the increased growth rate induced by VEGF-A ( Figure 25).
  • Figure 25 The results from cells treated with both VEGF-A and Link2 show that the dimeric Z variant significantly inhibits the increased growth rate induced by VEGF-A
  • VEGFR-2 binding polypeptides and controls to be used for in vivo studies, such as for imaging or therapeutic studies.
  • a C-terminal cysteine residue was incorporated to enable site-specific labeling.
  • An N-terminal (HE)3 sequence was added to non-ABD-fused polypeptides in order to simplify purification.
  • the polypeptides were conjugated and analyzed in terms of stability and binding to VEGFR-2. Materials and methods
  • NODAGA conjugation All buffers used during conjugation were supplemented with Chelex 100 to minimize occupied chelators.
  • the polypeptides were first re-suspended in PBS with 20 mM DTT and reduced for 30 min at 40 °C. Reduced polypeptides were buffer exchanged to 20 mM NH 4 Ac pH 5.5 using PD-10 columns (GE Healthcare) according to the manufacturer’s instructions.
  • the C-terminal cysteine of the respective polypeptide was conjugated by addition of 3x molar excess of maleimide- NODAGA (Chematech) and incubated at 40 °C for 1 h.
  • Circular dichroism spectroscopy The (HE) 3 -tagged NODAGA- conjugated polypeptides were analyzed using circular dichroism (CD) spectroscopy at 195-250 nm using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) at a concentration of 0.4 mg/ml. To determine the thermal stability of each sample, ellipticity was measured at 221 nm while heating the sample from 20 to 90 °C. After being subjected to 90 °C, the sample was left for five minutes at 20 °C and a new spectrum at 195-250 nM was recorded.
  • CD circular dichroism
  • Production of polypeptides The six specified polypeptides were successfully produced and used for conjugation.
  • NODAGA conjugation To enable radiolabeling of VEGFR-2 binding polypeptides and controls, coupling of a maleimide-NODAGA chelator was performed. Conjugated polypeptides were purified by RP-HPLC and the correct conjugation was verified by ESI-MS.
  • Circular dichroism spectroscopy CD analysis was performed to determine alpha-helical content, thermal stability and refolding capacity of (HE) 3 -tagged NODAGA-conjugated polypeptides. All four polypeptides showed alpha helical content and refolded after denaturation by heating to 90 °C.
  • This Example describes radiolabeling and in vivo imaging studies using VEGFR-2 binding polypeptides and controls administered to tumor bearing mice. For a rapid distribution of the imaging agent, as well as a rapid clearance of the non-bound fraction, the polypeptides are not fused to ABD. Materials and methods
  • Radiolabeling of polypeptides Chelator-conjugated VEGFR-2 binding polypeptides and controls such as the non-ABD fused polypeptides described and purified in Example 13 ((HE) 3 -Z17701-Cys-NODAGA, (HE) 3 -Z17701- (S 4 G)-Z17719-Cys-NODAGA, (HE) 3 -Z03638-Cys-NODAGA, (HE) 3 -Z03638- (S 4 G)-Z03638-Cys-NODAGA) are labelled with a suitable radiotracer.
  • labeling may be performed with generator-produced 68Ga in an NaAc buffered solution of approximately pH 4 and incubation for 15-60 min at 95 °C. Purification is carried out using size-exclusion NAP-5 columns (GE Healthcare). A small fraction is analyzed by instant thin-layer chromatography (iTLC; Biodex Medical Systems) to determine the radiochemical purity. In this system, free radiotracer migrates with the solvent front, while bioconjugates remain at the origin.
  • iTLC instant thin-layer chromatography
  • the MMTV-PyMT transgenic mouse model of metastatic breast cancer in which the expression of the polyoma middle T antigen (PyMT) oncoprotein is controlled by the mouse mammary tumor virus (MMTV), may be used to assess the ability of the disclosed VEGFR-2 binding polypeptides to detect levels of VEGFR-2 in vivo.
  • MMTV mouse mammary tumor virus
  • FVB Friend Virus B
  • In vivo imaging Tumor bearing MMTV-PyMT mice at different stages of disease progression (11-15 weeks) and wild-type control mice are injected with 0.5-5 ⁇ g radiolabeled VEGFR-2 binding polypeptides and control polypeptides. In vivo imaging is performed using a PET or SPECT scanner (depending on the radiotracer used for labeling of the polypeptides). For example, recordings of 68Ga-labeled polypeptides may be performed with a micro PET Focus 120 scanner (CTI Concorde Microsystems). The mice are placed with the whole body in the field of view and data is collected
  • the imaging evaluation is expected to show that the VEGFR-2 binding polypeptides, but not the control polypeptides, localize to VEGFR-2 expressing cells in vivo including VEGFR-2 expressing tumor tissue.
  • the expected result would support the use of VEGFR-2 binding polypeptides as a diagnostic tool for visualization of angiogenesis in primary tumors and during metastasis development.
  • This Example describes the use of VEGFR-2 binding polypeptides for therapeutic applications such as tumor therapy, where inhibition of new blood vessel formation may suppress tumor growth, and ophthalmology, where inhibition of uncontrolled blood vessel growth ultimately may prevent loss or impairment of vision.
  • tumor therapy where inhibition of new blood vessel formation may suppress tumor growth
  • ophthalmology where inhibition of uncontrolled blood vessel growth ultimately may prevent loss or impairment of vision.
  • ABD-fused polypeptides For extended circulatory half-life, which enables enhanced efficacy as well as lower doses and/or less frequent dosing, the use of ABD-fused polypeptides is warranted when administered systemically.
  • topical administration such as in the form of eye drops.
  • polypeptides not fused to ABD may alternatively be used.
  • Example 14 To investigate the therapeutic effect related to inhibition of new blood vessel formation and tumor growth, the same tumor model (MMTV-PyMT) as described in Example 14 may be used.
  • MMTV-PyMT tumor model
  • ABD-fused VEGFR-2 binding polypeptides and control polypeptides, such as those described in Example 13, are administrated by injection at a single or at multiple time points and the effect on new blood vessel formation and tumor growth is studied over several months.
  • VEGFR-2 binding polypeptides are instead formulated as eye drops and administered topically.
  • VEGFR-2 binding polypeptides to inhibit cell signaling in vitro, as confirmed in Example 12, can be translated to therapeutic effects in vivo. More specifically, administration of VEGFR-2 binding polypeptides to animals is expected to demonstrate inhibition of VEGF-induced angiogenesis in tumor and eye disease models. Besides reduced vessel density, the effect in tumor models is ultimately observed as reduced tumor size.
  • VEGFR-2 binding polypeptide comprising a VEGFR-2 binding motif BM, which motif consists of an amino acid sequence selected from: i) ENX 3 X 4 ASX 7 EIA X 11 LPNLX 16 DX 18 QY IAFIYX 26 LLX 29 wherein, independently from each other, X 3 is selected from L and Y;
  • X 4 is selected from E, F, I, K, M, V, W and Y;
  • X 7 is selected from K, N and R;
  • X 11 is selected from F, H, L and N;
  • X 16 is selected from N and T;
  • X 18 is selected from A, D, E, G, H, K, Q, S and T;
  • X 26 is selected from K and S;
  • X 29 is selected from D and R;
  • VEGFR-2 binding polypeptide according to item 1 wherein, in sequence i), X 3 is selected from L and Y;
  • X 4 is selected from E, F, I, M, V and Y;
  • X 7 is selected from K, N and R;
  • X 11 is selected from F, H, L and N;
  • X 18 is selected from A, D, E, K, S and T;
  • X 29 is D.
  • VEGFR-2 binding polypeptide according to any preceding item wherein X 3 X 7 X 11 is selected from LNH and LKN.
  • VEGFR-2 binding polypeptide according to any preceding item wherein X 3 X 4 X 7 is selected from LKN, LMN, YIK, YMK, LFK and LVK.
  • VEGFR-2 binding polypeptide according to any preceding item, wherein X 3 X 4 X 11 is selected from LKH, LMN, YIN, YMN, LFN, LVN.
  • VEGFR-2 binding polypeptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25.
  • sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
  • VEGFR-2 binding polypeptide according to item 8 wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:23.
  • VEGFR-2 binding polypeptide according to any preceding item, wherein said VEGFR-2 binding motif forms part of a three-helix bundle protein domain.
  • VEGFR-2 binding polypeptide according to item 10 wherein said VEGFR-2 binding motif essentially forms part of two helices with an
  • VEGFR-2 binding polypeptide according to item 11 wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
  • VEGFR-2 binding polypeptide according to item 12 wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
  • VEGFR-2 binding polypeptide according to any preceding item, which comprises a binding module BMod, the amino acid sequence of which is selected from:
  • [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9 provided that X 29 is D;
  • X a is selected from A and S;
  • X b is selected from N and E;
  • X c is selected from A, S and C;
  • X d is selected from E, N and S;
  • X e is selected from D, E and S;
  • X f is selected from A and S;
  • VEGFR-2 binding polypeptide according to any one of items 1-13, which comprises a binding module BMod, the amino acid sequence of which is selected from:
  • [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9 provided that X 29 is R;
  • X a is selected from A and S;
  • X b is selected from N and E;
  • X c is selected from A, S and C;
  • X d is selected from E, N and S;
  • X e is selected from D, E and S;
  • X f is selected from A and S;
  • VEGFR-2 binding polypeptide according to item 14, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25.
  • VEGFR-2 binding polypeptide according to item 16 wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5-25. 18. VEGFR-2 binding polypeptide according to item 17, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:5-25.
  • VEGFR-2 binding polypeptide according to item 19, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:23.
  • VEGFR-2 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
  • [BMod] is a VEGFR-2 binding module as defined in any one of items 14-20;
  • VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
  • [BMod] is a VEGFR-2 binding module as defined in any one of items 14-20;
  • VEGFR-2 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
  • VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
  • [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9;
  • VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
  • [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9;
  • VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
  • [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9;
  • VEGFR-2 binding polypeptide according to item 29, wherein sequence xi) is selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
  • VEGFR-2 binding polypeptide according to any preceding item, which is capable of binding to VEGFR-2 such that the K D value of the interaction with VEGFR-2 is at most 1 x 10-6 M, such as at most 1 x 10-7 M, such as at most 1 x 10-8 M, such as at most 1 x 10-9 M, such as at most 1 x 10-10 M.
  • VEGFR-2 binding polypeptide according to any preceding item, wherein said VEGFR-2 is human VEGFR-2.
  • VEGFR-2 binding polypeptide according to any one of items 1-32, wherein said VEGFR-2 is murine VEGFR-2.
  • VEGFR-2 binding polypeptide according to any preceding item, which has a melting temperature Tm of at least 40 °C, such as at least 45 °C.
  • VEGFR-2 binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end.
  • VEGFR-2 binding polypeptide according to item 36, wherein said additional amino acid(s) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
  • VEGFR-2 binding polypeptide according to any preceding item in multimeric form, comprising at least two VEGFR-2 binding polypeptide monomer units, whose amino acid sequences may be the same or different.
  • VEGFR-2 binding polypeptide according to item 38, wherein said VEGFR-2 binding polypeptide monomer units are covalently coupled together.
  • VEGFR-2 binding polypeptide according to any one of items 38-40, in dimeric form.
  • VEGFR-2 binding polypeptide according to item 41 in homodimeric form.
  • VEGFR-2 binding polypeptide according to item 43 comprising - a first monomer unit consisting of a VEGFR-2 binding polypeptide according to any one of the preceding items;
  • a second monomer unit consisting of a second VEGFR-2 binding polypeptide, comprising a second VEGFR-2 binding motif BM2, which motif consists of an amino acid sequence selected from: xvii) EFX 3 X 4 ADX 7 EIR X 11 LPNLX 16 HGQX 20 X 21 AFIX 25 X 26 LYX 29 wherein, independently from each other, X 3 is selected from Q and R;
  • X 4 is selected from A, D, H, K, L, M, R, S and V;
  • X 7 is selected from A, I and R;
  • X 11 is selected from A, D and G;
  • X 16 is selected from N and T;
  • X 20 is selected from F and W;
  • X 21 is selected from K and Y;
  • X 25 is selected from K and V
  • X 26 is selected from K, N and S;
  • X 29 is selected from D and R;
  • VEGFR-2 binding polypeptide according to item 44, wherein said first and second monomer units bind to different epitopes on VEGFR-2.
  • VEGFR-2 binding polypeptide according to item 44 or 45, wherein, in sequence xvii),
  • X 3 is selected from Q and R;
  • X 4 is selected from A, K, R, S and V;
  • X 7 is R
  • X 11 is selected from A and G;
  • X 20 is selected from F and W;
  • X 21 is Y
  • X 25 is V; X 26 is selected from K and N; and
  • VEGFR-2 binding polypeptide according to any one of items 44-46, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42.
  • VEGFR-2 binding polypeptide according to item 47, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:26-42.
  • VEGFR-2 binding polypeptide according to item 48, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:42.
  • VEGFR-2 binding polypeptide according to item 48 wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as from the group consisting of SEQ ID NO:34 and SEQ ID NO:41.
  • VEGFR-2 binding polypeptide according to any one of items 44-51, wherein said BM2 forms part of a three-helix bundle protein domain.
  • VEGFR-2 binding polypeptide according to item 52 wherein said BM2 essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
  • VEGFR-2 binding polypeptide according to item 53 wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
  • VEGFR-2 binding polypeptide according to any one of items 44-55, wherein said second VEGFR-2 binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:26-42.
  • VEGFR-2 binding polypeptide according to item 56 wherein said first monomer unit is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23.
  • VEGFR-2 binding polypeptide according to item 56 or 57, wherein said second monomer unit is selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39; SEQ ID NO:41 and SEQ ID NO:42.
  • VEGFR-2 binding polypeptide according to item 57 or 58, comprising SEQ ID NO:12 and SEQ ID NO:34; SEQ ID NO:12 and SEQ ID NO:39; SEQ ID NO:12 and SEQ ID NO:41; SEQ ID NO:12 and SEQ ID NO:42; SEQ ID NO:13 and SEQ ID NO:34; SEQ ID NO:13 and SEQ ID NO:39; SEQ ID NO:13 and SEQ ID NO:41; SEQ ID NO:13 and SEQ ID NO:42; SEQ ID NO:20 and SEQ ID NO:34; SEQ ID NO:20 and SEQ ID NO:39; SEQ ID NO:20 and SEQ ID NO:41; SEQ ID NO:20 and SEQ ID NO:42; SEQ ID NO:23 and SEQ ID NO:34; SEQ ID NO:23 and SEQ ID NO:39; SEQ ID NO:23 and SEQ ID NO:41; or SEQ ID NO:23 and SEQ ID NO:42.
  • VEGFR-2 binding polypeptide according to item 59 comprising SEQ ID NO:23 and SEQ ID NO:41.
  • VEGFR-2 binding polypeptide according to any one of items 44-60, which is capable of binding to VEGFR-2 such that the dissociation rate constant k d of the interaction with VEGFR-2 is at least 2 times, such as at least 5 times, such as at least 10 times, such as at least 15 times, such as at least 20 times, such as at least 30 times lower than the dissociation rate constant k d of a comparable polypeptide in homodimeric form.
  • Fusion protein or conjugate according to item 64 wherein said desired biological activity is a therapeutic activity.
  • Fusion protein or conjugate according to item 64 or 65 wherein said desired biological activity is a binding activity.
  • Fusion protein or conjugate according to item 67, wherein said second moiety comprises the albumin binding domain of streptococcal protein G or a derivative thereof.
  • PDGFR- ⁇ platelet-derived growth factor receptor ⁇
  • PDGFR- ⁇ platelet-derived growth factor A
  • PDGF-B platelet-derived growth factor B
  • PDGF-C platelet-derived growth factor C
  • PDGF-D platelet-derived growth factor D
  • epidermal growth factor receptor 1 EGFR
  • epidermal growth factor receptor 2 HER2
  • epidermal growth factor receptor 3 HER3
  • epidermal growth factor receptor 4 HER4
  • epidermal growth factor epidermal growth factor receptor 1
  • HER2 epidermal growth factor receptor 2
  • HER3 epidermal growth factor receptor 3
  • HER4 epidermal growth factor receptor 4
  • epidermal growth factor epidermal growth factor
  • tumor growth factor ⁇ epigen, epiregulin, neuregulins 1-4, c-kit, Raf kinases such as B-Raf and C-raf, rearranged during transfection (RET) receptor, colony stimulating factor 1 receptor (CSF-1R) and f
  • fibroblast growth factor FGF
  • FGF-1 fibroblast growth factor 1
  • basic FGF angiogenin 1
  • Ang-2 angiogenin 2
  • Angpt-1 angiogenin 2
  • Angpt-1 angiopoietin 1
  • Angpt-2 angiopoietin 2
  • Angpt-3 angiopoietin 4
  • TIE-1 tyrosine kinase with immunoglobulin-like domains 1
  • TIE-2 vascular endothelial growth factor receptor 1
  • VEGFR-3 vascular endothelial growth factor A
  • VEGF-A vascular endothelial growth factor B
  • VEGF-C vascular endothelial growth factor C
  • complex according to item 84 wherein said antigen is selected from the group consisting of CD3, CD28, T-cell receptor ⁇ (TCR ⁇ ), T-cell receptor ⁇ (TCR ⁇ ), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1) and the natural cytotoxicity receptors NKp30 and NKp40;
  • P-L1 programmed death-ligand 1
  • PD-L2 programmed death-ligand 2
  • B7 homolog 3 B7-H3
  • B7 homolog 4 B7-H4
  • HVEM herpes virus entry mediator
  • BTLA T-lymphocyte attenuator
  • KIR killer inhibitory receptor
  • LAG3 lymphocyte-activation gene 3
  • TIM3 galectin-9/T cell immunoglobulin mucin-3
  • A2aR adenosine/ alpha-2 adrenergic receptors
  • VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of said antibody or antigen binding fragment thereof.
  • VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the light chain of said antibody or antigen binding fragment thereof.
  • VEGFR-2 binding polypeptide is attached to the N-terminus and/or C- terminus of the light chain and heavy chain of said antibody or antigen binding fragment thereof.
  • Fusion protein or conjugate according item 93 wherein said linker is arranged between said first moiety and said second moiety.
  • VEGFR-2 binding polypeptide according item 93, wherein said linker is arranged between said first monomer unit and said second monomer unit.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according any one of items 93-96, wherein said linker is a flexible linker comprising amino acid residues selected from the group consisting of glycine, serine and threonine.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to item 98, wherein n 1-5.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to item 105 wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes,
  • radionuclides and radioactive particles are examples of radionuclides and radioactive particles.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any preceding item comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the VEGFR-2 binding polypeptide via a thiol group of a cysteine residue or an epsilon amine group of a lysine residue.
  • Expression vector comprising a polynucleotide according to item 108.
  • Host cell comprising an expression vector according to item 109.
  • Composition comprising a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 and at least one pharmaceutically acceptable excipient or carrier.
  • Composition according to item 112 further comprising at least one additional active agent, such as selected from an anti-cancer agent, an anti-angiogenic agent and an immune response modifying agent.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or composition according to any one of items 112-113 for use in the treatment, diagnosis or prognosis of a VEGFR-2 related disorder.
  • VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 119 wherein said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, melanomas, cancer of the oral cavity and any cancer manifested by solid tumors with VEGFR-2 expression.
  • said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma
  • Method of treatment of a VEGFR-2 related disorder comprising administering to a subject in need thereof an effective amount of a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or a composition according to any one of items 112-113.
  • Method according to item 122 wherein said VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition inhibits VEGFR-2 mediated signaling by binding to VEGFR-2 expressed on a cell surface.
  • Method of detecting VEGFR-2 comprising providing a sample suspected to contain VEGFR-2, contacting said sample with a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or a composition according to any one of items 112-113, and detecting the binding of the VEGFR-2 binding polypeptide, fusion protein, conjugate or composition to indicate the presence of VEGFR-2 in the sample.
  • Method for determining the presence of VEGFR-2 in a subject comprising the steps:
  • Method according to item 125 further comprising a step of comparing said value to a reference.
  • VEGFR-2 related disorder is cancer, a vascular disease or an angiogenesis related disorder.
  • angiogenesis related disorder is age-related macular degeneration.
  • Method according to item 130 wherein said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, melanomas, cancer of the oral cavity and any cancer manifested by solid tumors with VEGFR-2 expression.
  • said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for vascular endothelial growth factor receptor 2 (VEGFR-2), and provides a VEGFR-2 binding polypeptide comprising the sequence ENX 3 X 4 ASX 7 EIAX 11 LPNLX 16 DX 18 QYIAFIYX 26 LLX 29. The present disclosure also relates to the use of such a VEGFR-2 binding polypeptide as a diagnostic, prognostic agent and/or therapeutic agent.

Description

VEGFR-2 BINDING POLYPEPTIDES Field of the invention
The present disclosure relates to a class of engineered polypeptides having a binding affinity for vascular endothelial growth factor receptor 2 (in the following referred to as VEGFR-2). The present disclosure also relates to the use of such a VEGFR-2 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent. Background
The formation of new blood vessels from pre-existing ones, called angiogenesis, plays a central role in the process of solid tumor growth and metastasis as well as in healing and tissue remodeling. Tumor angiogenesis is a complex process dependent on a multi-faceted program of endothelial cell activation, stromal cell and endothelial-progenitor recruitment,
extracellular matrix remodeling, pro-angiogenic cytokine signaling, and activation of oncogenic signaling cascades (Weis SM et al., (2011) Nat Med;17(11):1359-70; Potente M et al., (2011) Cell; 146(6):873-87).
Angiogenesis is regulated in normal and malignant tissues by the balance of angiogenic stimuli and angiogenic inhibitors that are produced in the target tissue and at distant sites (Fidler IJ et al., (1998) Cancer J. Sci. Am vol.4, no.1; 58-66). A key angiogenesis signaling system, that regulates proliferation and migration of endothelial cells, comprises proteins of the vascular endothelial growth factor (VEGF) family and their receptors (vascular endothelial growth factor receptor 1, 2 and 3, abbreviated VEGFR-1, VEGFR- 2 and VEGFR-3, respectively). Vascular endothelial growth factor A (VEGF-A, also known as vascular permeability factor, VPF) is a primary stimulant of angiogenesis. VEGF-A is a multifunctional cytokine that is induced by hypoxia and oncogenic mutations and can be produced by a wide variety of tissues (Kerbel RS et al., (1998) Mol. Med., 4(5):286-295; Mazure NM et al., (1996) Cancer Res., 56:3436-3440). The recognition of VEGF-A as a primary stimulus of angiogenesis in pathological conditions has led to various attempts to block VEGF-A activity.
In addition to VEGF-A, the VEGF family of soluble agonists includes VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF). These factors bind to the three cognate receptors VEGFR-1 (also known as fms-like tyrosine kinase 1/Flt-1), VEGFR-2 (also known as Flk-1/KDR) and VEGFR-3 (also known as Flt-4) (Clarke JM et al., (2013) Expert Opin. Biol. Ther.13(8):1187-1196). VEGF-A binds to VEGFR-1 and VEGFR-2. VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF-A and is widely considered to be the main receptor driving angiogenesis. The VEGFRs belong to the immunoglobulin subclass of the receptor tyrosine kinase (RTK) super family and have seven Ig-like extracellular domains with a single transmembrane helix with an intracellular kinase region (Olsson AK et al., (2006) Nat Rev Mol Cell Biol 7(5):359-71; Ferrara N et al., (2004)
Oncologist 9:2-10). Upon binding of VEGF-A, VEGFR-2 can form either a homodimer or a heterodimer complex with VEGFR-1, resulting in intracellular tyrosine phosphorylation (Olsson AK et al., (2006) supra; Ferrara N et al., (2004) supra). VEGFR stimulation leads to downstream activation of the mitogen-activated protein kinase (MAPK) pathway and promotion of endothelial cell proliferation as well as phosphatidylinositol 3’ kinase (PI3K) activity, ultimately leading to increased cell survival through AKT/PKB, cell migration and vascular permeability via expression of endothelial nitric oxide synthase. Additionally, activation of VEGFR promotes vascular permeability, actin remodeling, and cell migration by way of the Src/TSAd and P38/MAPK pathways (Ferrara N et al., (2004) supra).
VEGFRs are expressed at high levels in many types of human solid tumors, including lung, breast, renal, ovarian, glioma and gastrointestinal tract carcinomas (Clarke JM et al., (2013) Expert Opin. Biol. Ther.13(8):1187- 1196), and the inhibition of VEGFR signaling has recently emerged as a potential therapy method for cancers. VEGFR-2 has been shown to play an important role in the direct regulation of angiogenesis and mitogenic signaling and therapies targeting VEGFR-2 signaling have been clinically validated with the FDA approvals of the VEGFR-2 inhibitor bevacizumab, a fully humanized mAb (Presta LG et al., (1997) Cancer Res.57:4593-4599), and the small- molecule VEGFR signaling inhibitors sorafenib (Wilhelm SM et al., (2004) Cancer Res.64:7099-101) and sunitinib (Mendel DB et al., (2003) Clin Cancer Res.9:327-337, O’Farrel AM et al., (2003) Blood.101:3597-3605) for the treatment of multiple malignancies including colorectal, non-small cell lung (NSCLC), renal cell and hepatocellular carcinomas (Hurwitz H et al., (2004) N Engl J Med.350(23):2335-42; Sandler A et al., (2006) N Engl J Med.
355(24):2542-50; Motzer RJ et al., (2007) N Engl J Med.356(2):115-24;
Llovet JM et al., (2008) N Engl J Med.359(4):378-90). These molecules have demonstrated a clear anti-tumor effect, clinical tolerability, and an ability to promote overall survival as well as progression-free survival (Clarke JM et al. (2013), supra). Some VEGFR antagonists (for example lenvatinib (Matsui J et al., (2008) Clin Cancer Res.14:5459-5465) and motesanib (Polverino A et al., (2006) Cancer Res.66:8715-8721)) are under investigation for treating various cancers, and pazopanib and regorafenib were recently approved for treatment of renal cell carcinoma and colorectal cancer, respectively.
Additionally, many antibodies have been evaluated as potent inhibitors and some are currently in clinical trials for other various angiogenic related disorders such as retinopathies and age related macular degeneration
(Clarke JM et al., (2013) supra) as well as inflammatory and infectious diseases involving endothelial barrier disruption such as sepsis and acute lung injury (Darwish I et al., (2013) Virulence 4(6):572-582).
To summarize, downstream effects of VEGFR-2 signaling which culminate in angiogenesis include a potent increase in vascular permeability and promotion of endothelial cell migration, proliferation and survival (Olsson AK et al., (2006) supra). Thus, anti-angiogenic therapy with the potential for both high affinity and high specificity blockade of VEGFR-2 is an attractive goal for the treatment of malignancies and various diseases associated with excessive angiogenesis and cell migration, such as cancers manifesting solid tumors.
However, monoclonal antibodies are not always optimal for targeting solid tumors (neither for diagnostic nor for therapeutic pay-load purposes). Therapeutic effect is dependent on an efficient distribution of the drug throughout the tumor and molecular imaging depends on a high ratio between tumor uptake and surrounding normal tissue. Since tumor penetration rate (including extravasation) is negatively associated with the size of the molecule, the relatively large antibody molecule inherently has poor tissue distribution and penetration capacity. Moreover, for molecular imaging, the extraordinarily long in vivo half-life of antibodies results in relatively high blood signals and thereby relatively poor tumor-to-blood contrasts.
The continued provision of agents with a high affinity for VEGFR-2 remains a matter of substantial interest within the field. Of high importance is also the provision of agents of dual or even multiple affinities, such as agents with a high affinity for VEGFR-2 and for one or more additional factor(s) associated with a disease state or disorder. Additionally, the provision of uses of such molecules in the treatment and diagnosis of disease is of great interest. Summary of the invention
It is an object of the present disclosure to provide new VEGFR-2 binding agents, which could for example be used for therapeutic, prognostic and diagnostic applications.
It is an object of the present disclosure to provide a new multispecific agent, such as a bispecific agent, which has affinity for VEGFR-2 and at least one additional antigen.
It is an object of the present disclosure to provide a molecule allowing for efficient therapy targeting various forms of cancer and angiogenesis related disorders while alleviating the abovementioned and other drawbacks of current therapies.
It is furthermore an object of the present disclosure to provide a molecule suitable for prognostic and diagnostic applications.
These, and other objects which are evident to the skilled person from the present disclosure, are met by the different aspects of the invention as claimed in the appended claims and as generally disclosed herein. Thus, in the first aspect of the disclosure, there is provided a VEGFR-2 binding polypeptide, comprising a VEGFR-2 binding motif BM, which motif consists of an amino acid sequence selected from: i) ENX3X4ASX7EIA X11LPNLX16DX18QY IAFIYX26LLX29 wherein, independently from each other, X3 is selected from L and Y;
X4 is selected from E, F, I, K, M, V, W and Y;
X7 is selected from K, N and R;
X11 is selected from F, H, L and N;
X16 is selected from N and T;
X18 is selected from A, D, E, G, H, K, Q, S and T;
X26 is selected from K and S; and
X29 is selected from D and R;
and ii) an amino acid sequence which has at least 82 % identity to the sequence defined in i). The above definition of a class of sequence related, VEGFR-2 binding polypeptides is based on a statistical analysis of a number of random polypeptide variants of a parent scaffold, that were selected for their interaction with VEGFR-2 in selection experiments. The identified VEGFR-2 binding motif, or“BM”, corresponds to the target binding region of the parent scaffold, which region constitutes two alpha helices within a three-helical bundle protein domain. In the parent scaffold, the varied amino acid residues of the two BM helices constitute a binding surface for interaction with the constant Fc part of antibodies. In the present disclosure, the random variation of binding surface residues and subsequent selection of variants have replaced the Fc interaction capacity with a capacity for interaction with
VEGFR-2.
As the skilled person will realize, the function of any polypeptide, such as the VEGFR-2 binding capacity of the polypeptide of the present disclosure, is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the disclosure encompasses modified variants of the VEGFR-2 binding polypeptide, which have retained VEGFR-2 binding characteristics.
In this way, encompassed by the present disclosure is a VEGFR-2 binding polypeptide comprising an amino acid sequence with 82 % or greater identity to a polypeptide as defined in i). In some embodiments, said polypeptide may comprise a sequence which is at least 86 %, such as at least 89 %, such as at least 93 %, such as at least 96 % identical to a polypeptide as defined in i). For example, it is possible that an amino acid residue belonging to a certain functional grouping of amino acid residues (e.g.
hydrophobic, hydrophilic, polar etc) could be exchanged for another amino acid residue from the same functional group.
In some embodiments, such changes may be made in any position of the sequence of the VEGFR-2 binding polypeptide as disclosed herein. In other embodiments, such changes may be made only in the non-variable positions, also denoted scaffold amino acid residues. In such cases, changes are not allowed in the variable positions, i.e. positions denoted with an“X” in sequence i). The term "% identity", as used throughout the specification, may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al., (1994) Nucleic Acids Research, 22: 4673-4680). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared and the percentage of positions in the query sequence that have identical
correspondences in the target sequence is reported as % identity. As used herein,“Xn” and“Xm” are used to indicate amino acids in positions n and m in the sequence i) as defined above, wherein n and m are integers which indicate the position of an amino acid within said sequence as counted from the N-terminal end of said sequence. For example, X3 and X7 indicate the amino acid in position three and seven, respectively, from the N- terminal end of sequence i).
In embodiments according to the first aspect, there are provided polypeptides wherein Xn in sequence i) is independently selected from a group of possible residues according to Table 1. The skilled person will appreciate that Xn may be selected from any one of the listed groups of possible residues and that this selection is independent from the selection of amino acids in Xm, wherein n≠m. Thus, any of the listed possible residues in position Xn in Table 1 may be independently combined with any of the listed possible residues any other variable position in Table 1.
The skilled person will appreciate that Table 1 is to be read as follows: In one embodiment according to the first aspect, there is provided a
polypeptide wherein amino acid residue“Xn” in sequence i) is selected from “Possible residues”. Thus, Table 1 discloses several specific and
individualized embodiments of the first aspect of the present disclosure. For example, in one embodiment according to the first aspect, there is provided a polypeptide wherein X4 in sequence i) is selected from E, F, I, M, V, W and Y, and in another embodiment according to the first aspect, there is provided a polypeptide wherein X4 in sequence i) is selected from F, I, K, M, V, W and Y. For avoidance of doubt, the listed embodiments may be freely combined in yet other embodiments. For example, one such combined embodiment is a polypeptide in which X4 is selected from F, I, K, M, V, W and Y, while X7 is selected from K and R, and X18 is selected from A, E, K and T.
Figure imgf000008_0001
Figure imgf000009_0001
In one particular embodiment according to the first aspect, there is provided a polypeptide wherein, in sequence i), X3 is selected from L and Y;
X4 is selected from E, F, I, M, V and Y;
X7 is selected from K, N and R;
X11 is selected from F, H, L and N;
X16 is T;
X18 is selected from A, D, E, K, S and T;
X26 is K; and
X29 is D. In some embodiments of a VEGFR-2 binding polypeptide according to the first aspect, X3X7X11 is selected from LNH and LKN. In some
embodiments, X3X4X7 is selected from LKN, LMN, YIK, YMK, LFK and LVK. In some embodiments, X3X4X11 is selected from LKH, LMN, YIN, YMN, LFN and LVN. As described in detail in the experimental section to follow, the selection of VEGFR-2 binding polypeptide variants has led to the identification of a number of individual VEGFR-2 binding motif (BM) sequences. These sequences constitute individual embodiments of sequence i) according to this aspect. The sequences of individual VEGFR-2 binding motifs correspond to amino acid positions 8-36 in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5- 25 presented in Figure 1. Hence, in one embodiment of the VEGFR-2 binding polypeptide according to this aspect, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:5-25. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:23. In some embodiments of the present disclosure, the BM as defined above“forms part of” a three-helix bundle protein domain. This is understood to mean that the sequence of the BM is“inserted” into or“grafted” onto the sequence of the original three-helix bundle domain, such that the BM replaces a similar structural motif in the original domain. For example, without wishing to be bound by theory, the BM is thought to constitute two of the three helices of a three-helix bundle, and can therefore replace such a two-helix motif within any three-helix bundle. As the skilled person will realize, the replacement of two helices of the three-helix bundle domain by the two BM helices has to be performed so as not to affect the basic structure of the polypeptide. That is, the overall folding of the Cα backbone of the polypeptide according to this embodiment of the invention is substantially the same as that of the three-helix bundle protein domain of which it forms a part, e.g. having the same elements of secondary structure in the same order etc.
Thus, a BM according to the present disclosure“forms part” of a three-helix bundle domain if the polypeptide according to this embodiment has the same fold as the original domain, implying that the basic structural properties are shared, those properties e.g. resulting in similar CD spectra. The skilled person is aware of other parameters that are relevant.
In particular embodiments, the VEGFR-2 binding motif (BM) thus forms part of a three-helix bundle protein domain. For example, the BM may essentially constitute two alpha helices with an interconnecting loop, within said three-helix bundle protein domain. In particular embodiments, said three- helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different three- helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof. In some embodiments, the three-helical bundle protein domain is a variant of protein Z, which is derived from domain B of staphylococcal Protein A. In some embodiments where the VEGFR-2 binding polypeptide as disclosed herein forms part of a three-helix bundle protein domain, the VEGFR-2 binding polypeptide may comprise a binding module (BMod), the amino acid sequence of which is selected from: iii) K-[BM]-DPSQSXaXbLLXcEAKKLXdXeXfQ; wherein
[BM] is a VEGFR-2 binding motif as defined herein
provided that X29 is D;
Xa is selected from A and S;
Xb is selected from N and E;
Xc is selected from A, S and C;
Xd is selected from E, N and S;
Xe is selected from D, E and S;
Xf is selected from A and S; and iv) an amino acid sequence which has at least 83 % identity to a
sequence defined in iii). In some embodiments, said polypeptides may beneficially exhibit high structural stability, such as resistance to chemical modifications, to changes in physical conditions and to proteolysis, during production and storage, as well as in vivo. Thus, in other embodiments where the VEGFR-2 binding polypeptide as disclosed herein forms part of a three-helix bundle protein domain, the VEGFR-2 binding polypeptide may comprise a binding module (BMod), the amino acid sequence of which is selected from: v) K-[BM]-QPEQSXaXbLLXcEAKKLXdXeXfQ, wherein
[BM] is a VEGFR-2 binding motif as defined herein
provided that X29 is R;
Xa is selected from A and S;
Xb is selected from N and E;
Xc is selected from A, S and C;
Xd is selected from E, N and S; Xe is selected from D, E and S;
Xf is selected from A and S; and vi) an amino acid sequence which has at least 83 % identity to a
sequence defined in v). As discussed above, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptide, are also within the scope of the present disclosure. Thus, in some embodiments, sequence iv) and vi) have at least 85 %, such as at least 87 %, such as at least 89 %, such as at least 91 %, such as at least 93 %, such as at least 95 %, such as at least 97 % identity to a sequence defined by iii) and v), respectively.
In one embodiment, Xa in sequence iii) or v) is A.
In one embodiment, Xa in sequence iii) or v) is S.
In one embodiment, Xb in sequence iii) or v) is N.
In one embodiment, Xb in sequence iii) or v) is E.
In one embodiment, Xc in sequence iii) or v) is A.
In one embodiment, Xc in sequence iii) or v) is S.
In one embodiment, Xc in sequence iii) or v) is C.
In one embodiment, Xd in sequence iii) or v) is E.
In one embodiment, Xd in sequence iii) or v) is N.
In one embodiment, Xd in sequence iii) or v) is S.
In one embodiment, Xe in sequence iii) or v) is D.
In one embodiment, Xe in sequence iii) or v) is E.
In one embodiment, Xe in sequence iii) or v) is S.
In one embodiment, XdXe in sequence iii) or v) is selected from EE, ES, SD, SE and SS.
In one embodiment, XdXe in sequence iii) or v) is ES.
In one embodiment, XdXe in sequence iii) or v) is SE.
In one embodiment, XdXe in sequence iii) or v) is SD.
In one embodiment, Xf in sequence iii) or v) is A.
In one embodiment, Xf in sequence iii) or v) is S.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is A and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is A and Xf is A. In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is S and Xf is S.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C and Xf is S.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is A; XdXe is ND and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is A; XdXe is ND and Xf is A.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is ND and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is S; XdXe is ND and Xf is S.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is ND and Xf is S.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is A; XdXe is SE and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is A; XdXe is SE and Xf is A.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is SE and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is S; XdXe is SE and Xf is S.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is SE and Xf is S.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is A; XdXe is ES and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is A; XdXe is ES and Xf is A.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is ES and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is S; XdXe is ES and Xf is S.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is ES and Xf is S. In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is A; XdXe is SD and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is A; XdXe is SD and Xf is A.
In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is SD and Xf is A.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is S; XdXe is SD and Xf is S.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is A; XdXe is SD and Xf is S.
In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is SD and Xf is S. In yet a further embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25 presented in Figure 1. In another embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5-25. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:5-25. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:23. Also, in a further embodiment, there is provided a VEGFR-2 binding polypeptide, which comprises an amino acid sequence selected from: vii) YA-[BMod]-AP;
wherein [BMod] is a VEGFR-2 binding module as defined herein; and viii) an amino acid sequence which has at least 83 % identity to a
sequence defined in vii). Alternatively, there is provided a VEGFR-2 binding polypeptide, which comprises an amino acid sequence selected from: ix) FN-[BMod]-AP;
wherein [BMod] is a VEGFR-2 binding module as defined herein; and x) an amino acid sequence which has at least 83 % identity to a
sequence defined in ix). As discussed above, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptide, also fall within the scope of the present disclosure. Thus, in some embodiments, sequence viii) and x) may for example be at least 84 %, such as at least 86 %, such as at least 88 %, such as at least 90 %, such as at least 92 %, such as at least 94 %, such as at least 96 %, such as at least 98 % identical to a sequence defined by vii) and ix), respectively. In some embodiments, the VEGFR-2 binding motif may form part of a polypeptide comprising an amino acid sequence selected from
ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK;
ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;
ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK;
ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK;
AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK;
VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;
AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP;
AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP;
AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP;
AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP;
AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;
AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP;
AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;
AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;
AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP;
AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;
AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;
AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;
VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;
VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;
VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;
VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;
VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;
VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;
VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;
AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and
ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined herein. In one embodiment, the VEGFR-2 binding polypeptide comprises an amino acid sequence selected from:
xi) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined herein; and xii) an amino acid sequence which has at least 82 % identity to the
sequence defined in xi). In one embodiment, the VEGFR-2 binding polypeptide comprises an amino acid sequence selected from:
xiii) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined herein; and xiv) an amino acid sequence which has at least 82 % identity to the
sequence defined in xiii). In one embodiment, the VEGFR-2 binding polypeptide comprises an amino acid sequence selected from:
xv) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined herein; and xvi) an amino acid sequence which has at least 82 % identity to the
sequence defined in xv). Again, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptide, also fall within the scope of the present disclosure. Thus, in some embodiments, sequence xii), xiv) and xvi) may for example be at least 84 %, such as at least 86 %, such as at least 87 %, such as at least 89 %, such as at least 91 %, such as at least 93 %, such as at least 94 %, such as at least 96 %, such as at least 98 % identical to a sequence defined by xi), xiii) and xv), respectively. Sequence xi) or xiii) in such a polypeptide may be selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25 presented in Figure 1. In one embodiment, said sequence is selected from the group consisting of SEQ SEQ ID NO:3 and SEQ ID NO:5-25. In another embodiment, said sequence is selected from the group consisting of SEQ ID NO:5-25. In one embodiment, said sequence is selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23. In one embodiment, said sequence is SEQ ID NO:23. In one embodiment of the VEGFR-2 binding polypeptide as disclosed herein, it consists of any one of the sequences disclosed herein, having from 0 to 15, such as from 0 to 7, additional N-terminal residues. In one
embodiment, the VEGFR-2 binding polypeptide consists of any one of the sequences disclosed herein, having from 0 to 15, such as from 0 to 4, additional C-terminal residues. In a combination of these embodiments, the VEGFR-2 binding polypeptide consists of any one of the sequences disclosed herein, having from 0 to 15, such as from 0 to 7, additional N-terminal residues, and from 0 to 15, such as from 0 to 4, additional C-terminal residues. The terms“VEGFR-2 binding” and”binding affinity for VEGFR-2” as used in this specification refer to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance technology. For example as described in the examples below, VEGFR-2 binding affinity may be tested in an experiment in which VEGFR-2, or a fragment thereof, is immobilized on a sensor chip of the instrument, and the sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing VEGFR-2, or a fragment thereof, is passed over the chip. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for VEGFR-2. If a quantitative measure is desired, for example to determine a KD value for the interaction, surface plasmon resonance methods may also be used. Binding values may for example be defined in a Biacore (GE Healthcare) or ProteOn XPR36 (Bio-Rad) instrument. VEGFR-2 is suitably immobilized on a sensor chip of the instrument, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected in random order. KD values may then be calculated from the results using for example the 1:1 Langmuir binding model of the BIAevaluation 4.1 software, or other suitable software, provided by the instrument manufacturer.
The terms“albumin binding” and“binding affinity for albumin” as used in this disclosure refer to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance technology, such as in a Biacore instrument or ProteOn XPR36 instrument, in an analogous way to the example described above for VEGFR-2. In one embodiment, the VEGFR-2 binding polypeptide is capable of binding to VEGFR-2 such that the KD value of the interaction with VEGFR-2 is at most 1 x 10-6 M, such as at most 1 x 10-7 M, such as at most 1 x 10-8 M, such as at most 1 x 10-9 M, such as at most 1 x 10-10 M.
Binding of a polypeptide as defined herein to VEGFR-2 may interfere either with signaling via VEGFR-2 in vivo or in vitro. Thus, in one
embodiment, there is provided a VEGFR-2 binding polypeptide as defined herein which is capable of blocking VEGFR-2 dependent signaling. Blocking activity may for example be quantified by the half maximal inhibitory concentration (IC50), which is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. This quantitative measure indicates how much of a particular substance is needed to inhibit a given biological process by half, and is commonly used in the art.
In one embodiment, said VEGFR-2 is human VEGFR-2. In another embodiment, said VEGFR-2 is murine VEGFR-2.
It may be beneficial in some embodiments of the present disclosure that the VEGFR-2 binding polypeptide disclosure herein retains its α-helical conformation at temperatures higher than the body temperature of a fever free human subject. Fever (also known as pyrexia or febrile response) is one of the most common medical signs and is characterized by an elevation of body temperature above the normal range of 36.5– 37.5 °C. Elevated body temperature can be classified into fever (>37.5– 38.3 °C), hyperthermia (>37.5– 38.3 °C) and hyperpyrexia (>40.0– 41.5 °C). In some embodiments, it may be beneficial that said VEGFR-2 binding polypeptide retains its conformation at temperatures above the hyperthermia range, such as above the hyperpyrexia range. Thus, in one embodiment, there is provided a
VEGFR-2 binding polypeptide which has a melting temperature of at least 40 °C, such as at least 45 °C. The skilled person will understand that various modifications and/or additions can be made to a VEGFR-2 binding polypeptide according to any aspect disclosed herein in order to tailor the polypeptide to a specific application without departing from the scope of the present disclosure.
For example, in one embodiment, there is provided a VEGFR-2 binding polypeptide as described herein, which polypeptide has been extended by and/or comprises additional amino acids at the C terminus and/or N terminus. Such a polypeptide should be understood as a polypeptide having one or more additional amino acid residues at the very first and/or the very last position in the polypeptide chain. Thus, a VEGFR-2 binding polypeptide may comprise any suitable number of additional amino acid residues, for example at least one additional amino acid residue. Each additional amino acid residue may individually or collectively be added in order to, for example, improve and/or simplify production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide. Such additional amino acid residues may comprise one or more amino acid residues added for the purpose of chemical coupling. One example of this is the addition of a cysteine residue. Additional amino acid residues may also provide a”tag” for purification or detection of the polypeptide, such as a His6 tag, a (HisGlu)3 tag (“HEHEHE” tag) or a ”myc” (c-myc) tag or a”FLAG” tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of a His6-tag.
The further amino acids as discussed above may be coupled to the VEGFR-2 binding polypeptide by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as
expression of the VEGFR-2 binding polypeptide as a fusion protein or joined in any other fashion, either directly or via a linker, for example an amino acid linker.
The further amino acids as discussed above may for example comprise one or more polypeptide domain(s). A further polypeptide domain may provide the VEGFR-2 binding polypeptide with another function, for example another binding function, an enzymatic function, a toxic function, a fluorescent signaling function or combinations thereof.
A further polypeptide domain may moreover provide another VEGFR-2 binding moiety. Thus, in a further embodiment, there is provided a VEGFR-2 binding polypeptide in a multimeric form. Said multimer is understood to comprise at least two VEGFR-2 binding polypeptides as disclosed herein as monomer units, the amino acid sequences of which may be the same or different. Multimeric forms of the polypeptides may comprise a suitable number of domains, each having a VEGFR-2 binding motif, and each forming a monomer within the multimer. These domains may have the same amino acid sequence, but alternatively, they may have different amino acid sequences. In other words, the VEGFR-2 binding polypeptide of the invention may form homo- or heteromultimers, for example homo- or heterodimers. In one embodiment, there is provided a VEGFR-2 binding polypeptide, wherein said monomer units are covalently coupled together. In another embodiment, said VEGFR-2 binding polypeptide monomer units are expressed as a fusion protein. In one embodiment, there is provided a VEGFR-2 binding polypeptide in dimeric form. In one particular embodiment, said dimeric form is a homodimeric form. In another embodiment, said dimeric form is a
heterodimeric form. In a particular embodiment of a multimeric VEGFR-2 binding
polypeptide, which in turn is an embodiment of the first aspect of the disclosure, there is provided a VEGFR-2 binding polypeptide, comprising a first monomer unit as defined herein and a second monomer unit consisting of a different VEGFR-2 binding polypeptide. In this embodiment, the VEGFR-2 binding polypeptide comprises
- a first monomer unit consisting of a first VEGFR-2 binding polypeptide as defined above; and
- a second monomer unit consisting of a second VEGFR-2 binding polypeptide, comprising a second VEGFR-2 binding motif BM2, which motif consists of an amino acid sequence selected from: xvii) EFX3X4ADX7EIR X11LPNLX16HGQX20 X21AFIX25X26LYX29 wherein, independently from each other, X3 is selected from Q and R;
X4 is selected from A, D, H, K, L, M, R, S and V;
X7 is selected from A, I and R;
X11 is selected from A, D and G;
X16 is selected from N and T;
X20 is selected from F and W;
X21 is selected from K and Y;
X25 is selected from K and V
X26 is selected from K, N and S; and
X29 is selected from D and R;
and xviii) an amino acid sequence which has at least 82 % identity to the
sequence defined in xvii). It may be beneficial that said second monomer unit, consisting of a second VEGFR-2 binding polypeptide, has affinity for a different epitope of VEGFR-2 than said first monomer unit. Thus, in one embodiment, said first and second monomer units bind to different epitopes on VEGFR-2.
The above definition of a second, different class of sequence related, VEGFR-2 binding polypeptides is also based on a statistical analysis of a number of random polypeptide variants of a parent scaffold. This group of second VEGFR-2 binding polypeptides were selected for their interaction with VEGFR-2 in several different selection experiments as described in the Examples section of the present disclosure. The identified second VEGFR-2 binding motif, or“BM2”, corresponds to the target binding region of the parent scaffold, which region constitutes two alpha helices within a three-helical bundle protein domain. In the parent scaffold, the varied amino acid residues of the two BM2 helices constitute a binding surface for interaction with the constant Fc part of antibodies. In the same fashion as discussed above, the random variation of binding surface residues and subsequent selection of variants replaced the Fc interaction capacity of the parent scaffold with a capacity for interaction with VEGFR-2.
As discussed above, the skilled person will realize that the function of any polypeptide, such as the VEGFR-2 binding capacity of the second monomer unit disclosed herein, is dependent on the tertiary structure of said unit. It is therefore possible to make minor changes to the sequence of amino acids in said unit without affecting the function thereof. Thus, the disclosure encompasses VEGFR-2 binding polypeptides in heterodimeric form, wherein the second monomer unit comprises second VEGFR-2 binding polypeptides which have retained VEGFR-2 binding characteristics.
In this way, also encompassed by the present disclosure is a VEGFR-2 binding polypeptide, wherein said second monomer unit comprises a second VEGFR-2 binding polypeptide comprising an amino acid sequence with 82 % or greater identity to a polypeptide as defined in xvii). In some embodiments, the second polypeptide may comprise a sequence which is at least 86 %, such as at least, 89 %, such as at least 93 %, such as at least 96 % identical to a polypeptide as defined in xvii). Thus, the present disclosure encompasses VEGFR-2 binding polypeptides in heterodimeric form, comprising any VEGFR-2 binding polypeptide defined by sequence i) or having at least 82 % identity to a sequence defined in i) as a first monomer unit, and a second VEGFR-2 binding polypeptide having a sequence defined in xvii) or having at least 82 % identity to a sequence defined in xvii) as a second monomer unit. The discussion regarding sequence identity of encompassed modified variants of VEGFR-2 binding polypeptides above is equally relevant for the second VEGFR-2 binding polypeptide and will not be repeated here for the sake of brevity.
Also, the discussion regarding amino acids in positions n and m in the sequence i) as defined above, is also equally relevant for amino acids in positions n and m in the sequence xvii) and will not be repeated. Briefly, VEGFR-2 binding polypeptides in heterodimeric form comprising second monomer units, wherein Xn in sequence xvii) is independently selected from a group of possible residues according to Table 2 are encompassed by the present disclosure. The skilled person will appreciate that Xn may be selected from any one of the listed groups of possible residues and that this selection is independent from the selection of amino acids in Xm, wherein n≠m. Thus, any of the listed possible residues in position Xn in Table 2 may be
independently combined with any of the listed possible residues any other variable position in Table 2.
Figure imgf000023_0001
Figure imgf000024_0001
In one particular embodiment, there is provided a VEGFR-2 binding polypeptide comprising a second monomeric unit, wherein, in sequence xvii), X3 is selected from Q and R;
X4 is selected from A, K, R, S and V;
X7 is R;
X11 is selected from A and G;
X16 is T;
X20 is selected from F and W;
X21 is Y;
X25 is V;
X26 is selected from K and N; and
X29 is D. In one embodiment, sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42. In one embodiment, sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:26-42. In one embodiment, sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as from the group consising of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:42. In one embodiment, sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as the group consisting of SEQ ID NO:34 and SEQ ID NO:41. In one embodiment, sequence xvii) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:41. As discussed above, in some embodiments of the present disclosure, a binding motif as defined above“forms part of” a three-helix bundle protein domain. This is equally valid for BM2 as for BM described above. Thus, in particular embodiments, the second VEGFR-2 binding motif (BM2) forms part of a three-helix bundle protein domain. For example, the BM2 may essentially constitute two alpha helices with an interconnecting loop, within said three- helix bundle protein domain. In particular embodiments, said three-helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different three-helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof. In some embodiments, the three-helical bundle protein domain is a variant of protein Z, which is derived from domain B of
staphylococcal Protein A. The skilled person would appreciate that the embodiments and context discussed in relation to BM are equally relevant for BM2. Thus, BM2 may replace BM in any one of the 49-, 53- or 58-mer contexts disclosed herein. In one particular embodiment, the VEGFR-2 binding polypeptide comprises
- a first monomer unit consisting of a VEGFR-2 binding polypeptide as defined herein; and
- a second monomer unit consisting of a second VEGFR-2 binding polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:26-42.
Thus, any VEGFR-2 binding polypeptide defined by sequence i) or having at least 82 % identity to a sequence define in i) may for example be combined with any one of the second VEGFR-2 binding polypeptides listed in Figure 1, i.e. any polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:26-42.
In one embodiment, said first monomer unit is selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23. In one particular embodiment, said first monomer unit is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23.
In one embodiment, said second monomer unit is selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:42. In another embodiment, said second monomer unit is selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39; SEQ ID NO:41 and SEQ ID NO:42.
The skilled person will appreciate that said first monomer unit and said second monomer unit may be independently combined. Thus, a VEGFR-2 binding polypeptide as defined herein may comprise any one of the listed first monomer units and any one of the listed second monomer units.
In one embodiment, said VEGFR-2 binding polypeptide is in
heterodimeric form and comprises SEQ ID NO:12 and SEQ ID NO:34; SEQ ID NO:12 and SEQ ID NO:39; SEQ ID NO:12 and SEQ ID NO:41; SEQ ID NO:12 and SEQ ID NO:42; SEQ ID NO:13 and SEQ ID NO:34; SEQ ID NO:13 and SEQ ID NO:39; SEQ ID NO:13 and SEQ ID NO:41; SEQ ID NO:13 and SEQ ID NO:42; SEQ ID NO:20 and SEQ ID NO:34; SEQ ID NO:20 and SEQ ID NO:39; SEQ ID NO:20 and SEQ ID NO:41; SEQ ID NO:20 and SEQ ID NO:42; SEQ ID NO:23 and SEQ ID NO:34; SEQ ID NO:23 and SEQ ID NO:39; SEQ ID NO:23 and SEQ ID NO:41; or SEQ ID NO:23 and SEQ ID NO:42.
In one embodiment, said VEGFR-2 binding polypeptide in
heterodimeric form comprises SEQ ID NO:23 and SEQ ID NO:41. As discussed above, it may be beneficial that the first and second monomer units of said VEGFR-2 binding polypeptide bind to different epitopes of VEGFR-2. As used herein, the term“different epitopes of VEGFR- 2” refers to non-overlapping epitopes, such that binding of the first monomer unit does not sterically interfere with the binding of the second monomer unit to VEGFR-2. For example, binding to two different epitopes may result in stronger binding, as measured by association rate ka, dissociation rate kd and/or affinity KD, than binding to one epitope or to two partially overlapping epitopes. Thus, in one embodiment, there is provided a VEGFR-2 binding polypeptide in heterodimeric form, which is capable of binding to VEGFR-2 such that the dissociation rate constant kd of the interaction with VEGFR-2 is at least 2 times, such as at least 5 times, such as at least 10 times, such as at least 15 times, such as at least 20 times lower, such as at least 30 times lower than the dissociation rate constant kd of a comparable polypeptide in homodimeric form.
In one embodiment, there is provided a VEGFR-2 binding polypeptide in heterodimeric form, which is capable of binding to VEGFR-2 such that the dissociation rate constant kd of the interaction with VEGFR-2 is at most 10-2 s-1 , such as at most 10-3 s-1 , such as at most 10-4 s-1. For the sake of clarity, throughout this disclosure, the term“VEGFR-2 binding polypeptide” is used to encompass VEGFR-2 binding polypeptides in all forms, i.e. monomeric and multimeric forms, for example dimeric forms, and in particular the heterodimeric form discussed immediately above. Furthermore, it may be beneficial that the VEGFR-2 binding
polypeptide as defined herein is part of a fusion protein or a conjugate comprising a second or further moieties. Second and further moiety/moieties of the fusion polypeptide or conjugate in such a protein may suitably have a desired biological activity.
Thus, in a second aspect of the present disclosure, there is provided a fusion protein or a conjugate, comprising a first moiety consisting of a
VEGFR-2 binding polypeptide according to the first aspect, and a second moiety consisting of a polypeptide having a desired biological activity. In another embodiment, said fusion protein or conjugate may additionally comprise further moieties, comprising desired biological activities that can be either the same as or different from the biological activity of the second moiety.
Non-limiting examples of a desired biological activity comprise a therapeutic activity, a binding activity and an enzymatic activity. In one embodiment, the second moiety having a desired biological activity is a therapeutically active polypeptide.
Non-limiting examples of therapeutically active polypeptides are biomolecules, such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.
Non-limiting examples of binding activities are binding activities which increase the in vivo half-life of the fusion protein or conjugate, and binding activities which act to block a biological activity. In one particular embodiment, the binding activity is an albumin binding activity which increases the in vivo half-life of the fusion protein or conjugate. In one embodiment, said albumin binding activity is provided by the albumin binding domain of streptococcal protein G or a derivative thereof. Thus, said fusion protein may for example comprise a VEGFR-2 binding polypeptide in monomeric or multimeric form (such as a homodimeric or heterodimeric form) as defined herein and an albumin binding domain of streptococcal protein G or a derivative thereof.
In one embodiment, said binding activity is binding to a cancer associated factor. Non-limiting examples of cancer associated factors include platelet-derived growth factor receptor α (PDGFR-α), platelet-derived growth factor receptor β (PDGFR-β), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B), platelet-derived growth factor C (PDGF-C), platelet-derived growth factor D (PDGF-D), epidermal growth factor receptor 1 (EGFR), epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor 3 (HER3), epidermal growth factor receptor 4 (HER4), epidermal growth factor, tumor growth factor α, epigen, epiregulin, neuregulins 1-4, c-kit, Raf kinases such as B-Raf and C-raf, rearranged during transfection (RET) receptor, colony stimulating factor 1 receptor (CSF- 1R) and fms-like tyrosine kinase 3 (Flt-3).
In one embodiment, said binding activity is binding to an angiogenesis associated factor. Non-limiting examples of angiogenesis associated factors include fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF-1), basic FGF, angiogenin 1 (Ang-1), angiogenin 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase with immunoglobulin-like domains 1 (TIE-1), tyrosine kinase with immunoglobulin-like domains 2 (TIE-2), vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor 3 (VEGFR-3), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor E (VEGF-E), placental growth factor (PlGF), transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF- β2), transforming growth factor β receptors (type I, type II and type III), matrix metalloproteinase (MMP), MET receptor tyrosine kinase (also denoted cMET and hepatocyte growth factor receptor (HGFR)), members of the Notch family of receptors and beta-catenin. In one embodiment, said binding activity is binding to an immune response associated factor. Non-limiting examples of immune response associated factors include T-cell recruitment factors such as CD3, CD28, T- cell receptor α (TCRα), T-cell receptor β (TCRβ), cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), and programmed cell death protein 1 (PD-1); NK-cell recruitment factors such as CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1) and the natural cytotoxicity receptors NKp30 and NKp40, programmed death- ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2) -, B7 homolog 3 (B7- H3), B7 homolog 4 (B7-H4), herpes virus entry mediator (HVEM)/B- and T- lymphocyte attenuator (BTLA), killer inhibitory receptor (KIR), lymphocyte- activation gene 3 (LAG3), galectin-9 (Gal9)/T cell immunoglobulin mucin-3 (TIM3) and adenosine/ alpha-2 adrenergic receptors (A2aR).
Furthermore, it may be beneficial to elicit an increased immune response to a cell that expresses VEGFR-2, for example a cancer cell that expresses VEGFR-2. In one embodiment of the second aspect of the present disclosure, the polypeptide comprises an immunostimulatory cytokine. In one embodiment, said immunostimulatory cytokine is selected from the group consisting of IL-2, IL-7, IL-9, IL-12, IL-15, IL-21 and interferons and G-CSF. In one embodiment of either the first and second aspect of the present disclosure, there is provided a VEGFR-2 binding polypeptide, fusion protein or conjugate which comprises an anti-cancer agent.
Non-limiting examples of anti-cancer agents include agents selected from the group consisting of auristatin, anthracycline, calicheamycin, combretastatin, doxorubicin, duocarmycin, the CC-1065 anti-tumor-antibiotic, ecteinsascidin, geldanamycin, maytansinoid, methotrexate, mycotoxin, taxol, ricin, bouganin, gelonin, pseudomonas exotoxin 38 (PE38), diphtheria toxin (DT), and their analogues, and derivates thereof and combinations thereof.
In one embodiment of either the first and second aspect of the present disclosure, there is provided a VEGFR-2 binding polypeptide, fusion protein or conjugate which comprises an anti-angiogenic agent.
Non-limiting examples of anti-angiogenic agents include agents selected from the group consisting of angiostatin, endostatin, tumstatin, angiopoietin, thrombospondin, IFN-α, IL-12, cartilage-derived angiogenesis inhibitors, matrix metalloproteinase inhibitors, and derivatives thereof and combinations thereof. Additional non-limiting examples of anti-angiogenic agents include bevacizumab, ranibizumab, etaracizumab, itraconazole, suramin, 2-methoxyestradiol, tasquinimod, linomide, axitinib, cediranib, motesanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, vandetanib and vatalanib.
Non-limiting examples of immune response modifying agents include agents selected from the group consisting of disease-modifying antirheumatic drugs (DMARDs), such as gold salts, azathioprine, methotrexate and leflunomide; calcineurin inhibitors, such as cyclosporin A or FK 506;
modulators of lymphocyte recirculation; mTOR inhibitors, such as rapamycin; an ascomycin having immuno-suppressive properties; glucocorticoids;
corticosteroids; cyclophosphamide; immunosuppressive monoclonal antibodies; adhesion molecule inhibitors, such as LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; anti-TNF agents, such as etanercept; blockers of proinflammatory cytokines; IL-1 blockers such as anakinra or IL-1 trap; IL-17 blockers; chemokine blockers; non steroidal anti-inflammatory drugs (NSAIDs) such as aspirin; and anti- infectious agents and other immune response modulating agents.
A skilled person would appreciate that the non-limiting examples of anti-cancer, anti-angiogenic and immune response modifying agents include all possible variants of said agents, for example the agent auristatin is intended to include for example auristatin E, auristatin F, auristatin PE, and derivatives thereof. Recently, considerable progress has been made in the development of multispecific agents, such as antibodies with the ability to bind to more than one antigen, for example through engineering of the complementarity determining regions (CDRs) to address two antigens in a single antibody combining site (Bostrom et al, 2009, Science 323(5921):1610-1614;
Schaefer et al, 2011, Cancer Cell 20(4):472-486), via construction of heterodimeric antibodies using engineered Fc units (Carter, 2001, J
Immunol Methods 248(1-2):7-15; Schaefer et al, 2011, Proc Natl Acad Sci USA 108(27):11187-11192) and via genetic fusion of auxiliary recognition units to N- or C-termini of light or heavy chains of full-length antibodies (Kanakaraj et al, 2012, MAbs 4(5):600-613; LaFleur et al, 2013, MAbs 5(2):208-218).
Thus, it may be beneficial for a polypeptide with affinity for VEGFR-2 as disclosed herein to also exhibit affinity for another factor, such as a factor associated with cancer or an angiogenesis related disorder, or an immune response associated factor. Thus, in a third aspect of the present disclosure, there is provided a complex comprising at least one VEGFR-2 binding polypeptide as defined herein and at least one antibody or an antigen binding fragment thereof.
When used herein to denote the third aspect of the disclosure, the term“complex” is intended to refer to two or more associated polypeptide chains, one having an affinity for VEGFR-2 by virtue of its VEGFR-2 binding motif as defined above, and the other being an antibody or an antigen binding fragment thereof. These polypeptide chains may each contain different protein domains, as described amply above for the VEGFR-2 binding polypeptide of the first aspect, and the resulting multiprotein complex can have multiple functions.“Complex” intends to refer to two or more polypeptides as defined herein, connected by covalent bonds, for example two or more polypeptide chains connected by covalent bonds through expression thereof as a recombinant fusion protein, or associated by chemical conjugation.
The third aspect provides a complex comprising an antibody or an antigen binding fragment thereof. As is well known, antibodies are immunoglobulin molecules capable of specific binding to a target (an antigen), such as a carbohydrate, polynucleotide, lipid, polypeptide or other, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term“antibody or an antigen binding fragment thereof” encompasses not only full-length or intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof, such as Fab, Fab′, F(ab′)2, Fab3, Fv and variants thereof, fusion proteins comprising one or more antibody portions, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies and covalently modified antibodies. Further examples of modified antibodies and antigen binding fragments thereof include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular
immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD- Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies and triomAbs. This listing of variants of antibodies and antigen binding fragments thereof is not to be seen as limiting, and the skilled person is aware of other suitable variants.
A full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH3). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). Depending on the amino acid sequence of the constant domain of its heavy chains, antibodies are assigned to different classes. There are six major classes of antibodies: IgA, IgD, IgE, IgG, IgM and IgY, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The term“full-length antibody” as used herein refers to an antibody of any class, such as IgD, IgE, IgG, IgA, IgM or IgY (or any sub-class thereof). The subunit structures and three-dimensional configurations of different classes of antibodies are well known.
An“antigen binding fragment” is a portion or region of an antibody molecule, or a derivative thereof, that retains all or a significant part of the antigen binding of the corresponding full-length antibody. An antigen binding fragment may comprise the heavy chain variable region (VH), the light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3. The three CDRs in VH or VL are flanked by framework regions (FR1, FR2, FR3 and FR4). As briefly listed above, examples of antigen binding fragments include, but are not limited to: (1) a Fab fragment, which is a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a Fab’ fragment, which is a Fab fragment with the heavy chain hinge region, (3) a F(ab′)2 fragment, which is a dimer of Fab’ fragments joined by the heavy chain hinge region, for example linked by a disulfide bridge at the hinge region; (4) an Fc fragment; (5) an Fv fragment, which is the minimum antibody fragment having the VL and VH domains of a single arm of an antibody; (6) a single chain Fv (scFv) fragment, which is a single polypeptide chain in which the VH and VL domains of an scFv are linked by a peptide linker; (7) an (scFv)2, which comprises two VH domains and two VL domains, which are associated through the two VH domains via disulfide bridges and (8) domain antibodies, which can be antibody single variable domain (VH or VL) polypeptides that specifically bind antigens.
Antigen binding fragments can be prepared via routine methods. For example, F(ab′)2 fragments can be produced by pepsin digestion of a full- length antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of F(ab′)2 fragments. Alternatively, fragments can be prepared via recombinant technology by expressing the heavy and light chain fragments in suitable host cells (e.g., E. coli, yeast, mammalian, plant or insect cells) and having them assembled to form the desired antigen- binding fragments either in vivo or in vitro. A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. For example, a flexible linker may be incorporated between the two variable regions. The skilled person is aware of methods for the preparation of both full-length antibodies and antigen binding fragments thereof.
Thus, in one embodiment, this aspect of the disclosure provides a complex as defined herein, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fc
fragments, Fv fragments, single chain Fv fragments, (scFv)2 and domain antibodies. In one embodiment, said at least one antibody or antigen binding fragment thereof is selected from full-length antibodies, Fab fragments and scFv fragments. In one particular embodiment, said at least one antibody or antigen binding fragment thereof is a full-length antibody.
In one embodiment of said complex as defined herein, the antibody or antigen binding fragment thereof is selected from the group consisting of monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and antigen-binding fragments thereof.
The term“monoclonal antibodies” as used herein refers to antibodies having monovalent affinity, meaning that each antibody molecule in a sample of the monoclonal antibody binds to the same epitope on the antigen, whereas the term“polyclonal antibodies” as used herein refers to a collection of antibodies that react against a specific antigen, but in which collection there may be different antibody molecules for example identifying different epitopes on the antigen. Polyclonal antibodies are typically produced by inoculation of a suitable mammal and are purified from the mammal’s serum. Monoclonal antibodies are made by identical immune cells that are clones of a unique parent cell (for example a hybridoma cell line). The term“human antibody” as used herein refers to antibodies having variable and constant regions corresponding substantially to, or derived from, antibodies obtained from human subjects. The term“chimeric antibodies” as used herein, refers to recombinant or genetically engineered antibodies, such as for example mouse monoclonal antibodies, which contain polypeptides or domains from a different species, for example human, introduced to reduce the antibodies’ immunogenicity. The term“humanized antibodies” refers to antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans, in order to reduce immunogenicity.
It may be beneficial for a complex as defined herein to, in addition to being capable of binding VEGFR-2, target at least one additional antigen, such as an antigen selected from the group consisting of an antigen associated with cancer, an antigen associated with an angiogenesis related disorder and an antigen associated with the immune response. In one embodiment, said additional antigen is associated with cancer. In another embodiment, said additional antigen is associated with angiogenesis. In one embodiment, said additional antigen is associated with the immune response.
Thus, in one embodiment there is provided a complex as defined herein, wherein said antibody or antigen binding fragment thereof has affinity for an additional antigen, for example associated with cancer or an
angiogenesis related disorder. In one embodiment, the antigen is selected from the group consisting of platelet-derived growth factor receptor α
(PDGFR-α), platelet-derived growth factor receptor β (PDGFR-β), platelet- derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B), platelet-derived growth factor C (PDGF-C), platelet-derived growth factor D (PDGF-D), epidermal growth factor receptor 1 (EGFR), epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor 3 (HER3), epidermal growth factor receptor 4 (HER4), epidermal growth factor, tumor growth factor α, epigen, epiregulin, neuregulins 1-4, c-kit, Raf kinases such as B-Raf and C-raf, rearranged during transfection (RET) receptor, colony stimulating factor 1 receptor (CSF-1R) and fms-like tyrosine kinase 3 (Flt-3). In one embodiment, said antibody or fragment thereof is selected from the group consisting of olaratumab, tovetumab, cetuximab, nimotuzumab, panitumumab, zalutumumab, trastuzumab, pertuzumab, MM-111,
seribantumab, duligotumab and patritumab.
In one embodiment, the antigen is associated with angiogenesis and is selected from the group consisting of fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF-1), basic FGF, angiogenin 1 (Ang-1), angiogenin 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase with immunoglobulin-like domains 1 (TIE-1), tyrosine kinase with immunoglobulin-like domains 2 (TIE- 2), vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor 3 (VEGFR-3), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor E (VEGF-E), placental growth factor (PlGF), transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2), transforming growth factor β receptors (type I, type II and type III), matrix metalloproteinase (MMP), MET receptor tyrosine kinase (also denoted cMET and hepatocyte growth factor receptor (HGFR)), members of the Notch family of receptors and beta-catenin. In one embodiment, said antibody or fragment thereof is selected from the group consisting of AMG 780, AMG 386, MEDI-3617, nesvacumab, CVX-241, bevacizumab, ranibizumab, VGX100, CVX-241, ABP 215, PF-06439535, fresolimumab, metelimumab, onartuzumab, emibetuzumab and tarextumab.
In one embodiment, the antigen is associated with the immune response and is selected from the group consisting of T-cell recruitment factors such as CD3, CD28, T-cell receptor α (TCRα), T-cell receptor β (TCRβ), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and programmed cell death protein 1 (PD-1); NK-cell recruitment factors such as CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1) and the natural cytotoxicity receptors NKp30 and NKp40; programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), herpes virus entry mediator (HVEM)/B- and T-lymphocyte attenuator (BTLA), killer inhibitory receptor (KIR), lymphocyte-activation gene 3 (LAG3), galectin-9 (Gal9)/T cell immunoglobulin mucin-3 (TIM3), adenosine/ alpha-2 adrenergic receptors (A2aR). In one embodiment, said antibody or fragment thereof is selected from the group consisting of visilizumab, otelixizumab, ipilimumab, tremelimumab, pembrolizumab, nivolumab, pidilizumab,
MPDL3280A, MEDI-4736, MPDL3280A and lirilumab. The complex as described herein may for example be present in the form of a fusion protein or a conjugate. Thus, said at least one VEGFR-2 binding polypeptide and said at least one antibody, or antigen binding fragment thereof, may be coupled by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the complex as a fusion protein or joined in any other fashion, either directly or via a linker, for example an amino acid linker.
Thus in one embodiment, there is provided a complex as defined herein, wherein said complex is a fusion protein or a conjugate. In one embodiment, said complex is a fusion protein. In another embodiment, said complex is a conjugate. In one embodiment of said complex, said VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of said antibody or antigen binding fragment thereof. In another embodiment, said VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the light chain of said antibody or antigen binding fragment thereof. In one embodiment, said VEGFR-2 binding polypeptide is attached to the N-terminus and/or C-terminus of the light chain and heavy chain of said antibody or antigen binding fragment thereof. For example, the VEGFR-2 binding polypeptide may be attached to only the N-terminus of the heavy chain(s), only the N-terminus of the light chain(s), only the C-terminus of the heavy chain(s), only the C-terminus of the light chain(s), both the N-terminus and the C-terminus of the heavy chain(s), both the N-terminus and the C- terminus of the light chain(s), only the C-terminus of the light chain(s) and the N-terminus of the heavy chain(s), only the C-terminus of the heavy chain(s) and the N-terminus of the light chain(s), of said antibody or antigen binding fragment thereof. As the skilled person would understand, the construction of a fusion protein often involves use of linkers between functional moieties to be fused. The skilled person is aware of different kinds of linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers. Linkers have been used to for example increase stability or improve folding of fusion proteins, to increase expression, improve biological activity, enable targeting and alter pharmacokinetics of fusion proteins. Thus, in one embodiment, the VEGFR-2 binding polypeptide, fusion protein, conjugate or complex as defined herein further comprises at least one linker, such as selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers. In one embodiment of a fusion protein or conjugate as disclosed herein, said linker is arranged between a first moiety consisting of a VEGFR-2 binding polypeptide as defined herein and a second moiety consisting of a polypeptide having a desired biological activity. In another embodiment, said linker is arranged within said first moiety. For example, one or more linker(s) may be arranged between monomeric units of the polypeptide as defined herein, such as between the monomer units in a homodimer or heterodimer as described above in connection with an embodiment of the first aspect. Thus, in one embodiment of a VEGFR-2 binding polypeptide as disclosed herein, said linker may be arranged between said first monomer unit and said second monomer unit. In one embodiment of a complex as disclosed herein, said linker is arranged between said VEGFR-2 binding polypeptide and said antibody or antigen binding fragment thereof. The skilled person will appreciate that the presence of linker arranged in any of above mentioned contexts does not exclude the presence of additional linkers in the same or any other context. Flexible linkers are often used when the joined domains require a certain degree of movement or interaction, and may be particularly useful in some embodiments of the VEGFR-2 binding polypeptide, fusion protein, conjugate or complex as defined herein. Such linkers are generally composed of small, non-polar (for example G) or polar (for example S or T) amino acids. Some flexible linkers primarily consist of stretches of G and S residues, for example (GGGGS)p and (SSSSG)p. Adjusting the copy number“p” allows for optimization of the linker in order to achieve appropriate separation between the functional moieties or to maintain necessary inter-moiety interaction. Apart from G and S linkers, other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues, such as T, A, K and E, to maintain flexibility, as well as polar amino acid residues to improve solubility.
In one embodiment, said linker is a flexible linker comprising glycine (G), serine (S) and/or threonine (T) residues. In one embodiment, said linker has a general formula selected from (GnSm)p and (SnGm)p, wherein, independently, n = 1-7, m = 0-7, n + m≤ 8 and p = 1-10. In one embodiment, n = 1-5. In one embodiment, m = 0-5. In one embodiment, p = 1-5. In a more specific embodiment, n = 4, m = 1 and p = 1-4. In one embodiment, said linker is selected from the group consisting of S4G, (S4G)3, (S4G)4 and (S4G)8. In one embodiment, said linker is selected from the group consisting of S4G, (S4G)3 and (S4G)4. In one embodiment, said linker is selected from the group consisting of S4G, (S4G)3 and (S4G)8. In one embodiment, said linker is S4G. In one embodiment, said linker is (S4G)3. In one embodiment, said linker is (S4G)8. In another embodiment, said linker is (S4G)4. In one embodiment, said linker is (G4S)3. In one embodiment, said linker is VDGS. With regard to the description above of fusion proteins, conjugates or complexes incorporating a VEGFR-2 binding polypeptide according to the disclosure, it is to be noted that the designation of first, second and further moieties is made for clarity reasons to distinguish between VEGFR-2 binding polypeptide or polypeptides according to the invention on the one hand, and moieties exhibiting other functions on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of the fusion protein, conjugate or complex. Similarly, the designations first and second monomer units are made for clarity reasons to distinguish between said units. Thus, for example, said first moiety (or monomer unit) may without restriction appear at the N-terminal end, in the middle, or at the C-terminal end of the fusion protein, conjugate or complex. The above aspects furthermore encompass polypeptides in which the VEGFR-2 binding polypeptide according to the first aspect, the VEGFR-2 binding polypeptide as comprised in a fusion protein or conjugate according to the second aspect or in a complex according to the third aspect, further comprises a label, such as a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides and radioactive particles. Such labels may for example be used for detection of the
polypeptide. For example, in some embodiments such labeled polypeptide may for example be used for labeling tumors with high expression of VEGFR- 2.
In other embodiments, the labeled VEGFR-2 binding polypeptide is present as a moiety in a fusion protein, conjugate or complex also comprising a second moiety having a desired biological activity. The label may in some instances be coupled only to the VEGFR-2 binding polypeptide, and in some instances both to the VEGFR-2 binding polypeptide and to the second moiety of the fusion protein or conjugate and/or the antibody or antigen binding fragment thereof the complex. Furthermore, it is also possible that the label may be coupled to a second moiety, or antibody or antigen binding fragment thereof only and not to the VEGFR-2 binding moiety. Hence, in yet another embodiment, there is provided a VEGFR-2 binding polypeptide comprising a second moiety, wherein said label is coupled to the second moiety only. In another embodiment, there is provided a complex as defined herein, wherein said label is coupled to the antibody or antigen binding fragment thereof only.
When reference is made to a labeled polypeptide, this should be understood as a reference to all aspects of polypeptides as described herein, including VEGFR-2 binding polypeptides, fusion proteins, conjugates and complexes comprising a VEGFR-2 binding polypeptide. Thus, a labeled polypeptide may contain only the VEGFR-2 binding polypeptide and e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide, or contain the VEGFR-2 binding polypeptide, a therapeutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy. A labeled polypeptide may contain a VEGFR-2 binding polypeptide in heterodimeric form and e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide, or contain the VEGFR-2 binding polypeptide in heterodimeric form, a therapeutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy. Also envisioned is a complex which contains a VEGFR-2 binding polypeptide as defined herein, an antibody or antigen binding fragment thereof and a e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the VEGFR-2 binding polypeptide or to the antibody or antigen binding fragment thereof. The skilled person is aware of other possible variants.
In embodiments where the VEGFR-2 binding polypeptide, VEGFR-2 binding polypeptide in heterodimeric form, fusion protein, conjugate or complex is radiolabeled, such a radiolabeled polypeptide may comprise a radionuclide. A majority of radionuclides have a metallic nature and metals are typically incapable of forming stable covalent bonds with elements presented in proteins and peptides. For this reason, labeling of proteins and peptides with radioactive metals is performed with the use of chelators, i.e. multidentate ligands, which form non-covalent compounds, called chelates, with the metal ions. In an embodiment of the VEGFR-2 binding polypeptide, fusion protein, conjugate or complex, the incorporation of a radionuclide is enabled through the provision of a chelating environment, through which the radionuclide may be coordinated, chelated or complexed to the polypeptide.
One example of a chelator is the polyaminopolycarboxylate type of chelator. Two classes of such polyaminopolycarboxylate chelators can be distinguished: macrocyclic and acyclic chelators.
In one embodiment, the VEGFR-2 binding polypeptide, fusion protein, conjugate or complex comprises a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the VEGFR-2 binding polypeptide via a thiol group of a cysteine residue or an epsilon amine group of a lysine residue.
The most commonly used macrocyclic chelators for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides and radiolanthanides are different derivatives of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid). In one embodiment, a chelating environment of the VEGFR- 2 binding polypeptide, VEGFR-2 binding polypeptide in heterodimeric form, fusion protein, conjugate or complex is provided by DOTA or a derivative thereof. More specifically, in one embodiment, a chelating polypeptide encompassed by the present disclosure is obtained by reacting the DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10- maleimidoethylacetamide (maleimidomonoamide-DOTA) with said
polypeptide. In one embodiment, a chelating polypeptide encompassed by the present disclosure is obtained by reacting the DOTA derivative DOTAGA (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid) with said polypeptide.
Additionally, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and derivatives thereof may be used as chelators. Hence, in one embodiment, a chelating environment of the VEGFR-2 binding polypeptide, VEGFR-2 binding polypeptide in heterodimeric form, fusion protein, conjugate or complex is provided by NOTA or a derivative thereof. In one embodiment, a chelating polypeptide encompassed by the present disclosure is obtained by reacting the NOTA derivative NODAGA (2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1- yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid) with said
polypeptide. The most commonly used acyclic polyaminopolycarboxylate chelators are different derivatives of DTPA (diethylenetriamine-pentaacetic acid).
Hence, polypeptides having a chelating environment provided by
diethylenetriaminepentaacetic acid or derivatives thereof are also
encompassed by the present disclosure. In further aspects of the present disclosure, there is provided a polynucleotide encoding a VEGFR-2 binding polypeptide, fusion protein or complex as described herein; an expression vector comprising said
polynucleotide; and a host cell comprising said expression vector.
Also encompassed by this disclosure is a method of producing
VEGFR-2 binding polypeptide, fusion protein or complex as described above, comprising culturing said host cell under conditions permissive of expression of said polypeptide from its expression vector, and isolating the polypeptide.
The VEGFR-2 binding polypeptide, fusion protein or complex of the present disclosure may alternatively be produced by non-biological peptide synthesis using amino acids and/or amino acid derivatives having protected reactive side-chains, the non-biological peptide synthesis comprising
- step-wise coupling of the amino acids and/or the amino acid derivatives to form a polypeptide, fusion protein or complex as described herein having protected reactive side-chains,
- removal of the protecting groups from the reactive side-chains of the polypeptide fusion protein or complex, and
- folding of the polypeptide in aqueous solution.
Said complex may also be produced by the conjugation of at least one VEGFR-2 binding polypeptide or fusion protein as described herein to at least one antibody or antigen binding fragment thereof. The skilled person is aware of conjugation methods, such as conventional chemical conjugation methods, for example using charged succinimidyl esters or carbodiimides. It should be understood that the VEGFR-2 binding polypeptide according to the present disclosure may be useful as a therapeutic, diagnostic and/or prognostic agent in its own right or as a means for targeting other therapeutic or diagnostic agents, with e.g. direct or indirect effects on
VEGFR-2. A direct therapeutic effect may for example be accomplished by inhibiting VEGFR-2 signaling. Thus, in another aspect, there is provided a composition comprising a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex as described herein and at least one pharmaceutically acceptable excipient or carrier. In one embodiment, said composition further comprises at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents. Non-limiting examples of additional active agents that may prove useful in such combination are anti-cancer agents, anti-angiogenic agents and immune response modifying agents as described herein.
The small size and robustness of the VEGFR-2 binding polypeptides of the present disclosure confer several advantages over conventional monoclonal antibody based therapies. Such advantages include advantages in formulation, modes of administration, such as alternative routes of administration, administration at higher doses than antibodies and absence of Fc-mediated side effects. Also, many diseases and disorders, such as cancers and angiogenesis related disorders, are associated with more than one factor and thus a complex as defined herein confers the advantage of targeting an additional antigen together with VEGFR-2. In another aspect of the present disclosure, there is provided a
VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein for use as a medicament, a prognostic agent and/or a diagnostic agent. In one embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the treatment, diagnosis or prognosis of a VEGFR-2 related disorder.
In one embodiment, said VEGFR-2 binding polypeptide, VEGFR-2 binding polypeptide in heterodimeric form, fusion protein, conjugate or composition is provided for use as a medicament. In a more specific embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein, for use as a medicament to modulate VEGFR-2 function in vivo. As used herein, the term “modulate” refers to changing the activity, such as rendering VEGFR-2 function hypomorph, partially inhibiting or fully inhibiting VEGFR-2 function.
In one embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the treatment of a VEGFR-2 related disorder. In one embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the diagnosis of a VEGFR-2 related disorder.
In one embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in the prognosis of a VEGFR-2 related disorder.
In one embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in diagnosis or prognosis to distinguish subjects who respond to anti angiogenic therapy from subjects who do not respond to said therapy. It is to be understood that said VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition may be used as the sole diagnostic or prognostic agent or as a companion diagnostic or prognostic agent.
In a related aspect, there is provided a method of treatment of a
VEGFR-2 related disorder, comprising administering to a subject in need thereof an effective amount of a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein. In a more specific embodiment of said method, the VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein modulates VEGFR-2 function in vivo. As used herein, the term“VEGFR-2 related disorder” refers to any disorder, disease or condition in which VEGFR-2 signalling plays a regulatory role. In one embodiment, said VEGFR-2 related disorder is selected from cancer and angiogenesis related disorders.
Non-limiting examples of VEGFR-2 related disorders are cancers selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, melanomas, cancer of the oral cavity and any cancer manifested by solid tumors with VEGFR-2 expression; as well as other VEGFR-2 related disorders including angiogenic related disorders including inflammatory diseases, retinopathies, age related macular degeneration, diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinopathy of prematurity and macular edema secondary to retinal vein occlusions. For example, VEGFR-2 has been linked to the prognosis and poor survival in patients suffering from squamous cell carcinoma of the lung and bladder cancer (Holtzer TR et al., (2013) PLoS One, Nov 14;8(11); Xia G et al., (2006) J Urol. Apr;175(4):1245-52).
Thus, in one embodiment, said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma,
astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, melanomas, cancer of the oral cavity and any cancer manifested by solid tumors with VEGFR-2 expression. In another embodiment, said angiogenic related disorder is selected from the group consisting of inflammatory diseases, retinopathies, age related macular degeneration, diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinopathy of prematurity and macular edema secondary to retinal vein occlusions.
In one embodiment, it may be beneficial to administer a therapeutically effective amount of a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein together with at least one second drug substance, such as an anti-cancer agent, anti-angiogenic agent or an immune response modifying agent.
In one embodiment, there is provided a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use in prognosis and/or diagnosis together with at least one cell proliferation marker. Non-limiting examples of contemplated cell proliferation markers are those selected from the group consisting of Ki-67, AgNOR, choline, claspin, cyclin A, CYR61, Cdk1, histone H3, HsMCM2, IL-2, Ki-S1, Ki-S2, LigI, MCM2, MCM6, MCM7, mitosin, p120, PCNA, PDPK, PLK, STK1, TK-1, topoisomerase II alpha and TPS. In another aspect of the present disclosure, there is provided a method of detecting VEGFR-2, comprising providing a sample suspected to contain VEGFR-2, contacting said sample with a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein, and detecting the binding of the VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition to indicate the presence of VEGFR-2 in the sample. In one embodiment, said method further comprises an
intermediate washing step for removing non-bound polypeptide, fusion protein, conjugate, complex or composition, after contacting the sample.
In one embodiment, said method is a diagnostic or prognostic method for determining the presence of VEGFR-2 in a subject, the method comprising the steps:
- contacting the subject, or a sample isolated from the subject, with a VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition as described herein, and
- obtaining a value corresponding to the amount of the VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition that has bound in said subject or to said sample.
In one embodiment, said method further comprises an intermediate washing step for removing non-bound polypeptide, fusion protein, conjugate or composition, after contacting the subject or sample and before obtaining a value.
In one embodiment, said method further comprises a step of comparing said value to a reference. Said reference may be by a numerical value, a threshold or a visual indicator, for example based on a color reaction. The skilled person will appreciate that different ways of comparison to a reference are known in the art and may be suitable for use.
In one embodiment of such a method, said subject is a mammalian subject, such as a human subject.
In one embodiment, said method is performed in vivo. In another embodiment, said method is performed in vitro. While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be
substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims. Brief description of the figures
Figure 1 is a listing of the amino acid sequences of examples of VEGFR-2 binding polypeptides of the present disclosure (SEQ ID NO:1, 3 and 5-25), second VEGFR-2 binding polypeptides (SEQ ID NO:2, 4 and 26- 42), albumin binding polypeptide ABD053 (SEQ ID NO:47); control polypeptides (SEQ ID NO:48, 49 and 50); as well as the amino acid sequences of human VEGFR-2 (SEQ ID NO:43), murine VEGFR-2 (SEQ ID NO:44), human VEGF-A (SEQ ID NO:45) and murine VEGF-A (SEQ ID NO:46) used for selection, screening and/or characterization of the invention. The deduced VEGFR-2 binding motifs (BMs and BM2s) of the VEGFR-2 binding polypeptides disclosed herein extend from residue 8 to residue 36 in the sequences with SEQ ID NO:1-42. The amino acid sequences of the 49 amino acid residues long polypeptides (BMod) predicted to constitute the complete three-helix bundle within each of these Z variants extend from residue 7 to residue 55.
Figure 2 shows results from evaluation of (A) Z05752 (SEQ ID NO:1) and (B) Z05993 (SEQ ID NO:2) in terms of binding specificity and epitope on VEGFR-2 using ELISA. The absorbance is represented on the Y axis and the different VEGFRs tested and their respective concentrations are indicated by numbers 1-10 on the X axis (see Example 1).
Figure 3 shows the result from flow-cytometric analysis of Z17681 (SEQ IN NO:3) and Z17682 (SEQ ID NO:4) displayed on the surface of S. carnosus. Z variants are represented on the X axis, and a ratio of FL-1 fluorescence intensity, corresponding to binding of (A) human VEGFR-2 or (B) murine VEGFR-2, to FL-6 fluorescence intensity, corresponding to surface expression level, is represented on the Y axis. Figure 3C shows a
representative scatter plot obtained from Z17681 displayed on S. carnosus and incubated with human VEGFR-2/Fc. The VEGFR-2 binding signal (FL-1) is represented on the Y axis and the surface expression level (FL-6) is represented on the X axis.
Figure 4 shows circular dichroism (CD) spectra of the first generation VEGFR-2-binding polypeptides. Figure 4A shows CD spectra of Z17681 and Z17682 at wavelengths ranging from 250 to 195 nm at 20 °C before and after variable temperature measurement (VTM). The spectra recorded before and after VTM overlap well for both variants. Figure 4B shows graphs of variable temperature measurement (VTM) spectra obtained at 221 nm while heating Z17681 and Z17682 from 20 to 90 °C. Figure 5 shows sensorgrams obtained from affinity measurements of VEGFR-2 binding polypeptides (A) Z17681 and (B) Z17682 for human and murine VEGFR-2 using surface plasmon resonance (SPR) analysis.
Sensorgrams obtained from injections of 50 nM (top), 100 nM, 200 nM, 350 nM and 500 nM (bottom) are shown. Z17681 and Z17682 over immobilized human or murine VEGFR-2-Fc are shown.
Figure 6 shows the results from SPR-based competition assays of Z17681 and Z17682 binding to human and murine VEGFR-2. Figure 6A shows sensorgrams obtained from a double injection, where 1 µM of Z17681 was injected (1), immediately followed by a second injection (2) of either a combination of 1 µM of Z17681 and 1 µM of Z17682, or 2 µM of Z17681, over immobilized human or murine VEGFR-2-Fc. Figure 6B shows sensorgrams obtained from a double injection, where 1 µM of Z17682 was injected (1), immediately followed by a second injection (2) of either a combination of 1 µM of Z17682 and 1 µM of Z17681, or 2 µM of Z17682 over immobilized human or murine VEGFR-2-Fc. An increase in response signal upon injection of a second Z variant indicates that Z17681and Z17682 can bind simultaneously to VEGFR-2.
Figure 7 shows the results obtained from SPR-based competition assays of Z17681 and Z17682 with human and murine VEGF-A.40 nM of human or murine VEGFR-2/Fc, which had been pre-incubated for 40 min with a 25 x molar excess of Z17681 or Z17682, was injected over a surface of immobilized human or murine VEGF-A. As shown in the SPR sensorgrams, pre-incubation with either of the VEGFR-2 specific Z variants blocked the interaction of VEGFR-2 with VEGF-A.
Figure 8 shows the result from flow-cytometric analysis of the alanine scan described in Example 5. The 13 residues in the VEGFR-2-binding Z variants that were substituted with alanine are represented on the X axis, and the fold change in normalized binding signal (a ratio of FL-1 fluorescence intensity, corresponding to VEGFR-2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level) compared to the corresponding non-mutated binder (Z17681 or Z17682) is represented on the Y axis. Binding to human (black bar) or murine (grey bar) VEGFR-2 is shown.
Figure 9A and 9B show scatter plots from fluorescence activated cell sorting of the affinity maturation library Z17681matlib and Z17682matlib, respectively, displayed on S. carnosus. The VEGFR-2 binding signal (FL-1) is represented on the Y axis and the surface expression level (FL-6) is represented on the X axis. The scatter plots show cells from the original unsorted library as well as cells isolated in the 1st, 2nd, 3rd and 4th selection round, respectively. For the 4th selection round, scatter plots are shown for selections including an off-rate step of 0 min, 30 min or 4.5 h. Scatter plots obtained for Z17681 and Z17682 are shown for comparison.
Figure 10A and 10B show the result from flow-cytometric analysis of the on-cell ranking experiment for maturated Z variants obtained from
Z17681matlib and Z17682matlib, respectively. Binding to human VEGFR-2 after an off-rate incubation step of 30 min or 4.5 h is presented as indicated.
The normalized binding signal to human VEGFR-2 after an off-rate incubation step of 30 min (black bar) or 4.5 h (grey bar) is shown. The Z variants are represented on the X axis and a ratio of FL-1 fluorescence intensity, corresponding to VEGFR-2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level, is represented on the Y axis.
Figure 11A and 11B show the result from flow-cytometric analysis of binding of the top ten ranked Z variants obtained from Z17681matlib and Z17682matlib, respectively, to murine VEGFR-2. The Z variants are
represented on the X axis and the fold change in normalized binding signal (a ratio of FL-1 fluorescence intensity, corresponding to VEGFR-2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level) compared to the corresponding non-mutated binder (Z17681 and Z17682, respectively) is represented on the Y axis.
Figure 12A and 12B show CD spectra obtained from VEGFR-2-binding Z variants. CD spectra of Z17701 (SEQ ID NO:23), Z17698 (SEQ ID NO:20), Z17691 (SEQ ID NO:13), Z17690 (SEQ ID NO:12), Z17720 (SEQ ID NO:41), Z17717 (SEQ ID NO:39), Z17733 (SEQ ID NO:34) and Z17719 (SEQ ID NO:40) at wavelengths ranging from 250 to 195 nm at 20 °C before and after the VTM.
Figure 13A and 13B show CD spectra from heat stability
measurements of affinity matured VEGFR-2-binding Z variants. VTM spectra obtained at 221 nm while heating the Z variants Z17701, Z17698, Z17691, Z17690, Z17720, Z17717, Z17712 and Z17719 from 20 to 90 °C are shown.
Figure 14 shows sensorgrams obtained for human VEGFR-2 of affinity maturated VEGFR-2 binding polypeptides. Injections of 5 nM, 10 nM and 20 nM Z17701 (SEQ ID NO:23), Z17698 (SEQ ID NO:20), Z17690 (SEQ ID NO:12) and Z17719 (SEQ ID NO:41) as indicated over immobilized human VEGFR-2-Fc were analyzed. Figure 15A and 15B show the results obtained for murine VEGFR-2 of affinity matured VEGFR-2 binding polypeptides evaluated by SPR. Injections of 5 nM, 10 nM and 20 nM murine VEGFR-2 over immobilized Z17701 (SEQ ID NO:23), Z17698 (SEQ ID NO:20), Z17690 (SEQ ID NO:12), or 5 nM, 10 nM and 20 nM Z17719 (SEQ ID NO:41), over immobilized human VEGFR-2- Fc were analyzed.
Figure 16 shows the results from an SPR-based competition assay of VEGFR-2 binding polypeptides Z17701 (SEQ ID NO:23) and Z17719 (SEQ ID NO:41). A) A double injection was performed, where 1 µM of Z11701 was injected (1), immediately followed by a second injection (2) of either a combination of 1 µM of Z17701 and 1 µM of Z17719, or 2 µM of Z17701 (as indicated), over immobilized human or murine VEGFR-2-Fc. B) A double injection was performed, where 1 µM of Z17719 was injected (1), immediately followed by a second injection (2) of either a combination of 1 µM of Z17719 and 1 µM of Z17701, or 2 µM of Z17719 (as indicated), over immobilized human or murine VEGFR-2-Fc.
Figure 17 shows the results from an SPR-based competition assay of VEGFR-2 binding polypeptides Z17701 and Z17719 with human and murine VEGF-A.40 nM of human or murine VEGFR-2/Fc, which had been pre- incubated for 40 min with a 25 x molar excess of Z17701 or Z17719, was injected over a surface of immobilized human or murine VEGF-A (as indicated). The SPR sensorgrams show that pre-incubation with either of the Z variant molecules blocked VEGFR-2 interaction with VEGF-A.
Figure 18 shows sensorgrams obtained from SPR analysis of dimeric VEGFR-2 binding Z variants as indicated.200 nM of Z17681-ABD053- Z17682, Z17682-ABD053-Z17681, Z17681-ABD053-Z17681 or Z17682- ABD053-Z17682 was injected over a surface of immobilized HSA followed by injections of (A) human or (B) murine VEGFR-2 at the indicated
concentrations.
Figure 19 shows the results of flow-cytometric analysis of binding of VEGFR-2-specifc dimeric Z variants (A) to VEGFR-2 expressing 293/KDR cells or (B) to non-VEGFR-2 expressing HEK293 cells in vitro. Binding of the Z variants (FL6) or the positive control antibody (FL1) is shown on the X axis and cell count is shown on the Y axis. Binding of heterodimers, homodimers or negative controls is indicated. A higher shift in fluorescence intensity was observed for the heterodimeric constructs than for the homodimeric constructs upon binding to VEGFR-2-expressing cells. Figures 20A and 20B show the results from flow cytometric analysis of the binding of dimeric Z variant constructs to VEGFR-2-expressing HUVEC and 293/KDR cells, respectively. The different constructs tested are shown on the X axis and the normalized binding signal (FL-6 fluorescence intensity of each sample divided by the FL-6 fluorescence intensity of the negative control construct Z03638-ABD053-Z03638) is shown on the Y axis.
Figure 21 shows the results from flow cytometric analysis of the IC50 evaluation of dimeric Z variant constructs blocking VEGF-A binding to
293/KDR cells. The concentrations of Z variant construct are shown on the X axis and the percentage of retained binding (FL-2 fluorescence intensity of each sample divided by the FL-2 fluorescence intensity of cells incubated with only VEGF-A) is shown on the Y axis.
Figure 22 shows the results from the VEGFR-2 phosphorylation blocking activity of dimeric Z variant constructs on 293/KDR cell. The different combinations of Z variant construct and stimulation with VEGF-A or PBS are shown on the X axis, and the percentage of phosphorylation (OD450 for each sample, compared to OD450 for cells stimulated with VEGF-A after treatment with no Z variant construct) is shown on the Y axis.
Figure 23 shows inhibition of VEGF-A induced downstream
ERK1/ERK2 phosphorylation by the dimeric Z variant Link2.
Figure 24 shows in vitro inhibition of sprout formation on HUVECs 3-4 days after the indicated treatments, in A) confocal microscopy images, B) a diagram showing the number of sprouts per bead and C) a diagram showing the average sprout length.
Figure 25 shows inhibition of VEGF-A induced proliferation by the dimeric Z variant Link2.
Figure 26 shows retained binding by a NODAGA-conjugated
polypeptide analyzed by SPR as described in Example 13. (HE)3-Z17701- (S4G)-Z17719-Cys-NODAGA (grey) and Link2 (black) were injected at concentrations of 50 nM, 100 nM and 500 nM. Examples Summary
The following Examples disclose the development of novel Z variant molecules targeted to VEGFR-2, based on phage display and staphylococcal display technology. The genes encoding the VEGFR-2 binding polypeptides described herein were sequenced, and the corresponding amino acid sequences are listed in Figure 1 and denoted by the identifiers SEQ ID NO:1- 42. The Examples also describe the characterization of VEGFR-2 binding polypeptides as well as their in vitro functionality.
Example 1
Selection and screening of VEGFR-2 binding polypeptides Materials and methods
Phage display selection of VEGFR-2 binding Z variants: A
combinatorial naive library of variants of protein Z displayed on
bacteriophage was subjected to four rounds of selection using human
VEGFR-2/Fc (hVEGFR-2/Fc, R&D Systems) essentially as described in Grönwall et al., 2007(Grönwall et al., (2007) J Biotechnol, 128:162-183). The library variants were fused to a gene encoding a Taq polymerase binding Z variant, denoted Ztaq (Gunneriusson E et al., (1999). Protein Eng. 12(10):873-8). The selection buffer was phosphate buffered saline (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4, PBS) supplemented with 0.1 % Tween 20 (PBST 0.1) and 3 % BSA (Sigma). PBST 0.1 was used as wash buffer. Pre-selections were performed in cycles 1-3 by incubation of phage stock with biotin-SP conjugated human IgG, Fc (Jackson Immuno Research Laboratories) immobilized on streptavidin coated paramagnetic beads (SA-beads, Invitrogen) and with biotin conjugated anti-IgG Z variant (Affibody AB) immobilized on SA-beads (anti IgG-Z conjugated SA-beads). The selection was performed in solution in four cycles, with one track in the first cycle and subsequently divided into six tracks in the last cycle. An overview of the selection strategy, describing an increased stringency in subsequent cycles obtained using a lowered target concentration and an increased number of washes, is shown in Table 3.
Phage particles bound to Fc fused target were captured on anti-IgG-Z conjugated SA-beads before washing and phage particles were finally eluted at pH 2.2 (50 mM glycine-HCl). A constant high amount of phage particles was used for each round in half of the tracks in cycles 2-4 (see Table 3). In the remaining tracks, the number of phage particles used in the selection was about 2000 times the number of eluted phage particles in the previous cycle. Table 3. Overview of the phage display selection using human VEGFR-2/Fc as tar et
Figure imgf000052_0001
Production of Z variants for ELISA: The Z variants were produced by inoculating single colonies of Escherichia coli (E. coli) containing phagemids from the selections into culture medium supplemented with 1 mM isopropyl- β-D-1-thiogalactopyranoside (IPTG). Bacteria containing Z variants expressed as fusion proteins with Ztaq were subjected to repeated freeze- thaw cycles. Cells were pelleted by centrifugation to extract the proteins in the periplasmic fractions.
ELISA-based screening for VEGFR-2 binding Z variants: Binding of Z05752 and Z05993 to hVEGFR-2/Fc and murine VEGFR-2/Fc (mVEGFR- 2/Fc) was investigated using a standard sandwich ELISA. In brief, half-area 96 well plates were coated with a polyclonal goat anti-Z IgG (Affibody AB) at a concentration of 4 µg/ml in PBSC (PBS supplemented with 0.5 % casein). After washing the plates in water, the periplasmic samples containing the soluble Z-Ztaq variants (VEGFR-2-binding Z variants fused to Ztaq) were added to the wells. As a blank control, PBS supplemented with 0.05 % Tween 20 (PBST 0.05) was added instead of the periplasmic sample.
Plates were washed 4 times in PBST 0.05 followed by addition of hVEGFR- 2/Fc or mVEGFR-2/Fc diluted in PBSC. The target concentrations used for screening were 3.6 nM or 7.2 nM hVEGFR-2/Fc and 0.9 nM or 9 nM mVEGFR-2/Fc. After washing as described above, the plates were developed by addition of a horseradish peroxidase-conjugated goat anti- human IgG (anti-human IgG-HRP, Southern Biotechnology) diluted 1:10000 in PBSC followed by addition of TMB substrate (Thermo Scientific) according to the supplier’s recommendations. Plates were measured at 450 nm using a microplate reader (Victor3, Perkin Elmer).
Sequencing: The DNA sequences of clones shown to be positive in ELISA were determined by PCR amplification of insert sequences and using an ABI PRISM® 3130xl Genetic Analyzer instrument (PE Applied
Biosystems) according to the manufacturer’s instructions.
ELISA-based specificity and epitope analysis: The same ELISA set up as for said screening was used to investigate the specificity of Z05752 and Z05993 as well as the ability of these variants to interfere with the binding of VEGF-A to hVEGFR-2. Selectivity to human and murine VEGFR- 2/Fc, human and murine VEGFR-1/Fc (R&D Systems) and human and murine VEGFR-3/Fc (R&D Systems) were assayed at target concentrations of 1.8 nM hVEGFR-2/Fc, 1.8 nM hVEGFR-2/Fc, 20 nM hVEGFR-1 or 20 nM hVEGFR-3/Fc according to the setup below. To investigate if binding of hVEGFR-2 to said Z variants could be blocked by VEGF-A, hVEGFR-2/Fc at a concentration of 1.8 nM was premixed with a 15-fold molar excess of human VEGF-A (R&D Systems) before addition to the ELISA plates. The ELISA assays were developed as described above and the plates were measured at 450 nm using a microplate reader (Victor3, Perkin Elmer) For each Z variant the experimental set up was a follows:
1. Z variant + mVEGFR-2/Fc (1.8 nM)
2. Z variant + mVEGFR-2/Fc (1.8 nM)
3. Z variant + hVEGFR-2/Fc (1.8 nM)
4. Z variant + hVEGFR-2/Fc (1.8 nM)
5. Z variant + mVEGFR-2/Fc (1.8 nM) + 15 x VEGF-A
6. Z variant + hVEGFR-2/Fc (1.8 nM) + 15 x VEGF-A
7. Z variant + hVEGFR-1/Fc (~20 nM)
8. Z variant + hVEGFR-3/Fc (~20 nM)
9. PBST + m VEGFR-2/Fc (1.8 nM)
10.PBST + h VEGFR-2/Fc (1.8 nM) Results
Phage display selection and screening of Z variants with affinity for VEGFR-2: A naive Z variant library of 1.4 × 1010 complexity displayed on M13 filamentous phage (monovalent phagemid system) was used to select binders to hVEGFR-2/Fc. After four selection rounds, a total of 279
candidate clones from all tracks were analyzed for binding to hVEGFR-2/Fc in ELISA. In total, 48 Z variants were found to give a response of 16 times the blank control or higher. Since cross-binding to murine VEGFR-2 was desirable for future in vivo studies, a total of 1193 clones were screened using murine VEGFR-2.
Two clones binding to both human and murine VEGFR-2 were identified and denoted Z05752 (SEQ ID NO:1) and Z05993 (SEQ ID NO:2). Comparison of the sequences of these two variants revealed a very low level of similarity, which may indicate that the two Z variant molecules do not bind to the same epitope on VEGFR-2.
Specificity and epitope analysis: The specificity of the two human- and murine VEGFR-2-binding clones Z05752 and Z05993 was tested using an ELISA setup similar to the one used in the VEGFR-2-binding screening. Both clones showed binding to human and murine VEGFR-2, but no binding to the closely related receptors VEGFR-1 and VEGFR-3 were observed (Figure 2).
It was further investigated if blocking of hVEGFR-2 binding to Z05752 and Z05993 could be achieved using VEGF-A, the natural ligand of VEGFR- 2, by pre-mixing hVEGFR-2/Fc with a 15-fold molar excess of human VEGF- A before addition to the ELISA plates. For both clones, this reduced the signal greatly, indicating that the Z variants bind to the same epitope as VEGF-A or to a nearby epitope. This may be an advantage for future strategies for blocking of VEGF-A interaction with VEGFR-2. Example 2
Flow-cytometric analysis of VEGFR-2 binding Z variants displayed on
S. carnosus Summary
This Example describes the subcloning of the amino acid residues corresponding to helix 1 and 2 of Z05752 and Z05993 into a staphylococcal display vector to obtain Z variants Z17681 (SEQ ID NO:3) and Z17682 (SEQ ID NO:4). Flow cytometric analysis of Z17681 and Z17682 displayed on S. carnosus as well as purification of said variants is described. Materials and methods
Sub-cloning of Z05752 and Z05993 to a staphylococcal display vector: E. coli RR1 ^M15 cells (Rüther U (1982) Nucleic acids research 10(19): 5765- 5772) containing pAffi1 plasmids encoding Z05752 and Z05993 were inoculated in 1 l of TSB medium (30 g/l; Merck) and cultivated over night at 37 °C at 150 rpm. Plasmids were prepared using a Jetstar MAXIprep kit (Genomed). The genes encoding said Z variants were amplified by PCR, cleaved by the restriction enzymes XhoI and SacI (New England Biolabs) and ligated into the staphylococcal display vector pSCZ1 (Kronqvist, N et al., (2008) Protein Eng Des Sel 21(4): 247-255) which had been cleaved with the same enzymes. Ligation was performed using T4 DNA ligase (New England Biolabs) followed by transformation to E. coli RR1 ^M15 cells. Plasmids encoding Z17681 (SEQ ID NO:3) and Z17682 (SEQ ID NO:4) were thus obtained. The plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen) and transformed into S. carnosus by electroporation as previously described in Löfblom et al., 2007 (Löfblom J et al., (2007) Journal of Applied Microbiology 102(3): 736-747).
Flow-cytometric analysis of Z17681 and Z17682 displayed on S.
carnosus: Recombinant S. carnosus cells were inoculated in TSB medium (30 g/l; Merck) supplemented with 20 µg/ml chloramphenicol, and cultivated for 16 h at 37 °C and 150 rpm. Approximately 106 cells were washed with 1 ml PBS supplemented with 0.1 % Pluronic F108 NF Surfactant (PBSP; pH 7.4; BASF Corporation), re-suspended in 200 nM of hVEGFR-2/Fc or 150 nM mVEGFR-2/Fc, and incubated at room temperature for 1 h under conditions of gentle mixing. Next, cells were washed with PBSP, re-suspended in goat anti-human IgG-Alexa Flour® 488 (1:500, Invitrogen) and 225 nM HSA labeled with Alexa Fluor® 647 succinimidyl ester (Invitrogen) labeled according to the supplier’s recommendations. Cells were incubated on ice for 40 min and then washed with PBSP and analyzed using a GalliosTM flow cytometer (Beckman Coulter).
Sub-cloning, protein production and purification of Z17681 and
Z17682: The genes encoding Z17681 and Z17682 were amplified by PCR using Phusion DNA polymerase (Finnzymes) and cleaved by restriction enzymes NdeI and XhoI (New England Biolabs). The genes were ligated into a pET-26b(+) vector (Merck), cleaved with the same enzymes, using T4 DNA ligase (New England Biolabs) and transformed into E. coli RR1 ^M15 cells (Rüther U (1982), supra). Plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen) and transformed into E. coli Rosetta (DE3) cells (Merck) using heat shock. Colonies were inoculated to TSB medium (30 g/l; Merck), supplemented with 5 g/l yeast extract (Merck) and 50 μg/ml kanamycin (Sigma-Aldrich Company Ltd, Dorset, UK), and cultivated over night at 37 °C and 150 rpm. After 16 h, the cultures were diluted to an OD value of 0.05-0.1 in TSB medium (30 g/l; Merck). When an OD value of 0.5-1 was reached, protein expression was induced with isopropyl-beta-D- thiogalactopyranoside (IPTG) (Apollo Scientific Ltd.) and cultures were incubated for 20 h at 25 °C and 150 rpm. Cells were harvested by
centrifugation (2400 g, 8 min 4 °C) and Z variants were purified under denaturing conditions by ion-metal affinity chromatography (IMAC) using HisPurTM Cobalt resins (Thermo Scientific) and buffer exchanged to PBS using PD-10 columns (GE Healthcare).. Results
Flow-cytometric analysis of Z17681 and Z17682 displayed on S.
carnosus: The amino acid residues corresponding to helix 1 and 2 of Z05752 and Z05993 were subcloned into a staphylococcal display vector and Z variants Z17681 and Z17682 were obtained. To investigate whether staphylococcal display of the affinity maturation libraries would be a suitable strategy for selection of high-affinity VEGFR-2 binding Z variants, Z17681 and Z17682 were displayed on the surface of S. carnosus (Kronqvist N et al., (2008), supra; Kronqvist N et al., (2011) Protein Eng Des Sel 24(4): 385-396). Recombinant staphylococcal cells were incubated with either 150 nM mVEGFR-2/Fc or 200 nM hVEGFR-2/Fc, together with fluorescently labeled goat anti-human IgG and human serum albumin (HSA) labeled with a different fluorophore. Both Z17681 and Z17682 were functionally expressed on the cell surface and showed binding to human as well as murine VEGFR-2 (Figure 5). The measured binding signal for both human and murine VEGFR- 2 was slightly higher for Z17681 than for Z17682. A VEGFR-2 binding Z variant displayed on S. carnosus was used as a negative control.
Protein production and purification of Z17681 and Z17682: The genes encoding Z17681 and Z17682 were sub-cloned into the expression vector pET-26b(+) for production of soluble protein. Proteins were expressed in the cytoplasm and recovered by sonication of the cells followed by IMAC purification. The purity of the soluble proteins was analyzed using SDS- PAGE, and single bands corresponding to the correct molecular weight (~6.5 kDa) were observed for both variants.
Example 3
Characterization of VEGFR-2 binding polypeptides Summary
In this Example, Z17681 and Z17682 were further characterized using circular dischroism (CD) spectroscopy and surface plasmon resonance analysis. Materials and methods
Circular dichroism analysis of Z17681 and Z17682: Purified Z variants Z17681 and Z17682 were analyzed by CD spectroscopy using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB). CD spectra were obtained at 250-195 nm and 20 °C. Thermal unfolding was analyzed at 221 nm and a temperature range of 20-90 °C.
Binding kinetics analysis of Z17681 and Z17682: Surface plasmon resonance experiments were performed using a ProteOn XPR36 instrument (Biorad Laboratories), employing PBS buffer with 0.1 % Tween (PBST) as a running buffer and 10 mM HCl or PBST for regeneration. Human and murine VEGFR-2/Fc (R&D Systems) were immobilized at 4200 and 4800 RU, respectively, by amine coupling on two surfaces of a GLM sensor chip (Biorad Laboratories). Binding of Z17681 and Z17682 to both human and murine VEGFR-2 was analyzed by injections of five different concentrations (50 nM, 100 nM, 200 nM, 350 nM and 500 nM) at 100 µl/min over the immobilized VEGFR-2/Fc for 60 s followed by a dissociation phase of 600 s. PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration. Results
Analysis of heat stability and folding of Z17681 and Z17682: The heat stability and folding of Z17681 and Z17682 was analyzed using CD
spectroscopy. Both Z variants had an alpha-helical content and refolded after heat-induced denaturation at 90 °C (Figure 4A). Melting temperatures were estimated to about 45 °C for Z17681 and about 50 °C for Z17682 (Figure 4B).
Binding kinetics analysis of Z17681 and Z17682: The kinetics of the binding of Z17681 and Z17682 to human and murine VEGFR-2 was analyzed by surface plasmon resonance. Binding of Z17681 and Z17682 to both human and murine VEGFR-2 was detected by injecting the Z variants over human and murine VEGFR-2/Fc immobilized by amine coupling. Equilibrium dissociation constants (KD) for human VEGFR-2 were determined to be around 160 nM for Z17681 and 80 nM for Z17682. The KD value for murine VEGFR-2 was determined to be around 260 nM for Z17681 and 90 nM for Z17682 (Figure 5).
Example 4
Further characterization of VEGFR-2 binding polypeptides Summary
In this Example, Z17681 and Z17682 were analyzed using SPR to determine if they could bind simultaneously to VEGFR-2, and whether they competed with VEGF-A for binding to VEGFR-2. Materials and methods
Surface plasmon resonance-based competition assays: Competition assays were performed using a BIAcoreTM 3000 instrument (GE Healthcare). For analysis of potential simultaneous binding of Z17681 and Z17682 to VEGFR-2, human and murine VEGFR-2/Fc (R&D Systems) was immobilized on a CM-5 sensor chip (GE Healthcare). First, 1 µM of Z17681 was injected, directly followed by an injection of a mixture of 1 µM Z17681 and 1 µM
Z17682, or an injection of 2 µM Z17681 as a control. In a second assay, Z17682 was injected first, followed a mixture of Z17681 and Z17682, or an injection of 2 µM Z17682 as a control. PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration.
For analysis of competition with VEGF-A, VEGF-A (R&D Systems) was immobilized on a sensor chip surface.40 nM of human or murine VEGFR- 2/Fc (R&D Systems), which had been pre-incubated with a 25-fold molar excess of Z17681, Z17682 or PBS (as a control) for 40 min, was injected over the surface. PBS buffer with 0.1% Tween (PBST) was used as a running buffer and 10 mM NaOH was used for regeneration. Results
SPR-based competition assays: Z17681 and Z17682 were analyzed using SPR to determine if they could simultaneously bind to VEGFR-2. First, a saturating concentration (1 µM) of Z17681 was injected, directly followed by an injection of a mix of 1 µM Z17681 and 1 µM Z17682, or an injection of 2 µM Z17681 (control). A large increase in response signal was observed for the mix of Z17681 and Z17682, but not for Z17681 alone, indicating that Z17681 and Z17682 were able to bind simultaneously to VEGFR-2 (Figure 6A). The results indicate that Z17681 and Z17682 bind to distinct and non- overlapping epitopes on VEGFR-2. The experiment was also conducted in the reverse order, i.e. Z17682 injected first, followed by injection of a mixture of Z17681 and Z17682, or an injection of 2 µM Z17682 (control). Again, there was a large signal increase upon simultaneous injection of Z17681 and Z17682, verifying the previous results (Figure 6B).
To verify that the two Z variant molecules could compete with VEGF-A for binding to VEGFR-2, human or murine VEGF-A was immobilized on the chip. Human or murine VEGFR-2/Fc, which had been pre-incubated for 45 min with Z17681, Z17682 or PBS, was passed over the surface. Injection of the control sample containing VEGFR-2 pre-incubated with PBS resulted in an increase in signal showing binding of VEGFR-2 to VEGF-A. Injection of the samples containing VEGFR-2 pre-incubated with Z17681 or Z17682 did not result in a substantial increase in signal, showing that the Z variant molecules blocked the interaction between VEGFR-2 and VEGF-A (Figure 7). Example 5
Alanine scan of VEGFR-2 binding Z variants Summary
In this Example, alanine scanning mutagenesis was used to analyze the individual contribution from residues in Z17681 and Z17682 to the interaction with VEGFR-2. Materials and methods
Construction of single-point mutations of Z17681 and Z17682 for alanine scan: Single-point mutations of Z17681 and Z17682 with codon substitution to alanine at residues 9, 10, 11, 13, 14, 17 (not in Z17681), 18 (not in Z17682), 24, 25, 27, 32 or 35 were constructed by PCR amplification using oligonucleotides encoding each mutation. Residues Ala17 in Z17681 and Ala18 in Z17682 were mutated to valine. The mutated genes were cloned into the staphylococcal display vector pSCZ1, using restriction enzymes XhoI and SacI (New England Biolabs) and T4 DNA ligase (New England Biolabs) according to the supplier’s recommendations. Plasmids were prepared from E. coli RR1∆M15 cells using a QIAprep Spin Miniprep kit (Qiagen) and sequences were confirmed by BigDye Thermo Cycle Sequencing using an ABI Prism 3700 instrument (Applied Biosystems). Plasmids were transformed into S. carnosus by electroporation as described in Example 2.
Flow-cytometric analysis of mutants obtained by alanine scan: A single colony of each alanine and valine mutant was inoculated to 3 ml Tryptic Soy Broth supplemented with yeast extract (TSB+YE; Merck) and 10 µg/ml chloramphenicol. The cultures were incubated at 37 °C under conditions of shaking at 150 rpm for 18 h.106 cells were washed by centrifugation (15000 g, 6 min, 4 °C) with 800 µl PBS containing 0.1 % Pluronic® F108 NF
Surfactant (PBSP; pH 7.4; BASF Corporation), re-suspended in 75 µl 100 nM human or murine VEGFR-2/Fc (R&D Systems) and incubated at room temperature with gentle mixing for 50 min. After washing with 200 µl ice-cold PBSP, the cells were re-suspended in 100 µl of Alexa Fluor® 488 goat anti- human IgG (H+L) antibody (Invitrogen) (1:1000 dilution) and 150 nM Alexa Fluor® 647-HSA conjugate. Cells were incubated on ice for 45 min in the dark. Next, the cells were washed with 200 µl ice-cold PBSP and re-suspended in 200 µl ice-cold PBSP for flow-cytometric analysis. Results
Alanine scan mutagenesis of Z17681 and Z17682: Alanine and valine scan mutagenesis was used to analyze the individual contribution from residues in Z variants Z17681 and Z17682 to the interaction with VEGFR-2. Thirteen mutants of each binder were created and all were mutated to alanine except position Ala17 in Z17681 and Ala18 in Z17682, which were mutated to valine. The 26 mutants were expressed and displayed on the surface of staphylococci and binding to VEGFR-2 was analyzed by flow cytometry (Figure 8). The results obtained were used for the design of two affinity maturation libraries as described in Example 6.
Example 6
Design and construction of affinity maturation libraries of VEGFR-2 binding Z variants Summary
In this Example, two new libraries, Z17681matlib and Z17682matlib, were designed based on the VEGFR-2-binding variants Z17681 and Z17682, derived from the previously selected variants Z05752 and Z05993,
respectively, as well as the result from the alanine scan described above. Z17681matlib contained approximately 4.1 x 107 individual clones, and
Z17682matlib contained approximately 4 x 107 individual clones. Materials and methods
Construction of affinity maturation libraries: Two new libraries were designed, in which 13 positions of the Z variant molecules were biased towards amino acid residues based on the sequences of Z17681 and
Z17682, respectively. Each position was randomized with 18 codons corresponding to amino acids: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, Y, V, W (excluding C and P in all positions) with the amino acid residues based on the sequences of the VEGFR-2 binding Z variants Z17681 and Z17682 spiked at a higher proportion to generate an average mutation frequency of approximately three mutations per molecule. The randomization frequency in each position was also normalized with the results from the alanine scan described above, resulting in less mutations in positions important for VEGFR-2 binding and more mutations in positions of less importance (Table 4). Tables 4A and B show the library design of Z17681matlib and
Z17682matlib, respectively. The percentages of the codons used in each of the thirteen randomized library positions (1-13) are indicated. Table 4A:
Figure imgf000062_0001
Table 4B:
Figure imgf000063_0001
Oligonucleotides encoding helix 1 and 2 of Z variants containing the randomizations described above were purchased from Isogenica. The libraries were amplified by 15 cycles of PCR using Phusion DNA polymerase (Finnzymes) and the PCR products were purified using QIAquick PCR purification kit (Qiagen GmbH). The PCR products were digested with restriction enzymes XhoI and SacI (New England Biolabs) followed by purification using preparative gel electrophoresis (2 % agarose gel) and a QIAquick gel extraction kit (Qiagen). The staphylococcal display vector pSCZ1 was digested with the same restriction enzymes and purified by preparative gel electrophoresis as described above. The libraries were ligated into pSCZ1 using T4 DNA ligase (New England Biolabs) according to the supplier’s recommendations. Plasmids were purified and concentrated using phenol chloroform extraction and ethanol precipitation, and subsequently transformed into E. coli SS320 (MC1061 F’) electrocompetent cells (Lucigen). Plasmids were prepared using a Jetstar Maxi Kit (Genomed) and transformed into S. carnosus by electroporation as previously described (Löfblom J et al., (2007), supra).192 colonies from each library were sequenced by BigDye Thermo Cycle Sequencing using an ABI Prism 3700 instrument (Applied Biosystems). Results
Design and construction of affinity maturation libraries: Two new libraries were designed based on the previously selected VEGFR-2 binding variants Z17681 and Z17682 as well as on the result from the alanine scan. The libraries of DNA fragments were cloned into a staphylococcal expression vector and transformed into S. carnosus, generating a diversity of
approximately 4.1 x 107 individual (unique) clones for Z17681matlib and approximately 4 x 107 individual (unique) clones for Z17682matlib. Sequence analysis of individual library members verified a distribution of codons in accordance with the theoretical design.
Example 7
Selection of affinity-maturated VEGFR-2 binding Z variants Summary
In this Example, the selection of maturated VEGFR-2-binding Z variants from Z17681matlib and Z17682matlib using flow-cytometric cell sorting is described. Four rounds of selection were performed. Materials and methods
Flow-cytometric cell sorting of Z variant libraries: Aliquots of the libraries containing 1.2 x 109 cells (30 times the estimated library size) were inoculated to 500 ml TSB+YE (Merck) with 10 µg/ml chloramphenicol, and incubated at 150 rpm and 37 °C for 24 h. Approximately 109 cells were harvested by centrifugation (3500 x g, 6 min at 4 °C) and washed twice with PBSP, followed by re-suspension in 1.5 ml of 20 nM hVEGFR-2/Fc (R&D Systems) in PBSP and incubation for 2 h at room temperature under conditions of gentle mixing. After washing twice with 200 µl of ice-cold PBSP, cells were re-suspended in 800 µl of Alexa Fluor® 488 goat anti-human IgG (H+L) antibody (Invitrogen) (1:1000 dilution) and 150 nM Alexa Fluor® 647- HSA conjugate. Cells were incubated on ice for 45 min in the dark. Before sorting, the cells were washed three times in 200 µl ice-cold PBSP and resuspended in 1.4 ml ice-cold PBSP. Four rounds of sorting of the libraries were performed using a MoFlo Astrios flow cytometer (Beckman Coulter). In sorting round 2-4, approximately 10 x the library size was labeled for sorting. Those approximately 0.5 % of the population that exhibited the highest VEGFR-2-binding signal were gated and sorted to TSB+YE (Merck). The cells were grown overnight in TSB+YE with 10 µg/ml chloramphenicol. In the first round, the libraries were incubated with 50 nM hVEGFR-2/Fc and in the second and third round with 5 nM hVEGFR-2/Fc. In the fourth round, an off- rate selection strategy was applied. First, cells were incubated with 20 nM hVEGFR-2/Fc (R&D Systems) as described above. After washes, cells were incubated with 100 nM of unlabeled VEGFR-2 (Sino Biological Inc.) at room temperature with gentle mixing for 30 min or 4 h. In parallel, the fourth sorting round was also performed without the off-rate incubation step. Next, the cells were incubated with labeled secondary antibody and HSA as described above. Colonies from the fourth sorting round were sequenced using BigDye Thermo Cycle Sequencing and an ABI Prism 3700 instrument (Applied Biosystems). Results
Selection of maturated VEGFR-2 binding Z variants using flow- cytometric cell sorting: Flow-cytometric cell sorting (FACS) was used for isolation of staphylococcal cells displaying Z variants with increased affinity for VEGFR-2. Four rounds of sorting were performed, using a starting concentration of 50 nM hVEGFR-2/Fc fusion and decreasing down to 20 nM in the third round (Figure 9). Target binding was detected using a
fluorescently labeled anti-human Fc antibody. Surface expression level was monitored using fluorescently labeled HSA as described previously (Kronqvist N et al., (2008), supra). In the fourth sorting round, an off-rate strategy was applied, by incubating for 30 min or 4.5 h with an excess of unlabeled hVEGFR-2 after incubation with hVEGFR-2/Fc. The fourth sorting round was also performed without the off-rate incubation step.
Sequencing 416 colonies from the sorted Z17681matlib identified 50 unique variants, and sequencing 301 colonies from the sorted Z17682matlib identified 18 unique variants. All but two clones from Z17682matlib contained the mutation K33N, which is a mutation in the scaffold and not included in the intended randomization. Position 11, 14 and 25 were the most frequently mutated in the output from Z17681matlib, in agreement with the alanine scan data. Position 11 was the most frequently mutated position in the output from Z17682matlib.
Example 8
On-cell affinity ranking of maturated VEGFR-2 binding Z variants Summary
In this Example, the affinity for VEGFR-2 of 21 maturated Z variants (denoted SEQ ID NO:5-25) from Z17681matlib and 17 maturated Z variants (denoted SEQ ID NO:26-42) from Z17682matlib was analyzed by flow- cytometry. Each individual clone was given a unique Z##### denotation. Materials and methods
On-cell ranking of maturated Z variants: Individual clones from the fourth sorting round occurring more than twice among the sequenced colonies from Z17681matlib and all unique variants from Z17862matlib were inoculated in 3 ml TSB+YE (Merck) and incubated overnight at 150 rpm and 37 °C. Cells were washed with 800 µl PBSP and incubated with 20 nM hVEGFR-2/Fc (R&D Systems) for 1.5 h. After washing with ice-cold PBSP, cells were incubated at room temperature with 50 nM unlabeled VEGFR-2 (Sino Biological) for 30 min or 4 h under conditions of gentle mixing. Next, the cells were incubated in PBSP with Alexa Fluor® 488 goat anti-human IgG (H+L) antibody (Invitrogen) (1:1000 dilution) and 150 nM Alexa Fluor® 647- HSA conjugate for 45 min on ice in the dark. The cells were washed once with ice-cold PBSP and then analyzed using a Gallios flow cytometer (Beckman Coulter). Cells displaying binders Z17681 and Z17682 were included for comparison and cells displaying the TNFα-binding Z variant Z00185 (SEQ ID NO:48) were included as a negative control.
The ten top ranked clones from Z17681matlib and the eight top ranked clones from Z17682matlib were also analyzed for binding to mVEGFR-2 by labeling with mVEGFR-2/Fc (R&D Systems) and using flow cytometry as described above. Results
On-cell affinity ranking of maturated VEGFR-2-binding Z variants: The affinities of individual clones were compared by flow-cytometric analysis of recombinant staphylococci.21 Z variants (denoted SEQ ID NO:5-25) from Z17681matlib which occurred more than twice among the sequences, and the 17 unique Z variants (denoted SEQ ID NO:26-42) from Z17682matlib were included in the assay (Figures 10A and 10B). In order to obtain a ranking influenced by the off-rates, the samples were subjected to 30 min or 4.5 h incubations with unlabeled VEGFR-2 after incubation with hVEGFR-2/Fc. The target binding signal was normalized against the expression level as described above. All analyzed clones showed a slower dissociation compared to the primary binders Z17681 and Z17682. The top nine binders from
Z17681matlib (Z17687 (SEQ ID NO:9), Z17688 (SEQ ID NO:10), Z17689 (SEQ ID NO:11), Z17690 (SEQ ID NO:12), Z17691 (SEQ ID NO:13), Z17694 (SEQ ID NO:16), Z17698 (SEQ ID NO:20), Z17699 (SEQ ID NO:21) and Z17701 (SEQ ID NO:23)) as well as the top eight ranked clones from Z17682matlib (Z17705 (SEQ ID NO:27), Z17706 (SEQ ID NO:28), Z17712 (SEQ ID NO:34), Z17713 (SEQ ID NO:35), Z17714 (SEQ ID NO:36), Z17716 (SEQ ID NO:38), Z17717 (SEQ ID NO:39) and Z17720 (SEQ ID NO:42)) were also analyzed using mVEGFR-2. These binders exhibited retained cross reactivity to mVEGFR-2 (Figures 11A and 11B).
Four candidates from each library (Z17690, Z17691, Z17698 and Z17701 (corresponding to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, respectively) from Z017681matlib and Z17712, Z17717, Z17719 and Z17720 (corresponding to SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, respectively) from Z17682matlib) were chosen for further characterization. The four Z variants from Z017681matlib were chosen because they showed the highest signals after 30 min and 4.5 h of off-rate incubation. From Z17682matlib, three (Z17712, Z17717 and Z17720) of the five top performing variants were selected along with the best performing (Z17719) of the variants that did not contain the K33N mutation. Next, these eight Z variants were subcloned and produced as His-tagged soluble proteins for further characterization experiments. Example 9
Characterization of maturated VEGFR-2 binding polypeptides Summary
In this Example, the eight selected maturated Z variants from Example 8 were further characterized using CD analysis and surface plasmon resonance analysis. Material and methods
Sub-cloning, expression and purification of second-generation binders: Z17690, Z17691, Z17698 and Z17701 (corresponding to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, respectively) from
Z017681matlib and Z17712, Z17717, Z17719 and Z17720 (corresponding to SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, respectively) from Z017682matlib were sub-cloned to pET26-b(+) and transformed into BL21(DE3) cells (Merck). Soluble proteins were produced and purified as described in Example 2 for binders Z17681 and Z17682.
Circular dichroism analysis of maturated Z variants: Circular dichroism analysis was performed at 195-205 nm and 20 °C using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) in a cell with an optical path- length of 1 mm. Variable temperature experiments were performed by measuring the ellipticity while heating the Z variants from 20 °C to 90 °C. A concentration of 0.2 mg/ml Z variant was used.
Analysis of binding kinetics of maturated Z variants using surface plasmon resonance: Human VEGFR-2/Fc was immobilized on a CM-5 sensor chip (GE Healthcare). Binding kinetics were analyzed by flowing different concentrations of Z variants (20 nM, 10 nM and 5 nM) over immobilized human VEGFR-2/Fc using a BIAcoreTM 3000 instrument (GE Healthcare). The affinity constant of Z17719 for mVEGFR-2 was determined by injection of 20 nM, 10 nM and 5 nM Z17719 over immobilized mVEGFR-2/Fc (R&D Systems) on a CM-5 sensor chip (GE Healthcare). For determination of the affinity constants of Z17701, Z17698 and Z17690 for mVEGFR-2, the Z variants were immobilized on a GLC sensor chip (Biorad Laboratories) using a ProteOn XPR36 instrument (Biorad Laboratories).5 nM, 10 nM or 20 nM mVEGFR-2 (Sino Biological) was passed over the surfaces. PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration. Results
Circular dichroism: Secondary structure content of the four selected candidates from each library was analyzed using CD spectroscopy and the melting temperature was determined by heating the samples from 20 °C to 90 °C (Figure 12). Refolding after heating to 90 °C was evaluated after allowing the samples to cool to 20 °C. All clones showed an alpha-helical content, although Z17691 (SEQ ID NO:13) had a lower degree of alpha- helical content than the other clones. All clones demonstrated similar secondary structure content after heating, indicating complete refolding (Figure 12). The melting temperatures of the clones obtained from
Z17681matlib were around 45°C. Notably, the three clones from the
Z17682matlib containing the K33N mutation (Z17720, Z17717 and Z17712) had a lower melting temperature than both the primary binder Z17682 and the second generation binder Z17719, which both lack the K33N mutation (Figure 13 and Table 6). The melting temperatures for these clones were around 37 °C, while the melting temperature for Z17719 was 45 °C. Table 6: Melting temperatures of maturated VEGFR-2-binding Z variants.
Figure imgf000069_0001
Binding kinetics analysis of second-generation binders: Binding kinetics of Z17701, Z17698, Z17690 and Z17719 were analyzed using surface plasmon resonance (Figure 14 and 15). Different concentrations (5 nM, 10 nM and 20 nM) of each Z variant were injected over immobilized human VEGFR-2/Fc. The equilibrium dissociation constant for Z17719 for mVEGFR-2 was determined by injection of Z17719 over immobilized mVEGFR-2/Fc. Z17701, Z17698 and Z17690 were immobilized on a sensor chip and 5 nM, 10 nM or 20 nM mVEGFR-2 (not fused to Fc in order to avoid avidity effects) was passed over the surfaces. The equilibrium dissociation constant (KD) was determined from the association and dissociation rates calculated from sensorgrams using a monovalent binding equation and non- linear regression. Equilibrium dissociation constants for human VEGFR-2 were determined to be 5.0 nM-10.9 nM for the four candidates (Table 7A), representing about a 30-fold and an 8-fold increase in affinity compared to the original binders Z17681 and Z17682, respectively. The affinities for mVEGFR- 2 were determined to be 7.8 nM-11.9 nM, representing around a 15-fold increase in affinity compared to primary binders (Table 7B). Table 7A: Affinit constants of maturated Z variants for human VEGFR-2.
Figure imgf000070_0001
Example 10
Further characterization of maturated VEGFR-2 binding polypeptides Summary
In this Example, maturated Z variants were analyzed using SPR to determine if they could simultaneously bind to VEGFR-2. Material and methods
Surface plasmon resonance based competition assay: Competition assays were performed using a BIAcoreTM 3000 instrument (GE Healthcare). Human or murine VEGFR-2/Fc (R&D Systems) was immobilized on a CM-5 sensor chip (GE Healthcare). For detection of simultaneous binding, a double injection was performed, where 1 µM Z17701 (SEQ ID NO:23) was first injected, directly followed by an injection of either a combination of 1 µM of Z17719 (SEQ ID NO:41) and Z17701, or an injection of 2 µM Z17701. In a separate experiment, the Z variants were injected in the opposite order, i.e.1 µM Z17719 was injected first, followed by a second injection of either a combination of 1 µM Z17701 and 1 µM Z17719, or of 2 µM Z17719. PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM HCl was used for regeneration.
For analysis of competition with VEGF-A, VEGF-A (R&D Systems) was immobilized on a sensor chip surface.40 nM of human or murine VEGFR- 2/Fc (R&D Systems), which had been pre-incubated for 40 min with a 25-fold molar excess of Z17701 or Z17719 (or PBS as a control), was injected over the surface. PBS buffer with 0.1 % Tween (PBST) was used as a running buffer and 10 mM NaOH was used for regeneration. Results
SPR based competition assays: Surface plasmon resonance
experiments had indicated that the primary binders Z17681 and Z17682 could simultaneously bind to VEGFR-2 and the results from the ELISA assay suggested that both binders could compete with VEGF-A (see Example 4). To confirm that also the maturated binders could bind simultaneously to VEGFR- 2 and compete with VEGF-A, a competition assay was performed using surface plasmon resonance. In this assay, the maturated binders Z17701 and Z17719 were analyzed.
VEGFR-2/Fc was immobilized on a sensor chip and detection of simultaneous binding was performed using a double injection.1 µM of the first Z variant was injected, immediately followed by a second injection of either a combination of 1 µM of the first Z variant and 1 µM of the second Z variant, or 2 µM of the first Z variant (as control). The second injection of 2 µM of the first Z variant did not give rise to any substantial increase in binding signal, whereas the addition of 1 µM of the second Z variant resulted in a substantial increase in signal (Figure 16). To verify that the affinity-matured Z variants could compete with VEGF-A for binding to VEGFR-2, human or murine VEGF-A was immobilized on the chip and human or murine VEGFR-2/Fc that had been pre-incubated for 45 min with Z17701, Z17719 or PBS was injected over the surface. Injection of the control sample containing VEGFR-2 pre- incubated with PBS resulted in an increase in signal showing binding of VEGFR-2 to VEGF-A. Injection of the samples containing VEGFR-2 pre- incubated with said Z variants did not result in a substantial increase in signal, showing that the Z variants blocked the interaction between VEGFR-2 and VEGF-A (Figure 17).
These results demonstrate that the two Z variants can bind
simultaneously to VEGFR-2 and that both of them compete with human as well as murine VEGF-A for binding to VEGFR-2.
Example 11
Cloning, production and characterization of dimeric Z variants Summary
In this Example, dimeric maturated Z variants were produced and characterized using SPR analysis. The dimeric Z variants were also shown to be able to bind to VEGFR-2 expressed on the surface of mammalian cells. Materials and methods
Subcloning, expression and purification of dimer constructs: The genes encoding Z17701 (SEQ ID NO:23) and Z17719 (SEQ ID NO:41) were cloned into the plasmid pET26b-Z02891-ABD053-Z03638 (Malm M et al., (2014) Biotechnol J (2014), Mar 27 [Epub ahead of print]). pET26b-Z02891-ABD053- Z03638 encoded a dimeric Z variant construct comprising the HER2-binding Z variant Z02891 (SEQ ID NO:49) and the Taq polymerase-binding Z variant Z03638 (SEQ ID NO:50), separated by the albumin binding domain ABD053 (SEQ ID NO:47). These Z variant molecules were replaced by VEGFR-2- binding Z variants to generate the following four constructs: Z17701-(S4G)4-ABD053-(S4G)4-Z17719,
Z17719-(S4G)4-ABD053-(S4G)4-Z17701,
Z17701-(S4G)4-ABD053-(S4G)4-Z17701, and
Z17719-(S4G)4-ABD053-(S4G)4-Z17719. The genes encoding the first Z variants were amplified using Phusion DNA polymerase (Finnzymes). PCR products and vector were digested with NdeI and AscI (New England Biolabs), ligated using T4 DNA ligase (New England Biolabs) and transformed into E. coli RR1ΔM15 cells (Rüther U (1982), supra). Plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen). Z03638 was replaced by the second respective VEGFR-2-binding Z variant using restriction enzymes SacII and XhoI essentially as described above. Next, plasmids were prepared using a Qiaprep plasmid preparation kit (Qiagen) and transformed into E. coli BL21 (DE3) cells (Merck) using heat shock. Colonies were inoculated to TSB medium (30 g/l; Merck),
supplemented with 5 g/l yeast extract (Merck) and 50 μg/ml kanamycin (Sigma-Aldrich) and cultivated over night at 37 °C and 150 rpm. After 16 h, the cultures were re-inoculated to an OD600 of 0.05-0.1 in TSB medium (30 g/l; Merck). When an OD600 of 0.5-1 was reached, protein expression was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) (Apollo Scientific Ltd) and cultures were incubated for 20 h at 25 °C and 150 rpm. Cells were harvested by centrifugation (2400 g, 8 min at 4 °C) and Z variants were purified using affinity chromatography with HSA immobilized on a sepharose matrix. The purity and size of the purified proteins were analyzed by SDS- PAGE and MALDI-time of flight (TOF) mass spectrometry using a LaserToF LT3 Plus instrument (Scientific Analysis Instruments).
SPR analysis of dimeric constructs: The affinity of the four dimeric constructs for human and murine VEGFR-2 was analyzed by SPR using a ProteON XPR36 instrument. HSA was immobilized on the surface and 200 nM of each dimer construct was passed over the immobilized HSA.40 nM human or murine VEGFR-2 was thereafter passed over the surface. The dissociation constants of the homodimeric and heterodimeric constructs were compared by injections of 15 nM, 30 nM and 60 nM of human or murine VEGFR-2 following an injection of 200 nM of each of the homodimeric constructs; or by injections of 5 nM, 15 nM and 45 nM human or murine VEGFR-2 following an injection of 200 nM of the heterodimeric constructs. The dissociation constants were calculated from sensorgrams using a monovalent binding equation and non-linear regression.
Cell-binding analysis: 293/KDR cells (Sibtech Inc.) and HEK293 cells (ATCC) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM;
Sigma-Aldrich) supplemented with 10 % FSB, and in the case of 293/KDR cells also with 2 mM L-glutamine. Cells grown on petri dishes were washed with PBS and harvested by pipetting. Next, the cells were incubated for 20 min on ice with 500 nM of each of the four dimer constructs or the negative control construct Z03638-(S4G)4-ABD053-(S4G)4-Z03638 in PBS with 1 % BSA (PBSB). The cells were pelleted by centrifugation at 300 rpm for 4 min, and re-suspended in 150 nM Alexa Fluor 647-HSA conjugate. After incubation for 20 min on ice in the dark, cells were pelleted by centrifugation and re-suspended in PBSB. Cell binding of the dimeric Z variants was detected by flow-cytometric analysis using a Gallios flow cytometer (Beckman Coulter). Results
Production of dimeric VEGFR 2 binding Z variants:
The results from the SPR competition assay, described in Example 10, demonstrated that Z17701 and Z17719 could bind simultaneously to VEGFR- 2. To investigate whether heterodimers would result in higher affinity due to avidity effects, heterodimers based on Z17701 and Z17719 were constructed. Schematically, the following constructs were designed: Z17701-ABD-Z17719, Z17719-ABD-Z17701, Z17701-ABD-Z17701 and Z17719-ABD-Z17719. The homodimeric constructs Z17701-ABD-Z17701 and Z17719-ABD-Z17719 were included as controls. The four constructs were expressed in E. coli and purified by affinity chromatography using HSA-sepharose. SDS-PAGE and MALDI-time of flight (TOF) mass spectrometry confirmed that the proteins were pure and of the correct size.
SPR analysis of dimeric Z variants: The binding of the four dimeric constructs to human and murine VEGFR-2 was analyzed by SPR. HSA was immobilized on the chip and the dimer constructs were injected over respective surface, resulting in a directed immobilization. Human or murine VEGFR-2 was thereafter injected over the surfaces. The results
demonstrated that the heterodimeric constructs had a faster association as well as a slower off-rate compared to the homodimeric and the monovalent controls. The slower dissociation rates for the heterodimeric constructs verified that Z17701 and Z17719 could bind simultaneously to VEGFR-2 (Figure 18, Table 8). Table 8A: Dissociation rate constants for dimeric constructs against human VEGFR-2.
Figure imgf000074_0001
Table 8B: Dissociation rate constants for dimeric constructs against murine VEGFR-2.
Figure imgf000075_0001
Cell-binding analysis: To verify that dimeric Z variants could bind to VEGFR-2 expressed on the surface of mammalian cells, a flow-cytometry based analysis was performed.293/KDR cells (Sibtech Inc.), which are derivatives of HEK293 human embryonic kidney cells engineered to express VEGFR-2, were incubated with the four dimeric constructs (Z17701-(S4G)4- ABD053-(S4G)4-Z17719, Z17719-(S4G)4-ABD053-(S4G)4-Z17701, Z17701- (S4G)4-ABD053-(S4G)4-Z17701 and Z17719-(S4G)4-ABD053-(S4G)4-Z17719). Cell binding of the dimeric Z variants was detected using fluorescently labeled HSA. The dimeric Z variant construct Z03638-(S4G)4-ABD053-(S4G)4-Z03638, which binds to Taq polymerase, was included as a negative control. In order to verify that the observed binding was specific for VEGFR-2, HEK293 cells with no expression of VEGFR-2 were incubated with the same dimeric Z variants.
A shift in fluorescent signal was observed for the 293/KDR cells incubated with all four of the dimeric VEGFR-2 specific Z variants compared to the negative control and to the HEK293 cells without VEGFR-2 expression, indicating specific binding to VEGFR-2 expressed on the surface of mammalian cells (Figure 19). Incubation with heterodimeric constructs resulted in higher fluorescence signal intensity compared to the monovalent and homodimeric constructs indicating that the heterodimeric constructs had higher affinity for VEGFR-2 expressed on cells. The heterodimeric constructs show a higher shift in fluorescence intensity than the homodimeric constructs upon binding to VEGFR-2-expressing cells, which may be due to the presence of two VEGFR-2-binding paratopes. Example 12
Production and characterization of dimeric Z variants with different linker lengths Summary
In this Example, dimeric maturated Z variants with different linker lengths were produced and characterized in terms of binding to VEGFR-2, blocking of VEGF-A binding to VEGFR-2 expressed on the surface of mammalian cells, as well as inhibition of VEGFR-2 mediated phosphorylation and angiogenesis. Materials and methods
Subcloning, expression and purification of dimer constructs with different linker lengths: The genes encoding Z17701 (SEQ ID NO:23) and Z17719 (SEQ ID NO:41) were fused together with ABD053 in the following four constructs, denoted Link1, Link2, Link3 and Link4: Link1: Z17701-(S4G)-ABD053-(S4G)-Z17719,
Link2: Z17701-(S4G)-Z17719-(S4G)3-ABD053,
Link3: Z17701-(S4G)3-Z17719-(S4G)3-ABD053, and
Link4: Z17701-(S4G)8-Z17719-(S4G)3-ABD053. The genes encoding these constructs in the vector pJ411 were ordered from DNA2.0. Colonies were inoculated to TSB medium (30 g/l; Merck), supplemented with 5 g/l yeast extract (Merck) and 50 μg/ml kanamycin (Sigma-Aldrich) and cultivated over night at 37 °C and 150 rpm. After 16 h, the cultures were re-inoculated to an OD600 of 0.05-0.1 in TSB medium (30 g/l; Merck). When an OD600 of 0.5-1 was reached, protein expression was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) (Apollo Scientific Ltd) and cultures were incubated for 20 h at 25 °C and 150 rpm. Cells were harvested by centrifugation (2400 g, 8 min at 4°C) and Z variants were purified using affinity chromatography with HSA immobilized on a sepharose matrix. The purity and size of the purified proteins were analyzed by SDS- PAGE.
Cell-binding analysis: 293/KDR cells (Sibtech Inc.) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10 % FBS and 2 mM L-glutamine. Cells grown on petri dishes were washed with PBS and harvested by pipetting. HUVECs (Lonza) were cultivated in Endothelial Growth Medium-2 (EGM-2) (Lonza) supplemented with EGM-2 Bullet Kit (Lonza). Cells grown on petri dishes were washed with PBS, harvested by trypsination, centrifuged at 220 rcf for 5 min and pellets were re-suspended in PBS with 1 % BSA (PBSB).
Next, the cells were incubated for 20 min on ice with 200 nM of each of the four dimer constructs, the negative control construct Z03638-(S4G)4- ABD053-(S4G)4-Z03638, or the positive control mouse IgG anti-human VEGFR-2 antibody (R&D Systems), in PBSB. The cells were pelleted by centrifugation at 300 rpm for 4 min, and re-suspended in 150 nM Alexa Fluor® 647-HSA conjugate (or 1 μg/ml Alexa Fluor® 488 Rabbit Anti-Mouse IgG (H+L) antibody (Invitrogen). After incubation for 20 min on ice in the dark, cells were pelleted by centrifugation and re-suspended in PBSB. Cell binding of the dimeric Z variants was detected by flow-cytometric analysis using a Gallios flow cytometer (Beckman Coulter).
IC50 evaluation for VEGF-A blocking: 293/KDR cells (Sibtech Inc.) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10 % FBS and 2 mM L-glutamine. Cells grown on petri dishes were washed with PBS and harvested by pipetting.
Next, the cells were incubated for 15 min on ice with 1 nM, 4 nM, 16 nM, 64 nM, 256 nM or 1024 nM of each of the four dimeric Z constructs, or 4 nM, 64 nM or 1024 nM of the negative control construct Z03638-(S4G)4- ABD053-(S4G)4-Z03638, or no Z variant, in PBSB. The cells were pelleted by centrifugation at 300 rpm for 4 min, re-suspended in 50 nM biotinylated VEGF-A (Acro Biosystems), and incubated on ice for 15 min. Thereafter, cells were again pelleted by centrifugation at 300 rpm for 4 min, and re-suspended in 1 μg/ml streptavidin-R-phycoerythrin conjugate (SAPE, Invitrogen). After incubation for 15 min on ice in the dark, the cells were pelleted by
centrifugation and re-suspended in PBSB. Cell binding of the biotinylated VEGF-A was detected by flow-cytometric analysis using a Gallios flow cytometer (Beckman Coulter). Calculation of IC50 values was performed by interpolation of sigmoidal curves using the GraphPad Prism software
(GraphPad Software, Inc.).
Inhibition of VEGF-A induced VEGFR-2 phosphorylation: 293/KDR cells (Sibtech Inc.) were seeded in Dulbecco’s Modified Eagle Medium
(DMEM; Sigma-Aldrich) supplemented with 10 % FSB and 2 mM L-glutamine. 10 h after seeding, cells were shifted to starvation media (DMEM supplemented with 0.5 % FBS), and incubated overnight. Next, 200 nM or 20 nM of each of the four dimeric Z constructs, the negative control construct Z03638-(S4G)4-ABD053-(S4G)4-Z03638, or no Z variant, was added to the culture dishes. After incubation for 1 h at 37 °C, 1 nM human VEGF-A (R&D systems), or PBS, was added, and cells were incubated for 10 min at 37 °C. Thereafter, cells were washed with PBS containing 1 mM sodium
orthovanadate (Sigma) and cell lysates were prepared by sonication for 1 min in Cell Lysis Buffer (Cell Signaling Technology), supplemented with phenylmethylsulfonyl fluoride (PMSF, Sigma) and PhosStop (Roche), and analyzed by ELISA using a PathScan® Phospho-VEGFR-2-Tyr1175
Sandwich ELISA kit (Cell Signaling Technology).
Inhibition of VEGF-A induced ERK1/ERK2 phosphorylation: The dimeric Z variant Link2 was further evaluated in a downstream ERK1/ERK2 phosphorylation assay.293/KDR cells were cultivated and treated essentially as described for VEGFR-2 phosphorylation above, with the exceptions that RIPA Lysis and Extraction Buffer (Cell Signaling Technologies),
supplemented with 1 µg/ml Pepstatin (Sigma Aldrich), 1 mM sodium orthovanadate, complete protease inhibitor cocktail (Roche; 1 tablet per 50 ml) and 1 mM PMSF were used and that the cell lysates were analyzed with an ELISA based Phospho-ERK1/ERK2 DuoSet IC kit (R&D Systems; used according to the manufacturer’s instructions). Cells were incubated with 200 nM Link2 prior to addition of 10 nM VEGF-A. Untreated cells were used as a negative control and cells treated with VEGF-A only were used as a positive control.
SPR analysis: SPR was used to analyze the binding of Link1-Link4 to VEGFR-2 using a ProteOn XPR36 system. HSA was immobilized on a GLM sensor chip (Bio-Rad Laboratories) at 700 RU and 2300 RU. Each dimeric construct was injected at a concentration of 100 nM over the HSA
immobilized surfaces, followed by immediate injection of 5, 10 and 20 nM monomeric hVEGFR-2 (Sino Biologics) or 1, 5, 10 and 20 nM mVEGFR-2 (Sino Biologics). Calculation of dissociation constants was done using non- linear regression and a monovalent binding equation.
In vitro HUVEC sprouting assay: PKH67 Green Fluorescent Cell linker kit (Sigma-Aldrich) was used to label HUVECs according to the
manufacturer’s instructions. Cells were then coated onto Cytodex 3 beads (Sigma-Aldrich) by 24 h incubation at 37 °C. Cell-coated beads were embedded in an EBM-2 (Lonza) fibrinogen gel (2.5 mg/ml fibrinogen) supplemented with 2 % FBS and 50 mg/ml aprotinin (Sigma-Aldrich). The fibrinogen solution was clotted with 1 U thrombin for 30 min at 37 °C.
Approximately 25000 WI38 cells in EBM-2 supplemented with 2 % FBS and 50 ng/ml VEGF-A were plated in each well and incubated for 24 h at 37 °C. After incubation, the wells were treated with 50 nM or 10 nM Link2, 50 nM or 10 nM of the VEGFR-2 binding monoclonal antibody ramucirumab (ImClone Systems) or 50 nM of the negative control variant Z03638-(S4G)4-ABD053- (S4G)4-Z03638. A positive control with only VEGF-A added and a negative control without any VEGF-A added were also included. The number of sprouts and average sprout length were imaged 3-4 days later using a Zeiss700 confocal microscope and analysis was done using Image J.
VEGF-A induced 293/KDR proliferation assay: Approximately 1000 293/KDR cells (cultivated in DMEM supplemented with 10 % FBS and 2 mM L-glutamine) per well were seeded in different 96 well plates and incubated for approximately 20 h. After 7 h, 10 μl of CCK8 agent (from cell counting kit- 8; Sigma Aldrich) was added to one of the plates, which was analyzed to confirm that cells were distributed evenly. After 20 h, medium was removed and 200 μl fresh medium was added. Cells were then treated with 100 nM Link2 + 10 nM VEGF-A, 10 nM VEGF-A only (positive control), or PBS
(negative control). One of the plates was incubated for 72 h and the other for 96 h at 37 °C.10 μl CCK 8 was added to each well, followed by incubation for 1.5 h. Analysis of the plates was performed by measuring the absorbance at 450 nm, and background absorbance was measured at 570 nm. Results
Production of dimeric VEGFR-2 binding Z variants with different linker lengths: The results from the cell binding and SPR analysis, shown in
Example 11, demonstrated that the heterodimeric constructs Z17701- ABD053-Z17719 and Z17719-ABD053-Z17701 could bind to VEGFR-2 with a higher apparent affinity than the homodimeric constructs, as a result of simultaneous binding of Z17701 and Z17719. To investigate whether different linker lengths would result in higher or lower affinity due to steric effects, new heterodimeric constructs based on Z17701 and Z17719 were constructed. The genes encoding Z17701 (SEQ ID NO:23) and Z17719 (SEQ ID NO:41) were fused together with ABD053 in the four constructs denoted Link1, Link2, Link3 and Link4. The four constructs were expressed in E. coli and purified by affinity chromatography using HSA-Sepharose. SDS-PAGE confirmed that the proteins were pure and of the correct size.
Cell-binding analysis: To verify that dimeric Z variants with different linker lengths could bind to VEGFR-2 expressed on the surface of
mammalian cells, a flow-cytometry based analysis was performed.293/KDR cells (Sibtech Inc.) were incubated with the four dimeric constructs Link 1-4. The dimeric Z variant construct Z03638-(S4G)4-ABD053-(S4G)4-Z03638, which binds to Taq polymerase, was included as a negative control and a mouse IgG anti-human VEGFR-2 antibody (R&D Systems) was included as a positive control. Cell binding of the dimeric Z variants was detected using fluorescently labeled HSA (or Alexa Fluor® 488 Rabbit Anti-Mouse IgG Antibody for the positive control).
A shift in fluorescent signal was observed for the 293/KDR cells incubated with all four of the dimeric VEGFR-2 specific Z variants compared to the negative control, indicating specific binding to VEGFR-2 expressed on the surface of both HUVEC and 293/KDR mammalian cells (Figure 20).
IC50 evaluation for VEGF-A blocking: To verify that dimeric Z variants with different linker lengths could block VEGF-A binding to VEGFR-2 expressed on the surface of mammalian cells, a flow-cytometry based analysis was performed.293/KDR cells (Sibtech Inc.) were incubated with concentrations varying from 1 nM to 1024 nM of the four dimeric constructs Link1-4. The dimeric Z variant construct Z03638-(S4G)4-ABD053-(S4G)4- Z03638, which binds to Taq polymerase, was included as a negative control. After incubation with dimeric Z constructs, cells were incubated with biotinylated VEGF-A (Acro Biosystems). Cell binding of the biotinylated VEGF-A was detected using streptavidin-R-phycoerythrin conjugate (SAPE, Invitrogen) (or Alexa Fluor® 488 Rabbit Anti-Mouse IgG Antibody for the positive control).
A concentration-dependent shift in fluorescent signal was observed for the 293/KDR cells incubated with all four of the dimeric VEGFR-2 specific Z variants compared to the negative control, indicating blocking of VEGF-A binding to VEGFR-2 expressed on the surface of 293/KDR mammalian cells (Figure 21). The IC50 values obtained from interpolation of sigmoidal curves using the GraphPad Prism software (GraphPad Software, Inc.) were: 35 nM for Link1, 36 nM for Link2, 60 nM for Link3, and 68 nM for Link4. The relatively small differences in IC50 values demonstrate that all constructs can block VEGF-A binding to VEGFR-2 equally well, and that a linker length as short as 5 amino acids can be used without the introduction of steric constraint, which may be useful for future in vivo imaging applications, where a small protein size is desirable.
Inhibition of VEGF-A induced VEGFR-2 phosphorylation: To verify that dimeric Z variants with different linker lengths could inhibit VEGF-A induced phosphorylation of VEGFR-2 expressed on the surface of mammalian cells, a phosphorylation assay was performed.293/KDR cells (Sibtech Inc.) were incubated with 20 nM or 200 nM of the four dimeric constructs Link1-4. The dimeric Z variant construct Z03638-(S4G)4-ABD053-(S4G)4-Z03638, which binds to Taq polymerase, was included as a negative control. After incubation with dimeric Z constructs, VEGFR-2 phosphorylation was stimulated by addition of human VEGF-A to the cells. After VEGF-A incubation, further phosphorylation was prevented by the presence of PhosStop (Roche), a phosphatase inhibitor cocktail, in the cell lysis buffer. De-phosphorylation was prevented by the presence of Na orthovanadate and protein degradation was prevented by the presence of phenylmethylsulfonyl fluoride (PMSF). Cell lysates were prepared and analyzed by ELISA using a PathScan® Phospho- VEGFR-2-Tyr1175 Sandwich ELISA kit (Cell Signaling Technology), which detects phosphorylation of Tyr1175 on VEGFR-2 using a Tyr1175
phosphorylation-specific antibody and a secondary HRP-conjugated antibody.
A decrease in ELISA signal was observed for all four dimeric Z variants, demonstrating that all variants could inhibit VEGFR-2
phosphorylation (Figure 22). These results suggest that the dimeric Z variants can inhibit VEGFR-2 mediated signaling.
Inhibition of VEGF-A induced ERK1/ERK2 phosphorylation: VEGFR-2 downstream activation is an important part of signaling for angiogenesis progression (Gourlaouen et al. (2013), J Biol Chem 288:7467-7480).
Therefore, the ability of a dimeric Z variant to inhibit VEGF-A induced downstream ERK1/ERK2 phosphorylation was investigated. A decrease in the ERK1/ERK2 phosphorylation was observed for 293/KDR cells treated with the dimeric Z variant denoted Link2 prior to induction with VEGF-A, compared to cells treated with VEGF-A only (Figure 23), which confirmed that VEGFR-2 binding Z variants can inhibit downstream signaling.
SPR analysis: SPR was used to determine the kinetics of binding between dimeric Z variants with different linker lengths and human or murine VEGFR-2. HSA was immobilized on a chip surface followed by injection of the dimeric Z variants and then of monomeric hVEGFR-2 or mVEGFR-2. This particular setup both allowed determination of dissociation constants for the different constructs (Table 9A and 9B) and confirmed their ability to bind to VEGFR-2 while interacting with HSA. The dimeric Z variant with the shortest linker, Link 2, showed the slowest dissociation rate. The KD values for the interaction of Link2 with hVEGFR-2 and mVEGFR-2 were determined to be 313 ± 30 pM and 292 ± 11 pM, respectively. Table 9A: Dissociation rate constants a ainst human VEGFR-2.
Figure imgf000082_0001
Table 9B: Dissociation rate constants a ainst murine VEGFR-2.
Figure imgf000082_0002
In vitro HUVEC sprouting assay: In this assay, the anti-angiogenic effect of a dimeric VEGFR-2 binding Z variant was investigated by analysis of inhibition of HUVEC sprout formation. An ample decrease in both length and number of sprouts were shown for cells treated with Link2 or with the VEGFR- 2 binding monoclonal antibody ramucirumab, included for comparison, compared to cells incubated with negative control dimeric Z variant or untreated cells (Figure 24).
VEGF-A induced 293/KDR proliferation assay: The anti-angiogenic effect of Link2 was further investigated in a proliferation assay using 293/KDR cells. Cells treated with VEGF-A exhibit a significant increase in growth rate compared to untreated cells. The results from cells treated with both VEGF-A and Link2 show that the dimeric Z variant significantly inhibits the increased growth rate induced by VEGF-A (Figure 25). Example 13
Production and characterization of conjugated polypeptides for in vivo studies Summary
This Example describes the production and characterization of
VEGFR-2 binding polypeptides and controls to be used for in vivo studies, such as for imaging or therapeutic studies. A C-terminal cysteine residue was incorporated to enable site-specific labeling. An N-terminal (HE)3 sequence was added to non-ABD-fused polypeptides in order to simplify purification. The polypeptides were conjugated and analyzed in terms of stability and binding to VEGFR-2. Materials and methods
Production of polypeptides: pET26b+ plasmids encoding the following polypeptides were ordered from DNA 2.0 Inc: (HE)3-Z03638-Cys
(HE)3-Z03638-(S4G)-Z03638-Cys
(HE)3-Z17701-Cys
(HE)3- Z17701-(S4G)-Z17719-Cys
Link2-Cys
Z03638-(S4G)-Z03638-(S4G)-ABD53-Cys The plasmids were heat shock transformed to E. coli BL21 Star cells.
Transformed colonies were inoculated to 5 ml TSB+Y supplemented with 30 µg/ml kanamycin and cultured overnight at 150 rpm 37 °C. After 17 h, 900 µl overnight-culture was re-inoculated to 600 ml TSB+Y supplemented with 30 µg/ml kanamycin and incubated at 37 °C until an OD600 of 0.8-1 was reached. Protein expression was then induced by adding IPTG and cultures were incubated at 25 °C 150 rpm for 20 h. Cells were harvested by 8 min centrifugation at 4000 rpm, 4 °C (JLA-16250). ABD-fused polypeptides were affinity purified using an anti-ABD agarose resin and (HE)3 tagged proteins were purified using a Ni +
2 Sepharose fast flow column. The purity of the samples was analyzed by SDS-PAGE.
NODAGA conjugation: All buffers used during conjugation were supplemented with Chelex 100 to minimize occupied chelators. The polypeptides were first re-suspended in PBS with 20 mM DTT and reduced for 30 min at 40 °C. Reduced polypeptides were buffer exchanged to 20 mM NH4Ac pH 5.5 using PD-10 columns (GE Healthcare) according to the manufacturer’s instructions. The C-terminal cysteine of the respective polypeptide was conjugated by addition of 3x molar excess of maleimide- NODAGA (Chematech) and incubated at 40 °C for 1 h. Samples were freeze dried and re-suspended in [20 % acetonitrile, 80 % H2O and 0.1 % TFA] followed by RP-HPLC purification. Confirmation of correct conjugation was performed by ESI-MS.
Circular dichroism spectroscopy: The (HE)3-tagged NODAGA- conjugated polypeptides were analyzed using circular dichroism (CD) spectroscopy at 195-250 nm using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) at a concentration of 0.4 mg/ml. To determine the thermal stability of each sample, ellipticity was measured at 221 nm while heating the sample from 20 to 90 °C. After being subjected to 90 °C, the sample was left for five minutes at 20 °C and a new spectrum at 195-250 nM was recorded.
SPR binding analysis: Functionality check of two NODAGA-conjugated polypeptides was done using a Biacore 3000 SLM chip.4 µg/ml monomeric mVEGFR-2 (Sino Biological) was immobilized on a chip at 1200 RU. (HE)3- Z17701-(S4G)-Z17719-Cys-NODAGA, (HE)3-Z17701-Cys-NODAGA and Link2 were injected at concentrations of 50 nM, 100 nM and 500 nM. Link2 was used as a reference to analyze functionality of the two NODAGA- conjugated polypeptides. Results
Production of polypeptides: The six specified polypeptides were successfully produced and used for conjugation.
NODAGA conjugation: To enable radiolabeling of VEGFR-2 binding polypeptides and controls, coupling of a maleimide-NODAGA chelator was performed. Conjugated polypeptides were purified by RP-HPLC and the correct conjugation was verified by ESI-MS.
Circular dichroism spectroscopy: CD analysis was performed to determine alpha-helical content, thermal stability and refolding capacity of (HE)3-tagged NODAGA-conjugated polypeptides. All four polypeptides showed alpha helical content and refolded after denaturation by heating to 90 °C. The melting temperatures of the two VEGFR-2 binding polypeptides, i.e. (HE)3-Z17701-Cys-NODAGA and (HE)3-Z17701-(S4G)-Z17719-Cys- NODAGA, were estimated to be approximately 45 °C.
SPR binding analysis: In order to ensure that the new and NODAGA- conjugated polypeptides were functional, their VEGFR-2 binding was analyzed using a Biacore instrument. Monomeric mVEGFR-2 was
immobilized on a chip followed by injection of (HE)3-Z17701-(S4G)-Z17719- Cys-NODAGA and (HE)3-Z17701-Cys-NODAGA as well as unconjugated Link2 analyzed as a control. The results confirmed that the two newly produced and conjugated polypeptides were able to bind to VEGFR-2. Figure 26 shows the binding responses for (HE)3-Z17701-(S4G)-Z17719-Cys- NODAGA and Link2. As expected, the higher mass of Link2 results in a higher response than for (HE)3-Z17701-(S4G)-Z17719-Cys-NODAGA.
Example 14
In vivo evaluation of radiolabeled VEGFR-2 binding polypeptides for imaging Summary
This Example describes radiolabeling and in vivo imaging studies using VEGFR-2 binding polypeptides and controls administered to tumor bearing mice. For a rapid distribution of the imaging agent, as well as a rapid clearance of the non-bound fraction, the polypeptides are not fused to ABD. Materials and methods
Radiolabeling of polypeptides: Chelator-conjugated VEGFR-2 binding polypeptides and controls such as the non-ABD fused polypeptides described and purified in Example 13 ((HE)3-Z17701-Cys-NODAGA, (HE)3-Z17701- (S4G)-Z17719-Cys-NODAGA, (HE)3-Z03638-Cys-NODAGA, (HE)3-Z03638- (S4G)-Z03638-Cys-NODAGA) are labelled with a suitable radiotracer. For example, labeling may be performed with generator-produced 68Ga in an NaAc buffered solution of approximately pH 4 and incubation for 15-60 min at 95 °C. Purification is carried out using size-exclusion NAP-5 columns (GE Healthcare). A small fraction is analyzed by instant thin-layer chromatography (iTLC; Biodex Medical Systems) to determine the radiochemical purity. In this system, free radiotracer migrates with the solvent front, while bioconjugates remain at the origin. Animal model: The MMTV-PyMT transgenic mouse model of metastatic breast cancer, in which the expression of the polyoma middle T antigen (PyMT) oncoprotein is controlled by the mouse mammary tumor virus (MMTV), may be used to assess the ability of the disclosed VEGFR-2 binding polypeptides to detect levels of VEGFR-2 in vivo. In the FVB (Friend Virus B) genetic background, invasive mammary tumors and subsequent pulmonary metastases develop over 12-15 weeks. The model demonstrates gradual progression and significant stromal infiltration. It has been well characterized and shown to have a good translational potential (Lin et al., (2003), Am J Pathol, 163:2113–2126). Outgrowth of micrometastases in the MMTV-PyMT model has been shown to be dependent on endothelial progenitor cell infiltration followed by a distinct growth acceleration which correlated with an increased vascularization (Gao et al., (2008), Science, 319:195–198).
In vivo imaging: Tumor bearing MMTV-PyMT mice at different stages of disease progression (11-15 weeks) and wild-type control mice are injected with 0.5-5 µg radiolabeled VEGFR-2 binding polypeptides and control polypeptides. In vivo imaging is performed using a PET or SPECT scanner (depending on the radiotracer used for labeling of the polypeptides). For example, recordings of 68Ga-labeled polypeptides may be performed with a micro PET Focus 120 scanner (CTI Concorde Microsystems). The mice are placed with the whole body in the field of view and data is collected
continuously over 60 min, starting at the time of injection. Data is then processed, images are reconstructed and the uptake of the polypeptides in tumor and other organs are quantified and compared by means of
standardized uptake values (SUVs).
Ex vivo phosphor imaging and immunofluorescence: Tumor tissue and organs with high levels of radioactive uptake are excised from the MMTV- PyMT mice and cut into sections for phosphor imaging and
immunofluorescence staining, in order to assess the correlation of the uptake of radiolabeled VEGFR-2 binding polypeptides with elevated VEGFR-2 expression. Tissues are snap frozen and sectioned using a cryomicrotome and fixed on microscope slides. For phosphor imaging, tissue sections are exposed for 60 min and subsequently read using a biomolecular imaging laser scanner, for example a Typhoon 7000 FLA equipped with imaging quantification software (GE Healthcare). For immunofluorescence analysis, the same cryosections are subjected to sequential multiplex staining procedures according to established protocols (Mulder et al., (2009), Mol Cell Proteomics, 8:1612-1622) including the use of an anti-VEGFR-2 antibody (e.g. rabbit mAb from Cell Signaling Technology, cat. no.55B11), other markers for angiogenesis and/or tumor specific antibodies (such as anti- PyMT, Abcam, cat. no. ab15085) as well as a combination of fluorescence conjugated secondary antibodies and the tyramide signal amplification (TSA) method. Immunostained sections are analyzed in a microscope and processed images are overlaid with the phosphor images. Results
The imaging evaluation is expected to show that the VEGFR-2 binding polypeptides, but not the control polypeptides, localize to VEGFR-2 expressing cells in vivo including VEGFR-2 expressing tumor tissue. The expected result would support the use of VEGFR-2 binding polypeptides as a diagnostic tool for visualization of angiogenesis in primary tumors and during metastasis development.
Example 15
In vivo evaluation of radiolabeled VEGFR-2 binding polypeptides for therapeutic applications Summary
This Example describes the use of VEGFR-2 binding polypeptides for therapeutic applications such as tumor therapy, where inhibition of new blood vessel formation may suppress tumor growth, and ophthalmology, where inhibition of uncontrolled blood vessel growth ultimately may prevent loss or impairment of vision. For extended circulatory half-life, which enables enhanced efficacy as well as lower doses and/or less frequent dosing, the use of ABD-fused polypeptides is warranted when administered systemically. However, for treatment of eye diseases, a more patient-friendly means of administration would be topical administration, such as in the form of eye drops. Then, polypeptides not fused to ABD may alternatively be used.
To investigate the therapeutic effect related to inhibition of new blood vessel formation and tumor growth, the same tumor model (MMTV-PyMT) as described in Example 14 may be used. Alternatively, there are many other applicable mouse models for studying angiogenesis described in the literature (reviewed in Eklund et al. (2013), Molecular Oncology 7:259-282), including the RIP1-Tag2 and Apc-Min mouse models. ABD-fused VEGFR-2 binding polypeptides and control polypeptides, such as those described in Example 13, are administrated by injection at a single or at multiple time points and the effect on new blood vessel formation and tumor growth is studied over several months.
To investigate the therapeutic effect related to inhibition of new blood vessel formation in eye disease, the oxygen-induced retinopathy mouse model described in detail by Connor et al. (2009, Nat Protoc, 4(11):1565– 1573) is used. In a first experiment, ABD-fused VEGFR-2 binding
polypeptides are injected directly into to the eye and the effect on vessel regrowth after the 75%-oxygen exposure is assessed. In a subsequent experiment, VEGFR-2 binding polypeptides are instead formulated as eye drops and administered topically. Results
The result is expected to show that the ability of VEGFR-2 binding polypeptides to inhibit cell signaling in vitro, as confirmed in Example 12, can be translated to therapeutic effects in vivo. More specifically, administration of VEGFR-2 binding polypeptides to animals is expected to demonstrate inhibition of VEGF-induced angiogenesis in tumor and eye disease models. Besides reduced vessel density, the effect in tumor models is ultimately observed as reduced tumor size.
ITEMIZED LIST OF EMBODIMENTS 1. VEGFR-2 binding polypeptide, comprising a VEGFR-2 binding motif BM, which motif consists of an amino acid sequence selected from: i) ENX3X4ASX7EIA X11LPNLX16DX18QY IAFIYX26LLX29 wherein, independently from each other, X3 is selected from L and Y;
X4 is selected from E, F, I, K, M, V, W and Y;
X7 is selected from K, N and R;
X11 is selected from F, H, L and N;
X16 is selected from N and T;
X18 is selected from A, D, E, G, H, K, Q, S and T;
X26 is selected from K and S; and
X29 is selected from D and R;
and ii) an amino acid sequence which has at least 82 % identity to the
sequence defined in i). 2. VEGFR-2 binding polypeptide according to item 1, wherein, in sequence i), X3 is selected from L and Y;
X4 is selected from E, F, I, M, V and Y;
X7 is selected from K, N and R;
X11 is selected from F, H, L and N;
Figure imgf000089_0001
X18 is selected from A, D, E, K, S and T;
Figure imgf000089_0002
X29 is D. 3. VEGFR-2 binding polypeptide according to any preceding item, wherein X3X7X11 is selected from LNH and LKN. 4. VEGFR-2 binding polypeptide according to any preceding item, wherein X3X4X7 is selected from LKN, LMN, YIK, YMK, LFK and LVK.
5. VEGFR-2 binding polypeptide according to any preceding item, wherein X3X4X11 is selected from LKH, LMN, YIN, YMN, LFN, LVN.
6. VEGFR-2 binding polypeptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25.
7. VEGFR-2 binding polypeptide according to item 6, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:5-25.
8. VEGFR-2 binding polypeptide according to item 7, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
9. VEGFR-2 binding polypeptide according to item 8, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:23.
10. VEGFR-2 binding polypeptide according to any preceding item, wherein said VEGFR-2 binding motif forms part of a three-helix bundle protein domain.
11. VEGFR-2 binding polypeptide according to item 10, wherein said VEGFR-2 binding motif essentially forms part of two helices with an
interconnecting loop, within said three-helix bundle protein domain.
12. VEGFR-2 binding polypeptide according to item 11, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
13. VEGFR-2 binding polypeptide according to item 12, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
14. VEGFR-2 binding polypeptide according to any preceding item, which comprises a binding module BMod, the amino acid sequence of which is selected from:
iii) K-[BM]-DPSQSXaXbLLXcEAKKLXdXeXfQ; wherein
[BM] is a VEGFR-2 binding motif as defined in any one of items 1-9 provided that X29 is D;
Xa is selected from A and S;
Xb is selected from N and E;
Xc is selected from A, S and C;
Xd is selected from E, N and S;
Xe is selected from D, E and S;
Xf is selected from A and S; and
iv) an amino acid sequence which has at least 83 % identity to a
sequence defined in iii). 15. VEGFR-2 binding polypeptide according to any one of items 1-13, which comprises a binding module BMod, the amino acid sequence of which is selected from:
v) K-[BM]-QPEQSXaXbLLXcEAKKLXdXeXfQ,
wherein
[BM] is a VEGFR-2 binding motif as defined in any one of items 1-9 provided that X29 is R;
Xa is selected from A and S;
Xb is selected from N and E;
Xc is selected from A, S and C;
Xd is selected from E, N and S;
Xe is selected from D, E and S;
Xf is selected from A and S; and
vi) an amino acid sequence which has at least 83 % identity to a
sequence defined in v). 16. VEGFR-2 binding polypeptide according to item 14, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25.
17. VEGFR-2 binding polypeptide according to item 16, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5-25. 18. VEGFR-2 binding polypeptide according to item 17, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:5-25.
19. VEGFR-2 binding polypeptide according to item 18, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
20. VEGFR-2 binding polypeptide according to item 19, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:23.
21. VEGFR-2 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
vii) YA-[BMod]-AP;
wherein [BMod] is a VEGFR-2 binding module as defined in any one of items 14-20; and
viii) an amino acid sequence which has at least 83 % identity to a
sequence defined in vii).
22. VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
ix) FN-[BMod]-AP;
wherein [BMod] is a VEGFR-2 binding module as defined in any one of items 14-20; and
x) an amino acid sequence which has at least 83 % identity to a
sequence defined in ix).
23. VEGFR-2 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK;
ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;
ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK;
ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK;
AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK;
VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;
AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP;
AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9.
24. VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
xi) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9; and
xii) an amino acid sequence which has at least 82 % identity to the
sequence defined in xi).
25. VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
xiii) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9; and
xiv) an amino acid sequence which has at least 82 % identity to the
sequence defined in xiii).
26. VEGFR-2 binding polypeptide according to any one of items 1-20, which comprises an amino acid sequence selected from:
xv) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined in any one of items 1-9; and
xvi) an amino acid sequence which has at least 82 % identity to the
sequence defined in xv).
27. VEGFR-2 binding polypeptide according to item 24 or 25, wherein sequence xi) or xiii) is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25.
28. VEGFR-2 binding polypeptide according to item 27, wherein sequence xi) is selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5-25.
29. VEGFR-2 binding polypeptide according to item 28, wherein sequence xi) is selected from the group consisting of SEQ ID NO:5-25.
30. VEGFR-2 binding polypeptide according to item 29, wherein sequence xi) is selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:23, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:23.
31. VEGFR-2 binding polypeptide according to item 30, wherein sequence xi) is SEQ ID NO:23.
32. VEGFR-2 binding polypeptide according to any preceding item, which is capable of binding to VEGFR-2 such that the KD value of the interaction with VEGFR-2 is at most 1 x 10-6 M, such as at most 1 x 10-7 M, such as at most 1 x 10-8 M, such as at most 1 x 10-9 M, such as at most 1 x 10-10 M.
33. VEGFR-2 binding polypeptide according to any preceding item, wherein said VEGFR-2 is human VEGFR-2.
34. VEGFR-2 binding polypeptide according to any one of items 1-32, wherein said VEGFR-2 is murine VEGFR-2.
35. VEGFR-2 binding polypeptide according to any preceding item, which has a melting temperature Tm of at least 40 °C, such as at least 45 °C.
36. VEGFR-2 binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end.
37. VEGFR-2 binding polypeptide according to item 36, wherein said additional amino acid(s) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
38. VEGFR-2 binding polypeptide according to any preceding item in multimeric form, comprising at least two VEGFR-2 binding polypeptide monomer units, whose amino acid sequences may be the same or different.
39. VEGFR-2 binding polypeptide according to item 38, wherein said VEGFR-2 binding polypeptide monomer units are covalently coupled together.
40. VEGFR-2 binding polypeptide according to item 38 or 39, wherein the VEGFR-2 binding polypeptide monomer units are expressed as a fusion protein.
41. VEGFR-2 binding polypeptide according to any one of items 38-40, in dimeric form.
42. VEGFR-2 binding polypeptide according to item 41, in homodimeric form.
43. VEGFR-2 binding polypeptide according to item 41, in
heterodimeric form.
44. VEGFR-2 binding polypeptide according to item 43, comprising - a first monomer unit consisting of a VEGFR-2 binding polypeptide according to any one of the preceding items; and
- a second monomer unit consisting of a second VEGFR-2 binding polypeptide, comprising a second VEGFR-2 binding motif BM2, which motif consists of an amino acid sequence selected from: xvii) EFX3X4ADX7EIR X11LPNLX16HGQX20 X21AFIX25X26LYX29 wherein, independently from each other, X3 is selected from Q and R;
X4 is selected from A, D, H, K, L, M, R, S and V;
X7 is selected from A, I and R;
X11 is selected from A, D and G;
X16 is selected from N and T;
X20 is selected from F and W;
X21 is selected from K and Y;
X25 is selected from K and V
X26 is selected from K, N and S; and
X29 is selected from D and R;
and xviii) an amino acid sequence which has at least 82 % identity to the
sequence defined in xvii). 45. VEGFR-2 binding polypeptide according to item 44, wherein said first and second monomer units bind to different epitopes on VEGFR-2.
46. VEGFR-2 binding polypeptide according to item 44 or 45, wherein, in sequence xvii),
X3 is selected from Q and R;
X4 is selected from A, K, R, S and V;
X7 is R;
X11 is selected from A and G;
Figure imgf000096_0001
X20 is selected from F and W;
X21 is Y;
X25 is V; X26 is selected from K and N; and
X29 is D.
47. VEGFR-2 binding polypeptide according to any one of items 44-46, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42.
48. VEGFR-2 binding polypeptide according to item 47, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:26-42.
49. VEGFR-2 binding polypeptide according to item 48, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34-36, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:42.
50. VEGFR-2 binding polypeptide according to item 48, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, such as from the group consisting of SEQ ID NO:34 and SEQ ID NO:41.
51. VEGFR-2 binding polypeptide according to item 49 or 50, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:41.
52. VEGFR-2 binding polypeptide according to any one of items 44-51, wherein said BM2 forms part of a three-helix bundle protein domain.
53. VEGFR-2 binding polypeptide according to item 52, wherein said BM2 essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
54. VEGFR-2 binding polypeptide according to item 53, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
55. VEGFR-2 binding polypeptide according to item 54, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
56. VEGFR-2 binding polypeptide according to any one of items 44-55, wherein said second VEGFR-2 binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:4 and SEQ ID NO:26-42, such as the group consisting of SEQ ID NO:26-42.
57. VEGFR-2 binding polypeptide according to item 56, wherein said first monomer unit is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23.
58. VEGFR-2 binding polypeptide according to item 56 or 57, wherein said second monomer unit is selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39; SEQ ID NO:41 and SEQ ID NO:42.
59. VEGFR-2 binding polypeptide according to item 57 or 58, comprising SEQ ID NO:12 and SEQ ID NO:34; SEQ ID NO:12 and SEQ ID NO:39; SEQ ID NO:12 and SEQ ID NO:41; SEQ ID NO:12 and SEQ ID NO:42; SEQ ID NO:13 and SEQ ID NO:34; SEQ ID NO:13 and SEQ ID NO:39; SEQ ID NO:13 and SEQ ID NO:41; SEQ ID NO:13 and SEQ ID NO:42; SEQ ID NO:20 and SEQ ID NO:34; SEQ ID NO:20 and SEQ ID NO:39; SEQ ID NO:20 and SEQ ID NO:41; SEQ ID NO:20 and SEQ ID NO:42; SEQ ID NO:23 and SEQ ID NO:34; SEQ ID NO:23 and SEQ ID NO:39; SEQ ID NO:23 and SEQ ID NO:41; or SEQ ID NO:23 and SEQ ID NO:42.
60. VEGFR-2 binding polypeptide according to item 59, comprising SEQ ID NO:23 and SEQ ID NO:41.
61. VEGFR-2 binding polypeptide according to any one of items 44-60, which is capable of binding to VEGFR-2 such that the dissociation rate constant kd of the interaction with VEGFR-2 is at least 2 times, such as at least 5 times, such as at least 10 times, such as at least 15 times, such as at least 20 times, such as at least 30 times lower than the dissociation rate constant kd of a comparable polypeptide in homodimeric form.
62. VEGFR-2 binding polypeptide according to item 61, wherein said VEGFR-2 is human VEGFR-2.
63. VEGFR-2 binding polypeptide according to item 61, wherein said VEGFR-2 is murine VEGFR-2.
64. Fusion protein or conjugate comprising
- a first moiety consisting of a VEGFR-2 binding polypeptide according to any preceding item; and
- a second moiety consisting of a polypeptide having a desired biological activity.
65. Fusion protein or conjugate according to item 64, wherein said desired biological activity is a therapeutic activity. 66. Fusion protein or conjugate according to item 64 or 65, wherein said desired biological activity is a binding activity.
67. Fusion protein or conjugate according to item 66, wherein said binding activity is an albumin binding activity, which increases the in vivo half- life of the fusion protein or conjugate.
68. Fusion protein or conjugate according to item 67, wherein said second moiety comprises the albumin binding domain of streptococcal protein G or a derivative thereof.
69. Fusion protein or conjugate according to item 66, wherein said binding activity acts to block a biological activity.
70. Fusion protein or conjugate according to item 66, wherein said binding activity acts to stimulate a biological activity.
71. Fusion protein or conjugate according to item 64, wherein said desired biological activity is an enzymatic activity.
72. Fusion protein or conjugate according to item 65, wherein the second moiety is a therapeutically active polypeptide.
73. Fusion protein or conjugate according to item 72, wherein the second moiety is an anti-cancer agent.
74. Fusion protein or conjugate according to item 72, wherein the second moiety is an anti-angiogenic agent.
75. Fusion protein or conjugate according to item 72, wherein the second moiety is an immune response modifying agent.
76. Fusion protein or conjugate according to any one of items 64-65 and 71-75, wherein the second moiety is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.
77. Complex, comprising at least one VEGFR-2 binding polypeptide according to any one of items 1-63 or at least one fusion protein or conjugate according to any one of items 64-76 and at least one antibody or an antigen binding fragment thereof.
78. Complex according to item 77, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fc fragments, Fv fragments, single chain Fv fragments, (scFv)2 and domain antibodies. 79. Complex according to item 78, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments and scFv fragments.
80. Complex according to item 79, wherein said at least one antibody or antigen binding fragment thereof is a full-length antibody.
81. Complex according to any one of items 77-80, wherein said antibody or antigen binding fragment thereof is a monoclonal antibody or an antigen binding fragment thereof.
82. Complex according to any one of items 77-81, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of human antibodies, humanized antibodies and chimeric antibodies, and antigen-binding fragments thereof.
83. Complex according to item 82, wherein said antibody or antigen binding fragment thereof is a human or humanized antibody, or an antigen binding fragment thereof.
84. Complex according to any one of items 77-83, wherein said antibody or antigen binding fragment thereof has affinity for an antigen, such as selected from the group consisting of an antigen associated with cancer, an antigen associated with an angiogenesis related disorder and an antigen associated with the immune response.
85. Complex according to item 84, wherein said antigen is selected from the group consisting of platelet-derived growth factor receptor α
(PDGFR-α), platelet-derived growth factor receptor β (PDGFR-β), platelet- derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B), platelet-derived growth factor C (PDGF-C), platelet-derived growth factor D (PDGF-D), epidermal growth factor receptor 1 (EGFR), epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor 3 (HER3), epidermal growth factor receptor 4 (HER4), epidermal growth factor, tumor growth factor α, epigen, epiregulin, neuregulins 1-4, c-kit, Raf kinases such as B-Raf and C-raf, rearranged during transfection (RET) receptor, colony stimulating factor 1 receptor (CSF-1R) and fms-like tyrosine kinase 3 (Flt-3).
86. Complex according to item 84, wherein said antigen is selected from the group consisting of fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF-1), basic FGF, angiogenin 1 (Ang-1), angiogenin 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase with immunoglobulin-like domains 1 (TIE-1), tyrosine kinase with immunoglobulin-like domains 2 (TIE-2), vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor 3 (VEGFR-3), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor E (VEGF-E), placental growth factor (PlGF), transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF- β2), transforming growth factor β receptors (type I, type II and type III), matrix metalloproteinase (MMP), MET receptor tyrosine kinase (also denoted cMET and hepatocyte growth factor receptor (HGFR)), members of the Notch family of receptors and beta-catenin.
87. Complex according to item 84, wherein said antigen is selected from the group consisting of CD3, CD28, T-cell receptor α (TCRα), T-cell receptor β (TCRβ), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1) and the natural cytotoxicity receptors NKp30 and NKp40;
programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), herpes virus entry mediator (HVEM)/B- and T-lymphocyte attenuator (BTLA), killer inhibitory receptor (KIR), lymphocyte-activation gene 3 (LAG3), galectin-9 (Gal9)/T cell immunoglobulin mucin-3 (TIM3), adenosine/ alpha-2 adrenergic receptors (A2aR).
88. Complex according to any one of items 77-87, which is a fusion protein or a conjugate.
89. Complex according to any one of items 77-88, wherein said VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of said antibody or antigen binding fragment thereof.
90. Complex according to any one of items 77-88, wherein said VEGFR-2 binding polypeptide is attached to the N-terminus or C-terminus of the light chain of said antibody or antigen binding fragment thereof.
91. Complex according to any one of items 77-88, wherein said VEGFR-2 binding polypeptide is attached to the N-terminus and/or C- terminus of the light chain and heavy chain of said antibody or antigen binding fragment thereof.
92. Complex according to any one of items 88-91, which is a fusion protein. 93. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-92, further comprising at least one linker, such as selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
94. Fusion protein or conjugate according item 93, wherein said linker is arranged between said first moiety and said second moiety.
95. VEGFR-2 binding polypeptide according item 93, wherein said linker is arranged between said first monomer unit and said second monomer unit.
96. Complex according to item 93, wherein said linker is arranged between said VEGFR-2 binding polypeptide and said antibody or antigen binding fragment thereof.
97. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according any one of items 93-96, wherein said linker is a flexible linker comprising amino acid residues selected from the group consisting of glycine, serine and threonine.
98. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to item 97, wherein said linker has a general formula selected from
Figure imgf000102_0001
wherein, independently,
n = 1-7,
m = 0-7,
n + m≤ 8 and
p = 1-10. 99. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to item 98, wherein n = 1-5.
100. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 98-99, wherein m = 0-5.
101. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 98-100, wherein p = 1-5.
102. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 99-101, wherein n = 4, m = 1 and p = 1-4.
103. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to item 98, wherein said linker is selected from the group consisting of S4G, (S4G)3 and (S4G)8. 104. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to item 101, wherein said linker is (S4G)4.
105. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any preceding item, further comprising a label.
106. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to item 105, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes,
chemiluminescent compounds, bioluminescent proteins, enzymes,
radionuclides and radioactive particles.
107. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any preceding item, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the VEGFR-2 binding polypeptide via a thiol group of a cysteine residue or an epsilon amine group of a lysine residue.
108. Polynucleotide encoding a polypeptide according to any one of items 1-104.
109. Expression vector comprising a polynucleotide according to item 108.
110. Host cell comprising an expression vector according to item 109. 111. Method of producing a polypeptide according to any one of items 1-104, comprising
- culturing a host cell according to item 110 under conditions
permissive of expression of said polypeptide from said expression vector, and - isolating said polypeptide.
112. Composition comprising a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 and at least one pharmaceutically acceptable excipient or carrier.
113. Composition according to item 112, further comprising at least one additional active agent, such as selected from an anti-cancer agent, an anti-angiogenic agent and an immune response modifying agent.
114. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or composition according to any one of items 112-113 for use as a medicament.
115. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or composition according to any one of items 112-114 for use in diagnosis. 116. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or composition according to any one of items 112-113 for use in prognosis.
117. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or composition according to any one of items 112-113 for use in diagnosis or prognosis to distinguish subjects who respond to anti angiogenic therapy from subjects who do not respond to said therapy.
118. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or composition according to any one of items 112-113 for use in the treatment, diagnosis or prognosis of a VEGFR-2 related disorder.
119. VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 118, wherein said VEGFR-2 related disorder is cancer or an angiogenesis related disorder.
120. VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 119, wherein said angiogenesis related disorder is age-related macular degeneration.
121. VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 119, wherein said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, melanomas, cancer of the oral cavity and any cancer manifested by solid tumors with VEGFR-2 expression.
122. Method of treatment of a VEGFR-2 related disorder, comprising administering to a subject in need thereof an effective amount of a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or a composition according to any one of items 112-113.
123. Method according to item 122, wherein said VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition inhibits VEGFR-2 mediated signaling by binding to VEGFR-2 expressed on a cell surface. 124. Method of detecting VEGFR-2, comprising providing a sample suspected to contain VEGFR-2, contacting said sample with a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or a composition according to any one of items 112-113, and detecting the binding of the VEGFR-2 binding polypeptide, fusion protein, conjugate or composition to indicate the presence of VEGFR-2 in the sample.
125. Method for determining the presence of VEGFR-2 in a subject, the method comprising the steps:
- contacting the subject, or a sample isolated from the subject, with a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-107 or a composition according to any one of items 112-113, and
- obtaining a value corresponding to the amount of the VEGFR-2 binding polypeptide, fusion protein, conjugate, complex or composition that has bound in said subject or to said sample.
126. Method according to item 125, further comprising a step of comparing said value to a reference.
127. Method according to any one of items 124-126, wherein the method is performed in vivo.
128. Method according to any one of items 124-126, wherein the method is performed in vitro.
129. Method according to any one of items 122-128, wherein said subject is a mammalian subject, such as a human subject.
130. Method according to any one of items 122-129, wherein said VEGFR-2 related disorder is cancer, a vascular disease or an angiogenesis related disorder.
131. Method according to item 130, wherein said angiogenesis related disorder is age-related macular degeneration.
132. Method according to item 130, wherein said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, uterine cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, glioma, glioblastomas, astrocytoma, gastric cancer, stomach cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, melanomas, cancer of the oral cavity and any cancer manifested by solid tumors with VEGFR-2 expression.

Claims

CLAIMS 1. VEGFR-2 binding polypeptide, comprising a VEGFR-2 binding motif BM, which motif consists of an amino acid sequence selected from: i) ENX3X4ASX7EIA X11LPNLX16DX18QY IAFIYX26LLX29 wherein, independently from each other, X3 is selected from L and Y;
X4 is selected from E, F, I, K, M, V, W and Y;
X7 is selected from K, N and R;
X11 is selected from F, H, L and N;
X16 is selected from N and T;
X18 is selected from A, D, E, G, H, K, Q, S and T;
X26 is selected from K and S; and
X29 is selected from D and R;
and ii) an amino acid sequence which has at least 82 % identity to the
sequence defined in i).
2. VEGFR-2 binding polypeptide according to claim 1, wherein, in sequence i), X3 is selected from L and Y;
X4 is selected from E, F, I, M, V and Y;
X7 is selected from K, N and R;
X11 is selected from F, H, L and N;
Figure imgf000106_0001
X18 is selected from A, D, E, K, S and T;
Figure imgf000106_0002
X29 is D.
3. VEGFR-2 binding polypeptide according to any preceding claim, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25, for example the sequence from position 8 to position 36 in SEQ ID NO:23.
4. VEGFR-2 binding polypeptide according to any preceding claim, which comprises a binding module BMod, the amino acid sequence of which is selected from:
iii) K-[BM]-DPSQSXaXbLLXcEAKKLXdXeXfQ;
wherein
[BM] is a VEGFR-2 binding motif as defined in any one of claims 1-3 provided that X29 is D;
Xa is selected from A and S;
Xb is selected from N and E;
Xc is selected from A, S and C;
Xd is selected from E, N and S;
Xe is selected from D, E and S;
Xf is selected from A and S; and
iv) an amino acid sequence which has at least 83 % identity to a
sequence defined in iii).
5. VEGFR-2 binding polypeptide according to claim 4, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25, for example the sequence from position 7 to position 55 in SEQ ID NO:23.
6. VEGFR-2 binding polypeptide according to any preceding claim, which comprises an amino acid sequence selected from:
xi) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a VEGFR-2 binding motif as defined in any one of claims 1-4; and
xii) an amino acid sequence which has at least 82 % identity to the
sequence defined in xi).
7. VEGFR-2 binding polypeptide according to claim 6, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5-25, for example SEQ ID NO:23.
8. VEGFR-2 binding polypeptide according to any preceding claim, which is capable of binding to VEGFR-2 such that the KD value of the interaction with VEGFR-2 is at most 1 x 10-6 M, such as at most 1 x 10-7 M, such as at most 1 x 10-8 M, such as at most 1 x 10-9 M, such as at most 1 x 10-10 M.
9. VEGFR-2 binding polypeptide according to any preceding claim in multimeric form, comprising at least two VEGFR-2 binding polypeptide monomer units, whose amino acid sequences may be the same or different.
10. VEGFR-2 binding polypeptide according to claim 9, comprising - a first monomer unit consisting of a VEGFR-2 binding polypeptide according to any one of claims 1-8; and
- a second monomer unit consisting of a second VEGFR-2 binding polypeptide, comprising a second VEGFR-2 binding motif BM2, which motif consists of an amino acid sequence selected from: xvii) EFX3X4ADX7EIR X11LPNLX16HGQX20 X21AFIX25X26LYX29 wherein, independently from each other, X3 is selected from Q and R;
X4 is selected from A, D, H, K, L, M, R, S and V;
X7 is selected from A, I and R;
X11 is selected from A, D and G;
X16 is selected from N and T;
X20 is selected from F and W;
X21 is selected from K and Y;
X25 is selected from K and V
X26 is selected from K, N and S; and
X29 is selected from D and R;
and xviii) an amino acid sequence which has at least 82 % identity to the
sequence defined in xvii).
11. VEGFR-2 binding polypeptide according to claim 10, wherein sequence xvii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, for example the sequence from position 8 to position 36 in SEQ ID NO:41.
12. VEGFR-2 binding polypeptide according to any one of claims 10- 11, wherein
- said second VEGFR-2 binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:26-42, for example selected from the group consisting of SEQ ID NO:26-42, for example selected from the group consisting of SEQ ID NO:34, SEQ ID NO:39; SEQ ID NO:41 and SEQ ID NO:42; and/or
- said first monomer unit is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 and SEQ ID NO:23; and/or
- said VEGFR-2 binding polypeptide comprises SEQ ID NO:12 and SEQ ID NO:34; SEQ ID NO:12 and SEQ ID NO:39; SEQ ID NO:12 and SEQ ID NO:41; SEQ ID NO:12 and SEQ ID NO:42; SEQ ID NO:13 and SEQ ID NO:34; SEQ ID NO:13 and SEQ ID NO:39; SEQ ID NO:13 and SEQ ID NO:41; SEQ ID NO:13 and SEQ ID NO:42; SEQ ID NO:20 and SEQ ID NO:34; SEQ ID NO:20 and SEQ ID NO:39; SEQ ID NO:20 and SEQ ID NO:41; SEQ ID NO:20 and SEQ ID NO:42; SEQ ID NO:23 and SEQ ID NO:34; SEQ ID NO:23 and SEQ ID NO:39; SEQ ID NO:23 and SEQ ID NO:41; or SEQ ID NO:23 and SEQ ID NO:42, for example SEQ ID NO:23 and SEQ ID NO:41.
13. VEGFR-2 binding polypeptide according to any one of claims 10- 12, which is capable of binding to VEGFR-2 such that the dissociation rate constant kd of the interaction with VEGFR-2 is at least 2 times, such as at least 5 times, such as at least 10 times, such as at least 15 times, such as at least 20 times, such as at least 30 times lower than the dissociation rate constant kd of a comparable polypeptide in homodimeric form.
14. Fusion protein or conjugate comprising
- a first moiety consisting of a VEGFR-2 binding polypeptide according to any preceding claim; and - a second moiety consisting of a polypeptide having a desired biological activity, for example selected from the group consisting of a therapeutic activity, a binding activity and an enzymatic activity.
15. Fusion protein or conjugate according to claim 14, wherein the second moiety is selected from the group consisting of a therapeutically active polypeptide, an anti-cancer agent, an anti-angiogenic agent, an immune response modifying agent, human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.
16. Complex, comprising at least one VEGFR-2 binding polypeptide according to any one of claims 1-13 or at least one fusion protein or conjugate according to any one of claims 14-15 and at least one antibody or an antigen binding fragment thereof.
17. Complex according to claim 16, wherein said antibody or antigen binding fragment thereof has affinity for an antigen, such as selected from the group consisting of an antigen associated with cancer, an antigen associated with an angiogenesis related disorder and an antigen associated with the immune response.
18. Composition comprising a VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any preceding claim and at least one pharmaceutically acceptable excipient or carrier.
19. VEGFR-2 binding polypeptide, fusion protein, conjugate or complex according to any one of claims 1-17 or composition according to claim 18 for use as a medicament, for use in diagnosis and/or for use in prognosis.
PCT/EP2015/073687 2014-10-13 2015-10-13 Vegfr-2 binding polypeptides WO2016059057A1 (en)

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