BERGGREN, Kristina (Fägelhundsvägen 28, Lund, S-226 53, SE)
HOLMGREN, Lars (Snäckvägen 14, Bromma, S-167 53, SE)
BERGGREN, Kristina (Fägelhundsvägen 28, Lund, S-226 53, SE)
CLAIMS
1. Use of an antibody or an antigen-binding fragment thereof, which binds specifically to either a p80-angiomotin molecule or a polynucleotide encoding a p80-angiomotin molecule, in the manufacture of a medicament for the treatment of an angiogenesis-related eye disease.
2. A method for treating a subject with an angiogenesis-related eye disease; the method comprising the step of administering to the subject an antibody or an antigen-binding fragment thereof, which binds specifically to either a p80-angiomotin molecule or a polynucleotide encoding a p80-angiomotin molecule.
3. A pharmaceutical composition for treatment of an angiogenesis-related eye disease; comprising an antibody or an antigen-binding fragment thereof which binds specifically to either a p80-angiomotin molecule or a polynucleotide encoding a p80-angiomotin molecule and a pharmaceutical carrier, excipient or diluent.
4. The use, method or pharmaceutical composition of any previous claim wherein the angiogenesis-related eye disease is age-related macular degeneration, proliferative ischaemic retinapthy or corneal neovascularisation.
5. The use, method or composition according to any one of the preceding claims wherein the antibody or antigen-binding fragment thereof is human or humanised.
6. The use, method or composition according to any one of the preceding claims wherein the antigen-binding antibody fragment is an scFv or Fab.
7. The use, method or composition according to any one of the preceding claims wherein the antibody or antigen-binding fragment thereof binds to an epitope of full length human p80-angiomotin.
8. The use, method or composition according to any one of claims 1 to 7 wherein the antibody or antigen-binding fragment thereof binds specifically to an epitope of a fragment of human p80-angiomotin, wherein the fragment has substantially the same angiogenic activity as full length p80-angiomotin.
9. The use, method or composition according to any one of claims 1 to 8 wherein the antibody or antigen-binding fragment thereof binds specifically to the Big3 fragment of angiomotin.
10. The use, method or composition according to any one of the preceding claims wherein the antibody or antigen-binding antibody fragment acts by reducing angiogenesis in the eye.
11. The use, method or composition according to any one of the preceding claims wherein the antibody or antigen-binding antibody fragment is administered to the eye.
12. The use, method or composition according to any one of the preceding claims wherein the antibody or antigen-binding antibody fragment is administered intravitreally or in the form of an eye bath or eye drop.
13. The use, method or composition of any previous claim wherein the antibody has V H and VL regions with the amino acid sequences of figure l(a) and/or l(b).
14. The use, method or composition of any previous claim wherein the antibody has V H CDR regions of the amino acid sequences FSNAWMS, AVSGSGGSTYYADSVKG and TTDRLDYFDY.
15. The use, method or composition of any previous claim wherein the antibody has V H CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
16. The use, method or composition of any previous claim wherein the antibody has V L CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
17. The use, method or composition of any previous claim wherein the antibody has V L CDR regions of the amino acid sequences SGSISMGVNTVT, DNNKRPS and AAWDDSLNGW.
18. The use, method or composition of any of claims 1 to 13 wherein the antibody has V H CDR regions described in Claim 14 and V L CDR regions described in Claim 16.
19. The use, method or composition of any of claims 1 to 13 wherein the antibody has V H CDR regions described in Claim 15 and V L CDR regions described in Claim 17.
20. A kit of parts comprising:
(i) a pharmaceutical composition as described in claims 3 to 13;
(ii) apparatus for administering the pharmaceutical composition to the eye; and (iii) instructions for use.
21. A kit of parts as claimed in claim 20 wherein the apparatus of (ii) is an intra- vitreal needle, an eye bath or an eye dropper.
22. An antibody having V H and V L regions with the amino acid sequences of figure l(a) and/or l(b).
23. An antibody as claimed in claim 22 wherein V H CDR regions have the amino acid sequences FSNAWMS, AVSGSGGSTYYADSVKG and TTDRLDYFDY.
24. An antibody as claimed in claim 22 wherein V H CDR regions have the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
25. An antibody as claimed in claim 22 wherein V H CDR regions have the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
26. An antibody as claimed in claim 22 wherein V H CDR regions have the amino acid sequences SGSISNIGVNTVT, DNNKRPS and
AAWDDSLNGW.
27. An antibody as claimed in claim 22 having V H CDR regions as described in Claim 23 and V L CDR regions as described in Claim 25.
28. An antibody as claimed in claim 22 having V H CDR regions as described in Claim 24 and V L CDR regions as described in Claim 26.
29. The use of an antibody as described in any of claims 22 to 28 in the manufacture of a medicament for the treatment of an angiogenesis-related disease.
30. A method of treating an individual with an angiogenesis-related disease comprising administering an effective amount of an antibody as described in any ofclaims 22 to 28.
31. A pharmaceutical composition comprising an antibody as described any of claims 22 to 28 and a pharmaceutically acceptable diluent, carrier or excipient.
32. A use of an antibody in the manufacture of a medicament substantially as described herein with reference to the examples.
33. A method of treatment substantially as described herein with reference to the examples.
34. A pharmaceutical composition substantially as described herein with reference to the examples.
35. A kit of parts substantially as described herein with reference to the examples. |
BIOLOGICAL MATERIALS AND USES THEREOF
The present invention relates to prophylactic or therapeutic treatments for angiogenic eye diseases.
Vasculogenesis is the differentiation of stem cells into endothelial cells which then form blood vessels. Angiogenesis is the formation of blood vessels from preexisting ones. Terms such as blood vessel formation, neovascularisation and vascularisation cover both vasculogenesis and angiogenesis.
Angiogenesis (an example of blood vessel formation) is the formation of new capillary blood vessels by a process of sprouting from pre-existing vessels and occurs during development as well as in a number of physiological and pathological settings (Folkman, 1995, Nature Medicine, 1 :27-31). Formation of new blood vessels by the process of angiogenesis involves a complex series of events including endothelial cell proliferation, migration, interaction and adhesion to form cords and tubes, and finally maturation. Physiologically, angiogenesis is necessary for tissue growth, wound healing, and female reproductive function and is a component of pathological processes such as retinal disease, atherosclerosis, endometriosis, rheumatoid arthritis and inflammatory conditions. However, much of the longstanding interest in angiogenesis comes from the notion that for solid tumours to grow beyond a critical size, they must recruit endothelial cells from the surrounding stroma to form their own endogenous microcirculation. In order to promote neo-vascularisation, tumours release variety of factors that stimulate proliferation and migration of endothelial cells. Such factors include vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF), interleukin-8 (IL-8) placental growth factor, and thymidine phosphorylase (platelet-derived endothelial cell growth factor; ReIf et al., 1997, Cancer Research, 57:963-9). Therefore, much effort has been dedicated to finding molecules that interfere with these signalling pathways and thereby block tumour angiogenesis.
Targeting angiogenesis has potentially several advantages compared to traditional oncolytic therapy. The most prominent of these is that: all solid tumours are
angiogenesis-dependent; the target endothelial cells are readily accessible for therapy; and they are genomically stable and less prone to generate resistance to therapy. One of the obvious disadvantages of targeting a cancer cell-expressed protein is the genetic variability and a large selection pressure due to rapid cell growth and division, which often renders such drugs ineffective due to resistance mechanisms and the onset and use of alternative pathways.
A large number of naturally-occurring angiogenic inhibitors have been identified such as angiostatin (a 38kDa proteolytic fragment of plasminogen), anti- angiogenic anti -thrombin III, endostatin (collagen XVIII fragment), interferon alpha/beta/gamma, prolactin 16kDa fragment and thrombospondin-1 (TSP-I) which show varying degrees of effect in in vitro and in vivo models. These inhibitors target endothelial cells and inhibit angiogenesis. The observed inhibition of, for example, angiostatin, is independent of which angiogenic factor the endothelial cells are stimulated by (Eriksson et al, 2003 FEBS Letts., 536:19- 24). This is in contrast to agents such as antibodies that bind to VEGF or low molecular compounds that inhibit VEGF-receptor kinase activity. Most tumours express a variety of angiogenic factors indicating that targeting one single angiogenesis pathway is not enough for inhibiting tumour expansion. Thus, therapies that target directly the endothelial cells have a potential to circumvent the problem of angiogenesis being controlled by a plurality of tumor-derived factors. However, on the other hand, such therapies have to deal with the problem of being able to target specifically endothelial cells that are involved in the process of neo-vascularisation while sparing mature blood vessels.
An 8OkDa molecule named angiomotin ("p80-angiomotin") was identified by its ability to bind to the angiogenesis inhibitor, angiostatin (Troyanovsky et al, 2001, J. Cell. Biol, 152:1247-1254; WO 99/66038). p80-angiomotin belongs to a new protein family with two additional members, angiomotin-like protein 1 (Amotl) and 2 (Amot2). These proteins are characterised by conserved coiled-coil domains and C-terminal PDZ binding motifs (Nishimura et al., 2002, J. Biol. Chem., 277:5583-5587; Bratt et al, 2002, Gene, 298:69-77). p80-angiomotin differs from its related proteins as it contains an extracellular angiostatin-binding domain. P80-angiomotin is primarily expressed in endothelial cells and mediates
the inhibitory effect of angiostatin on endothelial cell migration and tube formation in vitro (Troyanovsky et al., 2001, J. Cell. Biol., 152:1247-1254). Expression of p80-angiomotin in mouse aortic endothelial (MAE) cells increases the migratory response to chemotactic factors (Troyanovsky et al., 2001, J. Cell. Biol., 152:1247-1254). A role in migration is further emphasised by the findings from p80-angiomotin knock-out experiments in mice in which approximately 70% of knock-out mice died during embryonic day 7-8 due to a migratory defect in the anterior visceral endoderm (Shimono and Behringer, 2003, Current Biology, 13:613-7).
WO 99/66038 discusses p80-angiomotin and its use as, for example, a drug screening target.
A first aspect of the invention provides the use of an antibody or an antigen- binding fragment thereof, which binds specifically to either a p80-angiomotin molecule or a polynucleotide encoding a p80-angiomotin molecule, in the manufacture of a medicament for the treatment of an angiogenesis-related eye disease.
The terms "selective binding" and "binding selectivity" indicates that the variable regions of the antibodies of the invention recognise and bind polypeptides of the invention exclusively (i.e., able to distinguish the polypeptide of the invention from other similar polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding selectivity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N. Y. (1988), Chapter 6. Antibodies that recognise and bind fragments of the polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost selective for, as defined above, full- length polypeptides of the invention. As with antibodies that are selective for full
length polypeptides of the invention, antibodies of the invention that recognise fragments are those which can distinguish polypeptides from the same family of polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.
A second aspect of the invention provides a method for treating a subject with an angiogenesis-related eye disease; the method comprising the step of administering to the subject an antibody or an antigen-binding fragment thereof, which binds specifically to either a p80-angiomotin molecule or a polynucleotide encoding a p80-angiomotin molecule.
A third aspect of the invention provides a pharmaceutical composition for treatment of an angiogenesis-related eye disease; comprising an antibody or an antigen-binding fragment thereof which binds specifically to either a p80- angiomotin molecule or a polynucleotide encoding a p80-angiomotin molecule; and a pharmaceutical carrier, excipient or diluent.
The invention provides the use of antibodies against p80 -angiomotin molecule or fragments thereof, for the treatment of angiogenic eye diseases including age- related macular degeneration, proliferative ischemic retinapathies and corneal vascularisation. The invention further provides the use of the antibodies as a medicament for the treatment of such angiogenic eye diseases.
We have found that angiogenesis inhibition using antibodies against p80 angiomotin can improve certain eye diseases that result from angiogenesis within and around the eye. Examples include diabetic retinopathy and macular degeneration, which both result from an overgrowth of ocular blood vessels. In these disorders, the vessels interfere with normal structures in the eye, or block light from reaching the back of the eye. The new blood vessels are themselves the primary pathology, and stopping blood vessel growth could prevent blindness.
Age-related macular degeneration (AMD) is the most common cause of social blindness (visual acuity =< 0.1) in the Western world and typically affects the elderly (>65 years). It is estimated that in Sweden over 300,000 patients suffer
from AMD and approximately 45,000 of these have the more severe forms of the disease.
The oxygen tension in the macular region of the eye is the highest in the body creating an exceptionally high metabolic turnover in the area. In addition the macula is exposed to high light levels. The retinal cells in this oxygen rich environment are considered to be particularly vulnerable to the energy rich blue light part of electromagnetic spectrum.
The oxygen stress that is generated in this situation creates free radicals that are toxic and inhibit the normal turnover of the photoreceptor outer segments normally executed by the underlying retinal pigment epithelial (RPE) cells. This incomplete digestion leads to an accumulation of waste products (lipofuscin and lipid-rich drusen) in and around the RPE cells that ultimately causes RPE cell death and the development of AMD. AMD has two basic manifestation (atrophic and neovascular AMD) of which neo vascular AMD is the most severe.
Neovascular AMD is caused by an impaired function in the retinal pigment epithelium (RPE) and its basal membrane (Bruch's membrane that constitutes the outer blood-retinal barrier) placed under the neural retina of the macula. As a consequence, vessels from the underlying choroid may grow into the subretinal space (e.g. choroidal neovascularization or CNV) causing edema, hemorrhage and fibrosis under the macula resulting in rapid central vision loss. The driving force of CNV is not clear but most likely involves an inflammatory/immune reaction secondary to the altered Bruch's membrane. Neovascular AMD is often bilateral with a 5 year risk of 45-50% risk in the fellow eye.
Proliferative ischemic retinapathies are characterised by neovascularisation on the retinal surface (e.g. retinal neovascularisation) and such retinapathies, secondary to diabetic retinapathy are the most common cause of vision loss in younger individuals, hi retinal neovascularisation the underlying condition (i.e. diabetes) causes retinal hypoxia that stimulates new vessel formation. If left untreated, retinal neovascularisation often leads to intraocular (vitreous) haemorrhage and
retinal detachment. Retinal neovascularisation is also seen in other ischemic retinal conditions including central vein occlusion and retinopathy of prematurity.
Corneal neovascularisation is the neovascularisation of the normally avascular cornea which disturbs the visual pathway and leads to a more or less severe vision loss. It is relatively uncommon in the Western world, mostly caused by corneal infections or trauma, but frequently seen in under developed countries secondarily to trachoma, an ocular infection caused by Chlamydia trachomatis. Corneal neovascularisation is invariably caused by an in-growth of vessels from the surrounding conjunctiva and is generally accompanied by a marked inflammatory response.
By "p80-angiomotin molecule" we include a polypeptide which: has coiled-coil and C-terminal PDZ binding domains with an estimated molecular mass of 72 kDa; is considered to be a cell surface-associated protein; and is considered to bind to angiostatin and to mediate inhibitory effects of angiostatin on endothelial cell migration and tube formation. Examples of naturally-occurring angiomotin polypeptides are given in the following: Troyanovsky et al, 2001, J. Cell Biol, 152:1247-1254; WO 99/66038; Levchenko et al, 2003, J. Cell ScL, 116:3803- 3810; Bratt et al, 2002, Gene, 298:69-77; GenBank accession No: NP_573572 (human).
By "polynucleotide" we include single-stranded and/or double-stranded molecules of DNA (deoxyribonucleic acid) and/or RNA (ribonucleic acid) and derivatives thereof. By "encoding polynucleotide" we include a polynucleotide the sequence of which that may be translated to form a desired polypeptide.
Antibodies comprise two identical polypeptides of M r 50,000-70,000 (termed "heavy chains") that are linked together by a disulphide bond, each of which is linked to one of an identical pair of polypeptides of M r 25,000 (termed "light chains"). There is considerable sequence variability between individual N-termini of heavy chains of different antibody molecules and between individual light chains of different antibody molecules and these regions have hence been termed "variable domains". Conversely, there is considerable sequence similarity
between individual C-termini of heavy chains of different antibody molecules and between individual light chains of different antibody molecules and these regions have hence been termed "constant domains".
The antigen-binding site is formed from hyper-variable regions in the variable domains of a pair of heavy and light chains. The hyper-variable regions are also known as complementarity-determining regions (CDRs) and determine the specificity of the antibody for a ligand. The variable domains of the heavy chain (V H ) and light chain (V L ) typically comprise three CDRs, each of which is flanked by sequence with less variation, which are known as framework regions (FRs).
The variable heavy (V H ) and variable light (V L ) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al, 1984, Proc. Natl. Acad. Sd. USA, 81, 6851-6855).
That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al, 1988, Science, 240:1041). Fv molecules (Skerra et al, 1988, Science, 240, 1038); single-chain Fv (ScFv) molecules where the VH and V L partner domains are linked via a flexible oligopeptide (Bird et al., 1988, Science 242:423; Huston et al, 1988, Proc. Natl. Acad. Sd. USA, 85:5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al., 1989 Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter et al, 1991, Nature, 349, 293-299.
Figure 1 shows the positions of CDR regions in the antibody sequences of the invention. The position of short sequences such as CDR regions or even individual amino acid residues can be identified in any given antibody or antibody fragment
using the KABAT system. The sequences can be aligned manually according to the method of Kabat et al. (1991) Sequences of Proteins of Immunological Interest. NIH publication no. 91-3242. An alternative method is by submitting the Fv protein sequence online at http://www.bioinf.org.uk/abs/seqtest.html. The server for this site aligns the submitted sequence to all KABAT database entries and makes the accurate numbering of residues. Also, any "unusual" residues (i.e. occurrence at a given position <1%) are reported.
Preferably, the antibody is a human or humanised antibody or fragment thereof.
Conveniently the antibody fragment of the invention is an scFv molecule or Fab.
By "ScFv molecules" we mean molecules wherein the V H and V L partner domains are linked via a flexible oligopeptide.
The advantages of using antibody fragments which have antigen-binding activity, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from Escherichia coli (E. coli), thus allowing the facile production of large amounts of the said fragments.
Whole antibodies, and F(ab') 2 fragments are "bivalent". By "bivalent" we mean that the said antibodies and F(ab') 2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site.
Methods for generating, isolating and using antibodies for a desired antigen or epitope are well known to those skilled in the relevant art. For example, an antibody may be raised in a suitable host animal (such as, for example, a mouse, rabbit or goat) using standard methods known in the art and either used as crude antisera or purified, for example by affinity purification. An antibody of desired specificity may alternatively be generated using well-known molecular biology
methods, including selection from a molecular library of recombinant antibodies, or grafting or shuffling of complementarity-determining regions (CDRs) onto appropriate framework regions. Human antibodies may be selected from recombinant libraries and/or generated by grafting CDRs from non-human antibodies onto human framework regions using well-known molecular biology techniques.
Methods for formulating polypeptides, polynucleotides and antibodies into medicaments, pharmaceutical compositions and vaccines are well known to those in the relevant art. Preferred formulations of medicaments, pharmaceutical compositions and vaccines comprising the polypeptides, polynucleotides and antibodies of the invention are described in the Examples.
Antibodies may be used in therapy - for example, a medicament comprising therapeutic antibodies may be introduced into a subject to modulate the immune response of that subject. For example, a therapeutic antibody specific for an antigen in the subject will stimulate an immune response to that antigen, thereby inducing and/or promoting an immune response and aiding recovery. Methods for administering therapeutic antibodies to a patient in need thereof are well known in the art. It will be understood that an antibody of the present invention may be used as a therapeutic antibody to modulate the immune response in a subject to p80-angiomotin and preferably inhibit and/or reduce angiogenesis in that subject.
Preferably, the subject to be treated is human, for example a human with or at risk of an angiogenesis-associated disease or condition (as defined above). Alternatively, the recipient may be an animal with or at risk of such a condition, for example a domesticated animal (e.g. a cat or dog) or animal important in agriculture, for example cattle, sheep, goats, or poultry.
Advantageously the antibody or antigen-binding fragment thereof binds to an epitope of full length human p80-angiomotin.
Conveniently, the antibody or antigen-binding fragment thereof binds specifically to an epitope of a fragment of human p80-angiomotin, wherein the fragment has substantially the same angiogenic activity as full length p80-angiomotin. An example of a binding fragment of angiomotin is the Big 3 fragment. The sequence of the Big3 fragment is given in figure 5 and its expression and purification has been described in WO 99/66038.
Preferably the antibody or antibody fragment acts by reducing angiogenesis in the eye in order to treat the angiogenesis related eye-disease. Therefore the antibody or antibody fragments acts by reducing the number of excess blood vessels in the eye and/or by preventing the growth of further blood vessels.
Methods of administration of the antibody or antibody fragment are described in the examples. Advantageously the antibody or antibody fragment is administered to the eye itself. Preferably the route of administration is intravitreally or in the form of an eye bath or eye drop.
Preferably the invention provides a use, method or composition wherein the antibody has V H and V L regions with the amino acid sequences of figure l(a) and/or l(b).
Preferably, the antibody has V H CDR regions of the amino acid sequences FSNAWMS, AVSGSGGSTYYADSVKG and TTDRLDYPDY.
Alternatively, the antibody has V H CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
Preferably, the antibody has V L CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
Alternatively, the antibody has V L CDR regions of the amino acid sequences SGSISNIGVNTVT, DNNKRPS and AAWDDSLNGW.
Conveniently, the antibody has V H CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI and V L CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
Conveniently, the antibody has V H CDR regions of the amino acid sequences FSNAWMS, AVSGSGGSTYYADSVKG and TTDRLDYFDY and V L CDR regions of the amino acid sequences SGSISNIGVNTVT, DNNKRPS and AAWDDSLNGW.
In a fourth aspect of the invention there is provided a kit of parts comprising:
(i) a pharmaceutical composition as described in the third aspect of the invention;
(ii) apparatus for administering the pharmaceutical composition to the eye; and (iii) instructions for use.
The inventions of the first to fourth aspects may be used to treat the following eye diseases: age-related macular degeneration; proliferative ischaemic retinapathy; and/or corneal neovascularisation.
Preferably the kit of parts comprises administration apparatus that is an intra- vitreal needle, an eye bath or an eye dropper.
In a fifth aspect of the invention there is provided an antibody having V H and V L regions with the amino acid sequences of figure l(a) and/or l(b) i.e. the AM-18- A03 and AM-19-B06 antibodies.
Preferably, the antibody has V H CDR regions of the amino acid sequences FSNAWMS, AVSGSGGSTYYADSVKG and TTDRLDYFDY.
Alternatively, the antibody has V H CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
Preferably, the antibody has V L CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
Alternatively, the antibody has V L CDR regions of the amino acid sequences SGSISNIGVNTVT, DNNKRPS and AAWDDSLNGW.
Conveniently, the antibody has V H CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI and V L CDR regions of the amino acid sequences FDDYGMS, AISGSGGSTYYADSVKG and ASNSWAFDTFDI.
Conveniently, the antibody has V H CDR regions of the amino acid sequences FSNAWMS, AVSGSGGSTYYADSVKG and TTDRLDYFDY and V L CDR regions of the amino acid sequences SGSISNIGVNTVT, DNNKRPS and AAWDDSLNGW.
There is also provided the use of these antibodies in the manufacture of a medicament for the treatment of an angiogenesis-related disease, a method of treating an individual with an angiogenesis-related disease by administering an effective amount of an antibody and a pharmaceutical composition comprising the antibody as described and a pharmaceutically acceptable diluent, carrier or excipient.
Disease suitable for treatment using the AM-18-A03 and AM-19-B06 antibodies are cancer (particularly solid tumours), haemangioma, ocular neovascularisation, diabetic retinopathy, macular degeneration, rheumatoid arthritis, inflammatory conditions (such as psoriasis, chronic inflammation of the intestines, asthma) and endometriosis.
Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
Figure 1 - Nucleotide and amino acid sequences.
Nucleotide and amino acid sequences for the variable domains, VH and VL, of Angiomotin binding antibodies, a) AM-18-A03, b) AM-19-B06 isolated from n- CoDeR® library. CDR regions identified using the KABAT system in each sequence are underlined.
Figure 2 - Binding of Angiomotin specific antibodies to human p80 Angiomotin measured in ELISA.
Purified p80 hAmot was coated in the wells and the amount of bound anti-Amot antibodies was detected using polyclonal anti-human IgGl antibodies. The Angiomotin specific antibodies have a dose dependent binding to p80hAmot while the n-CoDeR ® control antibody does not bind to Angiomotin.
Figure 3 - The binding of IgGl AM-19-B06 and IgGl AM-18-A03 to p80 hAmot expressed on the surface of CHO cells, monitored by flow cytometry. The anti -Angiomotin antibodies were directly labeled using the Alexa Fluor 647 Zenon Human IgG labeling Kit. CHO cells were harvested using a non-enzymatic cell dissociation buffer and stained at 2x10 6 cells/ml in 0.2 ml for 60 minutes at 10° C. Cells were counter stained with the dead-cell stain SYTOX Green and nonviable cells were excluded from data analysis.
Figure 4 - Sequences of p80 angiomotin
Figure provides the human amino acid and nucleotide sequences of p80 angiomotin.
Figure 5 - Big3 sequence
The sequence of the Big3 fragment of angiomotin.
Figure 6 - Selection of scFv specific to human and mouse p80 Amot.
(A) Single-chain fragment-variable (scFv) antibodies with specificity for human p80 angiomotin (Amot) were isolated from the human scFv phage antibody library (n-CoDeR) after three consecutive rounds of selection. Selected scFv were screened for human p80 Amot binding in a catcher ELISA where scFv were immobilised in plastic wells and incubated with cell-lysates from Mouse
aortic endothelial cells (MAE) transfected with human p80 Amot. Bound protein was detected using anti-angiomotin rabbit polyclonal antibodies (TLE described in Bratt, et al, 2005). MAE-vector lysate was used as a negative control. Graph shows relative binding to human p80 Amot versus vector transfected cells. (B) Cross-reactivity with mouse Amot was tested by binding to mouse p80 angiomotin in a catcher ELISA. The relative binding to human or mouse angiomotin were then plotted against each other.
(C) MAE cells transfected with either human p80 angiomotin or empty vector were analysed by FACS for cell surface binding of scFv B06 or control scFv CTH.
Figure 7 - Amot-specific B06-scFv inhibits VEGF and FGF-2 induced endothelial migration.
(A) MAE cells transfected with vector or human p80 Amot were stimulated with fibroblast growth factor (FGF)-2 ; or
(B) vascular endothelial growth factor (VEGF) in combination with increasing concentrations of either B06-scFv or CTl 7 scFv (reactive to Cholera toxin). The addition of B06-scFv inhibited migration of FGF-2 or VEGF-stimulated migration. No inhibitory effect of B06-scFv was observed in MAE-vector cells. (C) B06 scFv inhibits migration of bovine capillary endothelial (BCE) cells (that express endogenous angiomotin).
Figure 8 - B06-scFv specifically inhibits tube formation of human p80 Amot cells in vitro. MAE cells spontaneously form tubes when plated on Matrigel extra-cellular matrix in vitro. MAE-vector or MAE-Amot transfected cells were incubated with 500 ng ml "1 CT17-scFv or B06-scFv at the time of plating on Matrigel. Treatment with B 06 inhibited the formation of cellular sprouts resulting in inhibition of tubulogenesis. Images show tube formation 16 h after seeding on matrigel (Size bar = 130 μm).
Figure 9 - B06-scFv inhibits FGF-2-induced angiogenesis in the matrigel plug assay in vivo.
(A) Mice were injected with 500 μl Matrigel and the resulting plugs were excised and photographed seven days later. Vascularisation was analysed by PECAM staining of cryosections. No angiogenesis was detected in plugs lacking FGF-2, which served as the negative control. Extensive ingrowth of vessels was observed in plugs containing FGF-2 alone or FGF-2 and CT17-scFv. In contrast, the plugs containing FGF-2 and B06-scFv were translucent and the vessel density was similar to the negative control lacking FGF-2. (Size bars: Bright field images = 10 mm, fluorescence images = 100 μm).
(B) Bar diagram showing the vascular density of the different groups.
Figure 10 - B06-scFv inhibits tumor-induced angiogenesis. Angiogenesis was induced by suspending 75,000 TUBO cells into 500 μl of matrigel together with 500 μg ml "1 of CT17-scFv or B06-scFv. Seven days later, matrigel plugs were extracted and the matrigel plugs are shown (A & B). A visibly lower amount of haemorrhages are seen in the treated plugs (B). Vessel infiltration into the Matrigel plugs was analysed by platelet endothelial cell adhesion molecule (PECAM) staining. The control CT17-scFv plugs contained numerous vessels surrounding the TUBO microtumors (C & E). In contrast, in B06-scFv treated plugs vascularisation was detected primarily in the surrounding normal tissues (D & F). Bar diagram (G) shows vascular density of matrigel plugs stained for the endothelial marker PECAM. (Size bars = 125 μm and 20 μm).
Figure 11 - B06 scFv inhibits endothelial migration and filopodial extension in the neonatal retina. CT17-scFv- or B06-scFv were injected intra-ocularly at P4 (post-natal day 4) (A) 24 hours after injections, retinas were harvested and flat mounted before staining with isolectin-B4. A doubled arrow indicates spreading distance as measured from the optic disc. No detectable difference in vascularisation was observed between scFv-CT17 injected eyes or un-injected contra-lateral eyes whereas significant inhibition of vessel migration was detected in B06-scFv treated eyes (A & C). Quantification of filopodial extensions showed that B06-scFv markedly inhibited the number of extensions/ 100 μm membrane (B & D) whereas no effect on the number of branchpoints was detected (E). (Size bar = 20 μm).
Figure 12 - B06-Fab PEG inhibits choroidal neovascularization and plaque formation.
(A) Photographs are shown of mouse choroids at 10 days after laser photocoagulation and intraperitoneal injections of pegylated control CT- 17 control or B06-Fab polyethylene glycol (PEG) antibodies. Vascularisation was visualised by PECAM staining. Isolectin-B4 shows the outline of the plaques. (B) Vascularisation was quantitated as described in Methods.
EXAMPLE 1 - Angiomoήn binding antibodies isolated from n-CoDeR library
Three consecutive selection rounds using the n-CoDeR ® antibody library (Biolnvent, Sweden) were performed on lysate from Angiomotin transfected cell lines in combination with purified Angiomotin and Angiomotin expressing cell lines. The selected antibodies displayed on phage were converted to the single chain (scFv) format and 10 000 clones were screened for binding on purified Angiomotin and Angiomotin expressing cells. More than 100 unique Angiomotin binders, determined by sequencing, were found. Figure 1 show the variable domains, VH and VL, for two Angiomotin binding clones, AM-19-B06 and AM- 18-A03, selected from the n-CoDeR ® library. The CDR regions identified by Kabat system are underlined.
In Figure 2, the binding of Angiomotin specific antibodies, IgGl AM-19-B06 and IgGl AM-18-A03, to human p80 Angiomotin (p80 hAmot) was measured in an ELISA. Purified p80 hAmot was coated in the wells and the amount of bound anti-Amot antibodies was detected using polyclonal anti-human IgGl antibodies. The Angiomotin specific antibodies have a dose dependent binding to p80hAmot while the n-CoDeR ® control antibody does not bind to Angiomotin. The specificity for Angiomotin for IgGl AM-19-B06 and IgGl AM-18-A03, analysed by flow cytometry, is shown by the low binding to mock transfected CHO cells and a specific binding to p80 hAmot expressed on the surface of CHO cells, Figure 3. In the flow cytometry, the antibodies were directly labeled using the Alexa Fluor 647 Zenon Human IgG labeling Kit. CHO cells were harvested using
a non-enzymatic cell dissociation buffer and stained at 2x10 6 cells/ml in 0.2 ml for 60 minutes at 10° C. Cells were counter stained with the dead-cell stain SYTOX Green and non-viable cells were excluded from data analysis.
EXAMPLE 2 - In vivo test of Fab and PEGFab in a CNV model
Experimental choroidal neovascularisation (CNV) in the mouse is induced through rupture of Bruch's membrane by krypton laser photocoagulation as follows.
C57BL/6J mice are fixed on a rack connected to the slit lamp delivery system. Three krypton laser photocoagulation burns (50 μm spot size, 0.1 s duration, 120 mW power) are induced in each eye using a handheld contact lens. Only eyes in which a subretinal bubble is formed for each burn, indicating rupture of Bruch's membrane, are analyzed. The subsequent CNV lesions develop 7-10 days after laser induction.
CNV lesions are analyzed 10-14 days after laser-induction. Quantification of lesions is done histological sections (mean relative height of lesions is measured on serial sections) or isolectin B4/CD31 -labelled choroidal flat-mounts (analyzed parameters are: height of the CNV lesion, isolectinB4 positive area, CD31 positive area and ratio between CD31/isolectinB4). Angiomotin is targeted with Fab (19B06 clone isolated in example 1) or PEGFab (19B06 clone isolated in example 1). The Fab was administered as 5 intravitreal injections for 10 days, first injection immediately after laser treatment, 8 μg Fab/injection. The PEGFab was administered as intraperitoneal injections every second day for 10 days, first injection the day before laser induction, 400 μg PEGFab/iηjection.
Ten days after laser-induction, CNV was found to have been inhibited (p<0.05) with both the Fab and the PEGFab.
These results in a clinically relevant mouse model indicate angiomotin as a potential target for inhibition and regression of CNV in patients with neovascular
AMD. These results show that an antibody against anti-p80 angiomotin inhibits CNV.
EXAMPLE 3 - Preferred pharmaceutical formulations and modes and doses of administration.
The polypeptides, polynucleotides and antibodies of the present invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
The polypeptides, polynucleotides and antibodies of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.
Electroporation therapy (EPT) systems can also be employed for administration. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
Polypeptides, polynucleotides and antibodies of the invention can also be delivered by electroincorporation (EI). EI occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
An alternative method of administration is the ReGeI injectable system that is thermosensitive. Below body temperature, ReGeI is an injectable liquid while at
body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.
Polypeptides, polynucleotides and antibodies of the invention can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient (Derossi et al, 1998, Trends Cell Biol, 8, 84-87).
Preferably, the pharmaceutical formulation of the present invention is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.
The polypeptides, polynucleotides and antibodies of the invention can be administered by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.
In human therapy, the polypeptides, polynucleotides and antibodies of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical exipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
The polypeptides, polynucleotides and antibodies of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the
solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Generally, in humans, oral or parenteral administration of the polypeptides, polynucleotides and antibodies of the invention is the preferred route, being the most convenient.
For veterinary use, the polypeptides, polynucleotides and antibodies of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
The formulations of the pharmaceutical compositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
A preferred delivery system of the invention may comprise a hydrogel impregnated with a polypeptides, polynucleotides and antibodies of the invention, which is preferably carried on a tampon which can be inserted into the cervix and withdrawn once an appropriate cervical ripening or other desirable affect on the female reproductive system has been produced.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question.
EXAMPLE 4 - Exemplary pharmaceutical formulations
Whilst it is possible for a polypeptides, polynucleotides and antibodies of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen-free.
The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a polypeptides, polynucleotides and /or antibody of the invention.
Example 4A: Ophthalmic Solution
Active ingredient 0.5 g
Sodium chloride, analytical grade 0.9 g
Thiomersal 0.001 g
Purified water to 100 ml pH adjusted to 7.5
Example 4B: Capsule Formulations
Formulation A
A capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule. Formulation B {infra) is prepared in a similar manner.
Formulation B mε/capsule
Active ingredient 250
Lactose B.P. 143
Sodium Starch Glycolate 25
Magnesium Stearate 2
420
Formulation C mε/capsule
Active ingredient 250
Macrogol 4000 BP 350
600
Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.
Formulation D mε/capsule
Active ingredient 250 Lecithin 100
Arachis Oil 100
450
Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.
Formulation E (Controlled Release Capsule)
The following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule.
me/capsule
Active ingredient 250
Microcrystalline Cellulose 125
Lactose BP 125
Ethyl Cellulose 13
513
Example 4C: Injectable Formulation
Active ingredient 0.200 g
Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml
The active ingredient is dissolved in most of the phosphate buffer (35-40 ° C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.
Example 4D: Intramuscular injection
Active ingredient 0.2O g
Benzyl Alcohol 0.10 g
Glucofurol 75 ® 1.45 g Water for Injection q.s. to 3.00 ml
The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).
Example 4E: Syrup Suspension
Active ingredient 0.2500 g
Sorbitol Solution 1.500O g Glycerol 2.0000 g
Dispersible Cellulose 0.0750 g
Sodium Benzoate 0.0050 g
Flavour, Peach 17.42.3169 0.0125 ml
Purified Water q.s. to 5.0000 ml
The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.
Example 4F: Suppository mg/suppository
Active ingredient (63 μm)* 250 Hard Fat, BP (Witepsol H 15 - Dynamit Nobel) 1770
2020
*The active ingredient is used as a powder wherein at least 90% of the particles are of 63 μm diameter or less.
One fifth of the Witepsol Hl 5 is melted in a steam-jacketed pan at 45 0 C maximum. The active ingredient is sifted through a 200 μm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol Hl 5 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 μm stainless steel screen and, with continuous stirring, is allowed to cool to 40°C. At a temperature of 38°C to 40°C 2.02 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.
Example 4G: Pessaries mε/pessary
Active ingredient 250 Anhydrate Dextrose 380
Potato Starch 363
Magnesium Stearate 7
1000
The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.
Example 4H: Creams and ointments
Described in Remington, The Science and Practise of Pharmacy, 19* ed., The Philadelphia College of Pharmacy and Science, ISBN 0-912734-04-3.
Example 41: Microsphere formulations
The compounds of the invention may also be delivered using microsphere formulations, such as those described in Cleland (1997, Pharm. Biotechnol. 10:1- 43; and 2001, J. Control. Release 72:13-24).
EXAMPLE 5 - Targeting angiomotin with therapeutic antibody B06 inhibits physiological and pathological angiogenesis in vivo
Material and methods
Generation of human recombinant antibodies to Amot
Single chain human antibody fragments with specificity for human Amot were selected from the single-chain fragment-variable (scFv) n-CoDeR phage display library, essentially as described earlier (Soderlind, et al, 2000).
hi brief, human p80 Amot was expressed in eukaryotic cells and binding assays were performed with either intact cells, cell lysate or in a purified form, in 3 consecutive rounds of selection. Selected scFv were screened for specific Angiomotin binding in an automated system with an ELISA format with luminescence as the readout.
The scFv identified as being specific for Angiomotin were also screened for cross reactivity to murine Amot with an ELISA format with luminescence as the readout. The recombinant scFv contained a C-terminal His tag that allowed for IMAC purification using Ni-NTA sepharose (Qiagen). The purity of the preparations exceeded 95 %, as determined by SDS-PAGE. Fab fragments and full length IgG antibodies were produced by cloning into modified pcDNA3
vectors followed by transient transfections into HEK293 cells with Lipofectamin (Invitrogen).
Fab fragments and full length IgG antibodies were purified on a MabSelect protein A column (Amersham Biosciences). The purity of the preparations exceeded 98 %, as determined from SDS-PAGE. The binding specificity of the scFv was tested using luminescence-based ELISA where dilutions of the scFv antibody fragments were incubated in test plate wells coated with purified Angiomotin.
The Fab fragments utilised for PEGylation contained a C-terminal cystein allowing for a single site specific PEGylation using a 2O kD PEG-maleimid compound (Nectar Therapeutics). 2-Mercaptoethylamine HCL (Fluka), 5 mM, pH 7.0 at 37 °C for 90 min, was used for selective reduction of the C-terminal cysteins of the Fab fragments and the molar ratio (PEG:Fab) in the PEGylation reaction was 5:1. The PEGylation reaction was conducted at 25°C, pH 7 under nitrogen for 2 h. MabSelect protein A column chromatograpy was used to purify the Fab fragments from unreacted PEG-maleimid. PEGylated Fab could be separated from non-PEGylated Fab using size exclusion chromatography (GE Healthcare, Sweden).
Cell culture
Spontaneously immortalised mouse aortic endothelial (MAE) cells (Bastaki, et al., 1997) transfected with angiomotin or vector (Troyanovsky, et al., 2001) were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma, Sweden) containing 10 % fetal bovine serum (FBS, Gibco, Sweden), 1 % penicillin and 1 % glutamine. The TUBO cell line, kindly provided by Dr. Guido Forni (Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy), was derived from a spontaneous mammary tumour that arose in a BALB NeuT transgenic mouse expressing a transforming rat neu oncogene (Rovero, et al., 2000). Cells were cultured in Iscoves Modified Dubecco's Medium (IMDM, Sigma, Sweden) with 10 % FBS. hTERT + -immortalised bovine capillary endothelial (BCE) cells (Veitonmaki, et al, 2003) were grown in DMEM (Sigma,
Sweden) with 10 % fetal bovine serum, 2 Hg HiT 1 FGF-2 (Peprotech, London, UK), 1 % glutamine and 1 % penicillin/streptomycin.
FACS analysis For evaluation of cell surface binding by flow-cytometry, MAE cells were incubated with individual scFv clones at a concentration of 10 μg ml "1 in PBS (Invitrogen) containing 0.5 % w/v BSA (DPBS-B) for 1,5 hr. Detection of scFv binding was achieved by incubation with anti-flag-biotin (Sigma, Sweden) followed by Streptavidin-Alexa 647 Fluor (Molecular probes). Living cells were defined as negative for SYTOX Green Nucleic Acid Stain (Molecular probes). All incubations were performed on ice.
Tissue cross reactivity
Antibodies were tested at concentrations of between 5 and 20 μg ml "1 towards acetone fixed frozen sections (8 μm) of normal human tissues (placenta from 3 individuals, liver, kidney, heart, pancreas, lymph node and cerebrum from 2 individuals). The human IgGs were pre-incubated in tubes with a biotinylated monovalent goat Fab anti-human IgG fragment (Jackson, cat. No. 109-067-003) prior to the staining procedure.
Staining was performed using the avidin-biotin complex (ABC) method. Slides were evaluated under light microscope (Nikon, Labophot-2) and photos were taken with a Leica DMR microscope. Monoclonal mouse anti-human CD34 class II, clone QB End (Dakocytomaiton, code no. M7165) was used as a positive control antibody while an nCoDeR derived IgG 1 directed towards FITC was used as a negative control.
Migration assay
Migration assays were performed in a modified Boyden chamber using a 48-well chemotaxis chamber (Neuroprobe Inc., Gaithersburg, MD) as described earlier (Kundra, et al., 1995). Briefly, 8 μm Nucleopore polyvinylpirrolidine-free polycarbonate filters were coated with 100 μg ml "1 of collagen type 1 (Cohesion, Palo Alto, CA) overnight. hTERT + -BCE cells were starved in 0.2 % FCS- DMEM for 16 h. The cells were trypsinised, resuspended in DMEM containing
0.1 % bovine serum albumin (BSA) and 30,000 cells were added with or without B06 scFv or control scFv to each well of the upper chamber.
FGF-2 (Peprotech EC Ltd, Rocky Hill, NJ) at 30 ng ml "1 or VEGF at 50 ng ml "1 (Peprotech EC Ltd, Rocky Hill, NJ) were used as chemo-attractants in the lower chambers. The chemotaxis chambers were incubated for 3-5 h at 37 °C with 10 % CO 2 to allow cells to migrate through the collagen-coated polycarbonate filter. Non-migrating cells on the upper surface of the filter were removed and the filter was stained with Giemsa Stain (VWR International Ltd, West Chester, PA).
The total number of migrated cells per field was counted at * 20 magnification; each sample was tested in quadruplicates in at least three independent experiments.
Matrigel assay in vitro
150 μl of liquid Matrigel (Becton Dickinson, Biosciences) was added to each well of 8-well chamber slides (BD Falcon ™) and incubated at 37 0 C for 30 min to allow the gel to polymerise. 1,5 x 10 5 mouse aortic endothelial cells (MAE) maintained in serum-free DMEM with 0,5 % BSA were pre-treated for 16 hours with 5 μg ml "1 of single chains (Bioinvent International, AB) and seeded on a layer of polymerised matrigel as previously described (Troyanovsky, et ai, 2001). After 24 hours the changes in cell morphology were examined using a phase- contrast microscope.
Matrigel plug assay in vivo
Matrigel plug assays were performed as described previously with some modifications (Passaniti, et al., 1992). BALB/c mice were anaesthetised with Isofluran (Forene ® , Abbot, Sweden) and injected with matrigel mixture subcutaneously in the abdominal midline.
In the FGF-2-induced angiogenesis model, every matrigel plug contained 0,5 ml of Matrigel (Becton Dickinson, Biosciences), 200 ng ml "1 FGF-2 (Peprotech, London, UK), 250 μg of control antibodies in one group and 250 μg of B06 scFv in another.
Two control groups of mice were injected either with matrigel containing only FGF-2 or matrigel alone. Matrigel plugs were excised seven days after implantation, photographed and processed for histological studies.
In the tumour-induced angiogenesis model every matrigel plug contained 75,000 TUBO cells and 500 μg of single chain antibodies in 0,5 nύ of liquid Matrigel (Becton Dickinson, Biosciences). On day seven after gel implantation matrigel plugs were removed and prepared for immunohistochemical examination. Monoclonal rat anti-mouse CD31 (BD Pharmingen™) and anti-rat-FITC- conjugated (Jackson, Immunoresearch) antibodies were used to visualise vascularisation of matrigel plugs.
To analyse the microvessel density three images from every matrigel plug were taken with a Zeiss Axioplan 2 fluorescence microscope. The vessels were counted under the microscope at 20 x magnification.
Retinal angioRenesis assay
Intraocular injections were performed as described previously (Gerhardt, et ai, 2003). Briefly, pups (P4) were anaesthetized by isofluran inhalation. Injections (0.5 μl of ~5 μg μl "1 ScFv BO6 or ScFv CTl 7 in PBS) were performed using 10 μl gastight Hamilton syringes equipped with 34 gauge needles attached to a micromanipulator. Three litters of C57B16/J mice were treated, 10 pups per group. The uninjected eyes served as additional control. After 24h, pups were euthanised, eyes collected and fixed in 4 % PFA, and retinas were dissected and treated as described previously (Gerhardt, et ah, 2003).
For immunohistochemistry, endothelial cells and microglial cells were visualised with biotinylated isolectin B4 {Bandeiraea simplicifolia; Sigma-Aldrich) followed by streptavidin Alexa Fluor 488 (Molecular Probes, Invitrogen). Retinas were flat-mounted and analysed using a Zeiss SVI l fluorescence stereomicroscope equipped with an Axiocam HRc. The distance from optic nerve to vascular front was measured using Axiovision 4.5 software (Zeiss, Imaging Associates Ltd).
Images for filopodia analysis were taken on a Zeiss LSM 510 confocal microscope using 40 x 1.2 NA lens (settings: pinhole 1 airy unit, 1024 x 1024 pixel). 10 z- images were collected at 0.4 μm interval and presented as projection. Projection- images were converted to grey scale and inverted to facilitate filopodia visibility. The outline of the vessels at the migration front was measured and filopodia counted using ImageJ 1.36b (NIH, public domain software). Data were analysed by unpaired two-tailed T-test and graphed using Prism 4 software (GraphPad).
Laser-induced CNV in Mice CNV was generated by krypton laser-induced rupture of Bruch's membrane, as previously described (Berglin, et ai, 2003). Briefly, three krypton laser photocoagulation burns (50 μm spot size, 0.1 s duration, 12O mW power) were induced in each eye of C57BL/6J mice using a handheld contact lens (647 nm, Spectra-Physics 265 Exciter, Lasertek, Helsinki, Finland).
Mice received IP injections with 400 μg PEG FabB06 or CTl 7 every second day with the first injection given one day before laser treatment. Eyes were enucleated 10 days after laser treatment and fixed in 4 % paraformaldehyde for 30 min, the cornea and lens were removed and the entire retina was carefully dissected from the eyecup. The RPE-choroid-sclera eyecups were rinsed in PBS, permeabilised in 0.5 % Triton X-100 and blocked with 3 % goat serum in PBS/Triton X-100.
The eyecups were incubated with biotinylated isolectin B4 (1: 100 dilution, lectin from Griffonia simplicifolia, Sigma, CA, USA) and anti-CD31 (BD Biosciences Pharmingen, CA, USA) overnight at 4 0 C, followed by incubation with Texas Red Streptavidin (1:100 dilution, Vector Laboratories, Burlingame, CA, USA) and Alexa 488 goat anti-rat (1:100, Molecular Probes, OR, USA). Radial cuts were made from the edge of the eyecup to the equator and the eyecup was flattened and mounted with antifade medium (Vectashield Mounting Medium, Vector Laboratories, Burlingame, CA, USA) with the sclera facing down and the choroid facing up. Flat mounts were examined with a fluorescence microscope (Axioskop 2, Carl Zeiss, Jena, Germany), images were captured with a digital camera (Carl
Zeiss, Jena, Germany) and farther analysed using Axio Vision LE software (Carl Zeiss, Jena, Germany).
Lesions were manually measured in a masked fashion and data from each lesion was treated as a single statistical point. The outline of isolectin B4 staining was used to estimate the total plaque area and the vascularisation was estimated from the PECAM staining by quantifying the number of PECAM-positive pixels per plaque.
Results
Recombinant human p80 angiomotin (Amot) protein was used for panning against a human scFv phage antibody library (n-CoDeR) (Soderlind, et al., 2000). Three consecutive selections generated over 93 unique phage antibody clones. The individual single chain clones were converted to scFv format, and expressed in Escherichia coli.
Binding of purified scFv to both human and mouse Amot was assessed by ELISA as described in materials and methods and as shown in Figure 6 A and B. Cross- reactivity to mouse Amot was of importance as future screenings included mouse angiogenesis model systems. Another criterion was the ability of Amot-specific scFv to bind extra-cellular epitopes of angiomotin. FACS analysis showed cell surface binding of the B06 single chain Fv (BO6 scFv) to p80 Amot transfected cells (Figure 6 C).
In order to further verify the specificity of binding, cross-reactivity was examined to a limited panel of human normal tissues (Table 1). The B06 antibody bound only to cytotrophoblasts of the placenta, cells that previously have been shown to express high levels of Amot (Troyanovsky, et al., 2001).
Table 1 - Analysis of B06 binding to normal human tissues.
Fixed frozen sections of normal human tissues were tested for anti-angiomotin IgG) binding at 5 and 20 μg ml "1 . The human IgGs were pre-incubated in tubes with a biotinylated monovalent Fab fragment goat anti-human IgG before adding to the sections. Staining was performed using the avidin-biotin complex (ABC) method.
Angiomotin specific single chains inhibit migration and tube formation in vitro. For functional analysis, C-terminal His-tagged scFv was produced in HEK 293 cells as described above. Transfected mouse aortic endothelial (MAE) cells were used for screening for inhibitory migratory activity of angiomotin-reactive single chains. The MAE cells lack detectable endogenous expression of angiomotin, whereas transfection of p80 Amot promotes migration and renders the cells responsive to angiostatin treatment (Troyanovsky, et al., 2001). Non-specific inhibition of motility is excluded by parallel testing of the MAE-vector control cell line.
Over 83 scFv were screened in the Boyden chamber assay where 40 % (37/83) of the single chains showed marked inhibition of fibroblast growth factor (FGF)- induced migration. The B06 scFv was chosen as a prime candidate due to its low cross-reactivity to normal human tissues and the relatively high potency in inhibiting both bFG-2 and vascular endothelial growth factor (VEGF)-induced migration (Figure 7A and B, B06 IC 50 =IOO pg/ml, angiostatin IC 50 =500 ng/ml, (Troyanovsky, et al., 2001)). In addition, B 06 scFv inhibited migration of bovine
capillary endothelial cells that express endogenous angiomotin (Figure 7C) (Bratt, et al., 2005).
The ability of B06 scFv to inhibit tube formation was also assessed using the matrigel assay in vitro. MAE-p80 cells were plated on matrigel in the presence of either B06 scFv or the control single chain (CTl 7 scFv). In this assay, cells adhere to the matrix and spontaneously migrate to form an interconnecting tubular network within six hours. B06 scFv treated cells adhered to the matrix and formed aggregates but did not migrate out to form tubes (Figure 8), similar to what had previously been observed with angiostatin treatment (Troyanovsky, et al., 2001). No inhibition of tube formation was detected in the angiomotin- negative control cells.
Inhibition of angiogenesis in vivo.
To assess anti-angiogenic efficacy in vivo, an assay in which host endothelial cells invade implanted Matrigel plugs to form a capillary network, was utilised. Matrigel is a solubilised basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) murine sarcoma, which is liquid at 4 °C but solidifies at 37 0 C and allows slow release of growth factors that stimulate ingrowth of vessels. The vessels inside the plug have previously been shown to express high levels of angiomotin whereas the vessels in surrounding tissues are negative (Holmgren, et al., 2006).
Each mouse was injected with 0.5 ml of matrigel containing 200 ng ml "1 of basic fibroblast growth factor (bFGF) in combination with 500 μg ml "1 of CTl 7 scFv or B06 scFv. The animals were euthanised seven days after matrigel implantation and were analysed by platelet endothelial cell adhesion molecule (PECAM) staining (Figure 9). Analysis of the vascular density showed an almost complete inhibition of angiogenesis B06-scFv containing plugs as compared to the CTl 7- scFv control plugs (Figure 9B).
Typically, VEGF has a modest stimulatory effect on angiogenesis in the matrigel plug assay even when applied at high concentrations. To circumvent this problem
angiogenesis was induced by embedding TUBO breast cancer cells into the matrigel. TUBO cells originate from a cloned cell line generated from a spontaneous mammary gland carcinoma of a Balb-neuT mouse and induce tumours that are very similar to the alveolar type human lobular mammary carcinomas (Di Carlo, et al., 1999). They have been shown to induce angiogenesis via VEGF-dependent pathways (Melani et al., (2003) Blood 102 pp2138-2145).
After excision, visual observation showed extensive vascularisation and hemorrhage of the control plugs containing the control scFv whereas those containing scFv B06 were more translucent (Figure 10 A and B). This was consistent with the PECAM staining of cryosections, which revealed a 70 % inhibition of vascular infiltration in B06 scFv treated plugs (Figure 1OC, E and G).
These data suggested that local administration of the B06 scFv efficiently inhibited FGF-2 and tumour (VEGF)-driven angiogenesis.
Inhibition of neonatal retinal angiogenesis.
Vascularisation of the mouse retina has been used as a model system to study physiological angiogenesis due to the relative ease of visualisation of the vessels. The mouse retinal vasculature develops a network originating from the optic nerve which spreads to vascularise the retina within eight days (Gerhardt, et al., 2003). The vessel migration is orchestrated by matrix bound VEGF which is detected by the filopodia of tip cells at the leading edge of the migrating vessels (Gerhardt, & Betsholtz, et al., 2005). Angiomotin expression has previously been found in blood vessels during vascularisation of the postnatal mouse retina (Bratt, et al., 2005).
In order to assess the effect of B06 scFv on the vessel expansion and migration, four-day old pups were treated with intra-ocular injections of either B06 scFv or CTl 7 scFv. Animals were euthanised 24 h later and the retinas were processed for whole mount immunofluorescent staining with isolectin B4.
Expansion of the retinal network was estimated by measuring the distance from the optical nerve to the leading edge. Treatment with B06 scFv significantly
inhibited the migration of vessels during the 24 h period (Figure 11). In contrast, no effect on the vascular expansion was observed in CTl 7 scFv treated eyes as compared to untreated eyes, demonstrating that the single chains or the intraocular injections per se, do not affect retinal angiogenesis (Figure 11 E). Analysis of the vessel morphology revealed no differences in the number of vessel branch points. However, treatment with B06 scFv resulted in a significant reduction in the number of filopodia extensions in the tip cells (Figure 1 1 B and D).
Inhibition of choroidal neovascularisation Age-related macular degeneration is a major cause of severe visual loss in elderly patients. Retinal damage is often associated with subretinal plaque formation as a consequence of choroidal neovascularisation (CNV).
In this example, the effect of angiomotin intervention in a mouse model where CNV is induced by disruption of the Bruch's membrane/retinal pigment epithelium complex by laser treatment was studied. Mice were treated systemically with intra-peritoneal injections every second day using B06 or CTl 7 Fab fragments conjugated to poly-ethylene glycol (PEG). PEGylation was used to increase the half-life of the Fab antibodies in circulation.
Mice were euthanised on day 10 and their eyes were processed for whole mount staining using both isolectin B4 and PECAM-I. Vascularisation was estimated from the PECAM staining by quantifying the number of PECAM-positive pixels per plaque. Systemic treatment with B06 PEG-Fab resulted in a 73 % reduction of plaque vessels as compared to the animals treated with the CTl 7 PEG-Fab control.
In this example of the current invention, it was shown that a recombinant human antibody raised against angiomotin mimics the effects of angiostatin. This antibody specifically inhibited endothelial migration in vitro and affected filopodia formation in vivo. Furthermore evidence was provided from three independent angiogenesis models in vivo that this angiomotin antibody efficiently inhibits angiogenesis when administered locally or systemically.
References
Bastaki, M. et al, (1997) Arterioscler Thromb Vase Biol 17: 454-464.
Berglin, L. et al, (2003) Invest Ophthalmol Vis Sci 44: 403-408. Bratt, A. et al., (2005) J Biol Chem 280: 34859-34869.
Di Carlo, E. et al, (1999) Lab Invest 79: 1261-1269.
Gerhardt, H. et al, (2003) J Cell Biol 161: 1 163-1177.
Gerhardt, H., and Betsholtz, C. (2005) Exs 94: 3-15.
Holmgren, L. et al, (2006) Proc Natl Acad Sci USA 103: 9208-9213. Kundra, V. et al, (1995) J Cell Biol 130: 725-731.
Passaniti, A. et al, (1992) Lab Invest 67: 519-528.
Rovero, S. et al, (2000) J Immunol 165: 5133-5142.
Soderlind, E. et al, (2000) Nat Biotechnol 18: 852-856.
Troyanovsky, B. et al, (2001) J Cell Biol 152: 1247-1254. Veitonmaki, N. et al, (2003) FASEB J 17: 764-766.
Next Patent: ANGIOMOTIN SIRNA MOLECULES AND USES THEREOF