Stable and potent anti-angiogenic scFv fragments and their use as VEGF antagonists

Abstract

A functional antibody fragment is disclosed and claimed as an anti-angiogenic scFv fragment, and also as an inhibitor of VEGF: VEGFR interaction by binding as a VEGF antagonist to the VEGF protein. In addition, the present invention relates to a method of treating intraocular neovascular disease using said functional fragment. In addition, a method for producing the antibody fragments of the invention is provided.

Description

Technical Field

[0001] The present invention discloses and claims a functional antibody fragment as an anti-angiogenic scFv fragment and also as an inhibitor of VEGF:VEGFR interaction by binding to VEGF protein as a VEGF antagonist. In addition, the present invention relates to a method for treating intraocular neovascularization disease using the functional fragment. Pharmaceutical compositions comprising the functional fragment and methods for preparing formulations are also disclosed and claimed. In addition, a method for producing the antibody fragment of the present invention is provided. Background Art

[0002] Vascular endothelial growth factor (VEGF or VEGF-A) is a signaling protein that plays an important role in angiogenesis. VEGF has been widely studied as a key therapeutic target for inhibiting angiogenesis. The main signaling event in angiogenesis is the binding of VEGF to VEGF receptor (VEGFR), and the activity of VEGF is the rate-limiting step in normal and pathological angiogenesis.

[0003] Angiogenesis is involved in the pathogenesis of neovascular diseases in the eye, such as age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is the most common cause of vision loss in the elderly population, especially those over 50 years old. Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of AMD. VEGF drives the development of choroidal neovascularization, in which new blood vessels grow through the retinal pigment epithelium. Wet AMD is caused by new blood vessels that leak fluid into the retina and cause it to become wet. VEGF accumulation in wet AMD can be treated with anti-VEGF therapeutics. This process can be weakened or stopped by administering drugs that bind to and inhibit VEGF into the eye (ocular injection). Currently, there are several anti-VEGF therapeutics available for the treatment of wet AMD, including brolucizumab Aflibercept Ranibizumab Pegaptanib Sodium

[0004] Single-chain variable fragments (scFv) are a type of recombinant antibody. Antibodies have two variable domains (heavy and light) in their structure that exert antigen binding activity. scFvs consist of two variable heavy and light chains connected by a peptide linker. They are a single polypeptide of approximately 25 kDa that contains the variable light (VL) and variable heavy (VH) chains of an antibody. The two chains are connected by a flexible linker peptide that is typically 15-20 amino acids long and consists of glycine (Gly or G) and serine (Ser or S) with hydrophilic residues to increase solubility [1]. It can be assumed that the variable domains of scFv fragments will be similar to those of antibodies (VL-linker-VH), but both VL-linker-VH and VH-linker-VL configurations can generate functional scFvs. In addition, some individual scFvs perform better in one configuration than the other [2].

[0005] Since scFvs are smaller than conventional antibodies, they have many advantages in therapeutic applications, such as improved tissue penetration (useful for therapeutic and imaging applications), rapid blood clearance (useful for imaging applications), reduced immunogenicity when used in vivo due to the lack of an Fc region, and less difficult production processes in microbial systems due to the lack of glycosylation. In addition, scFvs are cheaper and easier to manufacture because they can be expressed in microbial systems, whereas mAbs generally require mammalian expression systems [3,4].

[0006] Compared to antibodies, scFvs may have some disadvantages related to stability, production yield, potency, and activity. scFvs tend to have lower long-term stability, lower affinity, and are more susceptible to aggregation due to their smaller size [4]. These disadvantages still limit the generation of functional scFvs.

[0007] U.S. Patent No. US 9149427B2 discloses a cell line comprising ARPE-19 cells that have been genetically engineered to produce a therapeutically effective amount of one or more anti-angiogenic polypeptides or anti-angiogenic molecules. In this document, a novel cell line is described, but no technical advantages of scFv proteins are claimed. U.S. Patent No. US8936785B2 discloses a new therapeutic approach for eye drops for the scFv protein. scFv may have some disadvantages related to their stability, production yield and activity. In response to these technical issues, the in vitro and in vivo advantages of these scFv proteins are not described.

[0008] Therefore, there is still a need to design a new anti-VEGF antagonist based on scFv fragment to overcome the above technical problems. The present invention meets these needs and other needs. The scFv sequence involved in the present invention exhibits technical advantages: (i) higher thermal stability, (ii) higher production yield and (iii) more effective anti-angiogenic activity in an in vivo zebrafish animal model. Summary of the invention

[0009] Thus, broad embodiments of the present invention relate to stable, soluble and potent VEGF-binding functional antibody fragments.

[0010] According to a first aspect of the present invention, a functional fragment that specifically binds to vascular endothelial growth factor (VEGF) protein is provided.

[0011] Another aspect of the present invention relates to a functional fragment, wherein the functional fragment has (i) high thermal stability, (ii) high production yield and (iii) high anti-angiogenic activity in an in vivo zebrafish model.

[0012] Another aspect of the present invention is to provide a functional fragment for treating and / or preventing intraocular neovascularization diseases or disorders, wherein the fragment is characterized by binding to and inhibiting vascular endothelial growth factor A (VEGF).

[0013] The present invention can be used to prepare a medicament (pharmaceutical composition) for treating and / or preventing disorders caused by inhibiting VEGF:VEGFR interaction and anti-VEGF activity.

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a functional antibody fragment.

[0015] In another aspect, the present invention relates to a method of administering a pharmaceutical composition to a subject, and provides a pharmaceutical composition for administering the same to a subject.

[0016] Another embodiment of the present invention is directed to a method and pharmaceutical composition for treating age-related macular degeneration (AMD).

[0017] Thus, a broad embodiment of the invention relates to an anti-VEGF therapy method, i.e., the use of drugs that block vascular endothelial growth factor. This is done in the treatment of certain cancers and age-related macular degeneration. The anti-VEGF therapy method can be used in clinical applications for ocular disorders and systemic diseases in which VEGF plays a major role.

[0018] In addition, a method for producing the antibody fragment of the present invention by microbial fermentation is also provided.

[0019] This and other objects of the present invention will become apparent from the following detailed discussion of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention is illustrated in the accompanying drawings, in which:

[0021] Figure 1 is a schematic diagram showing the vector map (the gene was purchased from GenScript and cloned into the pPICZαA vector).

[0022] Figure 2 The production of scFv in the fermentor at different time points is shown.

[0023] Figure 3 is a graphic representation of the thermal stability analysis of scFv proteins.

[0024] Figure 4 The SE-HPLC and SDS-PAGE spectra of scFv proteins (scFv1, scFv2, scFv3) compared with the reference scFv are shown. Their purity percentages are also indicated.

[0025] Figure 5 SPR sensorgrams of scFv proteins (scFv1, scFv2, scFv3) compared with a reference scFv.

[0026] Figure 6 is a graphic representation of the in vivo efficacy analysis of scFv proteins (scFv1, scFv2, scFv3) compared to a reference scFv in a zebrafish angiogenesis model. DETAILED DESCRIPTION

[0027] The present invention relates to the field of functional antibody fragments, their production by microbial fermentation, their solubility / stability / binding characteristics and their in vivo testing in the zebrafish model.

[0028] Therefore, the term "functional fragment" or "functional antibody fragment" as described herein refers to a fragment of Fv, scFv, Fab, F(ab')2, F(ab'), scFv-Fc type or diabody. In a preferred embodiment, the functional antibody fragment is a single chain variable fragment (scFv).

[0029] In addition, the present invention provides a method for producing the antibody construct of the present invention, the medical use of the antibody construct and a kit comprising the antibody construct. The form of the antibody construct comprising scFv as a functional fragment is selected from the group consisting of: (scFv)2, scFv-single domain mAb, double antibody and oligomers of the above forms.

[0030] As used herein, the term "inhibit, inhibition or inhibiting" refers to a significant decrease in the basal activity of a given condition, symptom, or disorder, or disease, or biological activity or process (such as VEGF:VEGFR interaction). Inhibitors of VEGF:VEGFR interaction are promising drug candidates for the treatment of systemic diseases, cancer (solid tumors) and various eye disorders.

[0031] The present invention discloses and claims a functional antibody fragment that acts as an anti-angiogenic scFv fragment and also acts as an inhibitor of VEGF:VEGFR interaction by binding to VEGF protein as a VEGF antagonist. In addition, it is a humanized monoclonal single-chain Fv (scFv) antibody fragment targeting VEGF protein.

[0032] According to the present invention, a single-chain variable fragment (scFv) comprises a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker.

[0033] In addition, the choice of peptide linker and the sequence arrangement of variable heavy / light chains are also important factors for the in vitro and in vivo properties of scFv. In the present invention, an optimal domain orientation and a flexible peptide linker with optimal content and length were developed.

[0034] According to the present invention, the single-chain variable fragment (scFv) includes scFv1, scFv2 and scFv3 respectively:

[0035] - a first binding domain of a variable light chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 1, a linker comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 2, and a second binding domain of a variable heavy chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 3; or

[0036] - a first binding domain of a variable light chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 4, a linker comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 5, and a second binding domain of a variable heavy chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 6; or

[0037] - A first binding domain of a variable light chain comprising in sequence the amino acid sequence of SEQ ID NO: 7, a linker comprising in sequence the amino acid sequence of SEQ ID NO: 8, and a second binding domain of a variable heavy chain comprising in sequence the amino acid sequence of SEQ ID NO: 9.

[0038] In a first embodiment, the single chain variable fragment (scFv) comprises scFv1 of the amino acid sequence of SEQ ID NO: 10, which consists of SEQ NO: 1, SEQ NO: 2 and SEQ NO: 3, respectively.

[0039] In a second embodiment, the single chain variable fragment (scFv) comprises scFv2 of the amino acid sequence of SEQ ID NO: 11, which consists of SEQ NO: 4, SEQ NO: 5 and SEQ NO: 6, respectively.

[0040] In a third embodiment, the single chain variable fragment (scFv) comprises scFv3 of the amino acid sequence of SEQ ID NO: 12, which consists of SEQ NO: 7, SEQ NO: 8 and SEQ NO: 9, respectively.

[0041] The primary structures of these scFvs (scFv1, scFv2, scFv3) are identical, but only the four amino acids marked in each sequence (as shown in the sequence listing) can be changed in different combinations (at position 83, F (phenylalanine; Phe) is changed to the amino acid E (glutamic acid; Glu) or vice versa, at position 12, S (serine; Ser) is changed to the amino acid M (methionine; M) or vice versa).

[0042] Therefore, the present invention relates to scFv (scFv1, scFv2, scFv3) that specifically binds to vascular endothelial growth factor (VEGF) protein, wherein the scFv has a high binding affinity to the VEGF protein, and wherein the scFv thereof binds to the VEGF protein with a dissociation constant (K) of less than 5 nM. D ) binds to VEGF protein as measured by surface plasmon resonance (SPR). In a preferred embodiment, the dissociation constant is less than 1 nM, more precisely between 0.01 nM and 1 nM.

[0043] In another embodiment, the present invention provides a scFv that binds to a VEGF protein with an association rate constant (k on ) is 7×10 4 M -1 s -1 or larger.

[0044] In addition, the present invention provides a scFv that binds to the VEGF protein, and its dissociation rate constant (k off ) is 3×10 -6 s -1 or smaller.

[0045] Dissociation constant (K D ), association rate constant (k on ) and the dissociation rate constant (k off) values ​​were calculated by fitting the kinetic association and dissociation curves to a 1:1 binding model.

[0046] In another embodiment, the invention provides an anti-VEGF scFv according to any one of the preceding embodiments, wherein the VEGF protein is a mammalian VEGF protein; more particularly, it is a human VEGF protein (Gibco Catalog No. PHC9391).

[0047] Furthermore, a method for producing an antibody fragment of the invention is provided.

[0048] According to the present invention, a method for producing scFv antibody fragments by a microbial fermentation process is provided, the method comprising the following steps:

[0049] - cultivating a host cell transformed with the DNA encoding said scFv under conditions allowing expression and production of said fragment,

[0050] - inoculating the culture into bioreactors to produce recombinant scFv at large scale under appropriate conditions in a step-wise scale-up approach,

[0051] - adjusting the pH with a pH adjusting agent which also serves as a nitrogen source,

[0052] - glycerol depletion in the culture (dissolved oxygen spike),

[0053] - After the dissolved oxygen spike, the fed-batch fermentation phase was started with 100% methanol or ethanol or glucose containing trace metal solutions,

[0054] - During the first 72 hours of fermentation, the feed rate of methanol or ethanol or glucose solution was increased exponentially and then kept constant during the last 24 hours of fermentation.

[0055] - Optimize temperature and pH during the batch phase,

[0056] - and recovering the supernatant from the fermentation culture at different times.

[0057] The present invention relates to a pharmaceutical composition comprising a humanized anti-VEGF scFv as described in any one of the aforementioned embodiments, for use in anti-VEGF therapy by inhibiting the VEGF:VEGFR interaction of a mammal, wherein the drug can be administered to the mammal in a therapeutically effective amount. Preferably, the mammal is a human, more preferably, the mammal is a human suffering from a tumor (solid tumor; cancer) or an eye disorder.

[0058] The present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of scFv as described in any one of the aforementioned embodiments and a pharmaceutically acceptable carrier. The composition is an aqueous solution for ophthalmic application. More preferably, the pharmaceutical composition is an ophthalmic solution containing phosphate buffered saline administered as eye drops or ocular injections.

[0059] The anti-VEGF effect of the scFv antibody fragment was found to be concentration-dependent. The concentration of scFv ranged from 20 μM to 100 μM.

[0060] The term "therapeutically effective amount" of a functional fragment of the present invention refers to a non-toxic and sufficient amount of the fragment of the present invention that will elicit a biological or medical response in a subject, for example, reducing or inhibiting protein:protein or protein:receptor interactions, or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing a disease or disorder, etc.

[0061] The pharmaceutical composition of the present invention as described in any of the aforementioned embodiments can be used to treat any VEGF protein-related disease or disorder, such as intraocular neovascularization disease.

[0062] In another embodiment, the pharmaceutical composition of the present invention as described in any of the preceding embodiments may be used to treat age-related macular degeneration (AMD), more preferably to treat the wet form of age-related macular degeneration (wet AMD).

[0063] In additional embodiments, the present invention is directed to a method of administering a pharmaceutical composition to a subject, and provides a pharmaceutical composition for administering the same to a subject.

[0064] All of the various embodiments of the invention disclosed herein are directed to methods of treating and / or preventing the ocular neovascular diseases or disorders described herein.

[0065] Non-limiting examples of ocular neovascular diseases include age-related macular degeneration (AMD), wet form of age-related macular degeneration (wet AMD), diabetic retinopathy, retinal vascular occlusion, retinopathy of prematurity.

[0066] These examples are intended to represent specific embodiments of the invention and are not intended to limit the scope of the invention.

[0067] Specific implementation plan

[0068] Stability, production yield, and potency are very important for scFv development. In these embodiments, we engineered several scFv proteins to achieve highly stable, high-yield, and efficient scFv proteins for therapeutic applications. Three scFv proteins (scFv1, scFv2, scFv3) have favorable properties compared to our reference antibodies. The protein yield of scFv3 is nearly 10 times that of the reference ( Figure 2 ), with >200 mg of purified scFv (MW: approximately 26.8 kDa) per liter of culture. The thermal stability of scFv1 and scFv2 was nearly 5°C higher than that of the reference ( Figure 3 After only one step of protein L affinity chromatography, the purity of scFv1 and scFv2 was 97.3% and 93.3%, respectively, while the purity of the reference product was 65% (Table 1, Figure 4 According to the surface plasmon resonance results, scFv1, scFv2, and scFv3 bound to VEGF more strongly than the reference product (Table 2, Figure 5 ). The anti-angiogenic efficacy was tested using a transgenic zebrafish model that induced GFP expression in endothelial cells. The results showed that scFv1, scFv2, and scFv3 were more effective than the reference, and among them, scFv3 had the highest in vivo activity, reducing the SIV area by 76.8% ( Figure 6 ).

[0069] Table 1. Purity analysis of scFv proteins by SE-HPLC

[0070]

[0071] In summary, we engineered anti-VEGF scFv fragments with enhanced:

[0072] 1. Thermal stability (thermal stability increase> 5℃)

[0073] 2. Higher production yields in microbial systems (>200 mg purified scFv per liter of culture). In addition, we achieved a purity of more than 95% after one-step purification.

[0074] 3. Higher anti-angiogenic efficacy in an in vivo zebrafish model (more than two times the efficacy)

[0075] Example

[0076] Example 1 - Production

[0077] Komagataella phaffii (Pichia pastoris) is used to express and produce soluble scFv proteins. A 5-liter bioreactor is used for large-scale production of recombinant scFv. A small bottle of frozen culture is inoculated into 200 mL of culture medium and cultured for about 20 hours at 28°C and 225rpm. 125 mL of culture is inoculated into 2.5 L of culture medium. The pH is adjusted with 25% ammonium hydroxide, which is also used as a nitrogen source. The batch phase lasts for about 15 hours and is completed (dissolved oxygen spike) as glycerol in the culture medium is depleted. After the dissolved oxygen spike signal, the fed-batch fermentation phase is started by 100% methanol or ethanol or glucose solution containing trace metals. During the first 72 hours of fermentation, the feed rate increases exponentially, and during the last 24 hours of fermentation, the rate remains unchanged. The temperature and pH of the batch phase are optimized to 18°C-30°C and 3.0-7.0, respectively. Within the first hour of the fed-batch phase, the temperature and pH values ​​are linearly related to the spectrum. The dissolved oxygen level was controlled at 30% saturation by adding air and pure oxygen with constant stirring. During the fed-batch phase, the supernatant was collected at different times. These samples were analyzed to measure the biomass level and to determine the protein produced by SDS-PAGE.

[0078] Example 2 - Purification

[0079] The filtered supernatant was loaded onto an affinity chromatography column (Protein L). With this first purification step, a purity of greater than 93% was achieved. A second purification step was applied to obtain >95% purity.

[0080] Example 3 - Size Exclusion Chromatography (SE-HPLC)

[0081] SE-HPLC was performed using an HPLC system with a UV-VIS detector and an analytical size exclusion chromatography column. The scFv sample was injected into the column. The absorbance value was monitored at 280 nm. Molecular weight standards were used to verify the retention time of the scFv protein.

[0082] Example 4 - Thermal stability determination

[0083] The thermal unfolding profile of the purified scFv protein was determined using fluorescent dyes. Optimal concentrations of dye and protein were used. The transition midpoint (T m value).

[0084] Example 5 - Surface Plasmon Resonance (SPR)

[0085] Affinity measurements were performed using a Biacore instrument. VEGF protein was immobilized on the chip. A series of solutions ranging from 1 nM to 100 nM scFv protein were then injected onto the VEGF-coated surface. Data were corrected by double referencing a control flow cell without VEGF and a flow cell injected with buffer. Sensorgram curves were analyzed. The dissociation constant (K D ), association rate constant (k on ) and the dissociation rate constant (k off ) values ​​were calculated by fitting the kinetic association and dissociation curves to a 1:1 binding model.

[0086] Table 2. scFv protein binding kinetics by surface plasmon resonance.

[0087] sample <![CDATA[K D (pM)]]> <![CDATA[k on (x10 4 M -1 s -1 )]]> <![CDATA[k off (x10 -6 s -1 )]]> Reference scFv* 26.1±8.2 7.646±0.002 1.990±0.008 scFv1 20.5±4.4 9.094±0.002 1.870±0.004 scFv2 17.2±6.0 8.793±0.001 1.520±0.006 scFv3 23.6±7.8 9.756±0.006 2.300±0.005

[0088] *The 10 sequences are shown in the sequence listing as SEQ ID No 10-19 respectively. The reference sequence is SEQ ID No 20.

[0089] Example 6 - Zebrafish Experiment

[0090] The zebrafish used in this study were provided by the Zebrafish Facility of Izmir Biomedicine and Genome Center. All animal procedures were approved by the Ethics Committee of Izmir Biomedicine and Genome Center (HADYEK), Izmir, Turkey. In addition, all experiments involving live animals were performed in accordance with relevant guidelines and regulations and reported as recommended in the ARRIVE guidelines.

[0091] Adult zebrafish were raised under standard conditions at the Zebrafish Facility of the Izmir Biomedicine and Genomic Center. Transgenic lines were crossed with wild-type to obtain embryos.

[0092] The estimated injection volume was determined using a stage micrometer. ScFv proteins were microinjected into the yolk of each anesthetized zebrafish embryo 2 days after fertilization. Microinjected larvae were stored at 28°C and 1 day after injection. The development of the subintestinal vascular (SIV) was examined. Larvae were fixed and melanocytes were depigmented with a pigment depigmentation solution.

[0093] Larvae were mounted laterally in low melting point agarose in imaging dishes and imaged using confocal microscopy. SIV area was measured and statistically analyzed.

[0094] References

[0095] [1].Monnier P,Vigouroux R,Tassew N(2013)In Vivo Applications ofSingle Chain Fv(Variable Domain)(scFv)Fragments.Antibodies 2:193-208.

[0096] [2].Sandomenico A,Sivaccumar JP,Ruvo M(2020)Evolution of Escherichiacoli Expression Systemin Producing Antibody Recombinant Fragments.IJMS21:6324.

[0097] [3].Ahmad ZA,Yeap SK,Ali AM,Ho WY,Alitheen NBM,Hamid M(2012)scFvAntibody:Principles and Clinical Application.Clinical and DevelopmentalImmunology 2012:1-1.

[0098] [4].Bates A,Power CA(2019)David vs.Goliath:The Structure,Function,andClinical Prospects of Antibody Fragments.Antibodies 8:28.

Claims

1. A single-chain variable fragment (scFv) that specifically binds to a vascular endothelial growth factor (VEGF) protein, wherein the scFv has a high binding affinity to the VEGF protein and comprises: - a first binding domain of a variable light chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 1, a linker comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 2, and a second binding domain of a variable heavy chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 3; or - a first binding domain of a variable light chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 4, a linker comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 5, and a second binding domain of a variable heavy chain comprising in sequence a sequence of the amino acid sequence of SEQ ID NO: 6; or - A first binding domain of a variable light chain comprising in sequence the amino acid sequence of SEQ ID NO: 7, a linker comprising in sequence the amino acid sequence of SEQ ID NO: 8, and a second binding domain of a variable heavy chain comprising in sequence the amino acid sequence of SEQ ID NO:

9.

2. The single-chain variable fragment of claim 1, wherein the VEGF protein is a mammalian VEGF protein.

3. The single-chain variable fragment of claim 2, wherein the mammalian VEGF protein is a human VEGF protein.

4. The single-chain variable fragment of claim 3, wherein the scFv binds to the VEGF protein with a dissociation constant of less than 5 nM. The single-chain variable fragment of claim 4 , wherein the scFv binds to the VEGF protein with a dissociation constant of less than 1 nM.

6. The single-chain variable fragment of claim 5, wherein the scFv is expressed in 7×10 4 M -1 s -1 or greater association rate constant binds to the VEGF protein.

7. The single-chain variable fragment of claim 6, wherein the scFv is expressed in 3×10 -6 s -1 or smaller dissociation rate constant (k off ) binds to the VEGF protein.

8. A pharmaceutical composition comprising the humanized anti-VEGF scFv as claimed in any one of the preceding claims for use in anti-VEGF therapy by inhibiting VEGF:VEGFR interaction in a mammal, wherein the medicament can be administered to the mammal in a therapeutically effective amount.

9. The pharmaceutical composition of claim 8, wherein the mammal is a human.

10. The pharmaceutical composition of claim 9, wherein the mammal suffers from a tumor.

11. The pharmaceutical composition of claim 9, wherein the mammal suffers from an ocular disorder.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the scFv according to any one of the preceding claims.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is an aqueous ophthalmic solution.

14. The pharmaceutical composition of claim 13, wherein the composition is used to treat and / or prevent age-related macular degeneration (AMD).

15. The scFv according to any one of the preceding claims, for use in the treatment and / or prevention of age-related macular degeneration (AMD).

16. An antibody construct comprising the scFv as claimed in any one of the preceding claims, wherein the antibody construct is in a form selected from the group consisting of: (scFv)2, scFv-single domain mAb, diabodies and oligomers of the above forms.

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