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

Abstract

Stable and potent anti-angiogenic SCFV fragments and their use as VEGF antagonists The present invention discloses and claims functional antibody fragments as anti-angiogenic scFv fragments and as inhibitors of the VEGF:VEGFR interaction by binding to the VEGF protein as VEGF antagonists. Furthermore, the present invention relates to methods of using said functional fragments to treat intraocular neovascular diseases. Furthermore, methods for producing the antibody fragments of the present invention are provided.

Description

[Technical Field]

[0001] The present invention discloses and claims functional antibody fragments as anti-angiogenic scFv fragments and as inhibitors of the VEGF:VEGFR interaction by binding to the VEGF protein as VEGF antagonists. The present invention also discloses and claims methods for using said functional fragments in the treatment of intraocular neovascular diseases. Also disclosed and claimed are methods for preparing pharmaceutical compositions and formulations containing said functional fragments. Additionally, methods for producing the antibody fragments of the present invention are provided. [Background technology]

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

[0003] Angiogenesis is involved in the development of intraocular neovascular diseases, such as age-related macular degeneration (AMD), which is the most common cause of vision loss, especially in people over the age of 50. Vascular endothelial growth factor (VEGF) plays a key role in the development of AMD. VEGF promotes the development of choroidal neovascularization, in which new blood vessels grow through the retinal pigment epithelium. Wet AMD is caused by leakage of fluid from the new blood vessels into the retina, resulting in retinal irritation. VEGF accumulation in wet AMD can be treated with anti-VEGF therapeutic drugs. This process can be slowed or stopped by administering drugs to the eye (intraocular injection) that bind to and inhibit VEGF. Currently, several anti-VEGF medications are available for the treatment of wet AMD, including brolucizumab (Beovu®), aflibercept (Eylea®), ranibizumab (Lucentis®), and pegaptanib sodium (Macugen®).

[0004] Single-chain variable fragments (scFvs) are a type of recombinant antibody. Antibody structures contain two variable domains (heavy and light chains) with antigen-binding activity. scFvs consist of two variable heavy and light chains connected by a peptide linker. These form a single polypeptide of approximately 25 kDa, containing the antibody's variable light (VL) and variable heavy (VH) chains. These two chains are connected by a flexible linker peptide, typically 15–20 amino acids long and composed of hydrophilic residues, glycine (Gly or G) and serine (Ser or S), to enhance solubility [1]. While the variable domain of an scFv fragment may be expected to mirror that of an antibody (VL-linker-VH), functional scFvs can be generated in both VL-linker-VH and VH-linker-VL configurations. Furthermore, some individual scFvs perform better in one configuration than the other [2].

[0005] Because scFvs are smaller than conventional antibodies, they offer many advantages for therapeutic applications, including improved tissue penetration (benefiting both therapeutic and imaging applications), rapid blood clearance (benefiting imaging applications), reduced immunogenicity upon administration due to the absence of an Fc region, and a simplification of the microbial production process (benefiting from the lack of glycosylation). Furthermore, scFvs can be expressed in microbial systems, making them inexpensive and easy to produce, whereas mAbs generally require mammalian expression systems [3,4].

[0006] Compared to antibodies, scFvs may have several drawbacks related to stability, yield, potency, and activity. They tend to have poor long-term stability, low affinity, and a higher likelihood of aggregation due to their small size. [4] The production of functional scFvs is still limited by these drawbacks.

[0007] U.S. Patent Publication No. US9149427B2 discloses cell lines comprising ARPE-19 cells genetically engineered to produce therapeutically effective amounts of one or more antiangiogenic polypeptides or molecules. This document describes the novel cell lines without claiming the technical superiority of scFv proteins. U.S. Patent Publication No. US8936785B2 discloses a new therapeutic route: eye drops of the scFv proteins. scFvs may have several drawbacks related to their stability, production yield, and activity. Regarding these technical issues, no discussion is given of the in vitro or in vivo advantages of these scFv proteins.

[0008] Therefore, there remains a need to design new anti-VEGF antagonists based on scFv fragments that overcome the above-mentioned technical problems. These and other needs are met by the present invention. The present invention relates to scFv sequences that exhibit the technical advantages of (i) higher thermal stability, (ii) higher production yield, and (iii) more potent anti-angiogenic activity in an in vivo zebrafish animal model. Summary of the Invention

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

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

[0011] Another aspect of the present invention relates to functional fragments that have (i) high thermostability, (ii) high production yield, and (iii) highly potent 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 neovascular diseases or disorders, characterized in that the fragment binds to and inhibits vascular endothelial growth factor A (VEGF).

[0013] The present invention can be used to prepare medicaments (pharmaceutical compositions) useful for inhibiting VEGF:VEGFR interaction and for treating and / or preventing diseases caused by anti-VEGF activity.

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

[0015] In further aspects, the invention relates to methods of administering pharmaceutical compositions to a subject, and to pharmaceutical compositions for administration to a subject.

[0016] Another embodiment of the present invention relates to methods and pharmaceutical compositions for treating age-related macular degeneration (AMD).

[0017] Therefore, a broad embodiment of the present invention relates to anti-VEGF therapy using drugs that inhibit vascular endothelial growth factor, which is used to treat certain cancers and age-related macular degeneration. Anti-VEGF therapy can be used in clinical applications for ocular and systemic diseases in which VEGF plays a major role.

[0018] Additionally, methods for producing the antibody fragments of the present invention by microbial fermentation are provided.

[0019] This and other objects of the present invention will become apparent from the detailed description of the invention that follows. [Brief explanation of the drawings]

[0020] The invention is illustrated in the accompanying drawings, in which: [Figure 1]Figure 1 is a schematic diagram showing the vector map (the gene was purchased from GenScript and cloned into the pPICZαA vector). [Figure 2] Figure 2 shows the production of scFv in the fermentor at different time points. [Figure 3] FIG. 3 is a diagram of the thermostability analysis of scFv proteins. [Figure 4] Figure 4 shows the SE-HPLC and SDS-PAGE profiles of scFv proteins (scFv1, scFv2, scFv3) compared to a reference scFv. The purity percentages are also shown. [Figure 5] FIG. 5 shows SPR sensorgrams of scFv proteins (scFv1, scFv2, scFv3) compared to a reference scFv. [Figure 6] FIG. 6 shows in vivo efficacy analysis of scFv proteins (scFv1, scFv2, scFv3) compared to a reference scFv in a zebrafish angiogenesis model. Detailed Description of the Invention

[0021] The present invention relates to the field of functional antibody fragments, their production by microbial fermentation, their solubility / stability / binding properties, and their in vivo testing in the zebrafish model.

[0022] Thus, the term "functional fragment" or "functional antibody fragment" as used herein refers to a fragment of the Fv, scFv, Fab, F(ab')2, F(ab'), scFv-Fc type or diabody type. In a preferred embodiment, the functional antibody fragment is a single-chain variable fragment (scFv).

[0023] Furthermore, the present invention provides a method for producing the antibody construct of the present invention, a medical use of the antibody construct, and a kit containing the antibody construct. The antibody construct containing an scFv as a functional fragment is in a format selected from the group consisting of (scFv)2, scFv-single domain mAb, diabody, and oligomers of the above formats.

[0024] As used herein, the terms "inhibit," "inhibition," or "inhibiting" refer to the reduction or suppression of a particular condition, symptom, disorder, or disease; or a significant decrease in the baseline activity of a biological activity or process, such as the VEGF:VEGFR interaction. Inhibitors of the VEGF:VEGFR interaction are promising candidates for the treatment of systemic diseases, cancer (solid tumors), and various ocular diseases.

[0025] The present invention discloses and claims functional antibody fragments as anti-angiogenic scFv fragments and as inhibitors of the VEGF:VEGFR interaction by binding to the VEGF protein as VEGF antagonists, and further humanized monoclonal single-chain Fv (scFv) antibody fragments targeting the VEGF protein.

[0026] According to the present invention, a single chain variable fragment (scFv) comprises a variable heavy chain (VH) and a variable light chain (VL) linked by a peptide linker.

[0027] Furthermore, the selection of peptide linkers and the sequence lineup of variable heavy and light chains are important factors for the in vitro and in vivo properties of scFvs. In the present invention, flexible peptide linkers with optimal domain orientation, content, and length have been developed.

[0028] According to the present invention, single chain variable fragments (scFv) include scFv1, scFv2, and scFv3, respectively: - a first binding domain comprising, in order, a variable light chain having the amino acid sequence of SEQ ID NO: 1, a linker having the amino acid sequence of SEQ ID NO: 2, and a second binding domain comprising, in order, a variable heavy chain having the amino acid sequence of SEQ ID NO: 3, or - a first binding domain comprising, in order, a variable light chain having the amino acid sequence of SEQ ID NO: 4, a linker having the amino acid sequence of SEQ ID NO: 5, and a second binding domain comprising, in order, a variable heavy chain having the amino acid sequence of SEQ ID NO: 6, or - a first binding domain comprising, in order, a variable light chain having the amino acid sequence of SEQ ID NO: 7, a linker having the amino acid sequence of SEQ ID NO: 8, and a second binding domain comprising, in order, a variable heavy chain having the amino acid sequence of SEQ ID NO: 9.

[0029] In a first embodiment, the single chain variable fragment (scFv) comprises scFv1 having the amino acid sequence of SEQ ID NO: 10, which consists of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0030] In a second embodiment, the single chain variable fragment (scFv) comprises scFv2 having the amino acid sequence of SEQ ID NO: 11, which consists of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0031] In a third embodiment, the single chain variable fragment (scFv) comprises scFv3 having the amino acid sequence of SEQ ID NO: 12, consisting of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0032] These scFvs (scFv1, scFv2, scFv3) have the same primary structure, but only four marked amino acids (shown in the sequence listing) in each sequence can be changed in different combinations (from F (phenylalanine, Phe) to amino acid E (glutamic acid, Glu) or vice versa at position 83, and from S (serine, Ser) to amino acid M (methionine, M) or vice versa at position 12).

[0033] Thus, the present invention relates to scFvs (scFv1, scFv2, scFv3) that specifically bind to vascular endothelial growth factor (VEGF) protein, wherein the scFvs have high binding affinity to VEGF protein, and the scFvs bind to VEGF protein with a dissociation constant (KD) of less than 5 nM 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.

[0034] In a further embodiment, the present invention provides scFvs that bind to VEGF proteins with an association rate constant (k on ) of 7×10 4 M −1 s −1 or greater.

[0035] Furthermore, the present invention provides scFvs that bind to VEGF proteins with a dissociation rate constant (koff) of 3 x 10-6 s-1 or less.

[0036] The values ​​of the dissociation constant (KD), binding rate constant (kon), and dissociation rate constant (koff) were calculated by fitting the kinetic association and dissociation curves to a 1:1 binding model.

[0037] 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 a human VEGF protein (Gibco catalogue number PHC9391).

[0038] Additionally, methods for producing the antibody fragments of the present invention are provided.

[0039] According to the present invention, there is provided a method for producing scFv antibody fragments by a microbial fermentation process comprising the steps of: - culturing host cells transformed with DNA encoding the scFv under conditions allowing the expression and production of said fragment; - inoculating the culture into a bioreactor and producing the recombinant scFv at large scale under appropriate conditions by a stepwise scale-up method; - adjusting the pH with a pH adjuster that also serves as a nitrogen source; - Depleting glycerol in the culture medium (a sudden increase in dissolved oxygen), - after the dissolved oxygen spike signal, starting the fed-batch phase of the fermentation by feeding 100% methanol or ethanol or glucose containing trace metals solution; - exponentially increasing the feed rate of the methanol, ethanol, or glucose solution during the first 72 hours of fermentation, and then keeping the rate constant during the last 24 hours of fermentation; - Optimizing temperature and pH during the batch phase, and - harvesting the supernatant from the fermentation culture at different time points.

[0040] The present invention relates to a pharmaceutical composition comprising a humanized anti-VEGF scFv according to any one of the preceding embodiments for use in anti-VEGF therapy by inhibiting VEGF:VEGFR interaction in a mammal, said medicament being capable of being administered to the mammal in a therapeutically effective amount. Preferably, the mammal is a human, more preferably the mammal is a human with a tumor (solid tumor, cancer) or an eye disease.

[0041] The present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an scFv according to any of the preceding embodiments and a pharmaceutically acceptable carrier. The composition is an aqueous solution for ophthalmic use. More preferably, the pharmaceutical composition is an ophthalmic solution comprising phosphate buffered saline administered as eye drops or ophthalmic injections.

[0042] The anti-VEGF effect of the scFv antibody fragments was found to be concentration-dependent, with the scFv concentrations ranging from 20 μM to 100 μM.

[0043] The term "therapeutically effective amount" of a functional fragment of the invention refers to a non-toxic amount of a fragment of the invention sufficient to elicit a biological or medical response in a subject, such as, for example, reducing or inhibiting a protein:protein or protein:receptor interaction, ameliorating symptoms, alleviating a condition, slowing or delaying the progression of a disease, or preventing a disease or disorder.

[0044] The pharmaceutical composition of the present invention according to any one of the above embodiments can be used in the treatment of any VEGF protein-related disease or disorder, such as intraocular neovascular disease.

[0045] In another embodiment, the pharmaceutical composition of the present invention according to any one of the preceding embodiments can be used to treat age-related macular degeneration (AMD), more preferably the wet form of age-related macular degeneration (wet AMD).

[0046] In further embodiments, the present invention relates to a method for applying a pharmaceutical composition to a subject, and to a pharmaceutical composition for administering it to a subject.

[0047] The various embodiments of the invention disclosed herein all relate to methods of treating and / or preventing the intraocular neovascular diseases or disorders described herein.

[0048] Non-limiting examples of ocular neovascular diseases include age-related macular degeneration (AMD), the wet form of age-related macular degeneration (wet AMD), diabetic retinopathy, retinal occlusion, and retinopathy of prematurity.

[0049] These examples are representative of specific embodiments of the invention and are not intended to limit the scope of the invention.

[0050] (Specific embodiment) Stability, yield, and potency are crucial factors in scFv development. In these embodiments, several scFv proteins were engineered to yield highly stable, highly productive, and highly potent scFv proteins for therapeutic applications. Compared to a reference antibody, three scFv proteins (scFv1, scFv2, and scFv3) exhibited advantageous properties. The protein yield of scFv3 was over 200 mg of purified scFv (MW: approximately 26.8 kDa) per liter of culture medium, nearly 10-fold higher than that of the reference (Figure 2). scFv1 and scFv2 exhibited approximately 5°C higher thermal stability than the reference (Figure 3). After just one protein L affinity chromatography step, scFv1 and scFv2 exhibited 97.3% and 93.3% purity, respectively, while the reference exhibited only 65% ​​purity (Table 1, Figure 4). Surface plasmon resonance (SPMR) analysis showed that scFv1, scFv2, and scFv3 bound to VEGF more strongly than the reference (Table 2, Figure 5). To test their antiangiogenic efficacy, we used a transgenic zebrafish model in which GFP expression was induced in endothelial cells. scFv1, scFv2, and scFv3 demonstrated higher potency than the reference, with scFv3 demonstrating the highest in vivo activity, reducing SIV area by 76.8% (Figure 6).

[0051] Table 1. Purity analysis of scFv proteins by SE-HPLC [Table 1]

[0052] In summary, anti-VEGF scFv fragments were designed with the following enhancements: 1.Thermal stability (thermal stability improved by 5℃ or more) 2. Increased production in microbial systems (more than 200 mg of purified scFv per liter of culture medium). Also, a purity of more than 95% was achieved in a single purification step. 3. High anti-angiogenic effect (more than 2-fold) in an in vivo zebrafish model

[0053] (Example) Example 1 - Production Komagataella phaffii (Pichia pastoris) has been used for the expression and production of soluble scFv proteins. A 5L bioreactor was used to produce recombinant scFv at a large scale. A vial of frozen culture was inoculated into 200mL medium and incubated at 28°C and 225 rpm for approximately 20 hours. 125mL of the culture was inoculated into 2.5L medium. pH was adjusted with 25% ammonium hydroxide, which also served as a nitrogen source. The batch phase continued for approximately 15 hours and was completed when the medium was depleted of glycerol (dissolved oxygen spike). After the dissolved oxygen spike signal, the fed-batch fermentation phase was initiated by feeding 100% methanol, ethanol, or glucose containing a trace metal solution. The feed rate was increased exponentially for the first 72 hours of fermentation, and this rate was kept constant for the final 24 hours of fermentation. The temperature and pH during the batch phase were optimized between 18 and 30°C and between 3.0 and 7.0, respectively. The temperature and pH values ​​were set linearly with the profile during the first hour of the fed-batch phase. The dissolved oxygen level was controlled at 30% saturation by adding air and pure oxygen under constant agitation. During the fed-batch phase, supernatants were collected at various time points. These samples were analyzed to measure biomass levels and to determine the protein produced by SDS-PAGE.

[0054] Example 2 - Purification The filtered supernatant was applied to an affinity chromatography column (Protein L). This first stage of purification achieved a purity of >93%. A second stage of purification was applied, resulting in a purity of >95%.

[0055] Example 3 - Size Exclusion Chromatography (SE-HPLC) SE-HPLC was performed using an HPLC system equipped with a UV-VIS detector and an analytical size-exclusion chromatography column. The scFv sample was injected onto the column. The absorbance value was monitored at 280 nm. Molecular weight standards were used to verify the retention time of the scFv protein.

[0056] Example 4 - Thermostability Assay The thermal unfolding profile of purified scFv protein was determined using a fluorescent dye. Optimal concentrations of dye and protein were used. The transition midpoint (Tm) from the thermogram data was calculated based on the equation:

[0057] Example 5 - Surface Plasmon Resonance (SPR) Affinity measurements were performed using a Biacore instrument. VEGF protein was immobilized on a chip. A series of solutions of scFv protein ranging from 1 to 100 nM was then injected over the VEGF-coated surface. Data were corrected by double-referencing a control flow cell without VEGF and a flow cell with buffer injection. Sensorgram curves were analyzed. Values ​​for the dissociation constant (KD), binding rate constant (k), and dissociation rate constant (koff) were calculated by fitting the kinetic association and dissociation curves to a 1:1 binding model.

[0058] Table 2. Binding kinetics of scFv proteins by surface plasmon resonance [Table 2] *The 10 sequences are shown in the sequence listing as SEQ ID NOs: 10 to 19. The reference sequence is SEQ ID NO: 20.

[0059] Example 6 - Zebrafish Experiments The zebrafish used in this study were provided by the Zebrafish Facility of the Izmir Biomedicine and Genome Center. All animal experiments were approved by the Ethics Committee of the Izmir Biomedicine and Genome Center (HADYEK), Izmir, Turkey. All experiments using live animals were conducted in accordance with relevant guidelines and regulations and reported in accordance with the recommendations of the ARRIVE guidelines.

[0060] Adult zebrafish were maintained under standard conditions at the Izmir Biomedicine and Genome Center Zebrafish Facility. Embryos were obtained by crossing transgenic lines with wild-type lines.

[0061] The estimated injection volume was determined using a stage micrometer. The scFv protein was microinjected into the yolk of individual anesthetized zebrafish embryos on day 2 post-fertilization. Microinjected larvae were stored at 28°C for 1 day after injection. The development of the subintestinal vasculature (SIV) was examined. Larvae were fixed and melanocytes were bleached in a bleaching solution.

[0062] Larvae were mounted sideways on low-melting-point agarose in imaging dishes and imaged using a confocal microscope. SIV area was measured and statistically analyzed.

[0063] References [1]Monnier P, Vigouroux R, Tassew N (2013) In Vivo Applications of Single Chain Fv (Variable Domain) (scFv) Fragments. Antibodies 2:193-208. [2] Sandomenico A, Sivaccumar JP, Ruvo M (2020) Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. IJMS 21:6324. [3] Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M (2012) scFv Antibody: Principles and Clinical Application. Clinical and Developmental Immunology 2012:1-1. [4] Bates A, Power CA (2019) David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies 8:28.

Claims

1. A single-chain variable fragment (scFv) that specifically binds with high binding affinity to vascular endothelial growth factor (VEGF) protein: a first binding domain comprising, in order, a variable light chain having the amino acid sequence of SEQ ID NO: 1, a linker having the amino acid sequence of SEQ ID NO: 2, a second binding domain comprising, in order, a variable heavy chain having the amino acid sequence of SEQ ID NO: 3, or a first binding domain comprising, in order, a variable light chain having the amino acid sequence of SEQ ID NO: 4, a linker having the amino acid sequence of SEQ ID NO: 5, a second binding domain comprising, in order, a variable heavy chain having the amino acid sequence of SEQ ID NO: 6, or a first binding domain comprising, in order, a variable light chain having the amino acid sequence of SEQ ID NO: 7, a linker having the amino acid sequence of SEQ ID NO: 8, a second binding domain comprising, in order, a variable heavy chain having the amino acid sequence of SEQ ID NO: 9, A single chain variable fragment comprising:

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

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

6. 6. The single-chain variable fragment of claim 5, which binds to VEGF protein with a binding rate constant of 7 x 104 M-1s-1 or greater.

7. 7. The single-chain variable fragment of claim 6, which binds to the VEGF protein with a dissociation rate constant (koff) of 3×10 −6 s −1 or less.

8. A pharmaceutical composition comprising a humanized anti-VEGF scFv described in any of the preceding claims, which is used for anti-VEGF therapy by inhibiting VEGF:VEGFR interaction in a mammal and 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 has a tumor.

11. The pharmaceutical composition of claim 9, wherein the mammal has an eye disease.

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

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

14. 14. The pharmaceutical composition according to claim 13, which is used for the treatment and / or prevention of age-related macular degeneration (AMD).

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

16. An antibody construct comprising an scFv according to any one of the preceding claims, in a format selected from the group consisting of (scFv)2, scFv-single domain mAb, diabody, and oligomers of said formats.

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