The therapeutic positioning of monoclonal antibodies (Anti-sfltl, Anti-sflkl, Anti-seng, Anti-STIE2) and small peptides (Anti-fab, Anti-fc) prepared against soluble-trap receptors in some inflammatory and vascular diseases

Abstract

The feature of the invention is the use of monoclonal antibody (anti-sFltl, sFLKl, sENG, sTIE2) and small peptide (anti-Fab, anti-Fc) molecules prepared against solubul-trap receptors or all RNA types such as mRNA, miRNA and IncRNAs targeting associated DNA, mRNA or their DNA and mRNA in the treatment of inflammatory and vascular diseases as drugs.

Description

[0001] SPECIFICATION

[0002] THE THERAPEUTIC POSITIONING OF MONOCLONAL ANTIBODIES (Anti-sFLTl, Anti-sFLKl, Anti-sENG, Anti-sTIE2) AND SMALL PEPTIDES (Anti-Fab, Anti-Fc) PREPARED AGAINST SOLUBLE-TRAP RECEPTORS IN

[0003] SOME INFLAMMATORY AND VASCULAR DISEASES

[0004] Terminology:

[0005] A. Trap receptors: It is a defective, soluble receptor particle that is found in cell membranes and does not contain the intracellular and intramembranous parts of receptors consisting of intracellular, extracellular and intramembranous parts, but only the extracellular part of the receptor. This fragment circulates freely in the blood and tissues, binds to the relevant ligand (such as enzymes, hormones, and growth factors) like a trap receptor, preventing the ligand from binding to its own receptor. In this way, it disrupts cellular signaling pathways and biological functions.

[0006] B. B. Solubul trap receptors:

[0007] 1- sFLTl (sVEGFl): It is a soluble, defective fragment of the VEGF1 receptor that circulates freely in the blood and tissues. It is a soluble defective fragment consisting only of the extracellular part of the receptor, which does not contain the intracellular and inramembranous part of the receptor. This fragment binds to VEGF like a trapped receptor, disrupting its biological functions.

[0008] 2- 2. sFLKl(VEGFR2): This receptor is also known as kinase insert domain receptor (KDR). sFLKl is a soluble, defective fragment of the VEGF2 receptor that consists only of the extracellular part of the receptor, which circulates freely in blood and tissues. This fragment is the extracellular portion of the VEGF2 receptor and does not include the intracellular and intramembranous portion of the receptor. This soluble fragment binds to VEGF, impairing its biological functions.

[0009] 3- 3. sENG (Solubular Endoglin): It is the soluble defective fragment of the Endoglin receptor, which consists of the extracellular part of the endoglin receptor that circulates freely in the blood and tissues. This soluble fragment, which does not contain the intracellular and intramembranous part of the receptor, acts as a trap receptor and binds to Endoglin and disrupts its biological functions.

[0010] 4- 4. sTIE2 (also known as sTEK receptor): This decoy receptor is a defective, soluble fragment of the angiopoentin TIE2 receptor formed from the free- circulating extracellular part of the blood and tissues. This free fragment binds to angiopoentin like a decoy receptor, preventing the binding of angiopoentin to its receptor and impairs its biological functions.

[0011] C. Monoclonal antibodies: It describes monoclonal antibodies prepared against sVEGFl, sVEGF2, sTIE2 and sENG by recombinate gene technology and phage display technique. These antibodies will selectively and specifically bind to these receptor particles circulating freely in the blood and tissues, and this solubule, which is called a trap receptor, will ensure that the receptor particles are neutralized and removed from the environment. Each of these was defined according to the trap from which it was prepared, named anti-sFLTl for sFLTl, anti-sFLKl for sFLKl, anti- sTIA2 for sTIA2, and anti-sEng for sENG.

[0012] D. Small peptides: Solubul, which we refer to as trap receptors, refers to small antibodylike peptide fragments that will be prepared by recombinant gene technology against the Fc and Fab subregions of defective sFLTl, sFLKl, sTIA2 and sENG receptor fragments. Since these peptides will be prepared by targeting the specific regions of the defective receptor, they will be able to selectively neutralize the solubule trap receptors by binding more specifically to the Fc and Fab ends and subregions of the relevant solubul receptor fragment. In addition, since small peptides are much smaller than monoclonal antibodies, accumulation in tissues is less. Again, the possibility of unwanted contacts and interactions between normal receptors on cell membranes and other biological structures in small peptide applications is much lower than in monoclonal antibody applications.

[0013] Technical Field:

[0014] This invention proposes solubul trap receptors as new therapeutic targets for the treatment of many acute and chronic inflammatory, autoimmune and vascular diseases, especially Type-I and Type-II diabetes as a treatment modality. The invention is related to the use of some neutralizing-binding monoclonal antibodies (anti-sFltl, anti-sFLKl, anti-sENG, anti-sTIE2) and small peptides (anti-Fab, anti-Fc) developed against trap receptors as new biotechnological, therapeutic agents for the treatment of certain inflammatory and vascular diseases. These monoclonal antibodies and small peptides that will be prepared against soluble trap receptors can be used individually, in combination, or in combination with factors that regulate angiogenesis and endothelial structure and organization, such as VEGF, orf-VEGF, PDGF, FGF, TGF, Angiopoentin and Endoglin. With this invention, monoclonal antibodies and small peptides to be prepared against trap receptors are expected to be used as new therapeutic agents in the treatment of acute, chronic, inflammatory, autoimmune and vascular diseases listed below (tablet).

[0015] Diseases in which monoclonal antibodies and small peptides to be prepared against soluble decoy receptors will be used: Table 1

[0016] 1.Brain edema

[0017] 2. Pulmonary edema

[0018] 3. Lung fibrosis

[0019] 4. Pulmonary hypertension

[0020] 5. Fibrotic diseases of the liver, Cirrhosis

[0021] 6. ARDS, RDS

[0022] 7. SIRS, Sepsis, cytokine storm

[0023] 8. Macrophage activation syndrome

[0024] 9. Differentiation syndrome

[0025] 10. Familial Mediterranean fever (FMF) attack

[0026] 11. Amyloidiosis (kidney and other tissues)

[0027] 12. Parkinson's and Alzheimer's

[0028] 13. Atherosclerosis, coronary artery disease,

[0029] 14. Ischemic brain infarction

[0030] 15. OHSS syndrome

[0031] 16. Preeclampsia, IUGR

[0032] 17. Recurrent abortions 18. Unexplained infertility

[0033] 19. IVF-related recurrent implantation failure

[0034] 20. Type I and Type II diabetes and associated vascular complications

[0035] 21. Diabetic retinopathy, retinoblastoma and age-related macular degeneration

[0036] 22. Neonatal retinopathy (ROP)

[0037] 23. Diabetic foot sores

[0038] 24. Slow-healing or non-healing wounds

[0039] 25. Accelerating wound healing

[0040] 26. Treatment of ischemic damage to other tissues resulting in necrosis and gangrene

[0041] 27. Ischemic vascular anomalies in the retina of the eye due to old age and diabetes

[0042] 28. Renal diseases such as nephrotic syndromes in which the glomerular endothelial layer of the kidneys is disrupted and acute and chronic renal failure

[0043] 29. Vascular diseases such as Takayasu arteritis, Behcet's disease, Kawazaki disease

[0044] 30. Celiac disease

[0045] 31. Some autoimmune diseases such as Sjogren's syndrome, SLE, RA and Scleroderma

[0046] 32. Erectile dysfunction due to vascular pathologies

[0047] 33. Post-COVID syndromes / long-COVID syndromes

[0048] 34. Reynaud's syndrome / Reynaud's phenomenon

[0049] State of the Art:

[0050] The formation of new vessels by disrupting angiogenesis, vasculogenesis and lymphogenesis by binding to solubul-trap receptors such as sFltl, sFLKl, sENG and sTIE2 circulating freely in the blood and tissues, Vascular endothelial growth factor (VEGF), Placental growth factor (PIGF), Endoglin (ENG), TGFB and Angiopoentine (ANGPT), maintaining endothelial stability and adaptation, as well as the formation of new vessels by disrupting angiogenesis, vasculogenesis and lymphogenesis it is known that it disrupts implantation, placentation and embryo nutrition and development.

[0051] Ischemic and inflammatory pathogenetic changes such as edema, ischemia, necrosis and infarction develop in tissues in addition to intravascular proteins and fluids with disruption of endothelial permeability, as well as endothelial mitosis, endothelial migration and endothelial organization necessary for the formation of new vessels by binding of solubular receptors to angiogenetic factors. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) have been shown to play a role not only in physiological, but also in pathological angiogenesis, such as cancer.

[0052] VEGF-A has several functions, including pro-angiogenic activity, vascular permeability activity, and stimulation of cell migration in macrophage lineage and endothelial cells. In recent years, anti -VEGF -VEGFR antibodies, also defined as tyrosinkinase inhibitors, have been developed and used for the treatment of major solid tumors and vascular diseases of the eye. But this application has not been able to provide a complete treatment for cancer and retinovascular diseases of the eye. The development of resistance to these drugs in some tumors and their limited effectiveness in the treatment of vascular pathologies of the eye limit the use of anti-VEGF monoclonal antibodies. Anti-VEGF antibodies can also cause serious side effects in some patients. For these reasons, extensive research is underway to develop more effective treatments in this area.

[0053] Trophoblast invasion into spiral arteries, which are vitally important for embryo implantation and nutrition, depends on vasculogenesis, angiogenesis, lymphogenesis and endothelial reorganization provided by a healthy functioning VEGF, VGFR, PIGF, Endoglin and Angopoentin. Retinoid metabolism disorder observed during the course of some chronic inflammatory and autoimmune diseases and associated immune dysregulation, increased inflammatory cytokines in the uterine endometrial microenvironment and soluble pathological proteins that disrupt angiogenesis, lymphogenesis, endothelial reorganization and trafoblast invasion, such as sFltl, sFLT2 and sEND, disrupt implantation and placentation. This vascular development disorder and insufficiency in the fetomatemal junction causes implantation failure and early abortions, as well as pathogenetic disorders such as preeclampsia and intrauterine growth retardation in the later weeks of pregnancy. Increased sFLTl levels in the circulation lead to functional VEGF deficiency, resulting in endothelial dysfunction, decreased angiogenesis and neovascularization, impaired capillary repair, and ultimately hypertension and proteinuria. Hypertension and proteinuria seen in patients treated with tyrosine kinase inhibitors, one of the VEGF inhibitors used as a part of cancer chemotherapy, result from the deterioration of VEGF functions with these drugs. It has also been shown that genetic patterns of glomerular VEGF deficiency are associated with proteinuria and glomerular endothelial damage. Soluble VEGFR-1 (sFLTl) is abnormally and overexpressed in the placenta of preeclamptic patients. This soluble receptor particle, which circulates freely in the blood, blocks physiological VEGF-A and causes major pathological symptoms such as hypertension and renal dysfunction in the mother. VEGFR-1, including its soluble form, is implicated in a variety of human diseases. This makes VEGFR1 an important target for the development of new strategies to suppress the disease. Increased circulating sFltl levels lead to functional VEGF deficiency, resulting in endothelial dysfunction, decreased angiogenesis, impaired capillary repair, and ultimately endothelial dysfunction, hypertension, and proteinuria. It has been shown that the decrease in free-circulating VEGF and P1GF levels in patients with preeclampsia is associated with increased circulating sFltl levels. It has also been shown that the resulting endothelial dysfunction can be rescued by exogenous VEGF and PIGF. In addition, exogenous VEGF and PIGF also induce microvascular relaxation of in-vitro rat renal arterioles blocked by sFLTl.

[0054] The pathophysiology of preeclampsia, which continues to be an important cause of fetomatemal morbidity and mortality worldwide, is largely unknown. However, there are many studies in the literature showing that the basic pathophysiological disorder underlying the etiopathogenesis of preeclampsia may be impaired retinoid signaling and soluble entrapment receptors. For this reason, we predict that there will be a paradigm shift in treatment in this area with the placement of monoclonal antibodies and small peptides that we have developed against anti-decoy receptors in the treatment of preeclampsia. There are previous patent applications for the positioning of sFltl in this area. However, administration of sFltl alone may not be sufficient for the treatment of preeclampsia. We predict that the combined use of VEGF, PIGF, SFltl, sFlkl, sTiE2 and sENG will be more effective in this indication. Positioning anti-trapping pro-angiogenic agents against preeclampsia will yield significant human and socioeconomic benefits. The efficacy of a retinoic acid derivative in the in-vitro model developed by Chinese researchers for the treatment of retino-vascular diseases of the eye supports the mechanism of retinoic acid signaling disorder and soluble defective receptor mechanism that we developed for the pathogenesis of inflammatory and vascular diseases. Shen Y, Xu M, Ren L, Li X, Han X, Cao X, Yao J, Yan B. A novel retinoic acid drug, EYE-502, inhibits choroidal neovascularization by targeting endothelial cells and pericytes. Sci Rep. 2023 Jun 27;13(l):10439. doi: 10.1038 / s41598-023-37619-7. PMID: 37369771; PMCID: PMC10300120.

[0055] Use of anti-VEGF antibodies in retinal vascular disorders of the eye, a pathogenetic model for lupus-trap receptors: False drug target and low success rate:

[0056] Antibodies to VEGF have been used for years as Tyrosine Kinase inhibitors to interrupt the vascularization and nutrition of cancer tissue. In recent years, a similar treatment model has been used for the prevention and regression of aberrant vascularization seen in retinal diseases of the eye. However, in this case, VEGF itself is targeted instead of soluble decoy receptors, and monoclonal antibodies developed against VEGF are given as multiple injections into the retinal layer of the eye. However, in most of the patients, no results can be obtained from this treatment. This situation, which is mentioned in the literature as drug resistance, is not a drug resistance, but a misconstrued therapeutic target. Here, deterioration in the retina and vascular bed develops mainly due to the binding of entrapment receptors to angiogenetic factors that maintain vascular health. Here, VEGF was targeted in the treatment by holding the compensatory increase in VEGF responsible for the pathogenetic disorder by the researchers. This is the main reason for treatment failure.

[0057] Cornel S, Adriana ID, Mihaela TC, Speranta S, Algerino S, Mehdi B, Jalaladin HR. Anti- vascular endothelial growth factor indications in ocular disease. Rom J Ophthalmol. 2015 Oct Dec;59(4):235-242. PMID: 29450313; PMCID: PMC5712945. https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5712945 /

[0058] In this treatment modality, antibodies developed against VEGF are given as multiple intravitreal injections to achieve the desired therapeutic effect, typically in patients with retinal diseases, and complications up to vision loss may occur. The most feared of these complications is endophthalmitis, a serious eye infection with extremely dire consequences that can lead to blindness. Each injection given carries the risk of serious vision-threatening complications. Manousaridis K, Talks J. Macular ischaemia: a contraindication for anti-VEGF treatment in retinal vascular disease? Br J Ophthalmol. 2012 Feb;96(2): 179-84. doi: 10.1136 / bjophthalmol-2011-301087. PMID: 22250209. https: / / pubmed.ncbi.nlm.nih.gov / 22250209 /

[0059] The main thing in this treatment and we should consider is that anti-VEGF antibodies (anti-VEGF) did not give effective results in retinal vascular disorders. And unfortunately, researchers have yet to find any other way to circumvent this problem, other than dose adjustments. Looking at the current literature, it is seen that these manipulations do not give effective results.

[0060] Wallsh, J.O.; Gallemore, R.P. Anti-VEGF-Resistant Retinal Diseases: A Review of the Latest Treatment Options. Cells 2021, 10, 1049. https: / / doi.Org / 10.3390 / cellsl0051049https: / / www.mdpi.com / 2073-4409 / 10 / 5 / 1049

[0061] The main defect here is soluble receptor synthesis, which develops due to retinoid signal disorder and RA dimeric receptor modulation disorder, and these trap receptors impair vascular stability and endothelial permeability. The hypoxic microenvironment that develops with the disruption of vascular circulation causes a further compensatory increase in VEGF, resulting in aberrant vascularization and an increase in endothelial permeability. Here, a vicious circle occurs due to hypoxia. It has been previously shown that hypoxia increases VEGF synthesis in retinal cells.

[0062] Blocking VEGF with antibodies may further impair vascular functions, endothelial organization, vascularization, angiogenesis, and lymphogenesis, as VEGF elevation is not the main pathogenetic disorder here. Here, blocking VEGF from binding by decoy receptors with monoclonal antibodies and small peptides to be developed may yield more effective results.

[0063] VEGF and other angiogenetic factors released by the binding of decoy receptors such as soluble sFLTl, sFLT2, sENG and sTIE2 by monoclonal antibodies will continue to function normally. Blocking VEGF itself with antibodies impairs the nutrition of the tissue by disrupting the endothelial organization and vascularization, as in the treatment of solid tumors. This causes hypoxia to develop, more VEGF release and aggravation of the pathogenetic process.

[0064] Currently, targeted studies to reduce circulating sFLTl in clinical practice are not available in the literature. Clearance and removal of sFltl from the blood using dextran sulfate apheresis has recently been shown to be a possible new strategy in patients with very severe preterm preeclampsia. However, these treatments are still in the experimental stage and are far from correcting the underlying pathogenetic mechanism. Also, although recombinant VEGF or placental growth factor has proven to be effective in experimental models, no clinical studies have yet been conducted on them.

[0065] As a result, monoclonal antibodies and small paptides developed against trap receptors that can overcome the disadvantages mentioned above with this invention will be positioned as new pro-angiogenetic drugs for the treatment of numerous inflammatory and vascular diseases listed in Tablet, especially retinovascular diseases of the eye and inflammatory and vascular disorders that develop due to Type-I and Type-II diabetes.

[0066] Neutralizing-binding antibodies and small peptides to be developed against soluble decoy receptors will be positioned as a new pro-angiogenetic treatment modality in the treatment of vascular and inflammatory diseases. The diseases in Table 1 have been identified as therapeutic targets for neutralizing-binding antibodies and small peptides.

[0067] Description of The Invention:

[0068] This invention is related to the use of monoclonal antibodies (anti-sFLTl, sFLKl, sENG, sTIE2) and small peptides (anti -Fab, anti-Fc) prepared against solubul-trap receptors in the treatment of inflammatory and vascular diseases, the characteristic of which is related to the use of monoclonal antibodies (anti-sFLTl, sFLKl, sENG, sTIE2).; Determination of soluble trap receptors as new therapeutic targets for the treatment of inflammatory and vascular diseases in Tablet, including Type-I and Type-II diabetes mellitus, and positioning neutralizing-binding antibodies and small peptides against these trap receptors as a new pro-angiogenetic treatment modality for the treatment of vascular and inflammatory diseases.

[0069] With this invention, monoclonal antibodies and small peptides developed against solubule decoy receptors will be positioned as pro-angigenetic agents in the treatment of many acute and chronic, inflammatory and vascular diseases. This approach has the potential to lead to a paradigm shift in this field. Solubule trap receptors cause edema, ischemia and infarction with increased endothelial permeability by disrupting endothelial reorganization and integrity in acute inflammatory conditions such as Sepsis, SIRS, ARDS, cytokine storm, preeclampsia, OHSS, ROP and brain edema. Solubule trap receptors can also cause chronic inflammatory diseases such as lung and liver fibrosis, Amyloidiosis, Parkinson's, Alzheimer's, Atherosclerosis, coronary vascular disease, Ischemic brain infarction by disrupting angiogenesis, vasculogenesis, lymphogenesis and endothelial reorganization with the same pathogenetic mechanism. VEGF, PIGF, angipoentin, and endoglin are responsible for angiogenesis, vasculogenesis, and endothelial reorganization, as well as vascular adaptations required for implantation, placentation, and increased nutritional needs in later pregnancy. Solubular trap receptors disrupt these vascular functions and cause unexplained infertility, recurrent abortions, and recurrent implantation failure in infertility practice and IVF applications. This pathogenetic disorder, which starts in the early stages of pregnancy, is also responsible for pathologies such as preeclampsia and IUGR that occur in the later weeks of pregnancy.

[0070] In order to realize all the purposes mentioned above and which will be revealed from the detailed description below, the invention; soluble in pathological conditions caused by inflammatory and vascular disorders such as preeclampsia, IUGR, OHSS syndrome, recurrent abortions, unexplained infertility, IVF-related recurrent implantation failure, Type-1 and Type-2 diabetes and associated vascular complications, diabetic retinopathy neonatal retinopathy (ROP) and age-related macular degeneration and retinovascular diseases of the eye, such as sFLT-1, sFLKl, sENG and Which neutralizes STIE2, it is related to monoclonal antibodies and anti -Fab and Anti-Fc peptides that reduce their levels and / or terminate their pathological activity, individually or in combination, and related to pro-angiogenetic drugs developed for prophylactic and therapeutic purposes in combination with other angiogenetic factors (VEGR, PIGF, Angiopoentin and Endoglin).

[0071] The invention aims to provide the treatment opportunity that cannot be achieved adequately with current treatment modalities in some inflammatory and vascular diseases through some pro-angigenetic mediators such as soluble, decoy receptors, VEGF, VEGR receptors.

[0072] VEGF group growth factors are the main regulators of angiogenesis, vasculogenesis, lymphogenesis and endothelial reorganization and vascular permeability. During the course of some chronic and acute diseases and due to retinoid signal disorder, solubular trap receptors impair their functions by binding VEGF and other pro-angiogenetic factors. Angiogenesis and vascular functions are largely provided by the normal functional activity of VEGF via VEGF receptors. Vascular endothelial growth factor receptor- 1 (VEGFR-1) is a member of the VEGFR family and binds VEGF-A, PIGF, and VEGF-B. An important feature of VEGFR-1, unlike other VEGFR genes, is that it expresses two types of mRNA, one for a full-length receptor and the other for a soluble short protein known as soluble VEGFR-1 (sFlt-1). The binding affinity of VEGFR-1 for VEGF-A is one-fold higher than that of VEGFR-2, whereas the kinase activity of VEGFR-1 is approximately 10-fold weaker than that of VEGFR-2. VEGFR-1 plays a negative role in embryogenesis and angiogenesis through its ligand binding site and by capturing ligands. However, in adulthood, VEGFR-1 is expressed not only on endothelial cells, but also on monocytes and macrophages, and supports the pathogenesis of macrophages, inflammatory diseases, cancer metastasis, and atherosclerosis through its kinase activity. Soluble VEGFR-1 is abnormally overexpressed in the placenta of preeclamptic patients. VEGF-1 causes major pathological symptoms such as maternal hypertension and renal dysfunction by blocking physiological VEGF-A. VEGFR-1, including its soluble form, is implicated in a variety of human diseases. This makes it an important therapeutic target in the development of new strategies to suppress the disease. The difficult part of the technique is the possibility that monoclonal antibodies developed against soluble, defective receptors may adhere to receptors on cell surfaces as well as free-floating antibodies in the blood and disrupt cellular signaling through this mechanism. If this is observed in 'in vitro' and 'in vivo' cell culture studies after antibody synthesis, small peptide will be developed against the Fc fragment of soluble receptors to overcome this problem. For receptors on the cell surface, this region is embedded in the cell membrane. Developed small peptides bind to this liberated intramembranous part of the solubule receptor. Therefore, the small peptide developed in small peptide applications is not likely to interact with normal receptors attached to the cell surface. However, in the free-floating soluble receptor fragments, this region is completely open and the small peptide can easily bind to this area selectively. Therefore, in this pro- angiogenetic therapy, which was developed by targeting OLUbule trap receptors, small peptides will only bind to soluble, trap receptors more selectively than monoclonal antibodies.

[0073] The product, solubul, monoclonal antibody and small peptides to be prepared against trap receptors are predicted to be effective in a wide range of diseases, including cerebral edema, pulmonary edema, cirrhosis, ARDS, RDS, Parkinson's, Alzheimer's, diabetic foot wounds, recurrent implantation failure, recurrent abortions and retinal vascular diseases of the eye, listed in table 1 below.

[0074] Description of the Invention:

[0075] Feature of the invention; it is the use of monoclonal antibody (anti-sFltl, sFLKl, sENG, sTIE2) and small peptide (anti-Fab, anti-Fc) molecules prepared against solubul-trap receptors or all biotechnological therapeutic agents such as associated DNA, mRNA or miRNA targeting them, IncRNA, monoclonal antibodies and small peptides as drugs in the treatment of inflammatory and vascular diseases.

[0076] In the synthesis process of anti-trap antibodies (Anti-sFltl, Anti-sFLKl, anti-sENG, anti- sTIE2) and Anti-trap small peptides (Anti-Fab peptide, AntiFc peptide) prepared against these solubular trap receptors, Preeclampsia, IUGR, OHSS syndrome, recurrent abortions, unexplained infertility, IVF-related recurrent implantation failure described in Table 1 below, Type- soluble in pathological conditions caused by inflammatory and vascular disorders such as Type 1 and Type-2 diabetes and associated vascular complications, diabetic retinopathy and age-related macular degeneration sFlt-1, sFLKl, Monoclonal antibodies that reduce, neutralize sENG and sTIE2 levels and / or terminate their pathological activity, as well as methods for the use of anti-Fab and Anti-Fc peptides individually or in combination or in combination with angiogenetic factors for prophylactic and therapeutic purposes are described.

[0077] Synthesis process of Anti-Trap antibodies and small peptides:

[0078] Many synthesis processes have been described for monoclonal antibody synthesis, but among these techniques, the antibody phage display method developed using bacteriophage represents the most successful in-vitro antibody selection technology. Hybridoma technology, which allows the production of monoclonal antibodies by fusion of an immortal myeloma cell with the antibody-producing spleen cell, is the first described method in monoclonal antibody synthesis. However, hybridoma technology has some limitations: the possible instability of aneuploid cell lines in this technique, most notably its limited application to generate human antibodies, and its inability to provide antibodies against toxic or highly conserved antigens are known disadvantages. In addition, the murine antibodies generated in this technique also limit therapeutic applications. Repeated applications with antibodies prepared by this technique can cause an anti-mouse antibody reaction (HAMA). This risk can technically be reduced by immunogenicity, chimerization or humanization. However, the complexity and relatively difficult nature of this technique and the high purity and rapid production process provided by the phage display technique, as well as its cost-effective advantages, have currently brought the phage display technique to the fore in recombinant antibody synthesis.

[0079] The most widely used filamentous phage display today is based on the groundbreaking work of Georg P. Smith. Antibody phage display offers great potential for direct automation, representing a unique method of producing high-purity and rapidly recombinant human therapeutic antibodies as a robust technology with high yields and low costs as well as a robust technology. Here, the genotype and phenotype of the oligopeptides are fused to the small coat protein III gene of the filamentous bacteriophage Ml 3 of the corresponding gene fragments. The resulting peptide:pIII fusion protein is displayed on the surface of the phage, allowing affinity purification of the peptide and its corresponding gene. Likewise, antibody fragments fused to the hap can be presented on the surface of the Ml 3 phage.

[0080] Two different genetic systems have been developed for the expression of antibody pin fusion proteins for phage display. First, the antibody genes are inserted directly into the phage genome fused with the wild-type Ml 3 phage protein III (pill) gene. However, most successful systems separate antibody expression from phage propagation by providing genes encoding antibody:: pill fusion proteins on a separate plasmid (called a "phagemid"). This phagemid contains a phage morphogenetic signal to package the vector into assembled phage particles. Thus, antibody gene replication and expression is separated from the phage replication cycle, leading to higher genetic stability and simplification of antibody gene library amplification. Using the phagemid system, a helper phage is needed for the production of antibody phage particles. Due to the limitations of the folding mechanism in E. coli, only the corresponding light chain variants of antibody fragments that bind specifically, such as single chain fragment variant (scFv), fragment antigen binding (Fabs), camel (VHH), or human variable heavy domain (dAbs), domain is routinely used for antibody phage display. IgG phage display cannot compete with Fab or scFv display and the additional Fc portion does not benefit the display of linkers. Production of immunoglobulin G (IgG) in E. coli is possible only in rare cases. In this context, E.coli is ideal for the production of small peptides (Anti-fab and Anti-Fc) against soluble-defective receptors and E.coli is envisaged to be used in the project.

[0081] 1. In vitro isolation of antibody fragments from antibody gene libraries by binding activities is performed. This technique is called "scrolling" in reference to the gold washer tool. 2. The antigen is immobilized onto a solid surface such as nitrocellulose magnetic beads, column matrices, polystyrene tubes or plastic surfaces such as 96-cavity microtiter plates and incubated with the antibody phage of the antibody taken from the gene library.

[0082] 3. During this incubation step, it is necessary to select antibodies that can bind to the antigen under these defined conditions, using physical (e.g. temperature), chemical (e.g. pH) and biological (e.g. competitor) parameters to be controlled. Antibody phage particles that bind weakly to the antigen and the excess of nonbinding antibody phage are removed by strict washing.

[0083] DNA Cloning of Solubul Trap Receptors:

[0084] DNA sequences to be used in the synthesis of monoclonal antibodies and small peptides will be obtained separately from antibody gene banks such as the phage library (Clon- tech) and the National Library of Medicine Genbank.

[0085] Specifically bound antibody phages are differentiated (eg, by trypsin or pH shift) and reproliferated by E. coli infection. Next, the E. coli-bearing phagemid is infected with a helper phage to produce the new antibody phage, which can be used for further rounds of scrolling until achieved with a significant enrichment in the antigen-specific phage. Two or three scrolling rounds are usually sufficient to select specific binding antibody fragments, sometimes up to six scrolls are required.

[0086] After scrolling, soluble monoclonal antibody fragments or antibody phage are produced. Specific antigen binding is analyzed by ELISA to identify individual binders. These binders can then be sequenced and further biochemically characterized. This scrolling operation can also be performed with high efficiency. Because the linker's gene sequence is available, the antibody - depending on the desired application - can be converted into different antibody formats (eg scFv-Fc fusion or IgG) and produced in different production hosts. The affinity as well as the stability of antibodies selected by phage display can be enhanced by additional in vitro affinity maturation steps.

[0087] Limitations of the Invention and the Solution:

[0088] There are opinions that Solubul Trap receptors play a fine-tuning role in VEGF receptor interaction with VEGF, but despite the high affinity of VEGF 1 for angiogenetic growth factors (VEGF) during active signaling, the higher VEGF2 activity in the signaling cascade suggests that the main balancing role in the interaction of VEGF and VEGF receptors is provided by VEGF1 rather than by solubul defective receptors. In addition, it has been shown in numerous studies that soluble defective receptors disrupt angiogenesis by endothelial functions, which are produced during pathological processes. These defective trap receptor particles are produced as a result of retinoid signaling disorder during purely pathological processes and disrupt the function of these mediators by binding to angiogenetic factors. It has been shown that solubule trap receptors are produced during pathogenic processes and their value in the blood increases significantly during potological processes. There are no solubules, trap receptors in the blood and tissues of healthy people.

[0089] If monoclonal antibodies developed against soluble-defective receptors bind to normal receptors on cell surfaces as well as free-floating decoy antibodies in the blood, the possibility of disrupting cellular signaling arises. This is a possibility that should be considered in anti-decoy receptor applications. This crosslinking is possible in theory. After antibody synthesis, there is the possibility to test this possibility in in-vitro cell culture studies and animal experiments. The most important argument to overcome this problem is to develop small peptides against the Fc fragment of soluble receptors. For membrane-embedded receptors on the receptor cell surface, this region is embedded in the intramembranous portion of the receptor and is not susceptible to adhesion of any antibody or peptide. Likewise, in the free-floating solubul, decoy receptor fragments, this region is completely open and suitable for small peptide binding. Major Therapeutic Targets for anti-trapping monoclonal antibody and small peptide:

[0090] With this patent content, it is suggested that monoclonal antibodies and small peptides to be prepared against solubul and decoy receptors should be positioned as new pro- angiogenetic agents in the treatment of the following diseases and conditions.

[0091] Table 1: Diseases in which monoclonal antibodies and small peptides to be prepared against soluble decoy receptors will be used:

[0092] 1. Brain edema

[0093] 2. Pulmonary edema

[0094] 3. Lung fibrosis

[0095] 4. Pulmonary hypertension

[0096] 5. Liver fibrosis, Cirrhosis

[0097] 6. ARDS, RDS

[0098] 7. SIRS, Sepsis, cytokine storm

[0099] 8. Macrophage activation syndrome

[0100] 9. Differentiation syndrome

[0101] 10. Familial Mediterranean fever (FMF) attack

[0102] 11. Amyloidiosis (kidney and other tissues)

[0103] 12. Parkinson's and Alzheimer's

[0104] 13. Atherosclerosis, coronary artery disease

[0105] 14. Ischemic brain infarction

[0106] 15. OHSS syndrome

[0107] 16. Preeclampsia, IUGR

[0108] 17. Recurrent abortions

[0109] 18. Unexplained infertility

[0110] 19. Recurrent implantation failure (IVF-related)

[0111] 20. Type I and Type II diabetes and associated vascular complications

[0112] 21. Diabetic retinopathy and age-related macular degeneration

[0113] 22. Neonatal retinopathy (ROP) 23. Diabetic foot sores

[0114] 24. Slow-healing or non-healing wounds

[0115] 25. Accelerating wound healing

[0116] 26. Treatment of ischemic damage to other tissues resulting in necrosis and gangrene

[0117] 27. Ischemic vascular anomalies in the retina of the eye due to old age and diabetes

[0118] 28. Renal diseases such as nephrotic syndromes in which the glomerular endothelial layer of the kidneys is disrupted and acute and chronic renal failure

[0119] 29. Vascular diseases such as Takayasu arteritis, Behcet's disease, Kawazaki disease

[0120] 30. Celiac disease

[0121] 31. Some autoimmune diseases such as Sjogren's syndrome, SLE, RA and Scleroderma

[0122] 32. Erectile dysfunction due to vascular pathologies

[0123] 33. Post-COVID syndromes / long-COVID syndromes

[0124] 34. Reynaud's syndrome / Reynaud's phenomenon

Claims

CLAIMS1- The characteristics of the invention; in the treatment of inflammatory and vascular diseases, a predetermined amount of astragalus, monoclonal antibodies (anti-sFLTl, sFLT2, sENG, sTIE2) prepared against solubule-trap receptors (sFLTl, sFLKl, sTIE2 and sENG), small peptides (anti-Fab, anti-Fc) prepared against the Fab and Fc portions of sFLTl, sFLT2, sTIE2 and sENG decoy receptors, it is also the use of these molecules or all RNA types such as mRNA, miRNA and IncRNAs that target their associated DNA or mRNAs as drugs.2- According to claim 1 and it’ characterized by; cerebral edema, pulmonary edema, pulmonary fibrosis, pulmonary hypertension, cirrhosis, acute respiratory distress syndrome (ARDS), respiratory distress syndrome (RDS), systemic immune response syndrome (SIRS) and other diseases in tablet.3- According to claim 1 and it’ characterized by; sepsis is characterized by cytokine storm, macrophage activation syndrome, differentiation syndrome, familial Mediterranean fever (FMF) attack, amyloidiosis (kidney and other tissues), parkinson's and Alzheimer's disease, atherosclerosis, coronary vascular disease, ischemic cerebral infarction, OHSS syndrome, preeclampsia, IUGR, recurrent abortions, unexplained infertility.4- According to claim 1 and it’ characterized by; IVF-related recurrent implantation failure is characterized by Type I and Type II diabetes mellitus and associated vascular complications, diabetic retinopathy.5- According to claim 1 and it’ characterized by; age-related macular degeneration, neonatal retinopathy (ROP), diabetic foot wounds, late healing or non-healing wounds, treatment of ischemic damage to other tissues resulting in necrosis and gangrene, ischemic vascular anomalies in the retinal layer of the eye due to old age and diabetes,nephrotic syndromes in which the glomerular endothelial layer of the kidneys is disrupted, and kidney diseases such as acute and chronic kidney failure.6- According to claim 1 and it’ characterized by; vascular diseases such as Takayasu arteritis, Behcet's disease, Kawazaki disease, some autoimmune diseases such as celiac disease, Sjogren's syndrome, SLE, RA and scleroderma, and erectile dysfunction due to vascular pathologies.